Yeast Cell, Budding Yeast Cells and Pseudohyphae of Candida in Gram Stain of Sputum
Candida albicans in Gram Stain
Gram-stained sputum smear showing Gram-positive Candida albicans single yeast cells, budding yeast cells, and pseudohyphae as shown above picture. It was later confirmed as Candida albicans growing on SDA and performing biochemical tests. Germ tube test (GTT) was also positive.
It is a differential stain and thus used to differentiate Gram-positive and Gram-negative bacteria. It was originally devised by a Danish bacteriologist, Hans Christian Joachim Gram (1884) as a method of staining bacteria. Fungi are also gram-positive. Yeast cells( fungi) above are suggestive for this.
Principle of Gram Stain
The reaction is dependent on the permeability of the bacterial cell wall and cytoplasmic membrane, to the dye–iodine complex. In Gram-positive bacteria, the crystal violet dye iodine complex combines to form a larger molecule which precipitates within the cell. Also, the alcohol /acetone mixture which acts as a decolorizing agent causes dehydration of the multi-layered peptidoglycan of the cell wall. This causes a decrease in the space between the molecules causing the cell wall to trap the crystal violet iodine complex within the cell. Hence the Gram-positive bacteria do not get decolorized and retain primary dye appearing violet. Also, Gram-positive bacteria have more acidic protoplasm and hence bind to the basic dye more firmly. In the case of Gram-negative bacteria, the alcohol, being a lipid solvent, dissolves the outer lipopolysaccharide membrane of the cell wall and also damages the cytoplasmic membrane to which the peptidoglycan is attached. As a result, the dye-iodine complex is not retained within the cell and permeates out of it during the process of decolonization. Hence when a counterstain is added, they take up the color of the stain and appear pink.
Requirements for Gram Stain
Compound light microscope
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% safranine or dilute carbol fuchsin or neutral red
Preparation of smear
Take a clean and grease-free slide and label it.
Transfer mixed loopful sputum on the slide and make a smear.
Leave it for air drying.
Finally heat fix.
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 safranine for one minute.
Rinse the smear again gently under tap water and air dry it.
Observe the smear first under the low power (10 X) objective, and then under the oil immersion (100X) objective.
Gram-positive organism: Purple or violet in color
Gram-negative organism: Pink or red
Therefore, a Gram-stained smear of sputum under the microscope is showing yeast cells, budding yeast, and pseudohyphae Gram-positive ( above image).
Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, 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.
Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
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.
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