bacteria identification

• Bacterial Identification Tests

  Procedures used to identify bacteria to the genus and species level
• Bacterial Identification

  Staining of the isolated bacteria

  Motility testing

  Biochemical testing

  Serological tests

  Phage typing

  Identification disc testing

  Semiautomated and Automated identification systems

  Molecular techniques
• Staining

  The foremost and most important step in the identification of bacteria
• Staining techniques

  • Gram staining

  Albert staining

  Acid fast staining

  Special staining
• Gram staining

  Differentiates bacteria into Gram positive and Gram negative
• Gram positive bacteria


  Can be cocci, bacilli or vibrios. Examples: Staphylococci, Streptococci, Pneumococci
• Gram negative bacteria


  Can be cocci or bacilli. Examples: E.coli, Klebsiella, Salmonella, Shigella
• Albert staining


  Used for identification of Corynebacterium spp.
• Acid fast staining


  Used for identification of Mycobacterial infections like Tuberculosis, Leprosy
• Staphylococci, Streptococci, Pneumococci are examples of Gram positive bacteria
• E.coli, Klebsiella, Salmonella, Shigella are examples of Gram negative bacteria
• Motility testing


  Performed by preparing a wet mount and observing under a microscope. Can also be tested by inoculating in semisolid motility medium.
• Biochemical tests

  Used to get closer to the identification of bacteria. Examples: Catalase test, Coagulase test, Oxidase test, Sugar fermentation test, Indole test, Citrate test, Urease test
• Catalase test

  Detects the presence of the enzyme catalase. Useful in differentiating catalase-positive Micrococcaceae from catalase-negative Streptococcaceae.
• Catalase positive bacteria

  • Staphylococcus spp.
• Catalase negative bacteria

  • Streptococcus spp.
• Coagulase test


  Differentiates Staphylococcus aureus from other coagulase-negative Staphylococcus species. Can be performed as a slide test or tube test.
• Coagulase positive bacteria

  • Staphylococcus aureus
• Coagulase negative bacteria

  • Staphylococcus epidermis, Staphylococcus saprophyticus
• Oxidase test


  Assays for the presence of the enzyme cytochrome oxidase. Positive result indicates a color change to dark purple.
• Oxidase positive bacteria

  • Pseudomonas, Vibrio cholera
• Oxidase negative bacteria

  • E. coli, Klebsiella, Salmonella
• Indole test

  Screens for the ability of an organism to degrade the amino acid tryptophan and produce indole.
• Indole positive bacteria

  • E. coli, Vibrio cholera
• Indole negative bacteria

  • Klebsiella, Salmonella, Shigella spp.
• Citrate test


  Screens for the ability of an organism to utilize citrate as its carbon and energy source.
• Indole


  Used as part of the IMViC (indole, MR-Vp Citrate) procedures, a battery of tests designed to distinguish among members of the family Enterobacteriaceae
• Indole production test procedure

  1. Inoculate the tube of tryptone broth with a small amount of a pure culture

  2. Incubate at 37°C for 24 to 48 hours

  3. Add 5 drops of Kovác's reagent directly to the tube
• Positive indole test

  Formation of a pink to red color ("cherry-red ring") in the reagent layer on top of the medium within seconds of adding the reagent
• Indole negative test

  Reagent layer will remain yellow or be slightly cloudy
• Indole positive bacteria

  • E. coli
  • Vibrio cholera
• Indole negative bacteria

  • Klebsiella
  • Salmonella
  • Shigella spp.
• Citrate test


  Screens a bacterial isolate for the ability to utilize citrate as its carbon and energy source
• Citrate test procedure

  1. Use a fresh (16- to 18-hour) pure culture as an inoculation source

  2. Pick a single isolated colony and lightly streak the surface of the slant

  3. Incubate at 35oC (+/- 2oC) for 18 to 48 hours
• Citrate positive

  Growth will be visible on the slant surface and the medium will be an intense Prussian blue
• Citrate negative

  Trace or no growth will be visible, no color change will occur, the medium will remain the deep forest green color of the uninoculated agar
• Citrate positive bacteria

  • Klebsiella spp.
• Citrate negative bacteria

  • E. coli
• Urease test

  Identifies those organisms that are capable of hydrolyzing urea to produce ammonia and carbon dioxide
• Urease test procedure

  1. Use a heavy inoculum from an 18- to 24-hour pure culture to streak the entire slant surface

  2. Incubate tubes with loosened caps at 35oC

  3. Observe the slant for a color change at 6 hours, 24 hours, and every day for up to 6 days