Biochemical tests for bacterial identification

Why the order matters

Interpret each test in the context of the organism and the lab key. Coagulase is used here to distinguish staphylococci, not to identify a gram-negative rod. Oxidase is useful for gram-negative rods but is not how you sort streptococci. Running tests in the right order saves media and keeps you from over-reading a result that does not apply.

1. Start with a pure, fresh culture. Streak for isolated colonies of one organism and use fresh growth, usually 18 to 24 hours old for these organisms. A mixed culture gives mixed results.
2. Gram stain and shape. Gram-positive or gram-negative, cocci or rods, and the arrangement (clusters, chains, pairs). This picks which key you use.
3. One branching test. Catalase for gram-positive cocci; oxidase for gram-negative rods.
4. Narrow down. Follow only the branch your result sends you to, then confirm with one or two extra tests if your key allows.

Identification key: gram-positive cocci

Read this like a dichotomous key: each result selects a branch. Start at step 1, choose the line that matches your result, and follow the link to the next step.

1. Step 1. Catalase (3% hydrogen peroxide)

   • Bubbles (positive): Staphylococcus or Micrococcus. Go to step 2.
   • No bubbles (negative): Streptococcus or Enterococcus. Go to step 4.

2. Step 2. Tube coagulase (rabbit plasma)

   • Clot (positive): suggests Staphylococcus aureus in this key. Mannitol salt agar usually turns yellow.
   • No clot (negative): if confirmed as Staphylococcus, coagulase-negative staphylococci. Go to step 3.

   Micrococcus is also coagulase negative; exclude it before step 3 using your lab’s glucose fermentation or modified oxidase test. Clues: tetrads (groups of four cells), often yellow colonies, and no glucose fermentation.

3. Step 3. Novobiocin disk (5 µg; comparing S. epidermidis and S. saprophyticus)

   • Clear zone diameter greater than 16 mm (sensitive): Staphylococcus epidermidis.
   • Zone diameter 16 mm or less (resistant): Staphylococcus saprophyticus.

4. Step 4. Hemolysis on blood agar

   • Clear zone around colonies (beta): go to step 5.
   • Greenish discoloration (alpha): go to step 6.
   • No change (gamma): go to step 7.

5. Step 5. Bacitracin disk (A disk)

   • Sensitive: suggests Streptococcus pyogenes (group A); bacitracin alone is not specific, so confirm as your lab directs.
   • Resistant, and CAMP test positive (arrowhead of clearing): suggests Streptococcus agalactiae (group B).

6. Step 6. Optochin disk (P disk) and bile solubility

   • Sensitive and bile soluble: Streptococcus pneumoniae.
   • Resistant and bile insoluble: viridans group streptococci.

7. Step 7. Bile esculin agar and 6.5% NaCl broth

   • Bile esculin black and growth in 6.5% NaCl: Enterococcus (for example E. faecalis).
   • Bile esculin black but no growth in 6.5% NaCl: non-enterococcal group D streptococci (the S. bovis group, including Streptococcus gallolyticus).

   Enterococcus is usually gamma but some strains are alpha or beta, so some lab keys run bile esculin on every catalase-negative coccus.

Identification key: gram-negative rods

1. Step 1. Oxidase

   • Dark purple or blue within the method’s reading window (positive): consider Pseudomonas aeruginosa if it also makes colorless MacConkey colonies, ferments no sugars in triple sugar iron (TSI) agar, and produces a blue-green pigment. Oxidase alone does not identify it or exclude every Enterobacterales species.
   • No color change within the method’s reading window (negative): consider Enterobacterales, a group of gram-negative rods that ferment glucose. Other organisms can also be oxidase negative; this branch assumes glucose fermentation has been demonstrated. Go to step 2.

2. Step 2. Lactose on MacConkey agar

   • Pink or red colonies (lactose fermenter): go to step 3.
   • Colorless or pale colonies (non-lactose fermenter): go to step 5.

3. Step 3. IMViC (indole, methyl red, Voges-Proskauer, citrate)

   • ++−−: Escherichia coli.
   • −−++: typically Klebsiella pneumoniae, K. aerogenes or Enterobacter cloacae. Go to step 4.

4. Step 4. Motility (ability to move)

   • Nonmotile: Klebsiella pneumoniae.
   • Motile: Enterobacter (for example E. cloacae; Klebsiella aerogenes, formerly Enterobacter aerogenes, is also motile).

