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Conidia of Aspergillus in Gram Stain

Conidia of Aspergillus in gram stain as shown above picture. The specimen was ear discharge from a CSOM patient.

Requirements for Gram stain

a) Compound light microscope

b) Reagents and glass wares

• Bunsen flame
• Wire loop
• Clean grease-free slides
• Marker pen
• Crystal violet (Basic dye)
• Gram’s iodine(mordant)
• 95% ethanol (decolorizing agent)
• 1% safranin

C) Specimen (ear discharge)

Preparation of Smear

1. Take a clean, and grease-free slide for making a smear.
2. With an inoculating loop mix the specimen well and make a smear.
3. Allow the smear to air dry.
4. Heat fix the smear while holding the slide at one end, and by quickly passing the smear over the flame of Bunsen burner two  to three times.

Procedure of Gram stain

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 low power (10X) objective, and then under oil immersion (100X) objective.

Observation

Observe the stained smear for colour, shape, arrangement, evidence of capsule or spore of the organisms.

Result Interpretation of Gram stain

Gram-positive: purple or violet color

Gram-negative: Pink or red in color

Cocci: round in shape

Bacilli: rod in shape

Fungi are also Gram-positive and therefore  conidia of fungi are positive as shown above image.

Bibliography

1. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
2. 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.
3.  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
4.  Text book of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
5. Clinical Microbiology Procedure Hand book Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.