Take a clean, and grease-free slide for making a smear.
Clinician collects urethral discharge using a swab and he/she makes smear.
Allow the smear to air dry.
Smear sends to the laboratory as soon as possible.
Heat fix the smear while holding the slide at one end, and by quickly passing the smear over the flame of the Bunsen burner two to three times.
Cover the smear with crystal violet and allow it to stand for one minute.
Rinse the smear gently under tap water.
Cover the smear with Gram’s iodine and allow it to stand for one minute.
Rinse smear again gently under tap water.
Decolorize the smear with 95% alcohol.
Rinse the smear again gently under tap water.
Cover the smear again gently with safranin ( better to use weak neutral red in this condition) for one minute.
Rinse the smear again gently under tap water and air dry it.
Observe the smear first under the low power (10X) objective, and then under the oil immersion (100X) objective.
Positive Control: violet color, round in shape in single, pairs and cluster
Test: red color and rod in shape
Negative Control: red in color and rod in shape
Result and Interpretation
Gram-positive: purple or violet color
Gram-negative: Pink or red in color
Cocci: round in shape
Bacilli: rod in shape
Positive Control(PC): Gram-positive cocci in single, pairs and cluster
Test: Gram-negative diplococci intracellular as well as extracellular as shown above picture
Note: There are various modifications of Gram staining among them Jensen’s Gram method is for gonococci and meningococci. This method uses alcohol as a decolorizer and weak neutral red as counterstain.
Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
Manual of Clinical Microbiology. Editors: P.R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken, 7th ed 2005, Publisher ASM, USA
Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
Clinical Microbiology Procedure Handbook Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.