Gram Stain: Introduction, Principle, Test Requirements, Procedure, Result and Interpretation, Keynotes and Collection of Some Gram Stained Footages

Introduction of Gram Stain

Gram stain is the most commonly used routine stain in microbiology for the identification of microbes and also to know the evidence of etiological agents in clinical specimens. Gram staining is a differential stain and thus it applies 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. It gives ideas about organisms whether Gram positive or Gram negative or cocci or bacilli or coccobacilli, etc.

Principle

The reaction is based on the permeability of the organism’s cell wall and cytoplasmic membrane, to the dye–iodine complex. In Gram-positive organisms, the crystal violet dye iodine complex combines to form a larger molecule that precipitates within the cell. The alcohol or alcohol-acetone mixture which acts as a decolorizing agent causes dehydration of the multi-layered peptidoglycan of the cell wall. This causes a reduction in the space between the molecules causing the cell wall to trap the crystal violet iodine complex within the cell. Hence the Gram-positive organisms do not get decolorized and retain the primary dye appearing as violet. Similarly, Gram-positive bacteria have more acidic protoplasm and hence bind to the basic dye more strongly.

In the case of Gram-negative bacteria, the alcohol, being a lipid solvent, dissolves the outer lipopolysaccharide membrane of the cell wall and also breaks the cytoplasmic membrane to which the peptidoglycan attaches. As a consequence, 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 stain

a) Compound light microscope

b) Reagents and glassware

• Bunsen burner
• Inoculating 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 ( counter stain)
• Cedarwood oil
• Timer
• Water
• Tissue paper
• Slide rack (optional)

c) Quality control strains

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

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

d) Specimen/ test organism

Preparation of bacterial smear: from liquid culture

• Take a clean, and grease-free glass slide for making a smear.
• Pick up one or two loopful of the bacterial cell suspension and place them on the slide with an inoculating 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 glass slide for making a smear.
• Pick up 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 of Gram Stain

1. Cover the smear applying crystal violet and allow it to stand for one minute.
2. Rinse the smear gently under tap water.
3. Cover the smear applying Gram’s iodine and allow it to stand for one minute.
4. Rinse the smear again gently under tap water.
5. Decolorize the smear using 95% alcohol for 20-30 seconds while using acetone-alcohol-only treats 5-10 seconds.
6. Rinse the smear again gently under tap water.
7. Cover the smear gently with counter stain, and safranin for one minute.
8. Rinse the smear again gently under tap water and air dry it.
9. Observe the smear initially under the low power (10X) objective, and then under the oil immersion (100X) objective.

Observation of Gram Stain

Positive Control (PC): Violet color, round in shape in shingles, pairs, and clusters

Negative Control (NC): Red in color and rod in shape

Test: The reaction will appear on the nature of the test organisms.

Result and 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

Coccobacilli: Combination of cocci and bacilli forms

Positive Control(PC): Gram-positive cocci in singles, pairs, and cluster

Negative Control(NC): Gram-negative bacilli

Test: The reaction will appear on the nature of the test organisms.

Staphylococcus aureus in Gram stain

Escherichia coli in Gram stain

Diphtheroids in Gram stain

Neisseria gonorrhoeae in urethral discharge

Keynotes

• The purposes of fixation are as follows–

1. Vegetative forms of bacteria are killed.
2. Stuck the smear to the surface of the slide.
3. Render permeable to stain.
4. Preserve from undergoing autolytic little changes.

• Factor affecting Gram stain findings

1. Preparation of smear from the old culture.
2. Too thick smear
3. Using an old iodine solution
4. Over-decolorization of the smear
5. Cell wall damage is due to excessive heat fixation of the smear or antibiotic therapy.

• Gram stain Reporting

Gram staining reporting should include-

1. Number of organisms present-Numerous/ many, moderate, few, scanty/ Occasional
2. The gram reaction of organisms-Gram-positive or negative
3. Morphology and arrangement of microbes (bacteria and yeasts)- cocci, diplococci, streptococci, rods, coccobacilli, oval, etc.
4. Presence and number of pus cells and epithelial cells for host and microbial interaction
5. Presence of yeast cells
6. Evidence of capsules
7. Evidence of spores
8. Presence of pleomorphic organisms
9. Presence of evenly or unevenly stained bacteria

Collection of Some Gram Stained Footage

Yeast cells of Candida in Gram stain

Yeast cells of Cryptococcus neoformans in Gram stain

Streptobacillus in Gram stain

Enterococcus in Gram stain

Micrococcus in Gram stain

Beta-hemolytic streptococci in Gram stain

Viridans streptococci Gram stain

Klebsiella pneumoniae Gram stain

Haemophilus influenzae in sputum

Staphylococcus aureus in clinical sample

Streptococcus pneumoniae in sputum

Yeast cells and pseudohyphae of Candida

Streptococcus pneumoniae in Gram stain

Proteus in Gram stain

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 Francisco 1996.
2. Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
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. 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.
6. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC180726/
7. https://www.sigmaaldrich.com/catalog/product/sigma/ht90a?lang=en
8. https://www.asmscience.org/content/education/protocol/protocol.2886