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 Introduction 

Streptococcus pneumoniae  gram stain of sputum as shown above picture. Gram stain is a differential stain and therefore it uses to differentiate Gram-positive and Gram-negative bacteria. It was devised originally by a Danish bacteriologist, Hans Christian Joachim Gram (1884) as a method of staining bacteria in his laboratory.

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. 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 attaches. As a result, the dye-iodine complex does not retain within the cell and permeates out of it during the process of decolonization. Hence, when a counterstain uses, they take up the color of the stain and appear pink.

Requirements for Gram staining of Streptococcus pneumoniae

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

c) Quality control strains

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

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

d) Specimen ( sputum)

Preparation of bacterial smear for Streptococcus pneumoniae observation

• Take a clean, and grease-free slide for making a smear.
• Take one loopful of properly mixed sputum and place it 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.

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

Observation of Gram-stained smear of Streptococcus pneumoniae

Positive Control:   violet color, round in shape in single, pairs and cluster

Test: Purple color, round in shape, and arrangement in a number of two

Negative Control: red in color and rod in shape

Result and Interpretation of Gram stain for Streptococcus pneumoniae

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-positive diplococci s shown above picture

Negative Control(NC): Gram-negative bacilli as shown above image.

Bibliography

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