Gram Stain: Introduction, Principle, Procedure, Result and Interpretation

Gram stain result interpretation

Introduction of Gram Stain

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 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  decolorizing agent, cause dehydration of the multi-layered peptidoglycan of the cell wall. This causes decreasing  of 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 the  of 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 counter stain uses, they take up the color of the  stain and appear pink.

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 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

Preparation of bacterial smear: from liquid culture

  • Take a clean, and grease free slide for making smear.
  • Take one or two loopful of the bacterial cell suspension and place on the  slide  with  a bacteriological loop.
  • Then with 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 Bunsen burner two  to three times.

Preparation of bacterial smear: from the solid medium

  • Take a clean, and grease free slide for making 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 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 of Gram Stain

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 of Gram Stain

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

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

 E. coli under microscope|| Gram stain ||Gram Negative bacilli or Gram negative rods as shown below-

Variety of bacteria under the microscope showing
Gram positive bacteria, gram positive cocci in singles, pairs, clusters i.e. Staphylococcus aureus
Gram positive rods or bacilli
Bacillus species
Gram negative bacilli or rods
Salmonella Typhi
Diphtheroids
Sputum gram stained smear
having ideal smear
Gram positive cocci in pairs inside the pus cell
Gram positive cocci in chains

A patient was 53 years old with Chronic Otitis Media (COM) having failure of antibacterial drugs-
Pus swab from ear discharge sent to microbiology section for Gram staining- result found – Fungal spores with plenty of pus cells and lacking bacteria as shown in video.

Strongyloides stercoralis under Gram stained slide of sputum- a very very rare case-

Impression for this a rare case report from Gram stain of sputum While reporting gram stained slide, we always remember the nature of specimens and availability of organisms; and broad our vision for report variety of causative agents without missing them because as you know laboratory diagnosis is the third eye of clinicians that makes treatment easier as well as short hospital stay and reduce patient economic burden.

Growth of Candida albicans on SDA and its Gram stained smear under the microscope and germ tube test (GTT) positive as shown below-

References

  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.  Text book 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 Hand book 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.asmscience.org/content/education/protocol/protocol.2886
  8. https://www.sigmaaldrich.com/catalog/product/sigma/ht90a?lang=en

 

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