| Methyl Red (MR) | Acid products formed when glucose is metabolized by mixed acid fermentation pathway. Color changes pH indicator. | Pos = red color; pH ≤ 4.5 | (test becomes positive at pH ≤ 4.5) |
| Voges-Proskauer (VP) | Acetoin is produced from alternate pathway for glucose metabolism. Naphthol & KOH added. | Pos = red color after alpha-naphthol & KOH added. | Little acid produced by this pathway. Often inverse with MR. VP positive species typically include Klebsiella and Enterobacter. |
| Citrate | If organism can use citrate as sole source of carbon, pH ↑. pH indicator changes color. | Pos = green to blue or growth | If organism can use citrate as sole source of carbon, pH will increase, leading to a color change with a pH indicator. |
| Urease | Urease breaks down urea. Ammonia released, pH ↑. pH indicator changes color. | Pos = yellow to pink | Proeteae & Morganella are rapid urease producers. |
| Phenylalanine Deaminase (PD) | PD deaminates phenylalanine to phenylpyruvic acid, which reacts with ferric chloride to produce green color. | Pos = green color after addition of ferric chloride | PD deaminates phenylalanine to phenylpyruvic acid. Positive = green color after adding ferric chloride. Proteus, Providencia, Morganella are pos. |
| H₂S production | Organisms that possess S-containing enzymes produce color-less H₂S gas from sulfur-containing compounds. H₂S reacts with iron salt in medium to form black ferrous sulfide. | Black precipitate | Sulfur-containing compounds = sodium thiosulfate, cysteine, methionine. Good test to differentiate Salmonella (H₂S pos) from Shigella (H₂S neg). |
| Indole | Tryptophanase deaminates tryptophan. Indole produced. Kovacs reagent (paradimethylaminobenzaldehyde) forms pink-colored complex with indole. | Pos = pink | Tryptophanase breaks down tryptophan to indole. Indole is detected using Kovacs reagent, forming a pink complex. E. coli and Proteus vulgaris are pos. |
| Triple Sugar Iron (TSI) Agar | Sugar fermentation produces acid, changed color by pH indicator. | Yellow = acid (A), Pink = alkaline (K). Pink slant = glucose fermented. Yellow slant = lactose/sucrose not fermented. Yellow slant & butt = lactose and/or sucrose fermented. Black precipitate = H₂S produced. Bubbles = gas production. | Contains 0.1% glucose, 1% lactose, 1% sucrose, phenol red, sodium thiosulfate, iron salt. Leaves air slightly loose. Record as slant/butt, e.g., K/A (alkaline slant/acid butt). K/A (Same except no change) for non-fermenters. Black butt indicates H₂S production. Gas production is seen as bubbles or displacement of the agar. Spot indole test commercially available. Need source of tryptophan. Use colonies from SBA or Choc agar, not MAC. |
| Decarboxylase reactions | If organism has enzyme to decarboxylate amino acid (e.g., ornithine, lysine, arginine), pH ↑. pH indicator changes color. | Pos = yellow to purple | Test for the presence of enzymes that remove a carboxyl group from amino acids. Positive result indicates the production of alkaline amines, raising pH. Ornithine decarboxylase (ODC) is a common test. Most Enterobacteriales are pos, except Klebsiella & Shigella (neg). Differentiates Klebsiella & Enterobacter (pos) and differentiates Shigella (neg) & Salmonella (pos). |
| Motility | Motile organisms grow away from stab line in motility medium. | Pos = movement away from stab line or hazy appearance throughout medium after overnight incubation. | Observed in semi-solid agar. Motile bacteria will spread from the inoculation line, making the medium cloudy. Non-motile bacteria will only grow along the stab line. |
| Carbohydrate fermentation | When carbohydrate is fermented, acidic end products cause color change in pH indicator. | With phenol red indicator, color change from red to yellow. | Indicates the ability of the organism to ferment specific carbohydrates (e.g., lactose, sucrose, mannitol, etc.), producing acid that changes the pH indicator. |
| Oxidase | Tests for enzyme cytochrome c oxidase. Reagent changes color to blue or purple. | Pos = blue or purple | Good test to differentiate Enterobacteriales (oxidase negative) from other GNB (like Pseudomonas, which are oxidase positive). False pos from iron-containing wire. Use platinum loop or wooden stick. |
| Nitrate Reduction | If organism reduces nitrates to nitrites, red color develops when sulfanilic acid & alpha-naphthyl-amine added. If nitrites reduced further to N₂ gas, no color change after reagents, but gas bubble evident in Durham tube. | Pos = red color after adding reagents (nitrites are present), or no color after adding zinc dust (nitrites were reduced further to N₂). Neg = red color after adding zinc dust (nitrates are still present). | If color develops (red) after just adding reagents, nitrites are present. If no color after reagents, add zinc dust. If red develops after zinc dust, nitrates were still present (negative). If no color after zinc dust and a gas bubble is present, nitrates were reduced to N₂ gas (positive). All Enterobacteriales ferment glucose. |
| ONPG | ONPG is changed to ortho-nitrophenyl by Beta-galactosidase. | Pos = yellow | Test for slow lactose fermentation. ONPG is a substrate that detects the enzyme beta-galactosidase, which is required for lactose fermentation. Helpful in differentiating slow or non-lactose fermenters. Helpful in differentiating Citrobacter (pos) from most Salmonella (neg). |