1. Ex. 5: Acid-Fast and Endospore Stains
Objectives:
Explain the rationale and procedure for the acid-fast and endospore stains
Perform and interpret the acid fast and endospore stains
Review the structure and function of endospores
Basic dyes possess cationic functional groups such as -NR3+ or =NR2+. The name 'basic dye' refers to when these dyes were still used to dye wool in an alkaline bath. Protein in basic conditions develops a negative charge as the -COOH groups are deprotonated to give -COO-. In an electric field the chromophore ion travels to the cathode or negative pole; it is positively charged. Generally forms salts with negatively charged (acidic) substances in tissue (chromatin, ergastoplasm, cartilage matrix, some granules). Affinity for such dyes, is called basophilia. Basic dyes perform poorly on natural fibres, but work very well on acrylics.

2. For Acid Fast Bacteria Carbol fuchsin for AFB!! Use only Acid Alcohol
Methylene
Blue

3. Modified Kinyoun’s Acid-fast Stain – Cold Method
Heat fix smear
Primary stain: Carbol fuchsin for AFB, (5 min)
Rinse with water
Decolorize briefly (~10 drops) with acid alcohol (~ 30 sec)
Immediately rinse with water
Counterstain: Methylene blue (1 min)

4. Mycobacteriam phlei in sputum
Mycobacteriam phlei in sputum. In house picture done by Micro student from SS14 (Jesse Leonard)

5. Mycobacteriam phlei in sputum
Mycobacteriam phlei in sputum. In house picture done by Micro student from SS14 (Jesse Leonard)

6. Mycobacteriam phlei in sputum
Mycobacteriam phlei in sputum. In house picture done by Micro student from SS14 (Jesse Leonard)

7. Mycobacteriam phlei in sputum
Mycobacteriam phlei in sputum. In house picture done by Micro student from SS14 (Jesse Leonard)

8. Fig. 1 shows numerous bacilli representing a heavy TB infection
Fig. 1 shows numerous bacilli representing a heavy TB infection. Additionally, the number of bacilli in a sputum smear is very important for the diagnosis and prognosis of the infection.

9. Careful interpretation of Ziehl-Neelsen–stained slides may help show different stages of the tuberculosis disease. Many fields of the slide have to be read to make the final decision on the number of bacilli observed. The number of bacilli in the smear indicates the severity of the infection; therefore counting the number of bacilli accurately is mandatory for a valid report of the sputum acid-fast stain.  
Microbiologists are encouraged to perform direct microscopic examination on specimens submitted for culture. Not only may it be possible to provide the physician with a rapid presumptive diagnosis, but also the detection of specific microorganisms may serve as a guide for selecting appropriate culture media and provide a valuable quality control comparison with isolates recovered.

10. Endospore Stain Heat fix smear
Primary stain: Malachite green, steam (5 min)
Rinse thoroughly with water (~ 30 sec)
Counterstain: Safranin (1 min)

12. Acid fast stain from oral smear spiked with Mycobacterium phlei (In house pict)

14. Note the Gram negative Bacillus bacteria – old culture
Note the Gram negative Bacillus bacteria – old culture! All endospores are outside of the cell.s

15. the end