The regime of microbiological laboratory. Main groups of microorganisms. Microscopic method of examination. Microbiologic method of examination Modern methods of microscopic examination. Methods of preparation of smears for microscopic examination. Simple techniques of smear staining

Microscopic method of examination. Structural peculiarities of bacterial cell and methods of their detection. Complex methods of staining. Grams and Ziehl-Neelsens methods. Basic methods of bacteria studying Structure of bacterial cell

Complex methods of staining: Anjeskys, Neissers, Gins techniques.Morphology of Spirochetes, Rickettsia, Chlamydia, Mycoplasmas, Fungi and Protozoa

 

 

 

Principles of heaLth protection and safety rules in the microbiological laboratory. DESIGN, EQUIPMENT, AND WORKING REGIMEN OF A MICROBIOLOGICAL LABORATORY.

Depending on their designation, microbiological laboratories may be bacteriological, parasitological, mycological, virological, immunological, and special (for the diagnosis of particularly virulent infections). A microbiological laboratory usually comprises the following premises: (1) the preparatory room for preparing laboratory glassware, making nutrient media and performing other auxiliary works; (2) washroom; (3) autoclaving room where nutrient media and laboratory glassware are sterilized; (4) room for obtaining material from patients and carriers; (5) rooms for microscopic and microbiological studies comprising one or two boxes. Laboratory animals employed for biological sampling are kept in separate isolated premises (an animal unit).

It is preferable that laboratory rooms should have only one entrance. To facilitate such procedures as washing and treatment with disinfectants, the walls are painted with light-colored oil paint or lined with ceramic tiles, whereas the floors are covered with linoleum.

The infective material is examined in a separate room. The work requiring the observation of a microbiological regimen (inoculation of the material for sterility, contamination of tissue cultures, chicken embryos, etc.) requires special premises (box) whose floor space should be convenient for two workers. Prior to and after work the entire box is treated with disinfectant solutions and irradiated with bactericidal lamps.

Equipment of the laboratory. Laboratory furniture should be simple and convenient. Laboratory tables covered with special enamel, linoleum, or other easily disinfecting materials are placed near windows. Safe-refrigerators are used for storing microorganism cultures.

The main pieces of equipment in the bacteriological laboratory include apparatuses for different types of microscopy, apparatuses for heating (gas and alcohol burners, electrical stoves, etc.), incubators, refrigerators, sterilizing apparatuses (sterilizer, Koch apparatus, Pasteur stove, coagulator, etc.), a centrifuge, distillator, etc.

The used material is rendered safe in the way which is employed in bacteriological laboratories.

Immunological laboratory is furnished with incubators, refrigerators, glassware, and apparatuses necessary to run serological reactions on a wide scale.

The design and furnishing of a virological laboratory somewhat differ from those of a bacteriological one. The premises of a virological laboratory should include a box with pre-box inclosure separated by a glass partition where work with cell cultures and chicken embryos is conducted.

Apart from glassware and usual equipment, this type of laboratory should be furnished with chambers of deep and superdeep freezing (-30-70 C), refrigerator chambers (-20 C), centrifuges with a rotation velocity of 1500-3000 X g and over to ensure purification of the virus from ballast substances and its concentration. Other pieces of equipment include a homogenizer to comminute tissues, ovoscope, burners for ampoule soldering, and a vacuum pump.

Before starting the work, the premises are disinfected in a way which is employed for disinfecting the box of microbiological laboratories.

The premises are treated, using disinfectant solutions and bactericidal lamps.

 

Rules of work in the laboratory. 1. The personnel working at laboratories is supplied with medical coats and kerchiefs or caps. While working in boxes, one should wear a sterile coat, cap, and gauze mash. To make an autopsy of animals, put on an oil cloth apron, oversleeves, and rubber gloves. Special clothes protect the worker and also prevent contamination of the material to be studied with foreign microflora.

2. Eating and smoking in the laboratory are strictly forbidden.

3. Unnecessary walking about the laboratory, sharp movements, and irrelevant conversations should be discouraged.

4. In the process of examination the working place should be kept clean and tidy. Bacteriological loops are rendered harmless by burning them in the burner's flame; used spatulas, glass slides, pipettes, and other instruments are placed into jars with disinfectant solution.

5. Upon the completion of work the nutrient media with inoculated cultures are placed into an incubator; museum cultures, into safe-refrigerators; devices and apparatuses are set up in places specially intended for them. Wipe tables with disinfectant solution and thoroughly wash the hands.

6. If the material to be analyzed or the culture of microorganisms is accidentally spilt onto the hands, table, coat, or shoes, they should be immediately treated with 1 per cent solution of chloramine.

 

Rules of work at microbiological laboratory with special regime.

Before entering the laboratory, all personnel take off the overcoats at the cloakroom. In the next room with individual closets they take off the remaining clothes and the underwear, put pyjamas, medical coats, kerchief (or cap), socks, and slippers (Set 1 of protective clothes of the fourth type). When working in the autopsy room, put on an anti-plague suit (Set 2), a second autopsy coat, helmet, cotton wool-gauze mask, rubber gloves, oil-cloth apron, and oversleeves. To protect the eyes, one should wear goggles. An anti-plague suit of the first type is put on in the following order: (1) overalls; (2) socks; (3) high boots; (4) helmet; (5) anti-plague coat; (6) cotton wool-gauze mask (place cotton wool tampons over the wings of the nose); (7) goggles; (8) gloves; (9) oil cloth apron and oversleeves (these are put on while working in the autopsy room). A person working with infective material should have a towel soaked in 3 per cent solution of lysol. Upon completion of work immerse gloved hands into 5 per cent solution of lysol for 2 min and repeat this procedure after removing each item of the clothing. The anti-plague suit is taken off in the reverse order, with the exception of gloves which are the last to be taken off. Then. they are folded with the external surface inside and immersed into 5 per cent solution of lysol or I per cent solution of chloramine for 2 hrs. The goggles are put into 70 per cent alcohol.

Following autopsy, instruments and syringes are boiled in lysol for at least 40 min. All used material and corpses of animals are burnt or sterilized.

 

The Department of Microbiology is a place of work dangerous for health with the risk of professional infection.

Students are allowed to take off their clothes only in a cloakroom that must be locked and the key is placed in a reserved site to prevent any theft. If a student has any valuable things or larger sums of money with him, then he must announce it to his teacher who will secure its safe deposition. However, taking any valuables in is not recommended.

The students come into the hall through the entrance from the waiting room under the teacher's surveillance. They are lending protective coats which must be taken off before entering other departments. It is forbidden to damage these coats, to take away any infective material from the hall, as well as tools and coats. Students' own coats must not be worn.

Students must observe the principles of hygiene. They must disinfect and wash their hands always after contaminating them with a biological material and before leaving the hall. For disinfecting hands, 0.5 % chloramine is used for 2 minutes. Then the hands are to be rinsed with warm water and washed with soap. It is forbidden to eat, smoke and drink in the laboratory. It is also necessary to avoid rubbing one's eyes or nose, scratching one's head, biting nails, pencils, etc. The space in front of the building and in the waiting room must be kept clear and quiet.

 

PRINCIPAL MICROBIOLOGICAL PROCEDURES

A complex of bacterioscopic, bacteriological, serological, allergological, and biological techniques is used in the microbiological diagnosis of bacterial infections. Depending on the nature of the given infectious disease, one of these methods is used as the main one, while the others are supplementary. Such biological substances as blood, faeces, urine, cerebrospinal fluid, bile, etc. serve as the material for microbiological diagnosis.

The main microbiological techniques pertaining to the laboratory diagnosis of bacterial infection are outlined below. Interpretation and specification of each technique with regard to specific infections are presented in the respective sections.

 

BACTERIOSCOPIC EXAMINATION

: : : : Microscope

Modern methods of microscopic examination. Contemporary microbiological laboratories employ not only conventional methods of optical microscopy in transmitted light (Fig. 1) but also such special ones as dark-field microscopy and phase-contrast, luminescent, and electron microscopy.

 

Light microscopy. A light microscope is fitted with dry and immersion objectives. A dry objective with a relatively large focal distance and weak magnification power is ordinarily utilized for studying large biological and histological objects. In examining microorganisms, the immersion objective with a small focal distance and a higher resolving power is predominantly employed.

In microscopic examination with the help of an immersion objective the latter is immersed in oil (cedar, peachy, "immersiol", etc.) whose refractive index is close to that of glass. When such a medium is used, a beam of light emerging from the slide is not diffused and the rays arrive at the objective without changing their direction (Fig. 2). The resolving power of the immersion objective is about 0.2 mcm. The maximum magnification of modern light microscopes is as high as 2000-3000.

 

Figure 1. A light microscope

Figure 2. The course of rays in the dry (1) and oil-immersion (2) systems

 

: : : : Scheme_1

 

Table. Comparative sizes of different objects

 

Dark-field microscopy belongs to ultramicroscopic methods. Living objects 0.02-0.06 mcm in size are visualized in lateral illumination in a dark field of vision. In order to achieve bright lateral illumination, the usual condenser is replaced by a special parabolic condenser in which the central part of the lower lens is darkened, while the lateral surface is mirror (Fig. 3). This condenser intercepts the central portion of the parallel beam of rays forming a dark field of vision. The marginal rays pass through the circular slit, fall on the lateral mirror surface of the condenser, are reflected from it, and concentrate in the focus. On encountering in their path the cells of microorganisms or other optically non-homogeneous structures, the ray of light is reflected from them and gets into the objective. Cells of microorganisms and other objects are brightly illuminated in this case.

An electrical illuminator serves as a source of artificial light. To achieve lateral illumination, one needs a parallel beam of light which is created by means of a flat mirror of the microscope.

 

: : : : Dark-field

Figure 3. Diagram of a dark-field microscope showing the path of light. The dark-field ring in the condenser blocks the direct passage of light through the specimen and into the objective lens. Only light that is reflected off a specimen will enter the objective lens and be seen.

 

In dark-field microscopic examination a dry system is typically employed (objective 40). A small drop of the studied material is placed on the slide and covered with a cover-slip, taking care to prevent the formation of air bubbles. A drop of immersion oil is pipetted on the upper lens of the condenser. This oil should fill the space between the condenser and the slide.

Microscopy with an oil-immersion system makes use of a special objective with a diaphragm trapping the rays which pass unobstructed through a homogeneous medium.

Dark-field microscopy is employed for detecting unstained causative agents of syphilis, recurrent typhoid fever, leptospirosis, and other illnesses, as well as for investigating the motility of microorganisms. Yet, dark-field microscopic examination does not allow a good study of their form, to say nothing about their internal structure. Modified techniques of light microscopy are utilized for this purpose.

Phase-contrast microscopy is based on the fact that the optical length of the light traveling in any substance depends on its refractive index. Light waves transversing through optically denser sites of the object lag in their phase behind the light waves which do. not have to pass through these sites. The intensity of light in this case remains unaltered but the phase of fluctuation, detected by neither eye nor photoplate, is changed. To increase resolution of the-image, the objective is fitted with a special semi-transparent phase plate to create difference in the wave length between the rays of the background and the object. If this difference reaches one-fourth of the wave length, a visually tangible effect occurs when a dark object is clearly seen against a light background (positive contrast) or vice versa (negative contrast) depending on the structure of the phase plate.

Phase-contrast microscopy does not enhance the resolving power of the optical system but helps to elucidate new details of the structure of living microorganisms and to study different stages of their development, the effect on them of chemical agents, antibiotics, and other factors.

Luminescent microscopy. Luminescence (or fluorescence) is the ability of some objects and dyes to fluoresce upon their exposure to. ultraviolet and other short-wave rays of light.

It is commonly accepted to distinguish between inherent (primary) and secondary fluorescence. In primary fluorescence the test object contains substances capable to fluoresce upon their exposure to ultraviolet rays. Most of objects are not inherently fluorescent, so. prior to luminescent microscopy they have to be treated with dyes (fluorochromes) capable to fluoresce. The following substances are usually used as fluorochromes: auramine (for tuberculosis mycobacteria), acridine yellow (for gonococci), coryphosphine (for Corynebacteria of diphtheria), fluoresceinisothiocyanate (FITC) (for making labelled antisera), etc.

A specimen to be examined by luminescent microscopy is prepared in the usual manner, fixed in acetone or ethanol for 5-10 min and exposed to a fluorochrome for 20-30 min. Thereafter, the resultant preparation is washed with tap water for 15-20 min, covered by a cover-slip, and placed under a microscope.

Luminescent microscopes represent ordinary biological microscopes furnished with a bright source of illumination and a set of light filters which isolate a short-wave (ultraviolet or blue-violet) part of the spectrum inducing luminescence. Fluorochromes, binding "with nucleic acids or proteins, form stable complexes that give away yellow-green, orange-red, and brown-red light under the lumimescent microscope.

Fluorescence microscopy has the following advantages as compared with the conventional microscopic methods: colour image; considerable contrasting; possibility to study both live and dead microorganisms, transparent and non-transparent objects; detection of individual bacteria, viruses, and their antigens and possibility of their localization; differentiation of individual components of the cell.

Electron microscopy. In the electron microscope, a beam of electrons passing in vacuum and blocked by an anode is used instead of light. The source of electrons is an electron gun (a tungsten wire heated up to 2500-2900 C). The optic lenses are replaced by electromagnets, An electrical field of 30 000-50 000 V is generated between the tungsten wire and anode, which imparts high velocity to electrons which arrive at the first electromagnetic lens (a condenser) having passed through the anode opening. At their exit from the condenser the electron rays are accumulated in the plane of the studied object, deviate at different angles due to variable thickness and density of the preparation, and get into the electromagnetic lens of the objective equipped with a diaphragm. Electrons showing only little deviation upon entering the object pass through the diaphragm, while those deviating at a greater angle are retained, which ensures contrasting of the image- The lens of the objective gives an. intermediate enlarged image which is viewed through the viewing window. The projection lens may ensure a multiple magnification of the image; this image is perceived by a fluorescent screen and photographed. The most recent models of electron microscopes permit visualization of particles 1.4 nm in size.

Electron microscopy is extensively used in microbiology for detailed investigation into the structure of microorganisms. It is also employed in virology for diagnostic purposes.

To study preparations under the electron microscope, special films absorbing small numbers of electrons and fixed on supporting meshes are utilized instead of glass slides. Such films are made of collodium, aluminium oxide, and quartz. The material to be studied is thoroughly cleansed of various admixtures and placed on the film. A very thin layer remaining on the film after evaporation of the fluid is subjected to microscopic examination. The electron microscope may also be used for studying sections of tissues, cells, and microorganisms which are obtained with the help of an ultramicrotome. The preparations are contrasted by means of electron-dense (electron-capturing) substances, using such procedures as the spraying with heavy metals and treatment with phosphotungstic acid. uranyl acetate, salts of osmic acid, etc.

Scanning Electron Microscopy. The scanning electron microscope uses a fine beam or spot of electrons that is focused rapidly back and forth over the specimen. As the electrons strike the surface of particles in the sample, secondary electrons are emitted, which are collected by a detector to provide an image of the specimen's surface. This instrument does not require that the sample be sectioned and provides some spectacular three dimensional images. In addition, because the energy of the secondary emitted electron is determined by the identity of the scattering atom, the energy spectrum of these electrons provides information about the location and content of the different elements.

 

 

Table 1

Comparison of Various Types of Microscopes

 

Type of microscope

Maximum useful magnification

 

Resolution

 

Description

 

Bright-field

1,500x

100-200 nm

Extensively used for the visualization of micro organisms; usually necessary to stain specimens for viewing

Dark-field

1.500X

100-200 nm

Used for viewing live microorganisms, particularly those with characteristic morphology; staining not required; specimen appears bright on a dark background

Fluorescence

1,500X

100-200 nm

Uses fluorescent staining; useful in many diagnostic procedures for identifying microorganisms

Phase contrast

1.500X

100-200 nm

Used to examine structures of living microorganisms; does not require staining

TEM (trans- mission electron microscope)

500,000-1,000,000X

0.1 nm

Used to view ultrastructure of microorganisms, including viruses; much greater resolving power and useful magnification than can be achieved with light microscopy

SEM (scanning electron microscope)

10,000-100,000X

1-10 nm

Used for showing detailed surface structures of microorganisms, produces a three-dimensional image

 

 

Preparation and Staining of Smears

Preparation and staining of smears, as well as other microbiological procedures, are performed in a prepared working place. The working table should contain only those materials and objects which are necessary for the given examination, namely; the object to be studied (blood, pus, sputum, faeces, etc.), test tubes or dishes with a culture of microorganisms, sterile distilled water or isotonic sodium chloride solution, a stand for a bacteriological loop, a jar with clean glass slides, and felt tip pens. Other necessary items include a gas or alcohol burner, staining solutions, a basin with a supporting stand (bridge) for slides, a washer with water, forceps, filtering paper, a jar with disinfectant solution used for sterilizing preparations and pipettes.

Methods of the treatment of cover-slips. Now cover-slips are boiled in a 1 per cent solution of sodium hydrocarbonate. rinsed with water, immersed in a weak solution of hydrochloric acid, and then rinsed with water once again. The used glass slips and slides are placed in a concentrated sulphuric acid (technical grade) for 2 hrs or in a mixture of sulphuric acid, potassium bichromate, and water (100:50:1000), thoroughly washed with water, boiled in sodium hydrocarhonate solution or ill sodium hydroxide, then washed with water once more, dried with clean linen cloth, and stored in alcohol or an alcohol-ether mixture in jars with ground stoppers. Detergents are also utilized for washing slides. Prior to making smears take slides with a forceps from the solution whore they have been kept and blot them dry. Hold them by the edges with your fingers. A drop placed on the properly prepared glass spreads uniformly and does not assume a spherical form.

Preparation of a smear. Before making a preparation, glass slides are flamed to ensure their additional degreasing.

In preparing a smear from bacterial culture grown on a solid medium, a drop of isotonic saline or water is transferred onto the precooled glass. A test tube with the culture is taken by the thumb and the index finger of the left hand. The loop is sterilized in the flame. A cotton-wool plug is pinched by a small finger of the right hand, removed from the test tube, and left in this position. The edges of the test tube are flamed and then the loop is introduced into the test tube through the flame. Having cooled the loop against the inner wall of the tube, the loop is touched to the nutrient medium where it meets with the glass wall (if the loop is not sufficiently cooled, it induces cracking and melts the medium). Then the loop is touched to the culture of the microorganisms on the surface of the medium. Then the loop is withdrawn, the edges of the test tube are quickly flamed, the tube is closed with a stopper passed through the flame, and then replaced into the test tube rack. All the above described procedures are made above the flame. The culture sample is placed with the loop into a drop of water on the glass slide and spread uniformly with circular movements on an area of 1-1.5 cm in diameter, then the loop is flamed.

In preparing a smear from bacterial culture grown in a fluid nutrient medium, a drop of the culture is taken with a loop or a Pasteur pipette (the pipette is immersed in disinfectant solution), transferred onto the middle of the flamed glass slide and spread uniformly. On the other side of the glass slide the preparation is delineated by a wax pencil since very thin smears are almost invisible. The number of the analysis or culture is marked on the left side of the glass.

To prepare a smear from pus or sputum, two glass slides are used. A small amount of the material is transferred with a sterile loop or needle onto the middle of the glass slide and covered with a second one so that one-third of the surface of both slides remains free. Then, the glass slides are pulled gently aside (Fig. 4), which results in the formation of two large smears of the same thickness.

 

Figure 4. Preparation of a smear from sputum

 

 

 

 

Blood smear is prepared in the following way. Using a sterile needle, puncture a disinfected fourth finger of the left hand. Wipe away the first drop of blood with a piece of dry cotton wool and touch the thoroughly-cleansed glass slide to the second drop of blood. Quickly put the slide on the table, supporting it with the left hand. Place the end of a second narrower cover slide in touch with the drop of the blood at the 45-degree angle (Fig. 5).

Figure 5. Preparation of a smear from blood:

1-6 stages of thin smear preparation; 7-9 inadequately prepared smears

 

Putting some pressure on the cover slide, smoothly and rapidly move it along the glass slide in the leftward direction stopping it at a distance of 1-1.5 cm from the edge. The correctly prepared smear is yellowish in colour and semitransparent.

Impression preparations are made of the internal organs of cadavers and solid foodstuffs (meat, sausage, ham, etc.). The surface of the organs or of a foodstuff is burnt with a red-hot scalpel, and a piece of the material is cut off from this site. The surface of the section is touched to the glass in two-three places.

 

 

Drying and fixation of the smear. Thin smears usually dry rapidly in the air at room temperature; thicker ones are dried in an incubator or by holding them above the flame of a burner. The slide is held by the edges with the thumb and forefinger, the smear upward, while the middle finger is placed under the glass to regulate the degree of its heating and to prevent coagulation of the bacterial protein and destruction of the cell structure.

