Gram Stain

This is the most important stain in bacteriology and is so central to identification that it should be practised until the operator is fully competent. A number of different variations are found, and the laboratory should standardise on one method.

It is essential that young cultures (24 hrs) are used as old cultures may give false negative results.

Prepare a smear as described above.

a  a. Cover the slide with an ammonium oxalate, crystal violet preparation and leave for one minute. This is prepared by dissolving 20 g of crystal violet in 200 ml of methanol, and adding 800 ml of 1% ammonium oxalate.

b  b.Rinse well with water and blot.

c   c.Add an iodine preparation and leave for one minute. This is prepared by adding 1 g of iodine and 2 g of potassium iodide to 300 ml of distilled water.

d  d. Rinse well with water and blot.

e  e. Decolourise with 95%  ethanol for approximately 15 seconds, rinse with water and blot.

f.        Stain with 0.5% safranin for approximately 15 seconds, wash with water, blot dry, and examine with an oil     immersion objective.

Gram-positive cells will be seen as purple, whilst Gram-negative cells will be seen as pink. A few organisms are Gram-indeterminate, and the best way of examining these is to take a slide and divide it into three portions. A smear of the culture under examination is placed in the middle, whilst a smear of a known Gram-positive is placed on one side of it, and a smear of a known Gram-negative is placed on the other side. The entire slide is then stained and the central smear can be examined and compared with known organisms treated in exactly the same way.