4. Wash immediately with water.

5. Stain with water solution of fuchsine (Pfeiffer' fuchsine) for 1-2 minutes.

6. Wash with water and blot dry with filter paper.

7. Observe using oil immersion microscopy.

2. Study of live microorganisms (determination of motility).

In microbiological practice for study of morphology of bacteria sometimes non-stained preparation are used (native materials), prepared either from clinical specimens or colonies of grown microorganisms.

Native preparations are mostly used for studies of motility of live bacteria. Many bacteria are able to swim using flagella, which are made of a protein called flagellin, but too small to see without special staining techniques. Some bacteria have single flagella; others may have a bunch of flagella at one pole of the cell or covering the entire surface. There are four fundamental methods by which motility determination could be done: hanging drop, wet mount, tube method and microcameras with solid media.

Hanging drop

This technique requires a special microscope slide with an indentation in the middle and should be done as follows:

1. Place a cover glass on the bench surface.

2. Using bacteriological loop, place a small drop of a fresh culture in the

middle of the cover glass.

3. Place a small drop of Vaseline or immersion oil in the four corners of the cover glass.

4. Turn the hanging drop slide upside down, and lower the concave portion of the slide over the drop of culture.

5. Carefully pick up the slide with the cover slip, and quickly turn it over.

6. Observe the drop for motility at 40x.

Wet mount

1. Clean a new microscope slide.

2. Using bacteriological loop, place a small drop of water on the microscope slide.

3. Using bacteriological loop put small amount of a fresh culture and emulsify it in drop of water.

4. Carefully lay a cover glass on top of the drop of culture.

5. Blot it using filter paper to remove excess fluid.

6. Observe the drop for motility either at 40x or 90x.

Tube method (growth in semi-solid agar)

1. Using the inoculating needle, make a single stab down the middle of a tube with semi-solid agar (agar' concentration is 0.3-0.7%).

2. Incubate tube of agar at either 25°C or 35°C for 24-48 hours.

3. If the zone of growth spreads horizontally from the inoculation, organism is considered to be the motile.

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4. Non-motile organisms will grow only along the original stab of inoculation.

Microcameras with solid media

1. Sterile microscope slides with thin layer of solid are used.

2. Using Pasteur pipette, bacteria are placed.

3. Then agar is cut around the selected area and covered with cover glass.

4. To prevent drying, such preparations are incubated either in closed containers or with pressurized chinks between slide and cover glass by paraffin or wax filling.

For staining flagella, different methods are proposed. But majority of them share the common steps, including treatment with tannin, KAl(SO₄)₂ or HgCl₂, followed by staining with e.g. carbolic solution of fuchsine. As result, there is a precipitation of dye on flagella occurs which leads to their thickening and decrease of transparency.

Demonstrations

1. Gram stain technique.

2. Hanging drop preparation from live microorganisms (Vibrio spp.), followed by observation in the phase-contrast microscope.

Students' activities

1. To make smears from both gram-positive and gram-negative bacteria (Staphylococcus spp., anthracoid, E. coli, Vibrio spp.), make Gram stains, observe in microscope and draw the observations in the workbook.

2. To make a wet mount from dental deposit, to perform dark-field microscopy and draw the observations in the workbook.

Control questions

1. What are the major differences between eukaryotes and prokaryotes?

2. What bacterial features might be studied by simple and complex staining techniques?

3. What staining methods are called differential? Please, name them.

4. What bacterial features do allow on the capabilities to accept different dyes?

5. What staining method allows distinguishing all bacteria into two groups?

6. What is the structure of the cell wall of gram-positive and gram-negative bacteria?

7. Please, explain the steps and mechanism of Gram stain. What color bacteria might have and why?

8. What is the role of flagella in biology of bacteria?

9. What are the methods for determination of motility of bacteria?

10. What is the practical significance of studying of motility of bacteria?

11. Describe the method of preparation of hanging drop and wet mount and what are their peculiarities during the microscopy?

12. What is the classification of bacteria based on the arrangement of flagella?

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PRACTICAL SESSION No. 3

Structure of bacterial cell. Complex staining techniques (continuation).

Plan of the session

1. Acid-fast stain (Ziehl-Neelsen method).

2. Detection of spores in bacteria. Staining by Ozheshko method.

3. Detection of capsules in bacteria. Staining by Burry-Hins method.

4. Detection of inclusions by Neisser method.

Foreword notes

1. Acid-fast bacteria possess pronounced resistance to non-organic acids, alkalis and alcohols due to the presence in the cell walls a high proportion of neutral lipids, fatty waxes, hydroxy-acids and, namely, mycoloic acid. Such organisms are difficult to stain and also decolorize once they have been stained. For detection of acid-fast bacteria, concentrated dyes in carbolic acid solution applied with heat are used. Such combination allows the stain to penetrate the lipoid wall and reach the cell cytoplasm. Once the cytoplasm is stained it resists decolorization with sulfuric or nitric acids, which distinguish acid-fast from non-acid- fast bacteria. Among the clinically important bacteria, Mycobacterium spp. (including M. tuberculosis and M. leprae), and certain species of Nocardia spp. are acid-fast. The most widely used staining technique for acid-fast bacilli is Ziehl-Neelsen method.

