Ziehl-Neelsen Method
Materials:
nutrient agar slant culture of Mycobacterium smegmatis (48-hour culture)
nutrient broth culture of S. aureus electric hot plate and small beaker acid-fast staining kit (carbolfuchsin, acid
alcohol, and methylene blue)
Smear Preparation Prepare a mixed culture smear by placing two loopfuls of S. aureus on a slide and transferring a small amount of M. smegmatis to the broth on the slide with an inoculating needle. Since the smegma bacilli are waxy and tend to cling to each other in clumps, break up the masses of organisms with the inoculating needle. After air-drying the smear, heat-fix it.
Staining Follow the staining procedure outlined in figure 17.1.
Examination Examine under oil immersion and compare your slide with illustration 6, figure 15.3.
Laboratory Report Record your results on Laboratory Report 15–18.
Most bacteria in the genus Mycobacterium contain considerable amounts of waxlike lipoidal material, which affects their staining properties. Unlike most other bacteria, once they are properly stained with carbolfuchsin, they resist decolorization with acid-alcohol. Since they are not easily decolorized they are said to be acid-fast. This property sets them apart from many other bacteria.
This stain is used primarily in the identification of the tuberculosis bacillus, Mycobacterium tuberculo-sis, and the leprosy organism, Mycobacterium leprae.
After decolorization, methylene blue is added to the organisms to counterstain any material that is not acid-fast; thus, a properly stained slide of a mixture of acid-fast organisms, tissue cells, and non-acid-fast bacteria will reveal red acid-fast rods with bluish tis-sue cells and bacteria. An example of acid-fast stain-ing is shown in illustration 6 of figure 15.3.
The two organisms used in this staining exercise are Mycobacterium smegmatis, a nonpathogenic acid-fast rod found in soil and on external genitalia, and Staphylococcus aureus, a non-acid-fast coccus.
Benson: Microbiological Applications Lab Manual, Eighth Edition
III. Microscope Slide Techniques
18. Acid−Fast Staining:
Fluorescence Method
© The McGraw−Hill Companies, 2001
Acid-Fast Staining:
Fluorescence Method
18
In laboratories where large numbers of sputum, gas-tric washings, urine, and other body fluid samples are tested for pathogenic mycobacteria, fluorochrome acid-fast staining is used in conjunction with the Ziehl-Neelsen technique. The advantage of using a fluorescence method is that fluorochrome-stained slides can be scanned under lower magnification.
While a Ziehl-Neelsen prepared slide must be exam-ined under oil immersion (1000⫻ magnification), fluorochrome-stained slides can be examined with 60⫻ or 100⫻ magnification. In only a few minutes an entire fluorochrome prepared slide can be scanned.
Because of this fact, many laboratories use this faster technique as a screening tool. When they encounter a positive slide with this method, they use a Ziehl-Neelsen prepared slide as a means of confirmation.
The fact that dead or noncultivatable mycobacteria may fluoresce makes it necessary to use a confirma-tory technique.
The Truant method of fluorochrome staining (figure 18.1) consists of staining smears with au-ramine-rhodamine for 20 minutes, decolorizing with acid-alcohol, and “counterstaining” with potassium permanganate. As soon as the slides are dry, they are examined with a fluorescence microscope. Bacteria that are acid-fast will fluoresce as yellow-orange rods in a dark field. Areas of fluorescence that show up during scanning can be examined more critically un-der high-dry or oil immersion.
In this exercise you will stain a mixture of Staphylococcus aureus and Mycobacterium phlei by the Truant method. It will be examined with a fluorescence microscope. If the slide is prepared properly, only the acid-fast rod-shaped mycobacteria will fluoresce.
Materials:
broth culture of S. aureus
slant culture of M. phlei (Lowenstein-Jensen medium)
auramine-rhodamine stain
acid-alcohol (for fluorochrome staining) potassium permanganate (0.5% solution) microscope slides, inoculating loop, Bunsen
burner, wash bottle
1. Prepare a mixed smear of S. aureus and M. phlei by adding a small amount of M. phlei to two loop-fuls of S. aureus on a clean slide. The organisms should be well dispersed on the slide by vigorous manipulation of the inoculating loop on the clumps of organisms.
2. Allow the smear to air-dry completely.
3. Flame-fix the slide over a Bunsen burner. Avoid overheating.
In diagnostic work where pathogens are be-ing stained, the smear is usually heat-fixed on a slide warmer (65° C) for 2 hours.
4. Cover the smear with auramine-rhodamine stain and let stand for 20 minutes at room temperature.
5. Rinse off the stain with wash bottle.
6. Decolorize with acid-alcohol (2.5% HCl in 70%
ethanol) for 7–10 seconds.
7. Rinse thoroughly with wash bottle.
8. Cover the smear with potassium permanganate and let stand for 3 minutes. This solution elimi-nates background fluorescence (“quenching”).
Although this step is often referred to as “terstaining,” in actuality it is not. Excessive coun-terstaining must be avoided because fluorescence will be completely eliminated.
9. Rinse with water and air-dry.
O
BSERVATIONThe fluorescence microscope should be equipped with a BG12 exciter filter and an OG1 barrier filter.
Scan the slide with the lowest magnification that is possible on the microscope. On some instruments this may be high-dry, not low power. If high-dry is used, it is necessary to place a cover glass on the smear with immersion oil between the slide and cover glass. Be sure to use only oil that is specific for fluorescence viewing.
Once you have located areas of fluorescence (yellow-orange spots), add oil and swing the oil im-mersion lens into position for more critical observa-tion. Record your results on Laboratory Report 15–18.
Acid-Fast Staining: Fluorescence Method • Exercise 18
Figure 18.1 Fluorochrome acid-fast staining routine
Benson: Microbiological Applications Lab Manual, Eighth Edition
III. Microscope Slide Techniques
19. Motility Determination © The McGraw−Hill
Companies, 2001