Neisseria gonorrhoeae in Urethral Discharge: Introduction, Procedure and Result-Interpretation

Gram negative diplococci in urethral discharge

Urethral discharge in Gram stain

Urethral discharge in Gram stain is showing Gram-negative diplococci intracellular as well as extracellular as shown above picture. It denotes the probable pathogen Neisseria gonorrhoeae.

Requirements

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

Procedure

Preparation  smear

  • Take a clean, and grease-free slide for making a smear.
  • Clinician collects urethral discharge using a swab and he/she makes smear.
  • Allow the smear to air dry.
  • Smear sends to the laboratory as soon as possible.
  • 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.

Staining

  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 ( better to use weak neutral red in this condition)  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

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

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 diplococci intracellular as well as extracellular as shown above picture

Note: There are various modifications of Gram staining among them Jensen’s Gram method is for gonococci and meningococci. This method uses alcohol as a decolorizer and weak neutral red as counterstain.

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

 

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