The following points highlight the top two staining techniques for recognition of bacteria. The techniques are: 1. Gram Stain Method 2. Ziehl Neelsen Method.
Staining Technique # 1. Gram Stain Method:
Before staining, the position of the smear on the slide should be confirmed by making a scratch with the help of a needle or sharp glass slide, NEVER with the fingernail which may become contaminated; the slide should be placed in such as way that the smear is on the uppermost position on the staining rack or on glass rods levelled on the laboratory sink.
i. Cover the fixed smear with crystal violet or oxalated gentian violet stain which is allowed to act for 3 minutes;
ii. Pour off excess of the stain; wash away the remaining crystal violet stain with Gram’s iodine and allow the Gram’s iodine to act for 2 minutes;
iii. Decolorize with alcohol for about 5 seconds drop by drop till the violet colour is removed from the film;
iv. Wash with tap water;
v. Counterstain with basic fuchsin or safranin stain for 30 seconds;
vi. Wash with tap water, blot with blotting paper, dry in air and examine the smear under oil immersion objective (Fig. 3.3).
On the basis of Gram stain, bacteria are mainly grouped into: (1) Gram-positive bacteria and Gram-negative bacteria. Gram-positive bacteria retain the gentian violet in spite of decolourisation by alcohol and stained violet, whereas Gram-negative bacteria are decolorized by alcohol, loose the violet stain completely and are stained pink by the counterstain.
The clinician can choose antibiotics effective against any of the groups (Table 3.1).
Principles of Gram Stain:
Gram-positive organisms retain the basic dyes at higher hydrogen ion concentration (pH 2-3) than the Gram-negative bacteria. The iodine treatment enhances the cytoplasmic acid character which retains strongly the basic dye. Hence iodine acts as mordant. A dye iodine complex or “lake” is formed within the cell, which is soluble in alcohol used as decolouriser. This complex diffuses out of Gram-negative bacilli.
Gram-reaction depends upon a difference in the permeability of the cell wall or the cytoplasmic membrane and the presence of a specific magnesium ribonucleate protein complex in the cell. Gram-positive staining colours the whole cell including the cell wall. If the magnesium ribonucleate is removed by autolysis or the cell wall is ruptured, the Gram-positive bacilli will become Gram-negative.
Staining Technique # 2. Ziehl Neelsen Method:
i. Flood or fully cover the slide with carbol fuchsin and heat gently with spirit lamp from below till steam rises. Allow the stain to act for 5 minutes;
ii. Wash with tap water;
iii. Decolorize with 20% sulphuric acid for 1 minute till the yellowish brown or pink colour appears;
iv. Wash with tap water;
v. Counterstain with methylene blue for 15 seconds; ‘
vi. Wash, blot, dry and mount under oil immersion objective.
Acid fast bacilli are stained pink or red against the blue-back ground counterstain (Fig. 3.4).
Albert or Neisser method is used to demonstrate metachromatic granules; whereas India Ink method and Leifson’s method are used to observe capsules and flagella of bacteria, respectively; spores are stained by Gram’s method Or acid-fast stain, or malachite green stain.
Principles of Ziehl Neelsen Stain:
Some bacteria like tubercle bacilli cannot be easily stained by Gram stain because of the wax-like mycolic acid (fatty acid) in their cell wall; they can be stained with a strong reagent (basic fuchsin in aqueous 5% phenol), applied with heat; but subsequently they resist decolourisation by strong acid (20% sulphuric acid) and are stained pink or red.
Hence they are called “Acid fast bacilli” (AFB). Decolorized non-Acid fast organisms are counterstained with methylene blue.