The dried smears are flamed to kill and fix the bacteria on the glass slide, preventing thereby their washing off during staining. The dead microorganisms are more receptive to dyes and present no danger for the personnel working with them. The glass slide is grasped with a forceps or with the thumb and index finger of the right hand, the smear being in the upside position, and passed three times through the hottest part of the burner's flame. Fixation with this technique takes about 5-6 s, with the exposure to the Game being about 2 s.

Blood smears, impression smears, and smears from bacterial culture deforming at high temperature are treated with one of the following fixatives: (1) methyl alcohol (for 5 min); (2) ethyl alcohol (10-15 min); (3) Nikiforov's mixture: equal volumes of ethyl alcohol and ether (10-15 min); (4) acetone (5 min); (5) fumes of osmic acid and formalin (several seconds).

Staining of a smear. Smears are stained with aniline dyes. Chemically, acid, alkaline, and neutral dyes can be distinguished. Alkaline dyes, whose staining portion of the molecule is charged positively, more actively conjugate with a negatively charged bacterial cell.

Staining of bacteria is a complex physicochemical process. Interaction of the dye with the cell substances results in the formation of salts ensuring stability of staining. Relationship between various types of microorganisms and dyes is called a tinctorial property.

The following dyes are employed most extensively: (1) red (basic fuchsine, acid fuchsine, safranine, neutral red, Congo red); (2) blue (methylene blue, toluidine blue, trypan blue, etc.); (3) violet (gentian, methyl or crystal); and (4) yellow-brown (vesuvin, chrysoidine).

All the employed dyes are powder-like or crystalline. Such dyes as basic fuchsine, gentian violet, and methylene blue are usually used to prepare in advance saturated alcoholic solutions (1 g of the dye per 10 ml of 96 per cent alcohol). Saturated alcohol and phenol dye solutions are utilized to prepare water-phenol or water-alcohol solutions to be employed in staining by simple and complex techniques.

 

Simple techniques of staining make use of only one dye and demonstrate the form of bacteria.

Preparation of dye solutions for simple staining. Basic fuchsine is used for preparing Ziehl's phenol fuchsine which is stable upon storing. Ziehl's fuchsine is employed to stain in red colour the acid-fast microorganisms and spores.

Ziehl's phenol fuchsine

Basic fuchsine 1 g

95 per cent alcohol 10 mg

Crystal phenol 5 g

Glycerol several drops

Distilled water 100 ml

 

Fuchsine, together with phenol crystals and some drops of glycerol, is homogenized by grinding in a mortar, adding simultaneously small amounts of alcohol. Then, with the obtained mass being continuously stirred, distilled water is gradually added. The resultant dye is allowed to stand at room temperature for 48 hrs and is then filtered. Shelf life is prolonged.

 

Pfeiffer's fuchsine

Ziehl's fuchsine 1 ml

Distilled water 9 ml

In using Pfeiffer's fuchsine, the solution should be freshly prepared.

 

Saturated alcoholic solution of methylene blue

Methylene blue 10 g

95 per cent alcohol 100 ml

 

Alkaline solution of methylene blue as proposed by Loeffler

Saturated alcoholic solution of methylene blue 30 ml

Sodium hydroxide or potassium hydroxide (1 per cent solution) 1 ml

Distilled water 100 ml

 

 

Water-alcoholic solution of methylene blue

Saturated alcoholic solution of methylene blue 10 ml

Distilled water 100 ml

Old solutions of this dye have a better staining ability.

 

The fixed preparation is placed, the smear upward, on the support. A dye solution is pipetted onto the entire surface of the smear. With Pfeiffer's fuchsine the staining lasts 1-2 min, with alkaline solution of Loeffler's methylene blue or water-alcoholic solution of methylene blue, 3-5 min. Following the staining procedure the dye is dispensed, the preparation is washed with water, dried between sheets of filter paper, and then examined under the oil-immersion objective.

Live staining of microorganisms is made with methylene blue, neutral red, and other weakly poisonous dyes in a 1:10 000 dilution. For this purpose, a drop of the test material is mixed with the dye solution on a glass slide and covered with a cover-slip. Microscopic examination is carried out with a 40X objective.

In a negative method of living bacteria staining by Bum's technique, the bacteria remain unstained against a dark background. In a drop of Indian ink diluted with distilled water 1 to 10 the culture to be tested is introduced and spread uniformly with a loop or the edge of a glass slide. The smear is air dried. Nigrosin, Congo red, and other dyes may occasionally be utilized instead of Indian ink.

 

ii. bACTERIA CLASSIFICATION. GENERAL CHARACTERICTICS OF PROCARYOTIC CELLS. Grams method

http://www.innvista.com/health/microbes/bacteria/classif.htm

http://www.earthlife.net/prokaryotes/phyla.html

http://web.uct.ac.za/depts/mmi/lectures/bactax/ppframe.html

http://www.gsbs.utmb.edu/microbook/ch003.htm

http://www.bmb.leeds.ac.uk/mbiology/ug/ugteach/dental/tutorials/classification/introduction.html

http://www.microbiol.org/WPaper.Gram.htm

 

A. Morphology of Bacteria

Bacteria (Gk bakterion small staff) are, for the most part, unicellular organisms lacking chlorophyll Their biological properties and predominant reproduction by binary fission relates them to prokaryotes The size of bacteria is measured in micrometres (mcm) and varies from 0.1 mcm (Spiroplasma, Acholeplasma) to 16-18 mcm (Spirillum volutans). Most pathogenic bacteria measure 0.2 to 10 mm

The shape of spherical bacteria represents a certain ratio of surface area (As = 4π2) to volume (Vs = 4/πr3). For those cells having a proper cylindrical shape the formulae will be At = 2πb(b + 2a}; Vi = 2πab2, where a is equal to one-half the maximum length, b is equal to one-half the maximum width, and r is equal to the radius of the spherical cell. The shape as well as the dimensions of microbes is not absolutely constant Morphological differences are found in many bacterial species The organisms are subject to change with the surrounding environmental conditions However, in relatively stable conditions, the microbes are capable of retaining their specific properties (size, shape) inherited during the process of evolution

Morphologically, bacteria possess three main forms (Fig. 6). They are either spherical (cocci), rod-shaped (bacteria, bacilli, and clostridia) or spiral-shaped (vibriones and spirilla)

 

: : : : fig2_1

FIGURE 6 Typical shapes and arrangements of bacterial cells

 

Cocci (Gk. chokes berry). These forms of bacteria (Fig ) are spherical, ellipsoidal, bean-shaped, and lancelet Cocci are subdivided into six groups according to cell arrangement, cell division and biological properties

1 Micrococci (Monococci, Micrococcus) The cells are arranged singly or irregularly They are saprophytes, and live in water and in air (M agilis, M.roseus, M luteus, etc )

: : : : Ris_05_micrococcus

 

2. Diplococci (Gk. diplos double) divide in one plane and remain attached in pairs These include, meningococcus, causative agent of epidemic cerebrospinal meningitis, and gonococcus, causative agent of gonorrhoea and blennorrhoea.

: : : : Ris_06_diplococcus_gonococcus: : : : Ris_07_pneumococcus in mice liver Roman-Gimza

 

3. Streptococci (Gk. streptos curved, kokkos berry) divide in one plane and are arranged in chains of different length. Some streptococci are pathogenic for humans and are responsible for various diseases

: : : : Ris_08_streptococcus

 

4. Tetracocci (Gk. tetra four) divide in two planes at right angles to one another and form groups of ours. They very rarely produce diseases in humans

: : : : Ris_10_

 

5. Sarcinae (L. sarcio to tie) divide in three planes at right angles to one another and resemble packets of 8, 16 or more cells They are frequently found in the air Virulent species have not been encountered

: : : : Ris_11_sarcina

 

6. Staphylococci (Gk. staphyle cluster of grapes) divide in several planes resulting in irregular bunches of cells, sometimes resembling clusters of grapes Some species of Staphylococci cause diseases in man and animals.

: : : : Ris_09_staphylococccus

 

 

 

Rods. Rod-shaped or cylindrical forms (Fig. 7) are subdivided into bacteria, bacilli, and clostridia. Bacteria include those microorganisms which, as a rule, do not produce spores (colibacillus, and organisms responsible for enteric fever, paratyphoids, dysentery, diphtheria, tuberculosis, etc ) Bacilli and clostridia include organisms the majority of which produce spores (hay bacillus, bacilli responsible for anthrax, tetanus, anaerobic infections, etc).

Rod-shaped bacteria exhibit differences in form. Some are short (tularaemia bacillus), others are long (anthrax bacillus), the majority have blunted ends, and others have tapered ends (fusobacteria).

According to their arrangement, cylindrical forms can be subdivided into three groups (1) diplobacteria and diplobacilli occurring in pairs(bacteria of pneumonia); (2) streptobacteria or streptobacilli occurringin chains of different length (causative agents of chancroid, anthrax), (3) bacteria and bacilli which are not arranged in a regular pattern (these comprise the majority of the rod-shaped forms).

: : : : Image 2met2

 

Figure 7b

 

 

Figure 7. Rod-shaped bacteria and some spiral-shaped bacteria

Fig. 7a: 1 diplobacteria; 2- rods with rounded, sharpened and\or thickened ends; 3- different rod-shaped forms and streptobacteria

Fig. 7b: 1-vibriones 2spirilla

 

: : : : Ris_12_

: : : : Ris_14_Bac_subtilis

Some rod-shaped bacteria have pin-head thickenings at the ends(causative agents of diphtheria); others form lateral branchings (bacilli of tuberculosis and leprosy).

There is a significantly greater number of rod-shaped bacteria than coccal-shaped organisms. This is explained by the fact that in rod-shaped bacteria the ratio of surface area to volume is higher. Thus, a larger surface area is in direct contact with nutrient substances in the surrounding medium.

 

Spiral-shaped bacteria. Vibriones and spirilla belong to this group of bacteria (Fig. 7).

1. Vibriones (L. vibrio to vibrate) are cells which resemble a comma in appearance- Typical representatives of this group are , the causative agent of cholera, and aquatic vibriones which are widely distributed in fresh water reservoirs.

2. Spirilla (L. spira coil) are coiled forms of bacteria exhibiting twists with one or more turns. Only one pathogenic species is known (Spirillum minus) which is responsible for a disease in humans transmitted through the bite of rats and other rodents (rat-bite fever, sodoku). Microbes exhibit pleomorphism, they are subject to individual variations, unassociated with age or stage of development, causing the existence of different forms of cells in the same species. They are extremelylabile, and susceptible to changes which are associated with such factors as temperature, nutrients, salt concentration, acidity, metabolites, disinfectants, drugs, and body resistance.

3. Spirochetes: Treponema, Borrelia, Leptospira

: : : : Ris_20_vibrio cholerae

Vibrio comma

: : : : Ris_21_spirilla

Spirilla

 

: : : : Ris_23_borrelia

Spirochetes

 

 

PROKARYOTIC CELL STRUCTURE

 

The cytoplasm is enclosed within a lipoprotein cell membrane, similar to the prokaryotic cell membrane. Most animal cells have no other surface layers; many eukaryotic microorganisms, however, have an outer cell wall, which may be composed of a polysaccharide such as cellulose or chitin or may be inorganic, as in the silica wall of diatoms.

 

: : : : Scheme_8

 

The prokaryotic cell is simpler than the eukaryotic cell at every level, with one exception: the cell wall may be more complex.

 

 

: : : : Scheme_1

 

The prokaryotic nucleus can be seen with the light microscope in stained material. It is Feulgen-positive, indicating the presence of DNA. The negatively charged DNA is at least partially neutralized by small polyamines and magnesium ion, but histonelike proteins have recently been discovered in Cytoplasmic Structures

Prokaryotic cells lack autonomous plastids, such as mitochondria and chloroplusts. The electron transport enzymes are localized instead in the cell membrane; in photosynthetic organisms, the photosynthetic pigments are localized in lamellae underlying the cell membrane. In some photosynthetic bacteria, the lamellae may become convoluted and pinch off into discrete particles called chromatophores.

Bacteria often store reserve materials in the form of insoluble cytoplasmic granules, which are deposited as osmotically inert, neutral polymers. In the absence of a nitrogen source, carbon source material is converted by some bacteria to the polymer poly-β-hydroxybutyric acid and by other bacteria to various polymers of glucose such as starch and glycogen. The granules are used as carbon sources when protein and nucleic acid synthesis is resumed. Similarly, certain sulfur-oxidizing bacteria convert excess H2s from the environment into intracellular granules of elemental sulfur. Finally, many bacteria accumulate reserves of inorganic phosphate as granules of polymerized metaphosphate, called volutin. Volutin granules are also called metachromatic granules because they stain red with a blue dye. They are characteristic features of corynebacteria.

Microtubular structures, characteristic of eukaryotic cells, are generally absent in prokaryotes. In a few instances, however, the electron microscope has revealed bacterial structures that resemble microtubules.

STAINING

Stains combine chemically with the bacterial protoplasm; if the cell is not already dead, the staining process itself will kill it. The process is thus a drastic one and may produce artifacts.

The commonly used stains are salts. Basic stains consist of a colored cation with a colorless anion (eg, methylene blue chloride"); acidic stains are the reverse (eg, sodium + eosinate"). Bacterial cells are rich in nucleic acid, bearing negative charges as phosphate groups. These combine with the positively charged basic dyes. Acidic dyes do not stain bacterial cells and hence can be used to stain background material a contrasting color (see Negative Staining, below).

The basic dyes stain bacterial cells uniformly unless the cytoplasmic RNA is destroyed first. Special staining techniques can be used, however, to differentiate flagella, capsules, cell walls, cell membranes, granules, nuclei, and spores.

 

Gram Stain

An important taxonomic characteristic of bacteria is their response to Gram's stain. The gram-staining property appears to be a fundamental one, since the Gram reaction is correlated with many other morphologic properties in phylogenetically related forms. An organism that is potentially gram-positive may appear so only under a particular set of environmental conditions and in a young culture.

The gram-staining procedure begins with the application of a basic dye, crystal violet. A solution of iodine is then applied; all bacteria will be stained blue at this point in the procedure. The cells are then treated with alcohol. Gram-positive cells retain the crystal violet-iodine complex, remaining blue; gram-negative cells are completely decolorized by alcohol. As a last step, a counter stain such as the red dye safranin) is applied so that the decolorized gram-negative (cells will take on a contrasting color; the gram-positive cells now appear purple.

The Gram-Positive Cell

As previously mentioned, Gram-positive bacteria are characterized by their blue-violet color reaction in the Gram-staining procedure. The blue-violet color reaction is caused by crystal-violet, the primary Gram-stain dye, complexing with the iodine mordant. When the decolorizer is applied, a slow dehydration of the crystal-violet/iodine complex is observed due to the closing of pores running through the cell wall. Because the crystal-violet is still present in the cell, the counter stain is not incorporated, thus maintaining the cell's blue-violet color. If you recall, most cell walls contain peptidoglycan, a molecule made of amino acids and sugar. A distinguishing factor among Gram-positive bacteria is that roughly 90% of their cell wall is comprised of peptidoglycan and a Gram-positve bacteria can have more than 20 layers of peptidoglycan stacked together to form the cell wall. Examples of common Gram-positive cells include Staphylococcus aureus and Streptococcus cremoris, a bacterium used in dairy production.

 

The Bacterial Cell Wall

The bacterial cell wall is a unique structure which surrounds the cell membrane. Although not present in every bacterial species, the cell wall is very important as a cellular component. Structuraly, the wall is necessary for:

        Maintaining the cell's characteristic shape- the rigid wall compensates for the flexibility of the phospholipid membrane and keeps the cell from assuming a spherical shape

        Countering the effects of osmotic pressure- the strength of the wall is responsible for keeping the cell from bursting when the intracellular osmolarity is much greater than the extracellular osmolarity

        Providing attachment sites for bacteriophages- teichoic acids attached to the outer surface of the wall are like landing pads for viruses that infect bacteria

        Providing a rigid platform for surface appendages- flagella, fimbriae, and pili all emanate from the wall and extend beyond it

 

: : : : Scheme_9

 

The cell walls of all bacteria are not identical. In fact, cell wall composition is one of the most important factors in bacterial species analysis and differentiation. There are two major types of walls: Gram-positive and Gram-negative. The cell wall of Gram-positive bacteria consists of many polymer layers of peptidoglycan connected by amino acid bridges. A schematic diagram provides the best explanation of the structure. The peptidoglycan polymer is composed of an alternating sequence of N-acetylglucosamine and N-acetyl-muraminic acid. It's a lot easier to just remember NAG and NAMA. Each peptidoglycan layer is connected, or crosslinked, to the other by a bridge made of amino acids and amino acid derivatives. The particular amino acids vary among different species, however. The crosslinked peptidoglycan molecules form a network which covers the cell like a grid. Also, 90% of the Gram-positive cell wall is comprised of peptidoglycan.

The cell wall of Gram-negative bacteria is much thinner, being comprised of only 20% peptidoglycan. Gram-negative bacteria also have two unique regions which surround the outer plasma membrane: the periplasmic space and the lipopolysaccharide layer. The periplasmic space separates the outer plasma membrane from the peptidoglycan layer. It contains proteins which destroy potentially dangerous foreign matter present in this space. The lipopolysaccharide layer is located adjacent to the exterior peptidoglycan layer. It is a phospholipid bilayer construction similar to that in the cell membrane and is attached to the peptidoglycan by lipoproteins. The lipid portion of the LPS contains a toxic substance, called Lipid A, which is responsible for most of the pathogenic affects associated with harmful Gram-negative bacteria. Polysaccharides which extend out from the bilayer also contribute to the toxicity of the LPS. The LPS, lipoproteins, and the associated polysaccharides together form what is known as the outer membrane.

Keep in mind that the cell wall is not a regulatory structure like the cell membrane. Although it is porous, it is not selectively permeable and will let anything pass that can fit through its gaps.

The Gram-Negative Cell

Unlike Gram-positive bacteria, which assume a violet color in Gram staining, Gram negative bacteria incorporate the counter stain rather than the primary stain. Because the cell wall of Gram(-) bacteria is high in lipid content and low in peptidiglycan content, the primary crystal-violet escapes from the cell when the decolorizer is added. This is because primary stains like to bind with peptidoglycan- something the G(-) cell lacks. The pathogenic nature of Gram(-) bacteria is usually associated with certain components of their cell walls, particularly the lipopolysaccharide (endotoxin) layer. The Black Plague, which wiped out a third of the population of Europe, was caused by the tiny G(-) rod, Yersinia pestis. Most enteric (bowel related) illnesses can also be attributed to this group of bacteria.

I. Gram-staining Procedure

Gram-staining is a four part procedure which uses certain dyes to make a bacterial cell stand out against its background. The specimen should be mounted and heat fixed on a slide before you precede to stain it. The reagents you will need to successfully perform this operation are:

        Crystal Violet (the Primary Stain)

        Iodine Solution (the Mordant)

        Decolorizer (ethanol is a good choice)

        Safranin (the Counterstain)

        Water (preferably in a squirt bottle)

Before starting, make sure that all reagents, as well as the squirt-bottle of water, are easily accessible because you won't have time to go get them during the staining procedure. Also, make sure you are doing this near a sink because it can get really messy. Wear the appropriate lab attire.

 

STEP 1: Place your slide on a slide holder or a rack. Flood (cover completely) the entire slide with crystal violet. Let the crystal violet stand for about 60 seconds. When the time has elapsed, wash your slide for 5 seconds with the water bottle. The specimen should appear blue-violet when observed with the naked eye.

STEP 2: Now, flood your slide with the iodine solution. Let it stand about a minute as well. When time has expired, rinse the slide with water for 5 seconds and immediately precede to step three. At this point, the specimen should still be blue-violet.

STEP 3: This step involves addition of the decolorizer, ethanol. Step 3 is somewhat subjective because using too much decolorizer could result in a false Gram (-) result. Likewise, not using enough decolorizer may yield a false Gram (+) results. To be safe, add the ethanol dropwise until the blue-violet color is no longer emitted from your specimen. As in the previous steps, rinse with the water for 5 seconds.

STEP 4: The final step involves applying the counterstain, safranin. Flood the slide with the dye as you did in steps 1 and 2. Let this stand for about a minute to allow the bacteria to incorporate the safranin. Gram positive cells will incorporate little or no counterstain and will remain blue-violet in appearance. Gram negative bacteria, however, take on a pink color and are easily distinguishable from the Gram positives. Again, rinse with water for 5 seconds to remove any excess of dye.

After you have completed steps 1 through 4, you should dry the slide with bibulous paper or allow it to air dry before viewing it under the microscope (Gram's technique)

 

Tthe rules of work with immersion system of a microscope.

Positioning the slide

Place the slide specimen-side-up on the stage so that the specimen lies over the opening for the light in the middle of the stage. Secure the slide between (not under) the arms of the mechanical stage. The slide can now be moved from place to place using the 2 control knobs located on the right of the stage.

