Lab Exercise 6
Special Stains

After completing this exercise you should understand:

The acid fast stain, developed by Dr. Paul Ehrlich, is a differential stain for Mycobacterium. Mycobacterium is the causative agent in leprosy and tuberculosis, so it is useful clinically to have a stain for rapid identification of these organisms in sputum or skin scrapings.
Mycobacterium are unusual in that the cell wall has a mixture of waxy lipids called mycolic acids, that prevent the bacterium from staining by simple and gram stains, and also prevent the organisms from drying out. In the acid fast stain, a red stain (carbolfuchsin) is cooked into this cell wall, and cannot be removed with acidified alcohol. That is, it is acid-fast. Other bacterium will lose this red stain when washed with acid-alcohol, becoming colorless. They then are stained blue to see them, with a counterstain (methylene blue), just as in the Gram stain.

Some bacteria have characteristic surface structures (capsules or flagella) or internal components (endospores) which are of value in identifying these organisms. Special stains are available for these cell components, as well as for other cell components to aid in the study of microbes.

Some bacteria have capsules and these capsules may actually relate to pathogenicity in some strains, such as Streptococcus pneumoniae, Klebsiella pneumoniae, and Clostridium perfringens, in which the capsule protects the microbe from host defense mechanisms, especially endocytosis. These capsules are often identified immunologically, but can also be visualized with a simple negative stain. Material such as india ink is applied to the slide--ink particles will coat the slide but not penetrate the cell or capsule. A stain can be used to color the cells, then the capsules is seen as the clear region surrounding the cells against the dark background. There are also direct methods of staining capsules.

Flagella are tiny hairlike organelles of locomotion. Their fine protein structure requires special staining techniques. The number of flagella and their arrangement (monotrichous, amphitrichous, lophotrichous, peritrichous) may be useful in identifying a particular organism. A stain and mordant are used to react with the flagella, increasing its diameter to allow viewing in the microscope.

Endospore formation is most characteristic of two genera of bacteria, Bacillus and Clostridium. These spores provide a resistant form for survival of the organism in unfavorable conditions. Because of the tough coating, these spores are not readily stained and may appear as empty "holes" in simple or Gram stained bacteria. A special staining technique must be used to drive dye into these resistant spores. Usually malachite green is heated to stain the spores, then the bacteria are counterstained with safranin. In this case, spores appear green in red bacterial cells. You will perform this stain on Bacillus cultures.
The nuclear material (DNA) of bacteria can be viewed by the application of special staining techniques. By using a stain which reacts with nucleic acid in the nucleoid, this region can be specifically stained.

Prepared slides
acid fast stain of Mycobacterium
flagella stains
nuclear stains
Cultures of Bacillus and Klebsiella
Malachite green and Safranin stains
` congo red
ring stand and beaker of water
immersion oil

View the prepared slides, identifying the particular organism or component of the organism. Describe what you see and draw a sample.

Spore stain:
1. Make a heat fixed smear of Bacillus as done previously.

2. Cover the smear with a small piece of paper towel, not hanging over the edges of the slide.

3. Place the slide on top of a small beaker containing about 1-2 inches of boiling water, being certain that the slide is horizontal. Instructor will demonstrate the ring stand set up. Be careful of burning yourself or knocking down the ring stand.

4. Cover the smear and paper towel with malachite green, and steam. Don't let the slide get dry--keep adding stain it is appears to approach dryness. Steam gently for about 5-10 minutes. REASON: You are cooking the malachite green into the endospore wall, as they are very resistant to staining.

5. Remove the slide from the steaming beaker, turn off the flame, and dispose of the green paper in the wastebasket, using your forceps. Wash the slide gently in running water about 20 seconds, getting all the extra green off the slide.

6. Counterstain with safranin for one minute, at the staining sink. Gently rinse with water, and shake off excess water into the sink. REASON: The running water washed the green stain out of the vegetative cells and sporangia, and they became colorless. The counterstain now dyes them red.

7. Gently blot the slide dry, no rubbing, and let it air dry. Examine with oil immersion optics. Observe red vegetative cells and sporangia, and green endospores and free spores.

Spore stain of a overnight culture of Bacillus subtilis.

Note: The all red cells are vegitative cells. The free green "cells" are free spores. The cells that are red with a green interior structure are the cells with endospores.





Capsule Stain:(Negative stain)

1. Place a drop of congo red on a clean slide. Mix a loop of Klebsiella culture into the stain drop.

2. Using a second slide, spread the drop of stain/microbe across the first slide, making a smear, and feathering the end.

3. Allow slide to air dry and then view under oil immersion.

Congo Red Capsule stain of Klebsiella pneumonia.

Note: The red interior is the bacterial cell and the clear zone around the cell is the area where the capsule has excluded the congo red stain.







1. What is the clinical value of the acid-fast stain? the capsule stain?

2. Why must the spore stain include a heating step? What would happen if this were omitted?

3. Describe the different arrangements of flagella. What is their importance?

4. Why is staining bacterial components useful in strain identification?

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