2. A spore is a dormant form of the bacterium that allows it to survive non-optimal environmental conditions. Spores may be located in the middle of the cell (e.g. Bacillus anthracis), at the end (e.g. Clostridium tetani), or between the end and the middle of the cell (e.g. Clostridium perfringens). Spore shapes may be round or oval. Spore are formed in the presence of oxygen and at the temperature between + 15°C and + 43°C. They can be destroyed by autoclaving and application of the temperature + 120°C for 15-30 min. Spores have a touch outer covering made of the protein keratin and are resistant to heat and chemicals. The keratin also resists staining, so extreme measures must be taken to stain the spore. Bacterial spores can be detected by using both simple and complex staining techniques. For example, using 7% water solution of nigrosine, microorganism will have a green color, spores — colorless, background - black. More frequently, complex methods (Ozheshko and Shefer-Fulton) are used.

3. Capsule is located extracellular and is usually made either from polysaccharide or polypeptide. Production of the capsule contributes

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to the virulence of certain microbes by making them less vulnerable to phagocytosis and antibodies. Some bacteria produce capsule only inside the microorganism; others — both inside the human body and on the nutrient media, forming mucous colonies. For detection of capsule, special staining technique are used (e.g. by Burry-Hins method). The principle of method is as follows. The capsule stain technique stains around the capsule with a negative acidic stain. Then a basic stain will colorize the cell itself. The capsule appears as a white halo between the cell and the darker background.

4. Cytoplasm of some bacteria may contain inclusions different by their origin (e.g. lipoproteins, glycogen, sulfur, calcium, etc. ). Some microorganisms (e.g. Spirillum volutans, Bacillus subtilis, Bacillus anthracis, Corynebacterium diphtheriae) may have volutine in their cytoplasm. Volutine granules are made from polyphosphates (source of phosphate groups), have relatively large size and could be stained with different dyes, changing their color (metachromatic phenomenon). For example, during the staining by methylene blue, volutine is changing color to bright red. Presence of volutine granules may be used for laboratory diagnosis of diphtheria, because C. diphtheriae has bipolar location of granules. Neisser method is based on the selective fixation of acetic methylene blue (Neisser dye) by volutine granules. After application of vesuvine, Neisser dye is superseded by this dye from cytoplasm, which becomes yellow-stained, while volutine granules remain dark-blue (almost black) inside the yellow-stained bacteria.

Main objective of the session To learn methods of microscopic detection of structural elements of bacterial cell (spores, capsule, inclusions, cell wall of acid-fast bacteria).

Educational tasks

To know: 1. Structural elements of bacterial cell, their functions and role

in the identification of microorganisms.

1. Methods of microscopic studies of structural elements of bacterial cell:

• acid-fast stain (Ziehl-Neelsen method);

• spores stain (Ozheshko method);

• capsule stain (Burry-Hins method);

• volutine detection (Neisser method).

To be 1. To stain preparations by complex staining techniques for

capable: detection of acid-fast bacteria, spores, capsules and volutine

granules (in C. diphtheriae).

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Methodical guidelines

For detection of M. tuberculosis sputum is usually used. Using forceps or bacteriological needle, piece of sputum is placed on the middle of microscopic slide, which is then covered by second slide in the manner that 1/3 of both lower and upper slides will remain free. Then, free ends of slides are taken by lingers, and piece of sputum is pounded between slides. The microscopic slides are taken apart, thus two smears are obtained, which should be air-dried and flame-fixed.

For staining M. tuberculosis by Ziehl-Neelsen method, filter paper in size less than microscopic slide is placed on the surface of slide, and flood carbolic fuchsine (Ziehl fuchsine) onto filter paper. Then slide is flame heated until the formation of steam (please, do not allow fuchsine is dried off on the surface — add if necessary! ) for 4-5 minutes. Then allow slide to cool down, then throw away the paper and pour onto surface either 5% of sulfuric acid or 25% of nitric acid and leave until the appearance of yellow color. After that smear should be washed quickly and thoroughly with water. Then slide is counterstained with aqueous methylene blue for 2-3 minutes. After that, smear is washed, allowed to dry and examined microscopically under immersion objective.

M. tuberculosis will be stained a reddish-purple; non-acid-fast cells stain blue.

For staining of spores by Ozheshko method, rich smear is prepared on one side of microscopic slide, which then is air-dried. After that, 1% of hydrochloric acid is applied and flame-heated until the formation of steam for 1-2 min. Then smear is washed with water, dried, flame-fixed and stained by Ziehl-Neelsen method. Under immersion objective, spores will be red; vegetative cells will be blue.