Adjusting the illumination

Adjust the total light available by turning the flat mirror. Adjust the amount of light coming through the condenser using the iris diaphragm lever located below and to the front of the stage. Light adjustment using the iris diaphragm lever is critical to obtaining proper contrast. For oil immersion microscopy (900X), the iris diaphragm lever should be set almost all the way open (to your left for maximum light).

Obtaining different magnifications

The final magnification is a product of the 2 lenses being used. The eyepiece or ocular lens magnifies 7X, 10X, 15X. The objective lenses are mounted on a turret near the stage. They make magnifications: 10X; 40X, and 90X (black-striped oil immersion lens). Final magnifications are as follows:

 

Ocular lens

X

Objective lens

=

Total magnification

10X

X

10X

=

100X

10X</DH>

X

40X

=

400X

10X

X

100X (black)

=

900X

Focusing from lower power to higher power:

a. Rotate the 10X objective until it locks into place (total magnification of 100X).

b. Turn the coarse focus control (larger knob) all the way away from you until it stops.

c. Look through the eyepiece and turn the coarse focus control (larger knob) towards you slowly until the specimen comes into view.

d. Get the specimen into sharp focus using the fine focus control (smaller knob) and adjust the light for optimum contrast using the iris diaphragm lever.

e. If higher magnification is desired, simply rotate the 40X objective into place (total magnification of 400X) and the specimen should still be in focus. (Minor adjustments in fine focus and light contrast may be needed.)

f. For maximum magnification (900X or oil immersion), rotate the 40X objective slightly out of position and place a drop of immersion oil on the slide. Now rotate the black-striped 90X oil immersion objective into place. Again, the specimen should remain in focus, although minor adjustments in fine focus and light contrast may be needed.

Cleaning the microscope

Clean the exterior lenses of the eyepiece and objective before and after each lab using lens paper only. (Paper towel or kim-wipes may scratch the lens.) Remove any immersion oil from the oil immersion lens before putting the microscope away.

Reason for using immersion oil

Normally, when light waves travel from one medium into another, they bend. Therefore, as the light travels from the glass slide to the air, the light waves bend and are scattered (the "bent pencil" effect when a pencil is placed in a glass of water). The microscope magnifies this distortion effect. Also, if high magnification is to be used, more light is needed.

Immersion oil has the same refractive index as glass and, therefore, provides an optically homogeneous path between the slide and the lens of the objective. Light waves thus travel from the glass slide, into glass-like oil, into the glass lens without being scattered or distorting the image. In other words, the immersion oil "traps" the light and prevents the distortion effect that is seen as a result of the bending of the light waves.

 

Before making a preparation, glass slides are flamed to ensure their additional degreasing.

In preparing (Preparation of a smear) a smear from bacterial culture grown on a solid medium, a drop of isotonic saline or water is transferred onto the precooled glass. A test tube with the culture is taken by the thumb and the index finger of the left hand. The loop is sterilized in the flame. A cotton-wool plug is pinched by a small finger of the right hand, removed from the test tube, and left in this position. The edges of the test tube are flamed and then the loop is introduced into the test tube through the flame. Having cooled the loop against the inner wall of the tube, the loop is touched to the nutrient medium where it meets with the glass wall (if the loop is not sufficiently cooled, it induces cracking and melts the medium). Then the loop is touched to the culture of the microorganisms on the surface of the medium. Then the loop is withdrawn, the edges of the test tube are quickly flamed, the tube is closed with a stopper passed through the flame, and then replaced into the test tube rack. All the above described procedures are made above the flame. The culture sample is placed with the loop into a drop of water on the glass slide and spread uniformly with circular movements on an area of 1 1.5 cm in diameter, then the loop is flamed.

Then you should do drying an fixation of the smear.

The fixed preparation is placed, the smear upward, on the support. A dye solution is pipetted onto the entire surface of the smear. With Pfeiffer's fuchsine the staining lasts 1-2 min, with alkaline solution of Loeffler's methylene blue or water-alcoholic solution of methylene blue, 3-5 min. (Simple method of staining) Following the staining procedure the dye is dispensed, the preparation is washed with water, dried between sheets of filter paper, and then examined under the oil-immersion objective.

 

 

Structure of bacterial cell

The higher resolving power of the electron microscope not only magnifies the typical shape of a bacterial cell but also clearly resolves its prokaryotic organization (Fig. 1).

: : : : fig2_2

FIGURE 1. Electron micrograph of a thin section of Neisseria gonorrhoeae showing the organizational features of prokaryotic cells. Note the electron-transparent nuclear region (n) packed with DNA fibrils, the dense distribution of ribosomal particles in the cytoplasm, and the absence of intracellular membranous organelles.

 

: : : : Scheme_1

 

The Nucleoid

Prokaryotic and eukaryotic cells were initially distinguished on the basis of structure: the prokaryotic nucleoid the equivalent of the eukaryotic nucleus is structurally simpler than the true eukaryotic nucleus, which has a complex mitotic apparatus and surrounding nuclear membrane. As the electron micrograph in Fig. 2 shows, the bacterial nucleoid, which contains the DNA fibrils, lacks a limiting membrane. Under the light microscope, the nucleoid of the bacterial cell can be visualized with the aid of Feulgen staining, which stains DNA. Gentle lysis can be used to isolate the nucleoid of most bacterial cells. The DNA is then seen to be a single, continuous, "giant" circular molecule with a molecular weight of approximately 3 X 109. The unfolded nuclear DNA would be about 1 mm long (compared with an average length of 1 to 2 m for bacterial cells). The bacterial nucleoid, then, is a structure containing a single chromosome. The number of copies of this chromosome in a cell depends on the stage of the cell cycle (chromosome replication, cell enlargement, chromosome segregation, etc). Although the mechanism of segregation of the two sister chromosomes following replication is not fully understood, all of the models proposed require that the chromosome be permanently attached to the cell membrane throughout the various stages of the cell cycle.

Bacterial chromatin does not contain basic histone proteins, but low-molecular-weight polyamines and magnesium ions may fulfill a function similar to that of eukaryotic histones. Despite the differences between prokaryotic and eukaryotic DNA, prokaryotic DNA from cells infected with bacteriophage g, when visualized by electron microscopy, has a beaded, condensed appearance not unlike that of eukaryotic chromatin. Plasmids are small circular DNA molecules that can be thought of as carrying extra genes that can be used for special situations. They usually can be dispensed with when not required. There may be several different plasmids in one cell and the numbers of each may vary from only one to 100s in a cell .

 

Surface Appendages

Surface Layers

The surface layers of the bacterial cell have been identified by various techniques: light microscopy and staining; electron microscopy of thin-sectioned, freeze-fractured, and negatively stained cells; and isolation and biochemical characterization of individual morphologic components of the cell. The principal surface layers are capsules and loose slime, the cell wall of Gram-positive bacteria and the complex cell envelope of Gram-negative bacteria, plasma (cytoplasmic) membranes, and mesosomal membrane vesicles, which arise from invaginations of the plasma membrane. In bacteria, the cell wall forms a rigid structure of uniform thickness around the cell and is responsible for the characteristic shape of the cell (rod, coccus, or spiral). Inside the cell wall (or rigid peptidoglycan layer) is the plasma (cytoplasmic) membrane; this is usually closely apposed to the wall layer.

The topographic relationships of the cell wall and envelope layers to the plasma membrane are indicated in the thin section of a Gram-positive organism (Micrococcus lysodeikticus) in Figure 2-A and in the freeze-fractured cell of a Gram-negative organism (Bacteroides melaninogenicus) in Figure 2-B. The latter shows the typical fracture planes seen in most Gram-negative bacteria, which are weak cleavage planes through the outer membrane of the envelope and extensive fracture planes through the bilayer region of the underlying plasma membrane.

: : : : fig2_5a: : : : fig2_5b

FIGURE 2. (A). Electron micrograph of a thin section of the Gram-positive M lysodeikticus showing the thick peptidoglycan cell wall (cw), underlying cytoplasmic (plasma) membrane (cm), mesome (m), and nucleus (n). (B) Freeze-fractured Bacteriodes cell showing typical major convex fracture faces through the inner (im) and outer (om) membranes. Bars = 1 m; circled arrow in Fig. B indicates direction of shadowing.

 

Capsules and Loose Slime

Some bacteria form capsules, which constitute the outermost layer of the bacterial cell and surround it with a relatively thick layer of viscous gel. Capsules may be up to 10 m thick. Some organisms lack a well-defined capsule but have loose, amorphous slime layers external to the cell wall or cell envelope. The a hemolytic Streptococcus mutans, the primary organism found in dental plaque is able to synthesis a large extracellular mucoid glucans from sucrose. Not all bacterial species produce capsules; however, the capsules of encapsulated pathogens are often important determinants of virulence. Encapsulated species are found among both Gram-positive and Gram-negative bacteria. In both groups, most capsules are composed of highmolecular-weight viscous polysaccharides that are retained as a thick gel outside the cell wall or envelope. The capsule of Bacillus anthracis (the causal agent of anthrax) is unusual in that it is composed of a g-glutamyl polypeptide. Table 2 presents the various capsular substances formed by a selection of Gram-positive and Gram-negative bacteria. A plasma membrane stage is involved in the biosynthesis and assembly of the capsular substances, which are extruded or secreted through the outer wall or envelope structures. Mutational loss of enzymes involved in the biosynthesis of the capsular polysaccharides can result in the smooth-to-rough variation seen in the pneumococci.

The capsule is not essential for viability. Viability is not affected when capsular polysaccharides are removed enzymatically from the cell surface. The exact functions of capsules are not fully understood, but they do confer resistance to phagocytosis and hence provide the bacterial cell with protection against host defenses to invasion.

Capsules are usually demonstrated by the negative staining procedure or a modification of it. One such "capsule stain" (Welch method) involves treatment with hot crystal violet solution followed by a rinsing with copper sulfate solution. The latter is used to remove excess stain because the conventional washing with water would dissolve the capsule. The copper salt also gives color to the background, with the result that the cell and background appear dark blue and the capsule a much paler blue.

 

Cell Wall and Gram-Negative Cell Envelope

The Gram stain broadly differentiates bacteria into Gram-positive and Gram-negative groups; a few organisms are consistently Gram-variable. Gram-positive and Gram-negative organisms differ drastically in the organization of the structures outside the plasma membrane but below the capsule (Fig. 3): in Gram-negative organisms these structures constitute the cell envelope, whereas in Gram-positive organisms they are called a cell wall.

Most Gram-positive bacteria have a relatively thick (about 20 to 80 nm), continuous cell wall (often called the sacculus), which is composed largely of peptidoglycan (also known as mucopeptide or murein). In thick cell walls, other cell wall polymers (such as the teichoic acids, polysaccharides, and peptidoglycolipids) are covalently attached to the peptidoglycan. In contrast, the peptidoglycan layer in Gram-negative bacteria is thin (about 5 to 10 nm thick); in E coli, the peptidoglycan is probably only a monolayer thick. Outside the peptidoglycan layer in the Gram-negative envelope is an outer membrane structure (about 7.5 to 10 nm thick). In most Gram-negative bacteria, this membrane structure is anchored noncovalently to lipoprotein molecules (Braun's lipoprotein), which, in turn, are covalently linked to the peptidoglycan. The lipopolysaccharides of the Gram-negative cell envelope form part of the outer leaflet of the outer membrane structure.

The organization and overall dimensions of the outer membrane of the Gram-negative cell envelope are similar to those of the plasma membrane (about 7.5 nm thick). Moreover, in Gram-negative bacteria such as E coli, the outer and inner membranes adhere to each other at several hundred sites (Bayer patches); these sites can break up the continuity of the peptidoglycan layer. Table 2 summarizes the major classes of chemical constituents in the walls and envelopes of Gram-positive and Gram-negative bacteria.

The basic differences in surface structures of Gram-positive and Gram-negative bacteria explain the results of Gram staining. Both Gram-positive and Gram-negative bacteria take up the same amounts of crystal violet (CV) and iodine (I). The CV-I complex, however, is trapped inside the Gram-positive cell by the dehydration and reduced porosity of the thick cell wall as a result of the differential washing step with 95 percent ethanol or other solvent mixture. In contrast, the thin peptidoglycan layer and probable discontinuities at the membrane adhesion sites do not impede solvent extraction of the CV-I complex from the Gram-negative cell.

The above mechanism of the Gram stain based on the structural differences between the two groups has been confirmed by sophisticated methods of electron microscopy. The sequence of steps in the Gram stain differentiation is illustrated diagrammatically in Figure 4. Moreover, mechanical disruption of the cell wall of Gram-positive organisms or its enzymatic removal with lysozyme results in complete extraction of the CV-I complex and conversion to a Gram-negative reaction. Therefore, autolytic wall-degrading enzymes that cause cell wall breakage may account for Gram-negative or variable reactions in cultures of Gram-positive organisms (such as Staphylococcus aureus, Clostridium perfringens, Corynebacterium diphtheriae, and some Bacillus spp).

 

: : : : fig2_6

 

FIGURE 3. Comparison of the thick cell wall of Gram-positive bacteria with the comparatively thin cell wall of Gram-negative bacteria. Note the complexity of the Gram-negative cell envelope (outer membrane, its hydrophobic lipoprotein anchor; periplasmic space).

 

 

FIGURE 4. General sequence of steps in the Gram stain procedure and the resultant staining of Gram-positive and Gram-negative bacteria

 

: : : : Scheme_9

 

 

Peptidoglycan

Unique features of almost all prokaryotic cells (except for Halobacterium halobium and mycoplasmas) are cell wall peptidoglycan and the specific enzymes involved in its biosynthesis. These enzymes are target sites for inhibition of peptidoglycan synthesis by specific antibiotics. The primary chemical structures of peptidoglycans of both Gram-positive and Gram-negative bacteria have been established; they consist of a glycan backbone of repeating groups of β1, 4-linked disaccharides of β1,4-N-acetylmuramyl-N-acetylglucosamine. Tetrapeptides of L-alanine-D-isoglutamic acid-L-lysine (or diaminopimelic acid)-n-alanine are linked through the carboxyl group by amide linkage of muramic acid residues of the glycan chains; the D-alanine residues are directly cross-linked to the e-amino group of lysine or diaminopimelic acid on a neighboring tetrapeptide, or they are linked by a peptide bridge. In S aureus peptidoglycan, a glycine pentapeptide bridge links the two adjacent peptide structures. The extent of direct or peptide-bridge cross-linking varies from one peptidoglycan to another. The staphylococcal peptidoglycan is highly cross-linked, whereas that of E coli is much less so, and has a more open peptidoglycan mesh.

The diamino acid providing the e-amino group for cross-linking is lysine or diaminopimelic acid, the latter being uniformly present in Gram-negative peptidoglycans. The structure of the peptidoglycan is illustrated in Figure 5. A peptidoglycan with a chemical structure substantially different from that of all eubacteria has been discovered in certain archaebacteria. Instead of muramic acid, this peptidoglycan contains talosaminuronic acid and lacks the D-amino acids found in the eubacterial peptidoglycans. Interestingly, organisms containing this wall polymer (referred to as pseudomurein) are insensitive to penicillin, an inhibitor of the transpeptidases involved in peptidoglycan biosynthesis in eubacteria.

: : : : fig2_8

FIGURE 5. Diagrammatic representation of peptidoglycan structures with adjacent glycan strands cross-linked directly from the carboxyterminal D-alanine to the e-amino group of an adjacent tetrapeptide or through a peptide cross bridge ,N-acetylmuramic acid; N-acetylglucosamine.

 

The ß-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine is specifically cleaved by the bacteriolytic enzyme lysozyme. Widely distributed in nature, this enzyme is present in human tissues and secretions and can cause complete digestion of the peptidoglycan walls of sensitive organisms. When lysozyme is allowed to digest the cell wall of Gram-positive bacteria suspended in an osmotic stabilizer (such as sucrose), protoplasts are formed. These protoplasts are able to survive and continue to grow on suitable media in the wall-less state. Gram-negative bacteria treated similarly produce spheroplasts, which retain much of the outer membrane structure. The dependence of bacterial shape on the peptidoglycan is shown by the transformation of rod-shaped bacteria to spherical protoplasts (spheroplasts) after enzymatic breakdown of the peptidoglycan. The mechanical protection afforded by the wall peptidoglycan layer is evident in the osmotic fragility of both protoplasts and spheroplasts.

There are two groups of bacteria that lack the protective cell wall peptidoglycan structure, the Mycoplasma species, one of which causes atypical pneumonia and some genitourinary tract infections and the L-forms, which originate from Gram-positive or Gram-negative bacteria and are so designated because of their discovery and description at the Lister Institute, London. The mycoplasmas and L-forms are all Gram-negative and insensitive to penicillin and are bounded by a surface membrane structure. L-forms arising "spontaneously" in cultures or isolated from infections are structurally related to protoplasts and spheroplasts; all three forms (protoplasts, spheroplasts, and L-forms) revert infrequently and only under special conditions.

Teichoic Acids

Wall teichoic acids are found only in certain Gram-positive bacteria (such as staphylococci, streptococci, lactobacilli, and Bacillus spp); so far, they have not been found in gram- negative organisms. Teichoic acids are polyol phosphate polymers, with either ribitol or glycerol linked by phosphodiester bonds; their structures are illustrated in Figure 2. Substituent groups on the polyol chains can include D-alanine (ester linked), N-acetylglucosamine, N-acetylgalactosamine, and glucose; the substituent is characteristic for the teichoic acid from a particular bacterial species and can act as a specific antigenic determinant. Teichoic acids are covalently linked to the peptidoglycan. These highly negatively charged polymers of the bacterial wall can serve as a cation-sequestering mechanism.

Accessory Wall Polymers

In addition to the principal cell wall polymers, the walls of certain Gram-positive bacteria possess polysaccharide molecules linked to the peptidoglycan. For example, the C polysaccharide of streptococci confers group specificity. Acidic polysaccharides attached to the peptidoglycan are called teichuronic acids. Mycobacteria have peptidoglycolipids, glycolipids, and waxes associated with the cell wall.

Lipopolysaccharides

A characteristic feature of Gram-negative bacteria is possession of various types of complex macromolecular lipopolysaccharide (LPS). So far, only one Gram-positive organism, Listeria monocytogenes, has been found to contain an authentic LPS. The LPS of this bacterium and those of all Gram-negative species are also called endotoxins, thereby distinguishing these cell-bound, heat-stable toxins from heat-labile, protein exotoxins secreted into culture media. Endotoxins possess an array of powerful biologic activities and play an important role in the pathogenesis of many Gram-negative bacterial infections. In addition to causing endotoxic shock, LPS is pyrogenic, can activate macrophages and complement, is mitogenic for B lymphocytes, induces interferon production, causes tissue necrosis and tumor regression, and has adjuvant properties. The endotoxic properties of LPS reside largely in the lipid A components. Usually, the LPS molecules have three regions: the lipid A structure required for insertion in the outer leaflet of the outer membrane bilayer; a covalently attached core composed of 2-keto-3deoxyoctonic acid (KDO), heptose, ethanolamine, N-acetylglucosamine, glucose, and galactose; and polysaccharide chains linked to the core. The polysaccharide chains constitute the O-antigens of the Gram-negative bacteria, and the individual monosaccharide constituents confer serologic specificity on these components. Table 3 depicts the structure of LPS. Although it has been known that lipid A is composed of b1,6-linked D-glucosamine disaccharide substituted with phosphomonester groups at positions 4' and 1, uncertainties have existed about the attachment positions of the six fatty acid acyl and KDO groups on the disaccharide. The demonstration of the structure of lipid A of LPS of a heptoseless mutant of Salmonella typhimurium has established that amide-linked hydroxymyristoyl and lauroxymyristoyl groups are attached to the nitrogen of the 2- and 2'-carbons, respectively, and that hydroxymyristoyl and myristoxymyristoyl groups are attached to the oxygen of the 3- and 3'-carbons of the disaccharide, respectively. Therefore, only position 6' is left for attachment of KDO units.

 

Table. The three major, covalently linked regions that form the typical LPS.

: : : : fig2_10

LPS and phospholipids help confer asymmetry to the outer membrane of the Gram-negative bacteria, with the hydrophilic polysaccharide chains outermost. Each LPS is held in the outer membrane by relatively weak cohesive forces (ionic and hydrophobic interactions) and can be dissociated from the cell surface with surface-active agents.

As in peptidoglycan biosynthesis, LPS molecules are assembled at the plasma or inner membrane. These newly formed molecules are initially inserted into the outer-inner membrane adhesion sites.

