Special Stains

17. Special Stains: Endospore Stain, Capsule Stain, and Acid-fast Stain Introduction A few genera of bacteria such as the Gram positive rods of the genus Bacilli have the ability to produce resistant survival forms called endospores. They should not be confused with the spores of fungi and plants because they are not used for reproduction. Endospores are resistant to heat, dryness, radiation, and many disinfectants.

Endospore formation (sporulation) occurs through a complex series of events. One structure is produced within each vegetative or dividing cell. Once the endospore is formed, the vegetative portion of the bacterium is degraded and the dormant endospore is released. The green round structures are the endospores which trap malachite green. Vegetative cells stain red.

The endospore is able to survive for long periods of time until environmental conditions again become favorable for growth. The endospore germinates producing a single vegetative bacterium.

Some bacterial strains synthesize a capsule, a layer of polysaccharides or proteins that coats cells. The presence of a capsule is linked to virulence because it interferes with phagocytosis. Capsules are visualized by negative staining using dyes which are either too large to penetrate the cell or are negatively charged and repelled by the cell. Sample preparation for negative staining are not heat fixed because the heat would destroy the morphology of the capsule.

Acid-fast stain was developed to stain Mycobacteria, Gram positive bacteria that do not stain well in a Gram stain because of the waxes present in the outside cell wall. The waxy coats are an important virulence factor. The procedure can be used to detect the organisms that cause Hansen’s disease (Mycobacterium leprae) and tuberculosis (Mycobacterium tuberculosis). Mycobacterium smegmatis is a commensal organism of the human body and serves as model for mycobacteria.

Cords: because of their waxy outer membranes, Mycobacterium cells do not always separate after replication and form cords or rope-like aggregates. They appear as red clumps.

Relationship to Class Instruction: Chapters 3 and 10, and 16: stains, identification, and virulence factors

Lab worksheet due __________________________

Purpose of Laboratory Perform endospore stain using the Schaeffer-Fulton method Perform negative stain of Klebsiella pneumoniae Demonstration of acid-fast stain of Mycobacterium smegmatis and observation of prepared slides

Material and Methods Organisms: Bacillus subtilis Klebsiella pneumoniae Mycobacterium smegmatis Staphylococcus epidermidis

Inoculating loops

Kinyoun Carbol Fuchsin

Clothespins, goggles and gloves

20% sulfuric acid

Slides, Lens Paper, paper towels cut to size of slides,

Methylene blue

Nigrosin

Forceps

Malachite Green

Safranin

Handle malachite green carefully. Not only it does stain, but it is a suspected carcinogen.

Carbol fuchsin is another stubborn stain. It will stain skin and does not wash easily. Use clothespins.

Wipe immediately all stains from bench tops with paper towels soaked in ethanol.

Procedure For all the stains, sketch what you observed under oil immersion in your lab notebook.

Endospore Stain Special Safety Precautions Discard all biohazard material appropriately: Slides go in the metal tray.

Endospore stain of Bacillus subtilis

it with the drop of water until the water turns cloudy. Use an inoculating loop.

Don’t pick up too much material. You will have a thick smear and will not distinguish cells.

1. Heat-fix a smear of Bacillus after flaming the slide as follows: 2. Place a small drop of water on a clean slide by touching the dropper of the wash bottle to the slide. If the drop is large, it will take a long time to air dry!

4. Spread the suspension to form a thin film using the loop.

5. Burn the remaining bacteria off the loop.

3. Remove aseptically a small amount of the culture from the edge of the growth on the agar surface and generously mix

6. Air-dry this thin suspension completely.

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7. Pass the slide (film-side up) above the flame of the Bunsen burner 3 or 4 times to heat-fix.

discarded into the regular trash, not in the sink!

12. Wash excess stain off the slide with water. Don't forget to wash of any dye that got onto the bottom of the slide.

8. Cover the smears with a piece of absorbent paper cut to fit the slide.

Saturate the paper with malachite green.

13. Tap gently the slide on a paper towel to drain off excess water.

9. Let the malachite green sit on the slide for one minute and proceed to the next step.

14. Flood the smear with safranin and stain for 30 seconds.

15. Wash off the excess safranin with water.

10. Heat carefully the slide in the flame of a Bunsen burner holding the slide with a clothespin, until the stain just begins to steam. Remove the slide from the heat until steaming stops; then gently reheat. Continue steaming the smear in this manner for 3 minutes. As the malachite green evaporates, continually add more. Do not let the paper dry out.

16. Air-dry and observe under the light microscope using first the scanning and 40X objectives to locate samples, then the oil immersion objective.

Endospores stain green while vegetative bacteria stain pink. You will notice that some endospores are visible inside bacterial cells whereas other endospores have been released.

11. Remove the paper with a pair of forceps and rinse the slide thoroughly with tap water. The stained paper is

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Negative Stain

Kebsiella pneumoniae 1. Place a very small drop (more than a loop full--less than a free falling drop from the dropper) of nigrosin near one end of a well-cleaned and flamed slide.

toward the drop until it touches the drop and causes it to spread along the edge of the spreader slide. Push the spreader slide toward the clean end of the slide being stained dragging the drop behind the spreader slide and producing a broad, even, thin smear.

1. Remove a small amount of the culture of K pneumoniae with an inoculating loop and disperse it in the drop of stain without spreading the drop.

4. Allow the smear to dry without heating.

2. Use another clean slide to spread the drop of stain containing the organism using the technique below.

5. Focus on a thin area under oil immersion and observe the unstained cells surrounded by the gray stain.

3. Rest one end of the clean slide on the center of the slide with the stain. Tilt the clean slide toward the drop forming an acute angle and draw that slide

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Acid-fast Stain Demonstration

Mycobacterium smegmatis and Staphylococcus epidermidis 1. Prepare a bacterial smear as described dye that got onto the bottom of the under Endospore Stain. Two different slide. samples will be mixed.

8. Blot dry the underside of the slide and 2. Mix a small sample of M. smegmatis in a small drop of distilled water, Flame the inoculating loop and mix a small sample of S .epidermidis in the same drop.

tap the slide gently on a paper towel to remove excess water.

9. Flood with methylene blue and stain for 30 seconds.

10. Wash off the excess methylene blue with water

3. Allow to air dry and heat fix.

4. Flood the smear with Carbol-fuchsin.

Stain for 5 min.

11. Blot dry the underside of the slide and tap the slide gently on a paper towel to remove excess water.

5. Rinse with distilled water.

6. Decolorize by dripping 20% sulfuric acid across the slide 1 to 2 min. The effluent should appear pale or clear.

Wear goggles and gloves.

12. Air dry, and observe under the light microscope. The Staphylococcus cells will appear blue and the Mycobacterium cells will appear red or pale red.

7. Rinse with water. Rinse the underside of the slide. Don't forget to wash of any

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