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Introduction of Ziehl-Neelsen Stain for Mycobacterium leprae

 To demonstrate the presence of the causative organism, M. leprae, in the patient’s body to conform Leprosy also called Hansen disease.

 Principle of  Ziehl-Neelsen Stain

The presence of higher alcohol, glycerol, fatty acid and especially mycolic acid in the cell wall has been found responsible for keeping the acid-fast property of bacteria. Therefore, the Ziehl-Neelsen stain (AFB stain) is useful for Mycobacterium leprae, an etiological agent of Hansen disease.

Requirements for Ziehl-Neelsen Stain for Mycobacterium leprae

Requirements

a) Compound light microscope

b) Reagents and glasswares

• Bunsen flame/Sprit lamp
• Clean grease-free slides
•  Pencil
• Carbol fuchsin
• 5% Sulphuric acid/ 1% acid alcohol
• Methylene blue
• Staining and drying racks

c) Specimens

Skin smear

d) Quality control strains

• Positive control (PC): Mycobacterium leprae
• Negative Control: Escherichia coli

Procedure of  Ziehl-Neelsen Stain for Mycobacterium leprae

1. Take a skin smear slide that contains four smears.  (If possible also include positive and negative control strains for quality assurance  )
2.   Fix the stain before staining.
3. Flood slide with carbol fuchsin stain.  Heat the slide gently with a flame under the rack so that the stain ‘steams’.   Ensure that the slide does not boil. Wait for 15 minutes.
4. Rinse the stain with running tap water.
5. Decolourise with 1% acid alcohol (or 5% sulphuric acid) for less than 1 minute or until no more colour comes off into the reagent and only a faint pink colour remains in the stain.  If you are using 5% sulphuric acid, leave for 10 minutes.
6. Rinse the slide in running tap water.
7. Counterstain with methylene blue (or another counterstain) for about 1 minute.
8. Wash, drain off water and leave for drying for nearly  10 minutes.
9. Examine under the microscope.  Bacteria that stain deep pink are ‘acid fast’.  All host cells are not acid-fast.  Observe the length of the bacterium, whether they are curved or straight, whether they are slow beading or uniformly stained and the arrangement (grouped in bundles or scattered singularly).  Read across the slide in a uniform ‘zig-zag’ method.
10. First scan the slide under 10X objective and finally read under 100X objective (oil immersion lens).
11. Grade each smear according to Ridley’s logarithmic scale, as follows:

• 0 = Negative; no AFB in entire smear
• 1+ = 1-10 AFB in 100 microscopic field
• 2+ = 1-10 AFB in 10 microscopic field
• 3+ = 1-10 AFB in average microscopic field
• 4+ = 10-100 AFB in average micro field
• 5+ = 100-1000 AFB in average micro field
• 6+ = >1000 AFB, AFB in clumps (globi)

After all, smears are examined calculate the average bacteriological index (BI) of bacilli in all the sites examined.

Calculation

Average BI = Sum total on all sites examined/ Number of sites examined

1. Also, calculate the morphological index (MI) by estimating the typical bacilli (long 5x width and slender with rounded ends) in comparison with the atypical bacilli (fragmented or granular). Express this as a percentage.
2. Count 200 separate bacteria (i.e. not touching each other). Note the solidly stained bacteria and express them as a percentage.

MI Characteristics

• Uniformly stained.
• Length is 5 times width.
• The sides are parallel.
• Both ends are rounded.

Keynotes

1. Using gloves is mandatory.
2. Composition of concentrated carbol fuchsin: 1g basic fuchsin, 10mL ethyl alcohol (95%), 5gm phenol and makeup to 100mL in distilled water.
3. 1%Acid Alcohol: 1mL concentrated hydrochloric acid and 99mL ethyl alcohol (absolute or 99%).
4. 5% sulphuric acid preparation: 5mL concentrated sulphuric acid and makeup to 100mL distilled water (caution: Put 60-65mL distilled water into a flask and gradually add). sulphuric acid to the water, letting the acid run down the side of the flask.  This generates heat and should be done slowly, adding only a little acid at a time with constant stirring.  When the acid is added and mixed well, make up the volume with a small amount of additional distilled water.
5. Loeffler’s methylene blue (counterstain) preparation: 1g methylene blue and makeup to 150 mL with distilled water.