Outer Membrane of Gram-Negative Bacteria

In thin sections, the outer membranes of Gram-negative bacteria appear broadly similar to the plasma or inner membranes; however, they differ from the inner membranes and walls of Gram-positive bacteria in numerous respects. The lipid A of LPS is inserted with phospholipids to create the outer leaflet of the bilayer structure; the lipid portion of the lipoprotein and phospholipid form the inner leaflet of the outer membrane bilayer of most Gram-negative bacteria (Fig. 3).

In addition to these components, the outer membrane possesses several major outer membrane proteins; the most abundant is called porin. The assembled subunits of porin form a channel that limits the passage of hydrophilic molecules across the outer membrane barrier to those having molecular weights that are usually less than 600 to 700. Evidence also suggests that hydrophobic pathways exist across the outer membrane and are partly responsible for the differential penetration and effectiveness of certain β-lactam antibiotics (ampicillin, cephalosporins) that are active against various Gram-negative bacteria. Although the outer membranes act as a permeability barrier or molecular sieve, they do not appear to possess energy-transducing systems to drive active transport. Several outer membrane proteins, however, are involved in the specific uptake of metabolites (maltose, vitamin B12, nucleosides) and iron from the medium. Thus, outer membranes of the Gram-negative bacteria provide a selective barrier to external molecules and thereby prevent the loss of metabolite-binding proteins and hydrolytic enzymes (nucleases, alkaline phosphatase) found in the periplasmic space. The periplasmic space is the region between the outer surface of the inner (plasma) membrane and the inner surface of the outer membrane (Figure 3).

Thus, Gram-negative bacteria have a cellular compartment that has no equivalent in Gram-positive organisms. In addition to the hydrolytic enzymes, the periplasmic space holds binding proteins (proteins that specifically bind sugars, amino acids, and inorganic ions) involved in membrane transport and chemotactic receptor activities. Moreover, plasmid-encoded b-lactamases and aminoglycoside-modifying enzymes (phosphorylation or adenylation) in the periplasmic space produce antibiotic resistance by degrading or modifying an antibiotic in transit to its target sites on the membrane (penicillin-binding proteins) or on the ribosomes (aminoglycosides). These periplasmic proteins can be released by subjecting the cells to osmotic shock and after treatment with the chelating agent ethylenediaminetetraacetic acid.

Intracellular Components

Plasma (Cytoplasmic) Membranes

Bacterial plasma membranes, the functional equivalents of eukaryotic plasma membranes, are referred to variously as cytoplasmic, protoplast, or (in Gram-negative organisms) inner membranes. Similar in overall dimensions and appearance in thin sections to biomembranes from eukaryotic cells, they are composed primarily of proteins and lipids (principally phospholipids). Protein-to-lipid ratios of bacterial plasma membranes are approximately 3: 1, close to those for mitochondrial membranes. Unlike eukaryotic cell membranes, the bacterial membrane (except for Mycoplasma species and certain methylotrophic bacteria) has no sterols, and bacteria lack the enzymes required for sterol biosynthesis.

Although their composition is similar to that of inner membranes of Gram-negative species, cytoplasmic membranes from Gram-positive bacteria possess a class of macromolecules not present in the Gram-negative membranes. Many Gram-positive bacterial membranes contain membrane-bound lipoteichoic acid, and species lacking this component (such as Micrococcus and Sarcina spp) contain an analogous membrane-bound succinylated lipomannan. Lipoteichoic acids are structurally similar to the cell wall glycerol teichoic acids in that they have basal polyglycerol phosphodiester 1-3 linked chains. These chains terminate with the phosphomonoester end of the polymer, which is linked covalently to either a glycolipid or a phosphatidyl glycolipid moiety. Thus, a hydrophobic tail is provided for anchoring in the membrane lipid layers (Fig. 3). As in the cell wall glycerol teichoic acid, the lipoteichoic acids can have glycosidic and D-alanyl ester substituents on the C-2 position of the glycerol.

Both membrane-bound lipoteichoic acid and membrane-bound succinylated lipomannan can be detected as antigens on the cell surface, and the glycerol-phosphate and succinylated mannan chains appear to extend through the cell wall structure (Fig. 3). This class of polymer has not yet been found in the cytoplasmic membranes of Gram-negative organisms. In both instances, the lipoteichoic acids and the lipomannans are negatively charged components and can sequester positively charged substances. They have been implicated in adhesion to host cells, but their functions remain to be elucidated.

Multiple functions are performed by the plasma membranes of both Gram-positive and Gram-negative bacteria. Plasma membranes are the site of active transport, respiratory chain components, energy-transducing systems, the H+-ATPase of the proton pump , and membrane stages in the biosynthesis of phospholipids, peptidoglycan, LPS, and capsular polysaccharides. In essence, the bacterial cytoplasmic membrane is a multifunction structure that combines the mitochondrial transport and biosynthetic functions that are usually compartmentalized in discrete membranous organelles in eukaryotic cells. The plasma membrane is also the anchoring site for DNA and provides the cell with a mechanism (as yet unknown) for separation of sister chromosomes.

 

Mesosomes

Thin sections of Gram-positive bacteria reveal the presence of vesicular or tubular-vesicular membrane structures called mesosomes, which are apparently formed by an invagination of the plasma membrane. These structures are much more prominent in Gram-positive than in Gram-negative organisms. At one time, the mesosomal vesicles were thought to be equivalent to bacterial mitochondria; however, many other membrane functions have also been attributed to the mesosomes. At present, there is no satisfactory evidence to suggest that they have a unique biochemical or physiologic function. Indeed, electron-microscopic studies have suggested that the mesosomes, as usually seen in thin sections, may arise from membrane perturbation and fixation artifacts. No general agreement exists about this theory, however, and some evidence indicates that mesosomes may be related to events in the cell division cycle.

Other Intracellular Components

In addition to the nucleoid and cytoplasm (cytosol), the intracellular compartment of the bacterial cell is densely packed with ribosomes of the 70S type. These ribonucleoprotein particles, which have a diameter of 18 nm, are not arranged on a membranous rough endoplasmic reticulum as they are in eukaryotic cells. Other granular inclusions randomly distributed in the cytoplasm of various species include metabolic reserve particles such as poly-b-hydroxybutyrate (PHB), polysaccharide and glycogen-like granules, and polymetaphosphate or metachromatic granules (volutin granules). They possess high electron density. The volutin granules vary in size from several hundreds of 0.1 to 0.5 mcm.

A characteristic feature of the granules of volutin is their metachromatic stain. They are stained reddish-purple, with methylene blue while the cytoplasm is stained blue.

Volutin was first discovered in the cell of Spirillum volutans (from which it was named), then in Corynebacterium diphtheriae (Fig. 6) and other organisms. The presence of volutin is taken into account in laboratory diagnosis of diphtheria. Lipoprotein bodies are found quite frequently as droplets of fat in certain bacilli and spirilla. They disappear when the cells' are deprived of nutrients, and appear when bacteria are grown on nutrient media of a high carbohydrate content. They are discernible if stained with Sudan or fuchsin.

The presence of volutin granules and lipoprotein bodies is biologically important since they serve as sources of stored food for the bacterium in the case of starvation.

 

 

 

 

 

Figure 6. Granules of volutin in Corynebacterium diphtheriae

 

: : : : Ris_45_volutine

Volutins granules, Loefflers technique

 

: : : : Ris_47_corynebacterium_Neisser

Volutins granules, Neissers technique

 

 

Glycogen and granulose are intracellular inclusions which can be identified by treating the cell with Lugol's solution. Glycogen stains reddish-brown and granulose grey-blue. Glycogen granules are prominent in aerobic bacilli. Granulose is frequently found in butyric-acid bacteria, and especially in Clostridium pectinovorum.

Some bacteria contain crystals of a protein nature which have proved to be extremely toxic for certain insect larvae. In the cytoplasm of sulphur bacteria (Beggiatoa) which oxidize hydrogen sulphide, sulphur is deposited in the form of droplets of a colloidal nature. Energy derived from the sulphur is utilized in reducing carbon dioxide.

Granules of amorphic calcium carbonate, the physiological function of which is not yet known, are found in the cytoplasm of some sulphur bacteria [Achromatiun}.

Staining of volutin granules with alkaline methylene blue (by Loeffler's technique). On a fixed smear pour alkaline methylene blue to act for 3-5 min wash with water, dry with filter paper, and examine under the microscope. The cytoplasm of diphtheria corynebacteria is stained light-blue, while granules of volutin are dark-blue.

Staining with acetic-acidic methyl violet. A fixed smear is treated for 5-10 min with acetic-acidic methyl violet (methyl violet or crystal violet, 0.25 g, 5 per cent solution of acetic acid, 100 ml). The smear is washed with water and dried. In this case the cytoplasm of diphtheria corynebacteria is stained light lilac, while volutin granules appear dark-lilac.

In complex Neissers staining bacterial cells become yellow whereas volutin granules become brown-black.

Neisser's staining. Staining of volutin granules by this method includes the following stages.

1. A fixed smear is stained with acetic-acidic methylene blue for 1 min, then the dye is poured off, and smear is washed "with water.

2. Pour in Lugols solution to act for 20-30 s.

3. Without washing with water, stain the preparation with vesuvin for 1-3 min, then wash it with water and dry.

 

 

Ziehl-Neelsen staining is employed for detecting acid-fast tuberculosis and leprosy mycobacteria and some actinomycetes. The acid-fast nature of microorganisms is due to the fact that their cells contain lipids, wax, and oxyacids. Such microorganisms are poorly stained with diluted solutions of dyes. To facilitate the penetration of the stain into the cells of microorganisms, Ziehls phenol fuchsine applied onto the preparation is heated over the burner's flame.

Stained microorganisms do not decolorize with weak solutions of mineral acids and alcohol.

Staining of microorganisms by the Ziehl-Neelsen method includes the following stages.

1. Put a slip of filter paper on a fixed smear and pour Ziehl's phenol fuchsine on it (one can use filter paper saturated with a dye and then dried). Heat the smear over the flame until steam rises, then draw it aside for cooling and add a new portion of the dye. Repeat heating 2-3 times. Allow the smear to cool, take off the filter paper, and wash the preparation with water.

2. The preparation is decolorized by immersing it in or with 5 per cent solution of sulphuric acid and washed several times with water.

3. The preparation is stained with aqueous-alcoholic solution of methylene blue for 3-5 min, washed with water and dried.

Upon staining by Ziehl-Neelsen technique acid-fast bacteria acquire a bright red colour, while the remaining microflora is stained light-blue.

: : : : Ris_41_mycobacterium

 

To demonstrate bacterial nucleoid, one can use Feulgen's micro-chemical reaction in which weak acidic hydrolysis is employed. This is accompanied by the release of desoxyribose which subsequently transforms into aldehydes, reacting with colourless fuchsine-sulphurous acid of special Schiffs reagent. The nucleoid is stained red-violet. The nucleoid of microorganisms may also be detected by means of electron microscopic examination of ultrathin sections.

Due to high concentrations of metaphosphates and other phosphorous compounds volutin granules (inclusions in the cytoplasm) are characterized by metachromasia. "Upon staining with alkaline methylene blue and acetic-acidic methylene violet, their colour is more intensive as compared to that of the cytoplasm.

 

Endospores. Endospores are highly heat-resistant, dehydrated resting cells formed intracellularly in members of the genera Bacillus and Clostridium (fig. 7). Endospores are small spherical or oval bodies formed within the cell. A spore is formed at a certain stage in the development of some micro-organisms and this property was inherited in the process of evolution in the struggle for keeping the species intact. Some micro-organisms, principally rod-shaped (bacilli and clostridia), are capable of sporulation. These include the causative agents of anthrax, tetanus, anaerobic infections, botulism and also saprophytic species living in the soil, water and bodies of animals. Spore formation only rarely occurs in cocci {Sarcina lutea, Sarcina ureae) and in spiral forms (Desulfovibrio desulfuricans}. Sporulation occurs in the environment (in soil and on nutrient media), and is not observed in human or animal tissues.

 

: : : : Spore-6

Figure 7. Thin section through a sporulating cell of bacilli

 

: : : : Ris_53_spore_anthracis

 

The series of biochemical and morphologic changes that occur during sporulation represent true differentiation within the cycle of the bacterial cell. The process, which usually begins in the stationary phase of the vegetative cell cycle, is initiated by depletion of nutrients (usually readily utilizable sources of carbon or nitrogen, or both).

The cell then undergoes a highly complex, well-defined sequence of morphologic and biochemical events that ultimately lead to the formation of mature endospores. As many as seven distinct stages have been recognized by morphologic and biochemical studies of sporulating Bacillus species: stage 0, vegetative cells with two chromosomes at the end of exponential growth; stage I, formation of axial chromatin filament and excretion of exoenzymes, including proteases; stage II, forespore septum formation and segregation of nuclear material into two compartments; stage III, spore protoplast formation and elevation of tricarboxylic acid and glyoxylate cycle enzyme levels; stage IV, cortex formation and refractile appearance of spore; stage V, spore coat protein formation; stage VI, spore maturation, modification of cortical peptidoglycan, uptake of dipicolinic acid (a unique endospore product) and calcium, and development of resistance to heat and organic solvents; and stage VII, final maturation and liberation of endospores from mother cells (in some species).

When newly formed, endospores appear as round, highly refractile cells within the vegetative cell wall, or sporangium. Some strains produce autolysins that digest the walls and liberate free endospores. The spore protoplast, or core, contains a complete nucleus, ribosomes, and energy generating components that are enclosed within a modified cytoplasmic membrane. The peptidoglycan spore wall surrounds the spore membrane; on germination, this wall becomes the vegetative cell wall. Surrounding the spore wall is a thick cortex that contains an unusual type of peptidoglycan, which is rapidly released on germination. A spore coat of keratinlike protein encases the spore contained within a membrane (the exosporium). During maturation, the spore protoplast dehydrates and the spore becomes refractile and resistant to heat, radiation, pressure, desiccation, and chemicals; these properties correlate with the cortical peptidoglycan and the presence of large amounts of calcium dipicolinate.

Recent evidence indicated that the spores of Bacillus spharicus were revived which had been preserved in amber for more than 25 million years. Their claims need to be reevaluated. The thin section of the spore shows the ruptured, thick spore coat and the cortex surrounding the spore protoplast with the germinal cell wall that becomes the vegetative wall on outgrowth.

The spores of certain bacilli are capable of withstanding boiling and high concentrations of disinfectants. They are killed in an autoclave exposed to saturated steam, at a temperature of 115-125 C, and also at a temperature of 150-170 C in a Pasteur hot-air oven.

Sporulation: The sporulation process begins when nutritional conditions become unfavorable, depletion of the nitrogen or carbon source (or both) being the most significant factor Sporulation occurs massively in cultures that have terminated exponential growth as a result of such depletion. Sporulation involves the production of many new structures, enzymes, and metabolites along with the disappearance of many vegetative cell components. These changes represent a true process of differentiation: A series of genes whose products determine the formation and final composition of the spore is activated, while another series of genes involved in vegetative cell function is inactivated. These changes involve an alteration in the specificity of RNA polymerase. The sequence of events in sporulation is highly complex asporogenous mutants reveal at least 12 morphologically or biochemically distinguishable stages, and at least 30 operons (including an estimated 200 structural genes) are involved During the process, some bacteria release peptide antibiotics, which may play a role in regulating sporogenesis.

Morphologically, sporulation begins with the isolation of a terminal nucleus by the inward growth of the cell membrane. The growth process involves an infolding of the membrane so as to produce a double membrane structure whose facing surfaces correspond to the cell wall-synthesizing surface of the cell envelope. The growing points move progressively toward the pole of the cell so as to engulf the developing spore.

The 2 spore membranes now engage in the active synthesis of special layers that will form the cell envelope: the spore wall and cortex, lying between the facing membranes; and the coat and exosporium, lying outside of the facing membranes. In the newly isolated cytoplasm, or core, many vegetative cell enzymes are degraded and are replaced by a set of unique spore constituents.

In bacilli and clostridia, spores are located (1) centrally, in the centre of the cell (causative agent of anthrax); (2) terminally, at the ends of the rod (causative agent of tetanus); (3) subterminally, towards the ends (causative agents of botulism, anaerobic infections, etc.) (Fig. 8).

In some species of sporulating microorganisms, the spore diameter is greater than the width of the bacterial cell. If the spore is located subterminally, the microbes take on the form of a spindle (closter).

In tetanus clostridia the spore diameter is also greater than the width of the vegetative cell, but the spore is located terminally, and hence the drum-stick appearance.

This property of sporulation is important in characterizing and identifying spore-forming microbes, and also when selecting methods of decontaminating objects, housings, foodstuff's, and other substances. The microbe may lose its ability to sporulate by frequent cultivation on fresh media or by subjecting it to high temperatures.

: : : : Sporer-4

 

Figure 8. Shapes and arrangement of spores in bacilli and clostridia

 

Conclusion: Properties of Endospores:

1. Core-The core is the spore protoplast. It contains a complete nucleus (chromosome), all of the components of the protein-synthesizing apparatus, and an energy-generating system based on glycolysis. Cytochromes are lacking even in aerobic species, the spores of which rely on a shortened electron transport pathway involving flavoproteins. A number of vegetative cell enzymes are increased in amount (eg, alanine racemase), and a number of unique enzymes are formed (eg, dipicolinic acid synthetase). The energy for germination is stored as 3-phosphoglycerate rather than as ATP.

The heat resistance of spores is due in pan to their dehydrated state and in part to the presence of large amounts (5-15% of the spore dry weight) of calcium dipicolinate, which is formed from an intermediate of the lysine biosynthetic pathway. In some way not yet understood, these properties result in the stabilization of the spore enzymes, most of which exhibit normal heat lability when isolated in soluble form.

2. Spore wall-The innermost layer surrounding the inner spore membrane is called the spore wall. It contains normal peptidoglycan and becomes the cell wall of the germinating vegetative cell.

3. Cortex-The cortex is the thickest layer of the spore envelope. It contains an unusual type of peptidoglycan, with many fewer cross-links than are found in cell wall peptidoglycan. Cortex peptidoglycan is extremely sensitive to lysozyme, and its autolysis plays a key role in spore germination.

4. Coat-The coat is composed of a keratinlike protein containing many intramolecular disulfide bonds. The impermeability of this layer confers on spores their relative resistance to antibacterial chemical agents.

5. Exosporium-The exosporium is a lipoprotein membrane containing some carbohydrate.

 

Germination: The germination process occurs in 3 stages: activation, initiation, and outgrowth.

1. Activation-Even when placed in an environment that favors germination (eg, a nutritionally rich medium), bacterial spores will not germinate unless first activated by one or another agent that damages the spore coat. Among the agents that can overcome spore dormancy are heat, abrasion, acidity, and compounds containing free sulfhydryl groups.

2. Initiation-Once activated, a spore will initiate germination if the environmental conditions are favorable. Different species have evolved receptors that recognize different effectors as signalling a rich medium: thus, initiation is triggered by L-alanine in one species and by adenosine in another. Binding of the effector activates an autolysin that rapidly degrades the cortex peptidoglycan. Water is taken up, calcium dipicolinate is released, and a variety of spore constituents are degraded by hydrolytic enzymes.

3. Outgrowth-Degradation of the cortex and outer layers results in the emergence of a new vegetative cell consisting of the spore protoplast with its surrounding wall. A period of active biosynthesis follows; this period, which terminates in cell division, is called outgrowth. Outgrowth requires a supply of all nutrients essential for cell growth.

 

 

 

Flagella. Motile bacteria are subdivided into creeping and swimming bacteria. Creeping bacteria move slowly (creep) on a supporting surface as a result of wave-like contractions of their bodies, which cause periodic alterations in the shape of the cell. These bacteria include Myxobacterium, Beggiatoa, Thiothrix. Swimming bacteria move freely in a liquid medium. They possess flagella, thin hair-like cytoplasmic appendages measuring 0.02 to 0.05 mcm in thickness and from 6 to 9 mcm in length. In some spirilla they reach a length of 80 to 90 mcm. Investigations have confirmed that the flagella are made up of proteins the composition of which differs considerably from that of the bacterial cell proteins (keratin, myosin, fibrinogen).

Figure 9. The flagella of Proteus vulgaris demonstrated by electron microscopy

With the aid of paper chromatography, it has been discovered that the flagellate material contains several ammo acids: lysine, aspartic and glutamic acids, alanine, etc. It has been suggested that the flagella are attached to basal granules which are found in the outlying zones of the cytoplasm The flagella can be observed by dark-field illumination, by special methods involving treatment with mordants, adsorption of various substances and dyes on their surfaces, and by electron microscopy The latter has made it possible to detect the spiral and screw-shaped structure of the flagella. The axial filament of the flagellum consists of two entwined hair-like processes enclosed in a sheath.

