Chapter 8_Identification of Bacteria

• Methods used to identify bacteria

  • Phenotypic characteristics
  • Genotypic characteristics
• Phenotypic characteristics


  • Microscopic morphology
  • Staining reactions
  • Metabolic differences
• Microscopic morphology


  Characteristics noted: shape, size, side ends, arrangement and irregular forms, motility, flagella fimbriae, spores, capsule, staining

  
• Staining reactions

  Gram stain, Acid-fast stain, Ziehl-Neelsen stain, Fluorescent antibody technique
• Metabolic differences


  Requirements of oxygen, need for carbon dioxide, the capacity to form pigments, and the production of hemolysis
• Appearance of growth on solid media

  • Shape, size, elevation, margins, surface, color, structure, consistency, emulsifiability, differentiation
• Appearance of growth on liquid media

  • Degree, turbidity, deposit, surface growth
• Biochemical reactions


  • Sugar fermentation
  • Indole production
  • Methyl Red (MR) test
  • Voges-Proskauer (VP) test
  • Citrate utilization
  • Nitrate reduction test
  • Urease test
  • Catalase test
  • Oxidase test
  • Phenylalanine deaminase test
  • Hydrogen sulfide production
  • Potassium cyanide test
  • Triple sugar iron (TSI) agar
• Sugar fermentation


  Determines the ability of an organism to ferment sugar incorporated in a medium producing acid or acid with gas
• Indole production


  Demonstrates the ability of certain bacteria to decompose the amino acid tryptophane to indole, which accumulates in the medium
• Methyl Red (MR) test


  Detection of the production of sufficient acid during the fermentation of glucose so that pH of the medium falls, and it is maintained below 4.5
• Voges-Proskauer (VP) test


  Detects the presence of acetoin which is responsible to the conversion to diacetyl, and α-napthol serves as a catalyst to form a red complex
• Citrate utilization


  Test for the ability of an organism to utilize citrate as the sole carbon and energy source for growth and an ammonium salts as the sole source of nitrogen with resulting alkalinity
• Nitrate reduction test


  Test for the presence of the enzyme nitrate reductase which causes the reduction of nitrate to nitrite which can be tested for by an appropriate colorimetric reagent
• Urease test


  Used to identify bacteria capable of hydrolyzing urea using the enzyme urease which splits urea to ammonia. Ammonia makes the medium alkaline and thus phenol red indicator changes to pink/red in color
• Catalase test


  Demonstrates the presence of catalase, an enzyme that catalyses the decomposition of hydrogen peroxide into water and oxygen
• Nitrite reduction


  Indicates the presence of nitrite and hence the ability of the organism to reduce nitrate
• All members of Enterobacteriaceae are Nitrate Reduction Test Negative
• Urease test


  Used to identify bacteria capable of hydrolyzing urea using the enzyme urease which splits urea to ammonia
• Urease test method

  1. Test organism is inoculated on the entire slope of Christensen's medium which contains urea and phenol red indicator

  2. Incubated at 37C and examined after 4 hours and after overnight incubation
• Urease test positive


  Purple-pink color (Klebsiella sp., Proteus sp., Yersinia enterocolitica, Helicobacter pylori)
• Urease test negative


  Pale yellow color (E. coli, Providencia sp., Yersinia pestis)
• Catalase production


  Demonstrates the presence of catalase, an enzyme that catalyses the release of oxygen from hydrogen peroxide
• Catalase production test method


  1. 1mL of hydrogen peroxide solution is poured over a 24 hours nutrient agar slope culture of the test organism and the tube is held in a slanting position

  2. A small amount of the culture to be tested is picked from a nutrient agar slope with a clean sterile platinum loop and dip it in a drop of 10% hydrogen peroxide on a clean glass slide
• Catalase production positive


  Immediate bubbling (oxygen is formed); All members of Enterobacteriaceae except Shigella dystentriae type 1; Staphylococcus, Micrococcus, Bacillus
• Catalase production negative


  No bubbling (Shigella dystentriae type 1, Streptococcus, Clostridium)
• Oxidase test


  Determine the presence of bacterial cytochrome oxidase using the oxidation of the substrate, a redox dye, tetramethyl-p-phenylene-diamine dihydrochloride (oxidase reagent)
• Oxidase test plate method


  A freshly prepared 1% solution of tetramethyl-p-phenylene-diamine dihydrochloride is poured on to the plate so as to cover the surface, and is then decanted. Colonies of oxidase-positive organisms rapidly develop a purple color
• Oxidase test dry filter paper method


  A strip of filter paper soaked in the oxidase reagent is removed, laid in a petri dish and moistened with distilled water. Colony to be tested is picked up with a platinum loop and smeared over the moist area
• Oxidase test wet filter paper method


  Put a drop of freshly prepared 1% solution of oxidase reagent on a piece of filter paper. Then rub a few colonies of test organism on it
• Oxidase test positive


  Produce a deep purple color within 10 seconds; used to screen species of Neisseria, Aeromonas, Vibrio, Campylobacter, Pseudomonas
• Oxidase test negative


  No reaction; All members of the family Enterobacteriaceae
• Phenylalanine deaminase test


  Determines whether the organism possesses the enzyme phenylalanine deaminase that deaminates phenylalanine to phenylpyruvic acid, which reacts with ferric salts to give a green color
• Phenylalanine deaminase test method


  Agar slants of the medium containing phenylalanine is inoculated with a fairly heavy inoculum and incubated at 37C for overnight. A few drops of 10% ferri chloride solution are added directly to the surface of the agar
• Phenylalanine deaminase test positive


  Green color; Proteus sp., Morganella s.p., Providencia sp.
• Phenylalanine deaminase test negative


  No color change; All members of the rest of Enterobacteriaceae
• Hydrogen sulfide production


  Some organisms decompose sulfur-containing amino acids producing hydrogen sulfide (H2S) among the products
• Hydrogen sulfide production test method

  Organisms can be grown in culture tubes. Between the cotton plug and the tube insert a filter paper strip soaked in lead acetate solution and dried. Browning of the paper indicates H2S production. When cultured in media containing lead acetate or ferric ammonium citrate, they turn them black or brown.
• Hydrogen sulfide production positive

  Black color; Proteus mirabilis, Proteus vulgaris, Salmonella sp. with some exceptions
• Hydrogen sulfide production negative

  No color change; Morganella sp., Salmonella paratyphi A., S. choleraesuis