Cold staining for Mycobacterium lepare

Principle of Staining of Mycobacterium leprae

Mycobacterium leprae  (that cause leprosy) are extremely difficult to stain by ordinary methods because of the lipid-containing cell walls. They bind carbol -fuchsin tightly and resist destaining with strong decolorizing agents such as alcohol and strong acids. Acid-fast-negative bacteria readily lose the stain when treated with an acid-alcohol solution. The cold stain method is used for the detection of M. leprae and Nocardia asteroides (weakly acid-fast). Carbol- fuchsin as the primary stain and phenol as mordant. Following the counterstaining with methylene blue or malachite green the decolorized acid-fast negative organisms and other cells take blue color in contrast with the red colored acid-fast organisms.

Requirements for Staining of Mycobacterium leprae

• Pencil
• Glass slide
• Bunsen burner/ Sprite lamp
• Carbol fuchsin
• 5%(W/V) Sulphuric acid
• Methylene blue/Malachite green
• Microscope
• Cedarwood oil

Specimens

• Skin smears from visible lesions
• Biopsy of the nodular lesions and thickened nerves, and lymph nodes
• The puncture may be necessary in some cases.

Procedure for Staining of Mycobacterium leprae

• Flood the fixed  smear with a working carbol-fuchsin stain. Wait for 12-15 minutes without heating.
• Wash the smear with running tap water.
• Decolorize with 5% sulphuric acid for one minute.
• Counterstain with methylene blue for one minute.
• Wash with water, drain and blot dry.
• Observe the smear first under the low power (10X) objective, and then under the oil immersion (100X) objective.
  

  Result And Interpretation

  AFB: pink or red bacillus

  Background:  Blue ( note: according to counterstain used)

Grading of smear for Mycobacterium leprae

The grading of smear is based on the number of bacilli as follows:

1- 10 bacilli in 100 fields: 1+

1-10 bacilli in 10 fields: 2+

1-10 bacilli per field: 3+

10-100 bacilli per field: 4+

100-100 bacilli per field: 5+

more than 1000 bacilli, clumps, and globi in every field: 6+

From the above picture, the result of the grading smear is 6+ because of having following features-

More than bacilli

There is a presence of clumps.

There is also the presence of globi