According to a pattern in the attachment of flagella motile microbes can be divided into 4 groups: (1) monotrichates, bacteria having a single flagellum at one pole of the cell (cholera vibrio, blue pus bacillus), (2) amphitrichates, bacteria with two polar flagella or with a tuft of flagella at both poles (Spirillum volutans), (3) lophotrichales, bacteria with a tuft of flagella at one pole (blue-green milk bacillus,
Alcaligenes faecalis), (4) peritrichales, bacteria having flagella distributed over the whole surface of their bodies (colibacillum, salmonellae of enteric fever and paratyphoids A and B) (Fig. 10).

The above mentioned classification is provisional While studying the flagella under an electron microscope, it was revealed that the flagellum in some monotrichates is not located at the end of the cell, but at the point of transition of the lateral surface to the pole. It has been established that bacteria which once were considered to be monotrichous possess a number of flagella As to amphitrichates, their independent existence is a subject of controversy It has been suggested that the amphitrichate cell is actually comprised of two cells which have been separated incompletely, having flagella at their distal ends.

 

: : : : Flagella

Figure 10. Bacterial flagella

1 monotrichates, 2 amphitrichates, 3 lophotrichates 4 peritrichates

 

: : : : Ris_63_escherichia_coli_peritrich

: : : : Ris_61_pseudomonas_aeruginosa

 

The flagella are main locomotor organoid of bacteria. As the result of their vigorous movements, resembling the twiddling of a corkscrew, the fluid moves along them and the micro-organism moves at a rate of about 50 mcm/sec. The mechanism of the contraction is not quite clear. It has been suggested that the protein of the sheath surrounding the flagellum forms with flagella a bicomponent system
which contracts like actomyosin. The contraction of the flagella is due to the existence of two configurations of flagellin molecules differing in ammo acid composition.

The type of motility in bacteria depends on the number of flagella, age and properties of the culture, temperature, amount of chemical substances and on other factors. Monotrichates move with the greatest speed (60 mcm per second). Peritrichates move at rates ranging from 25 to 30 mcm per second. Certain species of motile microbes move at a rate of up to 200 mcm per second.

Motile bacteria also possess the power of directed movements, or taxis. According to the factors under the effect of which motion occurs, chemotaxis, aerotaxis, and phototaxis are distinguished.

Motility in bacteria can be observed by the hanging drop in wet conditions. The determination of motility in microbes is employed in laboratory practice as a means to identify cholera vibrio, dysentery, enteric fever, paratyphoid and other bacteria. However, although the presence of flagella is a species characteristic, they are not always essential to life, since a flagellate forms of motile bacteria exist.

Various types of microbes have pili (cilia, filaments, fimbriae), structures which are much shorter and thinner than the flagella (Fig. 11). They cover the body of the cell and there may be 100 to 400 of them on one cell. Pili are 0.3-1.0 mcm long and 0.01 mcm wide. It is supposed that cilia are not related to the organs of locomotion and that they serve to attach the microbial cells to the surface of some substrates. Nine different types of pili have been studied. They consist of protein. Just like in the case of flagella, it is not necessary that all bacterial cells have pili. Of most interest are the F-pili within which there is a canal through which the genetic material from the donor to the recipient is transferred during conjugation (see section on conjugation).

Figure 11. Cilia (pili) of Shigella flexneri demonstrated by electron microscopy

 

It is possible that the pili contribute to the nutrition of bacteria since they greatly increase the surface area of the bacterial cell. Besides actively moving by means of flagella or by cell contraction, microbes are capable of molecular, passive or brownian movement, due to the thermal molecular motion of the surrounding medium.

Two types of surface appendage can be recognized on certain bacterial species: the flagella, which are organs of locomotion, and pili (Latin hairs), which are also known as fimbriae (Latin fringes). Flagella occur on both Gram-positive and Gram-negative bacteria, and their presence can be useful in identification. For example, they are found on many species of bacilli but rarely on cocci. In contrast, pili occur almost exclusively on Gram-negative bacteria and are found on only a few Gram-positive organisms (e.g., Corynebacterium renale).

Some bacteria have both flagella and pili. The electron micrograph in Fig. 12 shows the characteristic wavy appearance of flagella and two types of pili on the surface of Escherichia coli.

: : : : fig2_3a

FIGURE 12. (A) Electron micrograph of negatively stained E coli showing wavy flagella and numerous short, thinner, and more rigid hairlike structures, the pili. (B) The long sex pilus can be distinguished from the shorter common pili by mixing E coli cells with a male bacterio phage that binds specifically to sex pili.

 

Morphology and Features structure Spirochaetes, Rickettsia,

Chlamydia, Mycoplasmas, Fungi, Protozoa

Morphology and Ultrastructure of Spirochaetes. Genetically Spirochaetes (L. spira curve, Gk. chaite cock, mane) differ from bacteria and fungi in structure with a corkscrew spiral shape. Their size varies considerably (from 0.3 to 1 5 mcm in width and from 7 to 500 mcm in length). The body of the spirochaete consists of an axial filament and cytoplasm wound spirally around the filament. No special membrane separates the nucleoid from the cytoplasm. Spirochaetes have a three-layer outer membrane. As demonstrated by electron microscopy, they possess a fine cytoplasmic membrane enclosing the cytoplasm The Spirochaetes do not possess the cell wall characteristic of bacteria, but electron microscopy has revealed that they have a thin cell wall (periplast) which encloses the cytoplasm. Spirochaetes do not produce spores, capsules, or flagella. Very delicate terminal filaments resembling flagella have been revealed in some species under the electron microscope.

In spite of the absence of flagella, Spirochaetes are actively motile due to the distinct flexibility of their bodies. Spirochaetes have a rotating motion which is performed axially, a translational motion forwards and backwards, an undulating motion along the whole body of the microorganism, and a bending motion when the body bends at a certain angle.

Some species stain blue, others blue-violet, and still others pink with the Romanowsky-Giemsa stain. A good method of staining Spirochaetes is by impregnation with silver.

Staining properties (reaction to stains) are used to differentiate between saprophytic and pathogenic representatives of Spirochaetes.

Classification of Spirochaetes. The order Spirochaetales, family Spirochaetaceae includes the saprophytes (Spirochaeta, Cristispira) representing large cells, 200-500 mcm long, some of which have crypts (undulating crests); the ends are sharp or blunt. They live on dead substrates, in foul waters, and in the guts of cold-blooded animals. They stain blue with the Romanowsky-Giemsa stain. Two pathogenic genera belong to the family Spirochaetaceae (Borrelia, Treponema), and one belong to family Leptospiraceae (Leptospira) [Fig. 13].

The organisms of genus Borrelia differ from Spirochaetes in that their cells have large, obtuse-angled, irregular spirals, the number of which varies from 3 to 10. Pathogenic for man are the causative agents of relapsing fever transmitted by lice (Borrelia hispanica}, and by ticks (Borrelia persica, etc.). These stain blue-violet with the Romanowsky-Giemsa stain.

 

: : : : Ris_23_borrelia

Borrelia

 

The genus Treponema (Gk. trepein turn, nema thread) exhibits thin, flexible cells with 6-14 twists. The micro-organisms do not appear to have a visible axial filament or an axial crest when viewed under the microscope. The ends of treponemas are either tapered or rounded, some species have thin elongated threads on the poles. Electron microscopy of ultrathin treponema sections revealed a thin, elastic, and poorly resistant membrane composed of lipids, poliosides, and proteins. The cytoplasmic membrane lends the treponemas a spiral shape. Besides the typical form, there may be treponemas seen as granules, cysts, L-forms, and other structures. The organisms stain pale-pink with the Romanowsky-Giemsa stain. A typical representative is the causative agent of syphilis Treponema pallidum.

 

: : : : Ris_22_treponema

 

: : : : Ris_25_treponema

 

Treponema

 

Organisms of the genus Leptospira (Gk. leptos thin, speira coil) are characterized by very thin cell structure. The leptospirae form 12 to 18 coils wound close to each other, shaping small primary spirals. The organisms have two paired axial filaments attached at opposite ends (basal bodies) of the cell and directed toward each other. The middle part of the leptospirae have no axial filament. Due to the presence of the two pairs of axial filaments the leptospirae are capable of quite complexand active movement. During movement the ends of the organisms rotate rapidly at a right angle to the main part of their body. At rest the ends are hooked while during rapid rotary motion they resemble but- tonholes. Secondary spirals give the leptospirae the appearance of brackets or the letter S. The cytoplasm is weakly refractive. They stain pinkish with the Romanowsky-Giemsa stain. Some serotypes which are pathogenic for animals and man cause leptospirosis.

 

: : : : Ris_27_Leptospira

 

Leptospira

 

: : : : met5 fig1

 

Figure 13. Morphology and structure of Spirochaetales (a Borrelia, b Treponema, cLeptospira)

 

 

 

 

 

 

Morphology and Ultrastructure of Actinomycetes

Actinomycetes (Gk. mykes fungus, aclis ray) are unicellular microorganisms which belong to the class Bacteria, the order Actinomycetales. The body of actinomycetes consists of a mycelium which resembles a mass of branched, thin (0.2-1.2 mcm in thickness), non-septate filaments hyphae. . In some species the mycelium breaks up into poorly branching forms. In young cultures the cytoplasm in the cells of actinomycetes is homogeneous, it refracts light to a certain extent, and contains separate chromatin grains. When the culture ages, vacuoles appear in the mycelial cells, and granules, droplets of fat and rod-shaped bodies also occur. The cell wall becomes fragile, breaks easily, and a partial lysis of the cells occurs. In actinomycetes, as in bacteria, differentiated cell nuclei have not been found, but the mycelial filaments contain chromatin

Classification and morphology of microorganisms granules. The actinomycetes multiply by means of germinating spores attached to sporophores (Fig. 14). and by means of fragmenation where they break up into hyphae.

 

 

Figure 14. Morphology and structure of actinomycetes

 

: : : : Ris_28_actinomyces

Actinomyces

 

The order Actinomycetales consists of 4 families: Mycobacteriaceae, Actinomycetaceae, Streptomycetaceae, Actinoplanaceae. The family Mycobactenaceae includes the causative agents of tuberculosis, leprosy, and the family Actinomycetaceae, the causative agents of actinomycosis and acid-fast species nonpathogenic for man.

Among the actinomycetes of the family Streptomycetaceae are representatives which are capable of synthesizing antibiotic substances. These include producers of streptomycin, chloramphenicol, chlortetracycline oxytetracycline, neomycin, nystatin, etc. No species pathogenic for animals and man are present in the family Actinoplanaceae.

 

 

 

: : : : Ris_28c_Streptomyces_Culture

Streptomyces

 

Morphology and Ultrastructure of Rickettsiae Rickettsiae are included in the order Rickettsiales of obligate intra- cellular bacteria containing DNA and RNA, and are pleomorphic organisms (Fig. 15). They live and multiply only within the cells (in the cytoplasm and nucleus) of the tissues of humans, animals, and vectors. Coccoid forms resemble very fine, homogeneous, or single-grain quite often they occur as the diploforms.

The Rickettsiae are small (0.3-0.5 x 0.8-2.0 um), Gram-negative, aerobic, coccobacilli that are obligate intracellular parasites of eucaryotic cells. They may reside in the cytoplasm or within the nucleus of the cell that they invade. They divide by binary fission and they metabolize host-derived glutamate via aerobic respiration and the citric acid (TCA) cycle. They have typical Gram-negative cell walls, and they lack flagella. The rickettsiae frequently have a close relationship with arthropod vectors that may transmit the organism to mammalian hosts. The rickettsiae have very small genomes of about 1.0-1.5 million bases. http://textbookofbacteriology.net/Rickettsia.html

 

Rickettsia prowazekii, the cause of epidemic typhus, is the prototypical rickettsia. Typhus has plagued humanity throughout history. The American bacteriologist, Hans Zinsser, to whom this textbook is dedicated, was able to grow the elusive intracellular pathogen and develop a protective vaccine for typhus fever. He wrote a book about the bacterium, published in 1935, Rats, Lice, and History: "being a study in biography, which, after 12 preliminary chapters indispensable for the preparation of the lay reader, deals with the life history of typhus fever".

Rickettsia prowazekii has made science news recently since it has been shown to be the probable origin of eucaryotic mitochondria. Its complete genome sequence of 1,111,523 base pairs has been shown to contain 834 protein-coding genes. The functional profiles of these genes show similarities to those of mitochondrial genes. No genes required for glycolysis are found in either R. prowazekii or mitochondrial genomes, but a complete set of genes encoding components of the tricarboxylic acid cycle and the respiratory-chain complex is found in both. In effect, ATP production in the rickettsia is the same as that in mitochondria. Many genes involved in the biosynthesis and regulation of biosynthesis of amino acids and nucleosides in free-living bacteria are absent from R. prowazekii and mitochondria. Such genes seem to have been replaced by homologues in the nuclear (host) genome. Phylogenetic analyses indicate that R. prowazekii is more closely related to mitochondria than it is to any bacterium on the Tree of Life.

Rickettsiae must be grown in the laboratory by co-cultivation with eucaryotic cells, and they have not been grown by in axenic culture. The basis of their obligate relationship with eucaryotic cells has been explained by rickettsial possession of "leaky membranes" that require the osmolarity and nutritional environment supplied by an intracellular habitat.

The rickettsiae, in spite of their small size and obligate intracellular habitat, are a group of alphaproteobacteria,  which include many well-known organisms such as Acetobacter, Rhodobacter, Rhizobium and Agrobacterium. Very few of the alphaproteobacteria are pathogens of humans. Brucella, Bartonella, Rickettsia, and a related intracellular parasite, Ehrlichia, are the main exceptions.

The genus Rickettsia is included in the bacterial family Rickettsiaceae of the order Rickettsiales. This genus includes many species associated with human disease, including those in the spotted fever group and the typhus group (figure 1). The rickettsiae that are pathogens of humans are subdivided into three major groups based on clinical characteristics of disease: 1. spotted fever group; 2. typhus group; and 3. scrub typhus group.

Figure 1.  Taxonomic classification of the order Rickettsiales

: : : : : http://textbookofbacteriology.net/RickettsialTaxonomy.gif

Spotted Fever Group (SFG)
Rickettsia rickettsii is the cause of Rocky Mountain spotted fever (RMSF) and is the prototype bacterium in the spotted fever group of rickettsiae.  Rickettsia rickettsii is found in the Americas and is transmitted to humans through the bite of infected ticks. The bacterium infects human vascular endothelial cells, producing an inflammatory response. The pathogenesis of RMSF is discussed in some detail below.

Other spotted fever group rickettsiae that produce human rickettsioses include R. conorii, R. mongolotimonae and R. slovaca (boutonneuse fever and similar illnesses), R. japonica (Japanese spotted fever), R. sibirica (North Asian tick typhus), R. africae (African tick bite fever),  R. helvetica (perimyocarditis), and R. honei (Flinders Island spotted fever). The spotted fever rickettsiae have been found on every continent except Antarctica.

Two "transitional group" (other) rickettsias cause spotted fever-like diseases: R. akari (rickettsial pox), and R. australis (Queensland tick typhus).

Typhus Group (TG)
Rickettsia prowazekii is the cause of epidemic or louse-borne typhus and is the prototypical bacterium from the typhus group of rickettsiae. R. prowazekii infects human vascular endothelial cells, producing widespread vasculitis. In contrast to RMSF, louse-borne typhus tends to occur in the winter. Infection usually is transmitted from person to person by the body louse and, therefore, tends to manifest under conditions of crowding and poor hygiene. The southern flying squirrel is apparently the reservoir in the United States, but the vector involved in transmission from the flying squirrel to humans is unknown. The disease has a worldwide distribution.

Other rickettsiae in the typhus group include R. typhi and R. felis. Murine typhus is caused by transmission of R. typhi from rats, cats and opossums to humans via a flea vector. Murine typhus is found worldwide and is endemic to areas of Texas and southern California in the United States. Although R. felis is phylogenetically more closely related to the spotted fever group of rickettsiae than the typhus group, it shares antigens with R. typhi and produces a murine typhus-like illness. Rickettsia felis has been detected in cat fleas and opossums.

Scrub Typhus Group (STG)
Orientia (Rickettsia) tsutsugamushi is the cause of scrub typhus. Originally called Rickettsia tsutsugamushi, this organism was given its own genus designation because it is phylogenetically distinct from the other rickettsiae, though closely related. Orientia tsutsugamushi is transmitted to humans by the bite of trombiculid mites (chiggers), which are the vector and host. Scrub typhus occurs throughout much of Asia and Australia.

The Rickettsiae are small (0.3-0.5 x 0.8-2.0 um), Gram-negative, aerobic, coccobacilli that are obligate intracellular parasites of eucaryotic cells. They may reside in the cytoplasm or within the nucleus of the cell that they invade. They divide by binary fission and they metabolize host-derived glutamate via aerobic respiration and the citric acid (TCA) cycle. They have typical Gram-negative cell walls, and they lack flagella. The rickettsiae frequently have a close relationship with arthropod vectors that may transmit the organism to mammalian hosts. The rickettsiae have very small genomes of about 1.0-1.5 million bases.

Rickettsia prowazekii, the cause of epidemic typhus, is the prototypical rickettsia. Typhus has plagued humanity throughout history. The American bacteriologist, Hans Zinsser, to whom this textbook is dedicated, was able to grow the elusive intracellular pathogen and develop a protective vaccine for typhus fever. He wrote a book about the bacterium, published in 1935, Rats, Lice, and History: "being a study in biography, which, after 12 preliminary chapters indispensable for the preparation of the lay reader, deals with the life history of typhus fever".

Rickettsia prowazekii has made science news recently since it has been shown to be the probable origin of eucaryotic mitochondria. Its complete genome sequence of 1,111,523 base pairs has been shown to contain 834 protein-coding genes. The functional profiles of these genes show similarities to those of mitochondrial genes. No genes required for glycolysis are found in either R. prowazekii or mitochondrial genomes, but a complete set of genes encoding components of the tricarboxylic acid cycle and the respiratory-chain complex is found in both. In effect, ATP production in the rickettsia is the same as that in mitochondria. Many genes involved in the biosynthesis and regulation of biosynthesis of amino acids and nucleosides in free-living bacteria are absent from R. prowazekii and mitochondria. Such genes seem to have been replaced by homologues in the nuclear (host) genome. Phylogenetic analyses indicate that R. prowazekii is more closely related to mitochondria than it is to any bacterium on the Tree of Life.

Rickettsiae must be grown in the laboratory by co-cultivation with eucaryotic cells, and they have not been grown by in axenic culture. The basis of their obligate relationship with eucaryotic cells has been explained by rickettsial possession of "leaky membranes" that require the osmolarity and nutritional environment supplied by an intracellular habitat.

The rickettsiae, in spite of their small size and obligate intracellular habitat, are a group of alphaproteobacteria,  which include many well-known organisms such as Acetobacter, Rhodobacter, Rhizobium and Agrobacterium. Very few of the alphaproteobacteria are pathogens of humans. Brucella, Bartonella, Rickettsia, and a related intracellular parasite, Ehrlichia, are the main exceptions.

The genus Rickettsia is included in the bacterial family Rickettsiaceae of the order Rickettsiales. This genus includes many species associated with human disease, including those in the spotted fever group and the typhus group (figure 1). The rickettsiae that are pathogens of humans are subdivided into three major groups based on clinical characteristics of disease: 1. spotted fever group; 2. typhus group; and 3. scrub typhus group.

Figure 1.  Taxonomic classification of the order Rickettsiales

: : : : : http://textbookofbacteriology.net/RickettsialTaxonomy.gif

 

Spotted Fever Group (SFG)
Rickettsia rickettsii is the cause of Rocky Mountain spotted fever (RMSF) and is the prototype bacterium in the spotted fever group of rickettsiae.  Rickettsia rickettsii is found in the Americas and is transmitted to humans through the bite of infected ticks. The bacterium infects human vascular endothelial cells, producing an inflammatory response. The pathogenesis of RMSF is discussed in some detail below.

Other spotted fever group rickettsiae that produce human rickettsioses include R. conorii, R. mongolotimonae and R. slovaca (boutonneuse fever and similar illnesses), R. japonica (Japanese spotted fever), R. sibirica (North Asian tick typhus), R. africae (African tick bite fever),  R. helvetica (perimyocarditis), and R. honei (Flinders Island spotted fever). The spotted fever rickettsiae have been found on every continent except Antarctica.

Two "transitional group" (other) rickettsias cause spotted fever-like diseases: R. akari (rickettsial pox), and R. australis (Queensland tick typhus).

Typhus Group (TG)
Rickettsia prowazekii is the cause of epidemic or louse-borne typhus and is the prototypical bacterium from the typhus group of rickettsiae. R. prowazekii infects human vascular endothelial cells, producing widespread vasculitis. In contrast to RMSF, louse-borne typhus tends to occur in the winter. Infection usually is transmitted from person to person by the body louse and, therefore, tends to manifest under conditions of crowding and poor hygiene. The southern flying squirrel is apparently the reservoir in the United States, but the vector involved in transmission from the flying squirrel to humans is unknown. The disease has a worldwide distribution.

