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Vibrio cholerae in Gram Stain

Vibrio cholerae in Gram stain showing gram-negative rod, curved or comma shape, no evidence of spore and capsule as shown above picture.

Requirements

a) Compound light microscope

b) Reagents and glasswares

• Bunsen flame
• Wire loop
• Clean grease-free slides
• Marker pen
• Crystal violet (Basic dye)
• Gram’s iodine(mordant)
• 95% ethanol (decolorizing agent)
• 1% safranin or dilute carbol fuchsin or neutral red ( in this case, better to use 1 in 10 carbol fuchsin as counterstain)

c) Quality control strains

Positive Control (PC) : Staphylococcus aureus (ATCC 25923)

Negative Control (NC): Escherichia coli (ATCC 25922)

d) Specimen (  Note:-Colony of Vibrio cholerae  from TCBS agar used and growth of this organism in alkaline peptone can also be taken.)

Preparation of bacterial smear: from liquid culture

• Take a clean, and grease-free slide for making a smear.
• Take one or two loopful of the bacterial cell suspension and place them on the slide with a bacteriological loop.
• Then with a circular movement of the loop, spread the cell suspension into a thin area.
• Allow the smear to air dry.
• 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.

Preparation of bacterial smear: from the solid medium

• Take a clean, and grease-free slide for making a smear.
• Take a loopful of 0.85% saline i. e. physiological saline and place it on the center of the slide.
• With a straight wire touch the surface of a well-isolated colony from the solid media and emulsify in the saline drop forming a thin film.
• Allow the smear to air dry.
• 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.

Procedure

1. Cover the smear with crystal violet and allow it to stand for one minute.
2. Rinse the smear gently under tap water.
3. Cover the smear with Gram’s iodine and allow it to stand for one minute.
4. Rinse smear again gently under tap water.
5. Decolorize the smear with 95% alcohol.
6. Rinse the smear again gently under tap water.
7. Cover the smear again gently with safranin for one minute.
8. Rinse the smear again gently under tap water and air dry it.
9. Observe the smear first under the low power (10X) objective, and then under the oil immersion (100X) objective.

Observation

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 bacilli and comma shape as shown above picture

Negative Control(NC): Gram-negative bacilli

Further Readings

1. 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.
2.  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
3.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
4. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
5. 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.