Theory of Metabolic Tests and Reactions Used to Identify Gram Neg. Enteric Bacteria
[Don’t use this sheet as instructions on how to set up or process your rest results!]
Test Name / Substance(s) or ability being tested for / Chemistry / Negative/Control / Positive ResultCarbohydrate Fermentation Tubes with Phenol Red
(Note this is different from our Kleyn and Bicknell lab manual that uses methyl red and different are produced) / Fermentation of a particular monosaccharide / Phenol red turns yellow when bacteria ferment sugars and produce acids (with or without gas) in the first 24 hrs. Gas is captured in the inner Durham tubes if it is produced during fermentation. After about 48 hours, when the sugar is exhausted, bacteria utilize the peptone in the medium, producing a hot pink (alkaline) product. Bacteria which cannot ferment the sugar will grow immediately on the peptone and may stay red or turn hot pink afteronly 24 hrs. / Red
(above pH 6.8) / Yellow
(below pH 6.8)
Phenol red turns hot pink/fuschia if the medium becomes alkaline or > pH 8.2
Methyl Red/Voges-Proskauer (MRVP) / Fermentation of glucose (if at all) to acetoin and onward to butanediol (uses the butanediol fermentation pathway) / MR and VP tubes are interpreted separately. Adding methyl red to a grown culture turns red if acid was produced. The VP reagent is added to a grown culture in the presence of O2 and makes a red ring at the broth surface if acetoin is present (typical of Enterobacter bacteria). MRVP must be read within 24-48 hours as nearly all cultures become alkaline after 72 hours due to catabolism of the peptone into ammonia. / Yellow MR test (no change)
Yellow VP test (no change) / Red MR test
Red ring at surface for VP test
Indole Production from Tryptone Broth / Presence of the enzyme tryptophanase / The amino acid tryptophan can be hydrolyzed into indole, ammonia, and pyruvate if the enzyme tryptophanase is present. Addition of Kovacs Reagent produces a red layer at the top of tryptone broth if indole is present. / No change in broth compared to control / Red layer at the top of the broth
Test Name / Substance(s) or ability being tested for / Chemistry / Negative/Control / Positive Result
Oxidase production / Presence of enzyme cytochrome oxidase in electron transport chain / Oxidase reagent turns purple-gray within 1 minute when dripped onto plated bacterial colonies / No change in colony color / Colony or streak turns pinkish-purple
Citrate utilization / Citrate permease enzyme / Green citrate medium with bromthymol blue indicator turns blue as citrate-utilizing bacteria convert citrate to alkaline products. Even though citrate is broken down into acidic produces, citrate-utilizers make an overwhelming amount of alkaline products. / Agar slant remains green throughout
(pH6.9) / Agar slant turns blue in some portion
(pH > 7.6)
Urease Test on Urea Agar / Urease enzyme / The urea in urea agar slants is deaminated when NH2 groups are removed, producing alakine ammonium ions (NH4+). Phenol red in the medium turns hot pink/fuschia in alkaline conditions. / Medium remains a peach color, or even turn yellow if bacteria produce acid / Medium turns fuschia/hot pink
MacConkey Agar for gram negatives and lactose utilization / Fermentation of lactose and selection for gram negatives / Only gram negative bacteria should grow (except Enterococcus and some Staphylococcus)as the bile salts and crystal violet in the medium inhibit gram positives. Neutral red, a colorless pH indicator in the agar, turns red when the lactose is fermented to produce acid. Lactose-fermenting gram neg. enterics are called coliforms / Medium remains clear to faintly pink and colonies are white or crème colored / Medium turns reddish and colonies themselves become pink or tinged with pink when lactose is fermented (pH < 6.8)
Motility Test / Flagellum/flagella present / Organisms that are motile “swim” away from inoculation tunnel, often creating an “inverted Christmas tree” effect in an agar deep. / Medium remains dull yellow with whitish-creamy opaque growth only within the inoculation tunnel / Wisps of and opaque growth extend away from the inoculation tunnel; “inverted Christmas tree”
[Don’t use this sheet as instructions on how to set up or process your rest results!]
Metabolic Tests and Reactions Used to Identify Gram-negative Enteric Bacteria pg. 1