Other rickettsiae in the typhus group include R. typhi and R. felis. Murine typhus is caused by transmission of R. typhi from rats, cats and opossums to humans via a flea vector. Murine typhus is found worldwide and is endemic to areas of Texas and southern California in the United States. Although R. felis is phylogenetically more closely related to the spotted fever group of rickettsiae than the typhus group, it shares antigens with R. typhi and produces a murine typhus-like illness. Rickettsia felis has been detected in cat fleas and opossums.

Scrub Typhus Group (STG)
Orientia (Rickettsia) tsutsugamushi is the cause of scrub typhus. Originally called Rickettsia tsutsugamushi, this organism was given its own genus designation because it is phylogenetically distinct from the other rickettsiae, though closely related. Orientia tsutsugamushi is transmitted to humans by the bite of trombiculid mites (chiggers), which are the vector and host. Scrub typhus occurs throughout much of Asia and Australia.

 

 

Figure 15. Pleomorphism in rickettsiae: 1 cocci forms; 2,3 small rod-shaped forms; 4 filamentous forms ovoids about 0.5 mm in diameter;

 

 

: : : : Ris_34_Rickettsia

Rickettsia

 

: : : : Ris_35_Rickettsia_prowazekii

Rickettsia_prowazeki

 

Rod-shaped rickettsiae are short organisms from 1 to 1.5 mcm in diameter with granules on the ends, or long and usually curved thin rods from 3 to 4 mcm in length. Filamentous forms are from 10 to 40 mcm and more in length: sometimes they are curved and multigranular filaments.

Rickettsiae are non-motile, do not produce spores and capsules and stain well by the Romanowsky-Giemsa stain and the Ziehl-Neelsen stain.

Electron microscopy and cytochemical study have shown that the rickettsiae have an inner (0.06 mcm) and an outer membrane acting as a wall and consisting of three layers. Granules of the ribosome type measuring 2-7 mcm and vacuole-like structures 0.06-0.08 mcm in diameter have been found in the cytoplasm or rickettsiae. Rickettsiae multiply by division of the coccoid and rod-shaped formswhich give rise to homogeneous populations of the corresponding type, and also by the breaking down of the filamentous forms giving rise to coccoid and rod-shaped entities.

Pathogenic rickettsiae invade various species of animals and man. The diseases caused by rickettsiae are known as rickettsioses. A typical representative is Rickettsia prowazekii (the name was given in honour of the scientists, the American Howard Ricketts and the Czech Stanislaus Prowazek), the causative agent of typhus fever.

Rickettsiae pertain to obligate parasites. They live and multiply only in the cells (in the cytoplasm and nucleus) of animals, humans, and vectors.

The order Rickettsiales consists of 3 families: Rickettsiaceae, which has been characterized above; Bartonellaceae, parasites of human erythrocytes; Anaplasmaceae, parasites of animal erythrocytes. The order

Genus Chlamydia, family Chlamydiaceae, order Chlamydiales include the causative agents of trachoma, conjunctivitis (inclusion blennorrhoea), inguinal lymphogranulomatosis (Nicolas-Favre disease), and ornithosis. The organisms contain DNA, RNA, nucleoproteins, lipids, and carbohydrates

Chlamydia are obligate intracellular parasites. They are coccal in shape and measure 0.2-1 5 mcm in diameter; reproduction occurs only in the cytoplasm of the cells of the vertebrates. The organisms are characterized by low metabolic activity and are cultivated at 33-41 C in the yolk sac of a chick embryo They are sensitive to antibiotics of the tetracycline series and are Gram-negative. Chlamydia organisms cause respiratory infections with generalization of the process in birds and affect the respiratory passages, placenta, joints, and the intestinal tract in mammals.

 

: : : : Ris_37_chlamidia

Chlamidia

 

Mycoplasmas

mashttp://www.ncbi.nlm.nih.gov/books/NBK7637/

w.rain-tree.com/mycoresearch.htm#.UUBkN3lkgUM

 

General Concepts

 

Clinical Manifestations

"Mycoplasmas are most unusual self-replicating bacteria, possessing very small genomes, lacking cell wall components, requiring cholesterol for membrane function and growth, using UGA codon for tryptophan, passing through "bacterial-retaining" filters, and displaying genetic economy that requires a strict dependence on the host for nutrients and refuge. In addition, many of the mycoplasmas pathogenic for humans and animals possess extraordinary specialized tip organelles that mediate their intimate interaction with eucaryotic cells. This host-adapted survival is achieved through surface parasitism of target cells, acquisition of essential biosynthetic precursors, and in some cases, subsequent entry and survival intracellularly. Misconceptions concerning the role of mycoplasmas in disease pathogenesis can be directly attributed to their biological subtleties and to fundamental deficits in understanding their virulence capabilities." (Baseman, 1997)

Mycoplasma pneumoniae infection is a disease of the upper and lower respiratory tracts. Cough, fever, and headache may persist for several weeks. Convalescence is slow. Ureaplasma urealyticum infection causes nongonococcal urethritis in men, resulting in dysuria, urgency, and urethral discharge.

Coplasmas

Mycoplasma pneumoniae_microscopy

Mycoplasmaculture

Structure, Classification, and Antigenic Types

Mycoplasmas are spherical to filamentous cells with no cell walls. There is an attachment organelle at the tip of filamentous M pneumoniae, M genitalium, and several other pathogenic mycoplasmas. Fried-egg-shaped colonies are seen on agar. The mycoplasmas presumably evolved by degenerative evolution from Gram-positive bacteria and are phylogenetically most closely related to some clostridia. Mycoplasmas are the smallest self-replicating organisms with the smallest genomes (a total of about 500 to 1000 genes); they are low in guanine and cytosine. Mycoplasmas are nutritionally very exacting. Many require cholesterol, a unique property among prokaryotes. Ureaplasmas require urea for growth, another unusual property. Mycoplasmas have surface antigens such as membrane proteins, lipoproteins, glycolipids, and lipoglycans. Some of the membrane proteins undergo spontaneous antigenic variation. Antibodies to surface antigens inhibit growth; various serological tests have been developed and are useful in classification.

 

The genomes of most Mycoplasma species encode about 600 proteins. For example, The M. genitalium and M. pneumoniae genomes contain 470 and 677 protein-coding gene sequences, respectively, compared with 1,703 protein genes in Haemophilus influenzae and about 4,000 genes in E. Coli. The genomes of M. genitalium and M. pneumoniae have lost the genes involved in certain biosynthetic pathways, such as the genes for amino and fatty acid and vitamin synthesis. Since they are cell wall-deficient bacteria, there is a major reduction in genetic information needed for cell wall biosynthesis. Although Mycoplasma species carry a minimal set of genes involved in energy metabolism and biosynthesis, they still have the essential genes for DNA replication, transcription, translation, and the minimal number of rRNA and tRNA genes. The reduction in mycoplasmal genomes explains their need for host nutritional molecules. A significant number of mycoplasmal genes appear to be devoted to cell adhesion and attachment organelles as well as variable membrane surface antigens to maintain parasitism and evade host immune and nonimmune surveillance systems. Mycoplasma species variably express structurally heterogeneous cell surface antigens. Variations in the genes encoding cell surface adherence molecules reveal distinct patterns of mutations capable of generating changes in mycoplasma cell surface molecular size and antigenic diversity. Variable surface antigenic structures and rapid changes in their expression are thought to play important roles in the pathogenesis of mycoplasmal infections by providing altered structures for escape from immune responses and protein structures that enhance cell and tissue colonization and penetration of the mucosal barrier." (Nicolson, GL 1999)

Pathogenesis

Mycoplasmas are surface parasites of the human respiratory and urogenital tracts. Mycoplasma pneumoniae attaches to sialoglycoproteins or sialoglycolipid receptors on the tracheal epithelium via protein adhesins on the attachment organelle. The major adhesin is a 170-kilodalton (kDa) protein, named P1. Hydrogen peroxide and superoxide radicals (O2) excreted by the attached organisms cause oxidative tissue damage. Pneumonia is induced largely by local immunologic and phagocytic responses to the parasites. Sequelae of M pneumoniae infection (mainly hematologic and neurologic) apparently have an autoimmune etiology. Several fastidious mycoplasmas may act as cofactors in activation of the aquired immunodeficiency syndrome (AIDS). Macrophage activation, cytokine induction, and superantigen properties of some mycoplasmal cell components can be considered as pathogenicity factors.

Host Defenses

IgM antibodies, followed by IgG and secretory IgA, are important in host resistance. The importance of cell-mediated immunity is unclear.

Epidemiology

Mycoplasma pneumoniae infection occurs worldwide and is more prevalent in colder months. It affects mainly children ages 5 to 9 years. It is spread by close personal contact and has a long incubation period. Ureaplasma urealyticum is spread primarily through sexual contact. Women may be asymptomatic reservoirs.

Diagnosis

Culture of M pneumoniae from sputum or a throat swab is possible, but very slow; therefore diagnosis is usually based on serologic tests. Tests using diagnostic DNA probes and amplification of specific genomic mycoplasma sequences by the polymerase-chain reaction (PCR) are being developed.

Control

There is no certified vaccine for M pneumoniae. Treatment with erythromycin or tetracyclines is effective in reducing symptoms in both M pneumoniae and U urealyticum infections.

Introduction

Mycoplasmas are the smallest and simplest self-replicating bacteria. The mycoplasma cell contains the minimum set of organelles essential for growth and replication: a plasma membrane, ribosomes, and a genome consisting of a double-stranded circular DNA molecule ( Fig. 37-1). Unlike all other prokaryotes, the mycoplasmas have no cell walls, and they are consequently placed in a separate class Mollicutes(mollis, soft; cutis, skin). The trivial term mollicutes is frequently used as a general term to describe any member of the class, replacing in this respect the older term mycoplasmas.

 

Electron micrograph of thin-sectioned mycoplasma cells. Cells are bounded by a single membrane showing in section the characteristic trilaminar shape. The cytoplasm contains thin threads representing (more...)

Mycoplasmas have been nicknamed the crabgrass of cell cultures because their infections are persistent, frequently difficult to detect and diagnose, and difficult to cure. Contamination of cell cultures by mycoplasmas presents serious problems in research laboratories and in biotechnological industries using cell cultures. The origin of contaminating mycoplasmas is in components of the culture medium, particularly serum, or in the flora of the technician's mouth, spread by droplet infection.

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Clinical Presentation

Mycoplasmal pneumonia

The term primary atypical pneumonia was coined in the early 1940s to describe pneumonias different from the typical lobar pneumonia caused by pneumococci. Several common respiratory viruses, including influenza virus and adenovirus, were shown to be responsible for a significant number of these pneumonias. From other cases, many of which developed antibodies agglutinating red blood cells in the cold (cold agglutinins), an unidentified filterable agent was isolated by Eaton and associates and was called Eaton agent. This agent was identified as a new Mycoplasma species after its successful cultivation on cell-free media in 1962. Named Mycoplasma pneumoniae, it was the first clearly documented mycoplasma pathogenic for humans.

The effects of M pneumoniae on humans include subclinical infection, upper respiratory disease, and bronchopneumonia. Most human infections do not progress to a clinically evident pneumonia. When pneumonia occurs, the onset generally is gradual and the clinical picture is one of a mild to moderately severe illness, with early complaints referable to the lower respiratory passages. Radiography frequently reveals evidence of pneumonia before physical signs are apparent. Involvement is usually limited to one of the lower lobes of the lungs, and the pneumonia is interstitial or bronchopneumonic. The course of disease varies; remittent fever, cough, and headache persist for several weeks. One of the most consistent clinical features is a long convalescence, which may extend from 4 to 6 weeks. Few fatal cases have been reported. Several unusual complications have been noted, including hemolytic anemia, polyradiculitis, encephalitis, aseptic meningitis, and central nervous system illness such as Guillain-Barré syndrome. In addition, pericarditis and pancreatitis have been observed. These sequelae may be related to the suspected immunopathology of M pneumoniae disease (see below).

Nongonococcal Urethritis and Salpingitis

Growing evidence suggests that Ureaplasma urealyticum causes nongonococcal urethritis in men free of Chlamydia trachomatis,an established agent of nongonococcal urethritis. The wide occurrence of U urealyticum in sexually active, symptom-free adults hampers research in this field. Evidence is based primarily on the production of nongonococcal urethritis symptoms in ureaplasma-free and chlamydia-free volunteers by intraurethral inoculation of U urealyticum and on a report that this disease could be cured in a chlamydia-free man only when he and his partner were treated simultanously with tetracycline, which eliminated U urealyticum from both. Ureaplasmas have also been associated with chorioamnionitis, habitual spontaneous abortion, and low-weight infants. Mycoplasma hominis, a common inhabitant of the vagina of healthy women, becomes pathogenic once it invades the internal genital organs, where it may cause pelvic inflammatory diseases such as tubo-ovarian abscess or salpingitis.

It has been suggested that Mycoplasma genitalium, isolated in 1981 from the urethral discharge of two homosexual men, may account for the tetracycline-responsive, nongonococcal urethritis cases in which chlamydias and ureaplasmas cannot be isolated (about 20 percent of all cases). However, M genitalium is so fastidious that very few clinical isolates have so far been made on the best mycoplasma medium available. Only the recent application of specific PCR amplification of the organism's DNA in clinical specimens has provided experimental proof for the relative prevalence of M genitalium in the human urogenital tract and its apparent role in male urethritis.

Mycoplasmas in AIDS and Immunocompromised Patients

The question of whether mycoplasmas act as co-factors in the development of AIDS has attracted much attention recently. Several mycoplasms have so far been incriminated: M fermentans, considered until recently a relatively rare mycoplasma of the human urogenital tract, and M penetrans , a newly-discovered human mycoplasma isolated from several AIDS patients. M pirum, a mycoplasma of an unknown host, has been recently isolated from the blood of a few AIDS patients. While, in vitro studies show that these mycoplasmas may markedly enhance pathogenicity of the human immunodeficiency virus, the possibility that the mycoplasmas may simply represent opportunistic agents found in high frequency in patients with AIDS, cannot be ruled out. Yet on the whole, with the increasing incidence of immunocompromised patients (due to AIDS, organ transplantation, etc.) evidence is accumulating for invasion of tissues and the intracellular location of some mycoplasmas, notably M fermentans and M penetrans. Extragenital infections by urogenital mycoplasmas are rather common in neonates, immunosuppressed and/or hypogammaglobulinemic patients; clinical symptoms are expressed frequently as arthritis.

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Structure, Classification, and Antigenic Types

Distinguishing Properties

The coccus is the basic form of all mycoplasmas in culture. The diameter of the smallest coccus capable of reproduction is about 300 nm. In most mycoplasma cultures, elongated or filamentous forms (up to 100 μm long and about 0.4 μm thick) also occur. The filaments tend to produce truly branched mycelioid structures, hence the name mycoplasma (myces, a fungus; plasma, a form). Mycoplasmas reproduce by binary fission, but cytoplasmic division frequently may lag behind genome replication, resulting in formation of multinuclear filaments.

Schematic presentation of the mode of mycoplasma reproduction. Cells may either divide by binary fission or first elongate to multinucleate filaments, which subsequently breakup to coccoid (more...)

Some mycoplasmas possess unique attachment organelles, which are shaped as a tapered tip in M pneumoniae and M genitalium. Mycoplasma pneumoniae is a pathogen of the respiratory tract, adhering to the respiratory epithelium, primarily through the attachment organelle. Interestingly, these two human mycoplasmas exhibit gliding motility on liquid-covered surfaces. The tip structure always leads, again indicating its importance in attachment.One of the most useful distinguishing features of mycoplasmas is their peculiar fried-egg colony shape, consisting of a central zone of growth embedded in the agar and a peripheral one on the agar surface Morphology of a typical fried-egg mycoplasma colony.

The lack of cell walls and intracytoplasmic membranes facilitates isolation of the mycoplasma membrane in a relatively pure form. The isolated mycoplasma membrane resembles that of other prokaryotes in being composed of approximately two-thirds protein and one-third lipid. The mycoplasma lipids resemble those of other bacteria, apart from the large quantities of cholesterol in the sterol-requiring mycoplasmas.

Membrane proteins, glycolipids, and lipoglycans exposed on the cell surface are the major antigenic determinants in mycoplasmas. Antisera containing antibodies to these components inhibit growth and metabolism of the mycoplasmas and, in the presence of complement, cause lysis of the organisms. These properties are used in various serologic tests that differentiate between mycoplasma species and serotypes and detect antibodies to mycoplasmas in sera of patients (see below).

Molecular Biology

The mycoplasma genome is typically prokaryotic, consisting of a circular, double stranded DNA molecule. The Mycoplasma and Ureaplasma genomes are the smallest recorded for any self-reproducing). Therefore, there are very few genes; in some mycoplasmas the number is estimated at fewer than 500, about one sixth the number of genes in Escherichia coli. Mycoplasmas accordingly express a small number of cell proteins and lack many enzymatic activities and metabolic pathways. Their nutritional requirements are correspondingly complex, and they are dependent on a parasitic mode of life.

 

Taxonomy and Properties of Mycoplasmas Capable of Infecting Humans a.

The dependence of mycoplasmas on their host for many nutrients explains the great difficulty of cultivation in the laboratory. The complex media for mycoplasma culture contain serum, which provides fatty acids and cholesterol for mycoplasma membrane synthesis. The requirement of most mycoplasmas for cholesterol is unique among prokaryotes. The consensus is that only a small fraction of mycoplasmas existing in nature have been cultivated so far. Some of the cultivable mycoplasmas, including the human pathogen M pneumoniae, grow very slowly, particularly on primary isolation. Ureaplasma urealyticum, a pathogen of the human urogenital tract, grows very poorly in vitro, reaching maximal titers of 107 organisms/ml of culture. Mycoplasma genitalium, another human pathogen, grows so poorly in vitro that only a few successful isolations have been achieved.

Glucose and other metabolizable carbohydrates can be used as energy sources by the fermentative mycoplasmas possessing the Embden-Meyerhof-Parnas glycolytic pathway. All mycoplasmas examined thus far possess a truncated, flavin-terminated respiratory system, which rules out oxidative phosphorylation as an ATP-generating mechanism. Breakdown of arginine by the arginine dihydrolase pathway has been proposed as a major source of ATP in nonfermentative mycoplasmas. Ureaplasmas have a requirement, unique among living organisms, for urea. Because they are non-glycolytic and lack the arginine dihydrolase pathway, it has been suggested, and later proven experimentally, that ATP is generated through an electrochemical gradient produced by ammonia liberated during the intracellular hydrolysis of urea by the organism's urease.

The mycoplasma genome is characterized by a low guanine-plus-cytosine content and by a corresponding preferential utilization of codons containing adenine and uracil, particularly in the third position. Most interesting is the use of the universal stop codon UGA as a tryptophan codon in many mycoplasmas, a rare property found so far only in mycoplasmas and in nonplant mitochondria. Resistance of mycoplasmal RNA polymerase to rifampicin is another property distinguishing mycoplasmas from the conventional eubacteria. However, apart from this resistance to rifampicin, the mycoplasmas are susceptible to antibiotics, such as tetracyclines and chloramphenicol, that inhibit protein synthesis on prokaryotic ribosomes.

Phylogeny

As the smallest and simplest self-replicating prokaryotes, the mycoplasmas pose an intriguing question: do they represent the descendents of exceedingly primitive bacteria that existed before the development of a peptidoglycan-based wall, or do they represent evolutionary degenerate eubacterial forms that have lost their cell walls? The balance of the molecular evidence, based largely on comparison of base sequences of the highly conserved ribosomal RNA (rRNA) molecules, particularly of the 16S rRNA type, favors the hypothesis of degenerative evolution. According to Woese and his colleagues, the mycoplasmas evolved as a branch of the low-guanine-plus-cytosine Gram-positive bacteria and are most closely related to two clostridia, Clostridium innocuum and C ramosum. However, the marked phenotypic and genotypic variability among mycoplasmas has led some workers to conclude that mycoplasmas evolved from a variety of walled bacteria and accordingly have a polyphyletic origin. Woese maintains that the origin of mycoplasmas is monophyletic and explains the great variety of mycoplasmas by a process of rapid evolution characteristic of the group.

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Pathogenesis

All mycoplasmas cultivated and identified thus far are parasites of humans, animals, plants, or arthropods. The primary habitats of human and animal mycoplasmas are the mucous surfaces of the respiratory and urogenital tracts and the joints in some animals. Although some mycoplasmas belong to the normal flora, many species are pathogens, causing various diseases that tend to run a chronic course Pathogenesis and disease sites of infection by M pneumoniae and U urealyticum.