For staining of spores by Shefer-Fulton method, fixed smear is covered by filter paper, flooded with 0.5% water solution of malachite green. Then slide is flame-heated until the formation of steam for 1-2 min. Then filter paper is thrown away, smear is washed with water and counterstained with 0.5% safranin solution and then again washed with water. Under immersion objective, spores will be green; vegetative cells will be red.

For detection of capsules by Burry-Hins method, one drop of water is applied onto microscopic slide. Then using sterile bacteriological loop, loopful of bacterial culture is placed into the above drop. Nearby to the first drop, one drop of India ink (1:10 dilution) is applied. After that, two drops are mixed together and, using another slide, by its edge smear is prepared (in the manner similar to preparation of blood smear). Then smear is allowed to dry on the air. Under immersion objective, pink cells of microorganisms, surrounded by colorless capsule, are visible on the dark background.

For detection of volutine granules by Neisser method, acetic methylene blue solution (Neisser stain) is placed onto fixed smear for 2-3 min. Then Lugol's iodine solution is applied for 1-2 min. The smear is washed with water and counterstained with vesuvin for 2-3 min. After that, smear is washed with water again, allowed to dry and examined under immersion microscope.

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Volutine granules which fix methylene blue stain firmly, stained dark blue, bacteria themselves have yellow color.

Demonstrations

1. Preparations of smear from sputum and staining by Ziehl-Neelsen method.

2. Stained smears from sputum of patient with tuberculosis and pure culture of C. diphtheriae.

3. Spore of anthracoids stained by Shefer-Fulton method.

Students' activities

1. To make smears from sputum, perform staining by Ziehl-Neelsen method, examine under microscope and draw the observations in the workbook.

2. To prepare a smear from Klebsiella pneumoniae or Klebsiella ozaenae culture, stain by Burry-Hins method, examine under microscope and draw the observations in the workbook.

3. To examine under microscope stained by Ozheshko method smears of anthracoid spores and draw the observations in the workbook.

Control questions

1. Which rod forms of bacteria are called bacilli?

2. In which conditions bacterial capsules can be formed?

3. In which conditions bacterial spores can be formed?

4. What is the role of capsule in bacteria?

5. What is the role of spores in bacteria?

6. What chemicals does capsule' composition include?

7. How may capsule be detected?

8. What types of bacteria are more often capable to form spores?

9. How can the spores be destroyed?

10. What does allow spores to be acid-fast?

11. What color produce acid-fast bacteria during the staining by Ziehl- Neelsen method?

12. What does allow some bacteria to be acid-fast?

13. What is the function of volutine granules?

14. How can volutine granules be detected?

15. Describe metachromatic phenomenon.

16. What is the significance of detection of volutine granules in bacteria?

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PRACTICAL SESSION No. 4

Morphology of spirochetes, fungi, rickettsia, chlamydia, protozoa, viruses (Including bacteriophages).

Plan of the session

1. Peculiarities of morphology of spirochetes.

2. Study of the morphology of yeasts and molds.

3. Morphology of obligate intracellular bacteria (rickettsia and chlamydia).

4. Study of morphology of protozoa.

5. Morphology of viruses. Bacteriophages: structure, receipt and titration.

Foreword notes

1. Spirochetes (Fig. 3) are a phylogenetically distinct group of bacteria which have a unique cell morphology and mode of motility. Spirochetes are very thin, flexible, spiral-shaped prokaryotes that move by means of structures called axial filaments or endoflagella. The flagellar filaments are contained within a sheath between the cell wall peptidoglycan and an outer membrane. The filaments flex or rotate within their sheath which causes the cells to bend, flex and rotate during movement. Most spirochetes are free living (in muds and sediments), or live in associations with animals (e.g. in the oral cavity or gastrointestinal tract).

General features of Spirochetes

• Gram-negative, but Gram stain not useful; the width of many spirochetes is at or just below the resolving power of the light microscope. They are observed by dark-field microscopy or by staining with special reagents.

• Long, slender, flexible spiral-shaped organisms; most appear as helical coils.

• The outer envelope is a membrane similar in structure to the outer

membrane of gram-negative bacteria and the larger organisms stain as gram-negative. However, no endotoxin has been demonstrated.

• Motile; move in corkscrew fashion; flagella, which lie between the outer membrane and the peptidoglycan layer, are referred to as periplasmic flagella or axial Fibrils. They are attached subterminally at each pole of the cell and extend along the body of the organism. Allow bacteria to move in corkscrew fashion; especially suited to movement in viscous environments.

• Some are free-living; others are obligate parasites

• many have not yet been cultivated in vitro.

Three genera are pathogenic for man: Treponema (T. pallidum is causing syphilis), Borrelia (B. burgdorferi is causing Lyme disease) and Leptospira (L. interrogans can cause leptospirosis). Each has a distinct morphology and method of movement.

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