Gram-Negative Bacteria Biochemical Tests: Introduction, Common Tests, Flow Chart and Keynotes

Gram-Negative Bacteria Biochemical Tests: Introduction, Common Tests, Flow Chart and Keynotes

Introduction of Gram-Negative Bacteria Biochemical Tests

Gram-Negative Bacteria Biochemical Tests is mainly concerned with  Biochemical Reactions of some Enterobacteria and other Enteric Organisms are listed below-

Common Gram-Negative Bacteria Biochemical Tests

  1. Triple Sugar Iron (TSI) Agar Test
  2. Motility Indole Urea (MIU) Test
  3. Citrate Utilization Test
  4. Oxidase Test


  1. is Negative Result
  2. + is Positive Result
  3. d is different strains give different Result
  4. ± Positive Or Negative Result
  5. R is Red color
  6. NC is No change in color
  7. Y is Yellow in color
  8. W is weak Positive
  9. S is slow

Lactose fermenting looks different on different media, In MacConkey agar lactose fermenting organisms are pink in color.

Citrate Test: Growth on the medium with color change from green to intense blue is positive and positive bacteria are Klebsiella pneumoniae, Citrobacter freundii, Enterobacter cloacae (a minority of strains gives negative result), Salmonella other than Typhi and Paratyphi A, Serratia marcescens, Proteus mirabilis(a minority of strains gives negative results), Providencia alcalifaeciens, Vibrio vulnificus, Euringella American and Achromobacter oxylosoxidans.

Motility: Spreading growth in inoculum is Motile. Diffused growth or turbidity extending away from the stab inoculation line in the case of motile organisms while non-motile organisms appear as restricted growth along the stab-line and positive organisms are E. coli,Salmonella Typhi,Salmonella Paratyphi A,Citrobacter freundii,Citrobacter koseri,Enterobacter spp., Serratia marcescens, Proteus vulgaris, Proteus mirabilis, Morganella morganii, Providencia alcalifaciens, Providencia stuartii, Providencia rettgeri, Yersinia enterocolitica, Vibrio cholerae,Vibrio parahaemolyticus, Pseudomonas aeruginosa, and Alcaligenes spp.

Indole Test: Red-colored rink after addition of Kovac’s reagent is positive and positive bacteria are Escherichia coli, Klebsiella oxytoca,Proteus vulgaris, Morgenella morganii, Vibrio cholerae, Providencia species.

Urea test: Red color is positive and positive organisms are Proteus vulgaris,Proteus mirabilis,Morganella morganii,Providencia rettgeri, etc.

H2S: Blackening is Positive. Hydrogen Sulfide is produced by the action of the bacteria with sodium Thiosulphate. This is detected by the reduction of ferric ions to produce a black precipitate. Positive bacteria are Salmonella Typhi,Citrobacter freundii,Proteus vulgaris and Proteus mirabilis.


Gas: Gas production from sugar fermentation is indicated by bubbles, the fracturing of the medium, or displacement of the medium. Gas forming organisms are Escherichia coli, Klebsiella pneumoniae, Salmonella Paratyphi, Citrobacter fruendii, C. koseri, Enterobacter species, etc.

Oxidase: Deep blue to purple color is Positive. Positive bacteria are seudomonas, Aeromonas, Vibrio, Brucella, Haemophilus, Alcaligenes, Neisseria, Campylobacter, and Pasteurella.


Further Readings on Gram-Negative Bacteria Biochemical Tests

  1. Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University Press.
  2. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  3. 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.
  4. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
  5. 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.
  6. 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.
  7.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
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