Most mycoplasmas that infect humans and other animals are surface parasites, adhering to the epithelial linings of the respiratory and urogenital tracts. Adherence is firm enough to prevent the elimination of the parasites by mucous secretions or urine. The intimate association between the adhering mycoplasmas and their host cells provides an environment in which local concentrations of toxic metabolites excreted by the parasite build up and cause tissue damage Moreover, because mycoplasmas lack cell walls, fusion between the membranes of the parasite and host has been suggested, and some experimental evidence for it has recently been obtained. Membrane fusion would alter the composition and permeability of the host cell membrane and enable the introduction of the parasite's hydrolytic enzymes into the host cell, events expected to cause serious damage. Recent studies have indicated the presence in mycoplasmas of antigenic variability systems. These systems, some of which are already defined in molecular genetic terms, are responsible for rapid changes in major surface protein antigens. The change in the antigenic coat of the parasite helps it to escape recognition by the immune mechanisms of the host.

 

: : Figure 37-5. Schematic presentation of a M pneumoniae organism attaching to the surface of the ciliary tracheal epithelium, as seen by electron microscopy of a thin section.

Schematic presentation of a M pneumoniae organism attaching to the surface of the ciliary tracheal epithelium, as seen by electron microscopy of a thin section. The clustering of the P1 adhesin on (more...)

Because attachment of M pneumoniae and M genitalium is affected by pretreatment of the host cells with neuraminidase, sialoglycoproteins and/or sialoglycolipids of the host cell membrane appear to be receptor sites for these mycoplasmas. There is evidence that several M pneumoniae membrane proteins act as adhesins and that they have high affinity for the specific receptors for M pneumoniae on host cells. Monoclonal antibodies to one of these proteins, protein P1 (molecular weight, 170,000 daltons), inhibit attachment of the parasite. Ferritin labeling of the antibodies has shown that P1 concentrates on the tip structure of the mycoplasma, a finding that further supports the notion that the tip serves as an attachment organelle.

The results obtained with M pneumoniae were essentially duplicated recently with M genitalium and showed that in this organism, which closely resembles M pneumoniae morphologically and physiologically, a major adhesin protein, named MgPa, is clustered at the tip organelle. The genes of the major adhesins of M pneumoniae (P1) and of M genitalium (MgPa) were cloned and sequenced, allowing the characterization of these proteins. The two adhesins are alike in many respects and in fact contain extensive areas of homology, as expressed also by shared epitopes. These two proteins may be the product of an ancestral gene that underwent a horizontal gene transfer event.

The nature of the toxic factors that damage the mucosal surfaces infected by mycoplasmas is still unclear. Toxins are rarely found in mycoplasmas. Consequently, researchers considered whether the end products of mycoplasma metabolism were responsible for tissue damage. Hydrogen peroxide (H2O2), the end product of respiration in mycoplasmas, has been implicated as a major pathogenic factor ever since it was shown to be responsible for the lysis of erythrocytes by mycoplasmas in vitro; however, the production of H2O2 alone does not determine pathogenicity, as the loss of virulence in M pneumoniae is not accompanied by a decrease in H2O2 production. For the H2O2 to exert its toxic effect, the mycoplasmas must adhere closely enough to the host cell surface to maintain a toxic, steady-state concentration of H2O2 sufficient to cause direct damage, such as lipid peroxidation, to the cell membrane. The accumulation of malonyldialdehyde, an oxidation product of membrane lipids, in cells exposed to M pneumoniae supports this notion. Moreover, M pneumoniae inhibits host cell catalase by excreting superoxide radicals (O2). This would be expected to further increase the accumulation of H2O2 at the site of parasite-host cell contact

 

Proposed mechanism of oxidative damage to host cells by adhering M pneumoniae. by increasing concentrations of H2O2 and O2. (Modified from Almagor M, (more...)

There is evidence that both organism-related and host-related factors are involved in the pathogenesis of mycoplasma infections. Mycoplasmas activate macrophages, and induce cytokine production and lymphocyte proliferation; the rat pathogen, Mycoplasma arthritidis, produces a potent superantigen. Thus, in the case of M pneumoniae, the host may be largely responsible for the pneumonia by mounting a local immune response to the parasite. Syrian hamsters inoculated intranasally with M pneumoniae show patchy bronchopneumonic lesions consisting of infiltration of mononuclear cells. The ablation of thymic function before the experimental infection prevents development of the characteristic pulmonary infiltration, but lengthens the period during which the organisms may be isolated from the lungs. When thymic animals are allowed to recover and then reinfected, an exaggerated and accelerated pneumonic process occurs. Epidemiologic data also suggest that repeated infections in humans are required before symptomatic disease occurs: serum antibodies to M pneumoniae can be found in most children 2 to 5 years of age, although the illness occurs with greatest frequency in individuals 5 to 15 years of age.

An immunopathologic mechanism also may explain the complications affecting organs distant from the respiratory tract in some patients infected with M pneumonia. Various autoantibodies have been detected in the sera of many of these patients, including cold agglutinins reacting with the erythrocyte I antigen, and antibodies reacting with lymphocytes, smooth muscle cells, and brain and lung antigens. Serologic cross-reactions between M pneumoniae and brain and lung antigens have been demonstrated, and these antigens are probably related to the glycolipids of M pneumoniae membranes, which are also found in most plants and in many bacteria. Clearly, host reaction varies markedly, as only about half of the patients develop cold agglutinins and complications are rare, even among individuals with anti-tissue globulins.

Host Defenses

Infection with M pneumoniae induces the development of serum antibodies that fix complement, inhibit growth of the organism and lyse the organism in the presence of complement. Generally, the first antibodies produced are of the IgM class, whereas later in convalescence the predominant antibody is IgG. Secretory IgA antibodies also develop and appear to be important in host resistance. The first infection in infancy usually is asymptomatic and generates a brief serum antibody response. Recurrent infections generate a more prolonged systemic antibody response and increasing numbers of circulating antigen-responsive lymphocytes. By late childhood, clinically apparent lower respiratory disease, including pneumonia, becomes more common. Therefore, mycoplasma respiratory disease manifestations appear to vary, depending on the state of local and systemic immunity at the time of reinfection. One hypothesis is that local immunity mediates resistance to infection and that systemic immunity contributes substantially to the pulmonary and systemic reaction characteristic of M pneumoniae pneumonia.

The relative importance of humoral and cell-mediated immunity in resistance to respiratory mycoplasma infections is still unclear. For many mycoplasma infections, such as bovine pleuropneumonia, resistance can be transferred with convalescent-phase serum, but this may not be true for all mycoplasma respiratory diseases. For example, resistance of rats to pulmonary disease induced by M pulmonis can be transferred only with spleen cells obtained from previously infected animals. Although IgA antibody may be important in resistance to mycoplasmas, other factors seem to be involved in resistance to pulmonary disease, and these factors may not be the same for all mycoplasma infections.

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Epidemiology

One of the most puzzling features of M pneumoniae pneumonia is the age distribution of patients. In a survey conducted between 1964 and 1975 of more than 100,000 individuals in the Seattle area, the age-specific attack rate was highest among 5- to 9-year-old children. Rates of M pneumoniae pneumonia in the youngest age group, 0 to 4 years old, were about one-half those in school-age children, but considerably higher than in adults. Mycoplasma pneumoniae pneumonia was rarely observed in infants younger than 6 months, suggesting maternally conferred immunity (Mycoplasma pneumoniae accounts for 8 to 15 percent of all pneumonias in young school-age children. In older children and in young adults, the organism is responsible for approximately 15 to 50 percent of all pneumonias. Infection with M pneumoniae occurs worldwide all year round but shows a predilection for the colder months, apparently because of the greater opportunity for transmission by droplet infection. Mycoplasma pneumoniae appears to require close personal contact to spread; successful spreading usually occurs in families, schools, and institutions. The incubation period ranges from 2 to 3 weeks.

 

Incidence of M pneumoniae pneumonia in Seattle by age, for two epidemics (1966-67 and 1974) and the endemic periods (1967-73). (From Foy HM, Kenny GE, Cooney MK, Allen ID: Long-term epidemiology (more...)

Ureaplasma urealyticum is spread primarily through sexual contact. Colonization has been linked to the frequency of sexual intercourse and the number of sexual partners. Women may be asymptomatic reservoirs of infection.

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Diagnosis

Culture is essential for definitive diagnosis (See below).

Culture

A routine mycoplasma medium consists of heart infusion, peptone, yeast extract, salts, glucose or arginine, and horse serum (5 to 20 percent). Fetal or newborn calf serum is preferable to horse serum. To prevent the overgrowth of the fast-growing bacteria that usually accompany mycoplasmas in clinical materials, penicillin, thallium acetate or both are added as selective agents. For Ureaplasma culture, the medium is supplemented with urea and its pH is brought to 6.0. Ureaplasm a and M genitalium are relatively sensitive to thallium, which is, therefore, omitted from their culture media. For M pneumoniae isolation, nasopharyngeal secretions are inoculated into a selective diphasic medium (pH 7.8) made of mycoplasma broth and agar and supplemented with glucose and phenol red. When M pneumoniae grows in this medium, it produces acid, causing the color of the medium to change from purple to yellow. Broth from the diphasic medium is subcultured to mycoplasma agar when a color change occurs, or at weekly intervals for a minimum of 8 weeks.

Identification

Colonies appearing on the plates can be identified as M pneumoniae by staining directly on agar with homologous fluorescein-conjugated antibody or by demonstrating that a specific antiserum to M pneumoniae inhibits their growth on agar. Colonies of ureaplasmas are usually minute (less than 100 μm in diameter); because of urea hydrolysis and ammonia liberation, the medium becomes alkaline. When manganous sulfate is added to the medium, the ureaplasma colonies stain dark brown. Isolates can be characterized in more detail by a variety of biochemical and serologic tests. More sophisticated tests, including electrophoretic analysis of cell proteins, DNA-DNA hybridization tests, mycoplasmal DNA cleavage patterns by restriction endonucleases, and PCR tests employing species-specific primers for amplification, may be performed in a research laboratory.

Serodiagnosis and Molecular Probes

Serodiagnosis consists of examining serum samples for antibodies that inhibit the growth and metabolism of the organism or fix complement with mycoplasmal antigens. A fourfold or greater rise in IgG titer is considered indicative of recent infection, whereas a sustained high antibody titer may not be significant, because a relatively high level of antibody may persist for at least 1 year after infection. A variety of rapid tests based on indirect hemagglutination of erythrocytes or latex particles coated with M pneumoniae antigens have been developed, and some are commercially available.The cold agglutinin test is less useful because only about one-half of patients develop cold agglutinins and because these antibodies also are induced by a great many other conditions.

Present techniques for laboratory diagnosis of M pneumoniae infections are of little use to the clinician because recovery by culture and identification of the mycoplasmas take at least 1 to 2 weeks. Methods for rapid laboratory diagnosis, such as direct demonstration of organisms in the respiratory specimens by nucleic acid amplification techniques, have promise but diagnostic kits are not yet commercially available.

Control

Prevention

Chemoprophylaxis of mycoplasma infections is not recommended, and no vaccine is available. Prior natural infection appears to provide the most effective resistance; however, evidence shows that M pneumoniae infections recur at intervals of several years. These observations suggest that immunity to a single natural infection is relatively short-term.

Treatment

The mycoplasmas are sensitive to tetracyclines, macrolides, and the newer quinolones, but are resistant to antibiotics that specifically inhibit bacterial cell wall synthesis. Tetracycline or erythromycin is recommended for treatment of M pneumoniae pneumonia, although effective treatment of the symptoms usually is not accompanied by eradication of the organism from the infected host. To prevent recurrence of nongonococcal urethritis caused by U urealyticum, sexual partners should be treated simultaneously with tetracycline. The incidence of tetracycline-resistant strains of U urealyticum and M hominis is on the rise.

Certain Mycoplasma species can either activate or suppress host immune systems, and they may use these activities to evade host immune responses. For example, some mycoplasmas can inhibit or stimulate the proliferation of normal lymphocyte subsets, induce B-cell differentiation and trigger the secretion of cytokines, including interleukin-1 (IL-1), IL-2, IL-4, IL-6, tumor necrosis factor-a (TNFa), interferons, and granulocyte macrophage-colony stimulating factor (GM-CSF) from B-cells as well as other cell types. Moreover, it was also found that M. fermentans-derived lipids can interfere with the interferon (IFN)-g-dependent expression of MHC class II molecules on macrophages. This suppression results in impaired antigen presentation to helper T-cells in an experimental animal model. Also, mycoplasmas are able to secret soluble factors that can stimulate proliferation or inhibit the growth and differentiation of immune competent cells.

Mycoplasmas can target the host white blood cells (lymphocytes/WBC) for intracellular infection, and these cells have the unique ability to cross the blood-brain barrier over into the spinal fluid and d into the host central nervous system (CNS).

Once inside the host CNS, certain pathogenic mycoplasmas have been reported to activate the CNS hypothalamus/pituitary/adrenal axis and neuroendocrine system. The hypothalamus and pituitary glands form part of the human endocrine system which produces hormones that regulate nearly every bodily function. This involvement is hypothesized to contribute to diseases such as fibromyalgia, chronic fatigue, and some AIDS-related symptoms.[Yirmiya R, 1999]

Mycoplasma species are known to secrete immune-modulating substances. For example, immune cells are affected by spiralin, a well-characterized mycoplasmal lipoprotein that can stimulate the in vitro proliferation of human peripheral blood mononuclear cells. This stimulation of immune cells results in secretion of proinflammatory cytokines (TNFa, IL-1 or -6). Spiralin can also induce the maturation of murine B-cells.

Mycoplasmas can escape immune recognition by undergoing surface antigenic variations thus rapidly altering their cell surface structures. Such antigenic variability, the ability to suppress host immune responses, slow growth rates and intracellular locations may explain the chronic nature of mycoplasmal infections and the common inability of a host to suppress mycoplasmal infections with host immune and nonimmune responses.

Rapid adaptation to host microenvironments by mycoplasmas is usually accompanied by rapid changes in cell surface adhesion receptors for more successful cell binding and entry as well as rapid structural protein changes to mimic host antigenic structures (antigen mimicry). For example, during chronic, active arthritis the size and antigenic diversity of the surface lipoprotein Vaa antigen changes in structure and expression in vivo. Antigenic divergence of Vaa can affect the adherence properties of M. hominis and enhance evasion of host-mediated immunity. Variations in the Vaa genes reveal a distinct pattern of mutations that generate mycoplasma surface variations and thus avoid host immune responses.

Mycoplasmas can directly suppress host immune responses by initiating or enhancing apoptosis. For example, M. fermentans, a recently discovered mycoplasma found in the urine of HIV and AIDS positive patients, can initiate or enhance concanavalin A-induced apoptosis (programmed cell death) of T-cells. Relatively large amounts of nucleases are also expressed by Mycoplasma species, and these can be released intracellularly to cause degradation of host DNA. Mycoplasmal nucleases may also be involved in secondary necrosis seen in advanced mycoplasmal infections, as indicated by the occurrence of morphological characteristics of apoptosis (chromatin condensation) and necrosis (loss of membrane integrity and organelle swelling). Although mycoplasmas can release activated oxygen species that may be involved in initiating apoptosis, some Mycoplasma species, such as M. fermentans, express a novel cytolytic activity in a nonlipid protein fraction that has a cytocidal effect not mediated by the known mycoplasmal cytokines like TNFa.

In addition to apoptosis, mycoplasmas can also release growth inhibitory molecules into their surroundings, such as arginine deaminase. This enzyme can act as a growth-inhibitory substance that suppresses IL-2 production and receptor expression in T cells stimulated by non-specific mitogens, and it can induce the morphologic features of dying cells and DNA fragmentation indicative of apoptosis.

Hydrogen peroxide and superoxide radicals are generated by adhering mycoplasmas, which induces oxidative stress, including host cell membrane damage.

Competition for and depletion of nutrients or biosynthetic precursors by mycoplasmas, which disrupts host cell maintenance and function.

Existence of capsule-like material and electron-dense surface layers or structures, which provides increased integrity to the mycoplasma surface and confers immunoregulatory activities

High-frequency phase and antigenic variation, which results in surface diversity and possible avoidance of protective host immune defenses

Secretion or introduction of mycoplasmal enzymes, such as phospholipases, ATPases, hemolysins, proteases, and nucleases into the host cell milieu, which leads to localized tissue disruption and disorganization and chromosomal aberrations and tumor formation.

Intracellular residence, which sequesters mycoplasmas, establishes latent or chronics

The mycoplasmas belong to the class Mollicutes, order Mycoplasmatales. These bacteria measure 100-150 nm, sometimes 200-700 nm, are non-motile and. do not produce spores

Three stages are observed in the developmental cycle of organisms: (1) small (0.2-0 4 mcm) elementary bodies containing in a compact state the nucleoid genetic material and ribosomes enclosed within a three-layer wall; (2) primary, large (0.8-1.5 mcm), bodies with nucleoid fibrils and ribosomal elements; they are covered with a thin wall and reproduce by fission; the daughter cells reorganize into elementary bodies which may be extracellular and penetrate other cells; (3) intermediate (transitory) stage between the primary and the elementary bodies. Small (elementary) bodies have infectious properties, large (primary) bodies accomplish vegetative function.

Growth, reproduction, and maturation of Chlamydia organisms are completed in 40 hours, microcolonies develop within the cytoplasm. Five or six antigens have been detected in the cell wall, which are responsible for the virulent properties of the different strains.

The mycoplasmas belong to the class Mollicutes, order Mycoplasmatales. These bacteria measure 100-150 nm, sometimes 200-700 nm, are non-motile and. do not produce spores.

 

 

: : : : Ris_38a_Mycoplasma

Mycoplasma

 

Mycoplasmas are the smallest microorganisms. They were first noticed by Pasteur when he studied the causative agent of pleuropneumonia in cattle. However, at the time he was unable to isolate them in pure culture on standard nutrient media, or to see them under a light microscope. Because of this, these micro-organisms were regarded as viruses. In 1898 Nocard and Roux established that the causative agent of pleuropneumoniacan grow on complex nutrient media which do not contain cells from tissue cultures. Elford using special filters determined the size of the microbe to be within the range of 124-150 nm. Thus, in size mycoplasmas appeared to be even smaller than some viruses.

Since they do not possess a true cell wall, mycoplasmas are characterized by a marked pleomorphism. They give rise to coccoid, granular, filamentous, cluster-like, ring-shaped, filterable forms, etc. Pleomorphism is observed in cultures and in the bodies of animals and man. No two forms are alike. The nuclear apparatus is diffuse. There are both pathogenic and non-pathogenic species. The most typical representative of the pathogenic species is the causative agent of pleuropneumonia in cattle (see section on pathogenic mycoplasmas).

At the present time more than 36 representatives of this order have been isolated, the most minute of all known bacteria. They are found in the soil, sewage waters, different substrates and in the bodies of animals and humans. Since mycoplasmas pass through many filters, and yet grow on media which do not contain live tissue cells, they are considered to be microorganisms intermediate between bacteria and viruses. Chemically, mycoplasmas are closer to bacteria. They contain up to 4 per cent DNA and 8 per cent RNA.

The most typical representatives of the pathogenic species are the causative agents of pleuropneumonia in cattle (Mycoplasma mycoides), acute respiratory infections (Mycoplasma hominis) and atypical pneumonia in humans (Mycoplasma pneumoniae).

 

Fungi http://www.microbiologyonline.org.uk/about-microbiology/introducing-microbes/fungi

Fungi can be single celled or very complex multicellular organisms. They are found in just about any habitat but most live on the land, mainly in soil or on plant material rather than in sea or fresh water. A group called the decomposers grow in the soil or on dead plant matter where they play an important role in the cycling of carbon and other elements. Some are parasites of plants causing diseases such as mildews, rusts, scabs or canker. In crops fungal diseases can lead to significant monetary loss for the farmer. A very small number of fungi cause diseases in animals. In humans these include skin diseases such as athletes foot, ringworm and thrush.

 

Types of fungi

Fungi are subdivided on the basis of their life cycles, the presence or structure of their fruiting body and the arrangement of and type of spores (reproductive or distributional cells) they produce.

The three major groups of fungi are:

  • multicellular filamentous moulds
  • macroscopic filamentous fungi that form large  fruiting bodies. Sometimes the group is referred  to as mushrooms, but the mushroom is just the part of the fungus we see above ground which is also known as the fruiting body.
  • single celled microscopic yeasts

Multicellular filamentous moulds

Moulds are made up of very fine threads (hyphae). Hyphae grow at the tip and divide repeatedly along their length creating long and branching chains. The hyphae keep growing and intertwining until they form a network of threads called a mycelium. Digestive enzymes are secreted from the hyphal tip. These enzymes break down the organic matter found in the soil into smaller molecules which are used by the fungus as food.

Some of the hyphal branches grow into the air and spores form on these aerial branches. Spores are specialized structures with a protective coat that shields them from harsh environmental conditions such as drying out and high temperatures. They are so small that between 500 1000 could fit on a pin head.

Spores are similar to seeds as they enable the fungus to reproduce. Wind, rain or insects spread spores. They eventually land in new habitats and if conditions are right, they start to grow and produce new hyphae. As fungi cant move they use spores to find a new environment where there are fewer competing organisms.

Macroscopic filamentous fungi

Macroscopic filamentous fungi also grow by producing a mycelium below ground. They differ from moulds because they produce visible fruiting bodies (commonly known as mushrooms or toadstools) that hold the spores. The fruiting body is made up of tightly packed hyphae which divide to produce the different parts of the fungal structure, for example the cap and the stem. Gills underneath the cap are covered with spores and a 10 cm diameter cap can produce up to 100 million spores per hour.

Yeasts

Yeasts are small, lemon-shaped single cells that are about the same size as red blood cells. They multiply by budding a daughter cell off from the original parent cell. Scars can be seen on the surface of the yeast cell where buds have broken off. Yeasts such as Saccharomyces, play an important role in the production of bread and in brewing. Yeasts are also one of the most widely used model organisms for genetic studies, for example in cancer research. Other species of yeast such as Candida are opportunistic pathogens and cause infections in individuals who do not have a healthy immune system.

 

Molds consist of long, branching filaments of cells called hyphae (singular, hypha). A tangled mass of hyphae visible to the unaided eye is a mycelium (plural, mycelia). In some molds, the cytoplasm passes through and among cells of the hypha uninterrupted by cross walls. These fungi are said to be coenocytic fungi. Those fungi that have cross walls are called septate fungi, since the cross walls are called septa.

Yeasts are microscopic, unicellular fungi with a single nucleus and eukaryotic organelles. They reproduce asexually by a process of budding. In this process, a new cell forms at the surface of the original cell, enlarges, and then breaks free to assume an independent existence.

Some species of fungi have the ability to shift from the yeast form to the mold form and vice versa. These fungi are dimorphic. Many fungal pathogens exist in the body in the yeast form but revert to the mold form in the laboratory when cultivated.

Reproduction in yeasts usually involves spores. Spores are produced by either sexual or asexual means. Asexual spores may be free and unprotected at the tips of hyphae, where they are called conidia (Figure 1 ). Asexual spores may also be formed within a sac, in which case they are called sporangiospores.

: : : : : http://media.wiley.com/Lux/71/8371.nfg024.jpg

The microscopic structures of a septate fungus showing asexually producedconidia that leave the fungus and germinate to produce a new mycelium.

Nutrition. Fungi grow best where there is a rich supply of organic matter. Most fungi are saprobic (obtaining nutrients from dead organic matter). Since they lack photosynthetic pigments, fungi cannot perform photosynthesis and must obtain their nutrients from preformed organic matter. They are therefore chemoheterotrophic organisms.

Most fungi grow at an acidic pH of about 5.0, although some species grow at lower and higher pH levels. Most fungi grow at about 25C (room temperature) except for pathogens, which grow at 37C (body temperature). Fungi store glycogen for their energy needs and use glucose and maltose for immediate energy metabolism. Most species are aerobic, except for the fermentation yeasts that grow in both aerobic and anaerobic environments.

 

(L. fungus a mushroom) belong to plant heterotrophic organisms (eukaryotes) devoid of chlorophyll. The cells of fungi have a differentiated nucleus and many of them multiply by sporulation.They differ greatly from bacteria.

The fungi are marked by various morphology. The main structural component of the vegetative body is the mycelium which is composed of branching colourless filaments (hyphae). In some species the mycelium is non-septate, i. e. formed of a single cell (Mucor mould), in others (higher fungi) it is polycellular (septate). Yeasts are oval or rounded and lack mycelium. The fungus Claviceps purpurea forms a sclerotium which is a firm network of mycelial hyphae.

: : : : Scheme_4

 

MODErN classificatyin of Fungi

Division Zygomycota. Members of the division Zygomycota are known as zygomycetes. Zygomycetes produce sexual spores known as zygospores (Figure 1 ), as well as asexual sporangiospores.

: : : : : http://media.wiley.com/Lux/72/8372.nfg025.jpg

Figure 1

Sexual reproduction in the mold Rhizopus stolonifer. Plus and minus mycelia produce sexually opposite hyphae that fuse and give rise to zygospores, which germinate to form new mycelia.

A familiar member of the division is Rhizopus stolonifer, a fungus found on fruits, vegetables, and breads. It is the familiar bread mold. It anchors itself to the substratum with special hyphae known as rhizoids. Rhizopus is used in the industrial production of steroids, meat tenderizers, industrial chemicals, and certain coloring agents.

Division Ascomycota. Members of the division Ascomycota are referred to as ascomycetes. After sexual fusion of cells has taken place, these organisms form their sexual spores within a sac called an ascus. Therefore, they are called sac fungi.

Ascomycetes include the powdery mildews and the fungi that cause Dutch elm disease and chestnut blight disease. The research organism Neurospora crassa is found within this group. Asexual reproduction in the ascomycetes involves conidia.

Many yeasts are classified in the division Ascomycota. Of particular interest is the fermentation yeast Saccharomyces. This yeast is used in the production of alcoholic drinks, in bread making, and as a source of growth factors in yeast tablets. It is an extremely important research organism as well.

Division Basidiomycota. Members of the division Basidiomycota are referred to as basidiomycetes and are called club fungi. After the sexual cells have united, they undergo division and produce a clubshaped structure called a basidium. Sexually produced basidiospores form at the tips of the basidia. Basidia are often found on huge, visible, fruiting bodies called basidiocarps. The typical mushroom is a basidiocarp.

Basidiomycetes are used as food (for example, mushrooms), but some basidiomycetes are pathogens. One of the organisms of meningitis is the basidiomycete Cryptococcus neoformans. The mushroom Amanita is poisonous to humans.

Division Deuteromycota. Members of the Deuteromycota division are called deuteromycetes. These fungi lack a known sexual cycle of reproduction and are said to be imperfect. When its sexual cycle is discovered, a fungus from this division is usually reclassified in one of the other divisions. Among the imperfect fungi are the organisms of athlete's foot and ringworm.

 

Fungi resemble algae in structure. They have a firm membrane consisting of cellulose, pectin substances, and carbohydrates. Various inclusions are found in the cytoplasm: glycogen, volutin, drops of fat. The cells of fungi may be mononuclear and polynucleate. The nuclei undergo both direct and indirect division. Fungi reproduce by rupture of the mycelium into pieces capable of germinating, by means of chlamydospores and conidia, by sporulation, and by the sexual way. The group of fungi includes saprophytes, parasites, and facultative parasites of plants, animals, and humans.

Chytridiomycetes. Most species inhabit water reservoirs. They lack mycelium or it is present in a rudimentary state. They move by means of pseudopodia. The cells are polynucleate. The Chytridiomycetes undergo a complex developmental cycle. They reproduce by simple division and sporulation. When occurring on a moist substrate, the spores of these fungi absorb water, swell, rupture the membrane, and divide with the production of amoeboid-like cells some of which coalesce and form zygotes which divide and develop into a polynucleate mucous mass. Some species which are pathogenic for plants induce, in particular, cabbage disease ('blackleg') and wart disease of potatoes.

Oomycetes are fungi with non-cellular (non-septate) mycelium. Some species live in water, others in the soil. Water inhabiting oomycetes cause diseases among fish and destroy the roe of fish and frogs. "Other oomycetes parasitize on plants and cause phytophtorosis of potatoes and the fruit of grapes and peronosporosis of sugar beet. The genus Mucor or bread mould belongs to the class Oomycetes (Fig. 16). It consists of a non-septate mycelium in the shape of a much branched cell, from which branch out the fruiting hyphae - sporangiophores with round dilatations at the tips sporangia. The latter are filled with endospores which provide a means of reproduction. Mucor mould may also reproduce sexually. It is widespread in nature, is often found on vegetables, moist surfaces of objects, and in manure.

A typical representative of Mucor mould is Mucor mucedo.

 

: : : : : Ris_29_Mucor

Mucor

 

Pathogenic species of this mould may cause infections of the lungs and middle ear, and a general severe infectious process in humans, Zygomycetes are soil fungi with a non-cellular mycelium. They reproduce by means of sporangios pores, less frequently by means of conidia. Enzymes secreted by these fungi are used for clarifying juices and pre- paring alcoholic beverages. The class Zygomycetes includes the order Entomophilies, parasites of insects: they cause the death of the larvae of mosquitoes and flies and are used as insecticides. Ascomycetes or sac fungi (35000 species) have a multicellular mycelium. They reproduce sexually by means of ascospores (spores which develop in special spore cases, asci). The organisms reproduce asexually by means of conidia (exospores which bear the function of asexual reproduction in many fungi).

 

 

Fungi resemble algae in structure. They have a firm membrane consisting of cellulose, pectin substances, and carbohydrates. Various inclusions are found in the cytoplasm: glycogen, volutin, drops of fat. The cells of fungi may be mononuclear and polynucleate. The nuclei undergo both direct and indirect division. Fungi reproduce by rupture of the mycelium into pieces capable of germinating, by means of chlamydospores and conidia, by sporulation, and by the sexual way. The group of fungi includes saprophytes, parasites, and facultative parasites of plants, animals, and humans.

Chytridiomycetes. Most species inhabit water reservoirs. They lack mycelium or it is present in a rudimentary state. They move by means of pseudopodia. The cells are polynucleate. The Chytridiomycetes undergo a complex developmental cycle. They reproduce by simple division and sporulation. When occurring on a moist substrate, the spores of these fungi absorb water, swell, rupture the membrane, and divide with the production of amoeboid-like cells some of which coalesce and form zygotes which divide and develop into a polynucleate mucous mass. Some species which are pathogenic for plants induce, in particular, cabbage disease ('blackleg') and wart disease of potatoes.

Oomycetes are fungi with non-cellular (non-septate) mycelium. Some species live in water, others in the soil. Water inhabiting oomycetes cause diseases among fish and destroy the roe of fish and frogs. "Other oomycetes parasitize on plants and cause phytophtorosis of potatoes and the fruit of grapes and peronosporosis of sugar beet. The genus Mucor or bread mould belongs to the class Oomycetes (Fig. 16). It consists of a non-septate mycelium in the shape of a much branched cell, from which branch out the fruiting hyphae - sporangiophores with round dilatations at the tips sporangia. The latter are filled with endospores which provide a means of reproduction. Mucor mould may also reproduce sexually. It is widespread in nature, is often found on vegetables, moist surfaces of objects, and in manure.

A typical representative of Mucor mould is Mucor mucedo.

 

: : : : Ris_29_Mucor

Mucor

 

Pathogenic species of this mould may cause infections of the lungs and middle ear, and a general severe infectious process in humans, Zygomycetes are soil fungi with a non-cellular mycelium. They reproduce by means of sporangios pores, less frequently by means of conidia. Enzymes secreted by these fungi are used for clarifying juices and pre- paring alcoholic beverages. The class Zygomycetes includes the order Entomophilies, parasites of insects: they cause the death of the larvae of mosquitoes and flies and are used as insecticides. Ascomycetes or sac fungi (35000 species) have a multicellular mycelium. They reproduce sexually by means of ascospores (spores which develop in special spore cases, asci).

 

The genus Aspergillus belongs to the class Ascomycetes. The fungi have divided septate mycelium, and a unicellular conidiophore which terminates in a fan-like row of short sterigmata from which the spores are pinched off in chains conidia (Gk. konidion particle of dust).

 

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Aspergillus niger

 

Microscopic investigations have revealed that the fruiting part of the aspergillus (arrangement of endospores) resembles a jet of water from a watering can, and hence the name 'sprinkler' mould.

A typical representative of aspergilla is Aspergillus niger which is widespread in nature. It is found on moist objects, on bread and jam. Certain species may cause aspergillosis of the lungs, ear, and eye in humans or may infect the whole body.

The genus Penicillium belongs to the class Ascomycetes. The mycelium and conidiophore are multicellular while the fruiting body is in the shape of a brush. The conidiophore branches towards its upper part and terminates in sterigmata from which even-rowed chains of conidia are pinched off.

 

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Penicillium roqueforti

 

This genus of fungi is widespread in nature. It is found in fodder, milk products, ink and jam, on moist objects, and old leather. The type species is: Penicillium glaucum. Certain species [Penicillium notation, Penicillium chrysogenium, etc.) are used for producing penicillin which is widely employed in treating many infectious diseases. Some species of this genus of fungi are pathogenic for humans, They cause infections of the skin, nails, ears, upper respiratory tract, lungs. and other organs.

To the class Ascomycetes, the order Saccharomycetales (primary sac fungi) belong the yeasts which are large, oval, round, and rod-shaped cells (Fig. 17).

 

Figure 17. Yeast

 

 

Yeast cells have a double-cell wall and a well defined nucleus. The cytoplasm is homogenous, sometimes of a fine granular structure. It contains inclusions (glycogen, volutin, lipid) and vacuoles, and also filamentous bodies chondriosomes, which are involved in synthetic processes in the cell. Yeasts multiply by budding, fission, sporulation. Some species of yeasts reproduce sexually. Daughter cells produced by budding from the parent cell transform into independent individuals.

True yeasts are capable of reproducing by sporulation. When there is alack of nutrition. 2. 4, 8 or 16 endospores are formed inside the cells of some species of yeast. The yeast cell forming the ascospores is called the ascus (sac), while sporulating yeasts are known as Ascomyceles.

Many species and varieties of this genus of yeasts are capable of fermenting different carbohydrates. They are widely used in brewing beer, wine making, and baking bread. Typical representatives of these yeasts are Saccharomyces cerevisiae, and Saccharomyces ellipsoides.

A widely used object of genetic research is Neurospora crassa which develops on some bread products as a fluffy, flake-like white or pink mass. The presence of two outwardly indistinguishable forms between which sexual crossbreeding occurs makes it possible to isolate the ascospores and produce pure neurospora lines. Numerous mutants arise under the effect of irradiation which require a definite metabolite for their development (see section 'Variation in Requirement in Metabolites').

The groups of asporogenic yeasts (family Saccharomycetaceae) includes species pathogenic for humans, which cause severe diseases such as thrush in infants and blastomycosis. They occur due to the suppression of the normal microflora by antibiotics used in the treatment of some infectious diseases and inflammatory processes, as well as in severe diseases in which the protective body forces are weakened.

Claviceps purpurea developing on the grains of rye, wheat, etc. Form a commonly encountered group of Ascomycetes. During flowering the ascospores in the young plants develop into mycelium. The hyphae form a sclerotium (ergot) which takes the place of the grain in the ear and resembles a dark-violet horn. The ergots contain the alkaloid cornutine and sphacelic and ergotic acids which, occurring in rye bread-cause a most severe disease in humans and animals called spasmodic ergotism.

Basidiomycetes, fungi with a multicellular mycelium. These organisms predominantly reproduce sexually by basidiospores (basidia reproductive organs in which a certain number of spores develop usually 4). The majority of them live on decaying humus and vegetable matter, Certain species are tree parasites. Two hundred species of mushrooms are edible. The fruiting bodies which are commonly known as mushrooms are used as food. Twenty-five species of mushrooms are poisonous. Smut fungi invade grain crops causing a disease known as smut. Rust fungi affect sunflowers, and other plants. They produce orange-coloured sports on infected plants.

Deuteromycetes (Fungi inperfecti) are a rather large group of fungi consisting of a multicellular mycelium without either the asco- or basidio-sporangiophore. but only with conidia. Reproduction is sexual. sexual reproduction is unknown. Among the hyphomycetes which maybe of interest to physicians are: Fusarium graminearum causing intoxication in humans ('drunken bread'), and Fusarium sporotrichiella causing intoxication in man and domestic animals who have eaten the grain crops which had remained in the fields during the winter.

Pathogenic species of imperfect fungi are causative agents of dermatomycoses: favus {Trichophyton schoenleini}, trichophytosis (Trichophyton violaceum), microsporosis {Microsporum canis}, epidermophytosis (Epidermophylon floccosum}.

Protozoa (Gk. protos first, zoon animal) are unicellular animal organisms more highly organized than bacteria. They have a cytoplasm, a differentiated nucleus, a cell wall which differs in optical properties and primitive organelles. Protozoa reproduce by simple and mullicellular division, sexually, and also by a more complicated process sexually and asexually (malarial plasmodium). Amoebae, lamblias, and balantidia can produce cysts which are more resistant forms for.survival. Representatives of certain species have two or more nuclei.

A more detailed description and characteristic of protozoa is given in the biology course. The main information on pathogenic species is given in the section on special microbiology.

 

 

Anjesky technique

Spores are most simply observed as intracellular refractile bodies in unstained cell suspensions or as colorless areas in cells stained by conventional methods. The spore wall is relatively impermeable, but dyes can be made to penetrate it by heating the preparation. The same impermeability then serves to prevent decolorization of the spore by a period of alcohol treatment sufficient to decolorize vegetative cells. The latter can finally be counterstained. Spores are commonly stained with malachite green or carbolfuchsin.

To demonstrate bacterial spores, special staining methods proposed by Anjesky, Peshkov, Bitter, Schaeffer-Fulton, and others are used.

Anjesky's staining. A thick smear is dried in the air, treated with 0.5 per cent sulphuric acid, and heated until it steams. Then, the preparation is washed with water, dried, fixed above the flame, and stained by the Ziehl-Neelsens technique. Spores stain pink-red, the cell appears blue.

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Wet-mount and hanging drop technique

Study of living microorganisms using the wet-mount and hanging-drop techniques. Using living microorganisms, one can study the processes of their propagation and spore formation, as well as the effect on them of various chemical and physical factors. In clinical laboratories living microorganisms are investigated to determine their motility, i.e., indirect confirmation of the presence of flagella. Preparations in this case are made using wet-mount or hanging-drop techniques and then subjected to dry or immersion microscopy. Results are better when dark-field or phase-contrast microscopy is employed.

Wet-mount technique. A drop of the test material, usually 24-hour broth culture of microorganisms, is placed into the centre of a glass slide. The drop is covered with a cover slip in a manner preventing the trapping of air bubbles; the fluid should fill the entire space without overflowing.

An inherent drawback of the wet-mount technique is its rapid drying. In prolonged microscopy it is recommended that the edges of a cover slip be sealed with petrolatum.

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Hanging drop technique. To prepare this kind of preparation, special glass slides with an impression (well) in the centre are utilized. A small drop of the test material is put in the middle of the cover slip. The edges of the well are ringed with petrolatum. The glass slide is placed onto the cover slip so that the drop is in the centre of the well. Then. it is carefully inverted and the drop hangs in the centre of the sealed well, which prevents it from drying.

The prepared specimens are examined microscopically, slightly darkening the microscopic field by lowering the condenser and regulating the entrance of light with a concave mirror. At first low power magnification is used (objective 8 X ) to detect the edge of the drop, after which a 40 x or an oil-immersion objective is mounted.

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Occasionally, molecular (Brownian) motility is mistaken for the motility of microorganisms. To avoid this error, it should be borne in mind that microorganisms propelled by flagella may traverse the entire microscopic field and make circular and rotatory movements.

After the examination the wet-mount and hanging-drop preparations should be immersed in a separate bath with disinfectant solution to kill the microorganisms studied.

 

 

Addition materials

http://en.wikipedia.org/wiki/Bacterial_cell_structure

http://www.microbiologytext.com/index.php?module=Book&func=displayarticlesinchapter&chap_id=35

http://student.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/glyco.html

http://www.ucmp.berkeley.edu/bacteria/spirochetes.html

http://en.wikipedia.org/wiki/Spirochaete

http://en.wikipedia.org/wiki/Actinobacteria

http://pathmicro.med.sc.edu/mycology/mycology-2.htm

http://en.wikipedia.org/wiki/Rickettsia

http://www.cehs.siu.edu/fix/medmicro/ricke.htm

http://pathmicro.med.sc.edu/mayer/ricketsia.htm

ttp://www.kcom.edu/faculty/chamberlain/Website/Lects/RICKETT.HTM

http://en.wikipedia.org/wiki/Chlamydia

http://pathmicro.med.sc.edu/mayer/chlamyd.htm

http://pathmicro.med.sc.edu/mayer/myco.htm

http://pathmicro.med.sc.edu/book/mycol-sta.htm

http://pathmicro.med.sc.edu/book/parasit-sta.htm

http://www.innvista.com/health/microbes/bacteria/classif.htm

http://www.earthlife.net/prokaryotes/phyla.html

http://web.uct.ac.za/depts/mmi/lectures/bactax/ppframe.html

http://www.gsbs.utmb.edu/microbook/ch003.htm

http://www.bmb.leeds.ac.uk/mbiology/ug/ugteach/dental/tutorials/classification/introduction.html

http://www.microbiol.org/WPaper.Gram.htm