5. Step 5. Hydrogen sulfide, H₂S (black in TSI, Kligler iron agar [KIA] or sulfide-indole-motility [SIM] medium)

   • Black precipitate: go to step 6.
   • No black: go to step 7.

6. Step 6. Urease

   • Pink or magenta, often within hours; very motile, may swarm: Proteus (P. mirabilis is indole negative, P. vulgaris indole positive).
   • Negative: Salmonella.

7. Step 7. Motility

   • Nonmotile, urease negative: Shigella.
   • Motile: check your lab key for the other non-lactose fermenters it includes.

What each test detects

Knowing what a test detects makes the colors easier to remember. A color change is the indicator responding to a product the cells made, or a reagent reacting with it. A zone of inhibition is a clear area without bacterial growth. For growth-dependent tests, no growth does not establish a negative metabolic result.

Two worked unknowns

Unknown A: gram-positive cocci in clusters

1. Pick the key. Gram-positive cocci, so use the gram-positive key. For this exercise, the candidates are S. aureus, S. epidermidis and S. saprophyticus; clusters fit staphylococci.
2. Catalase positive. This supports staphylococci, but catalase alone would not distinguish them from Micrococcus in a broader key.
3. Mannitol salt agar. It grew in 7.5% salt, which fits a staphylococcus. The medium stayed red, so mannitol was not fermented. That argues against S. aureus.
4. Coagulase negative. No clot at the scheduled readings through 24 hours favors a coagulase-negative staphylococcus over typical S. aureus.
5. Novobiocin sensitive. Of the two coagulase-negative candidates, S. epidermidis is sensitive and S. saprophyticus is resistant.

Answer: Staphylococcus epidermidis.

Unknown B: gram-negative rods from a urine culture exercise

1. Pick the key. Gram-negative rods, so use the gram-negative key.
2. Oxidase negative. This argues against typical P. aeruginosa; glucose fermentation in TSI supports following the Enterobacterales branch.
3. Colorless on MacConkey. No lactose fermentation was detected at the reading time, making typical lactose-fermenting E. coli, K. pneumoniae and E. cloacae less likely.
4. TSI: red over yellow with black. Of the three sugars in TSI, only glucose was fermented, and H₂S was made. That leaves Salmonella or Proteus in this key.
5. Urease strongly positive. Proteus is urease positive and Salmonella is not, so this is Proteus. The spreading film is swarming, a classic Proteus clue.
6. Indole negative. P. mirabilis is indole negative; P. vulgaris is indole positive.

Answer: Proteus mirabilis.

Common mistakes that give wrong results

• Gram staining an old culture. Older cells can have damaged walls and look gram-negative even when the species is gram-positive. Use fresh growth, usually an 18- to 24-hour culture for these organisms.
• Leaving the decolorizer on too long. Over-decolorizing washes crystal violet out of gram-positive cells too, so they look pink.
• Testing catalase with growth from blood agar. Red blood cells contain catalase, so carrying over any agar can give a false positive. Take growth carefully from the top of the colony, or use a plate without blood.
• Using a nichrome or steel loop for oxidase. Nickel, steel and other metal wires can give a false positive. Use a platinum loop, a plastic loop or a wooden stick.
• Reading oxidase late. Use the reading window for your reagent and method. Some protocols recognize delayed positives; color developing after the allowed window can be reagent oxidation and must not be scored as positive.
• Reading MR or VP too early. Many lab manuals incubate MR-VP broth for about 48 hours before adding reagents. VP color also needs time after the reagents are added and the tube is shaken, so follow your manual's wait time.
• Trusting a negative slide coagulase. The slide test can miss some S. aureus. Confirm a negative with the tube test, which can take up to 24 hours.
• Skipping controls. Run a known positive and negative organism alongside a test you are unsure of. Expired reagents and wrong media are common causes of odd results.

Practice: identify the unknown

Choose the best match among the listed options, using typical results, and press Check answer. The feedback explains which results support or argue against each choice; these patterns are not definitive identifications. Questions 7 and 8 ask which test you would run next.

Frequently asked questions

What is the first biochemical test for gram-positive cocci?

Catalase. Bubbles when the cells meet 3% hydrogen peroxide point to Staphylococcus or Micrococcus. No bubbles point to Streptococcus or Enterococcus, and the next step is the hemolysis pattern on blood agar.

What do catalase, coagulase and oxidase results tell you?

In this teaching key, catalase helps distinguish typically positive staphylococci and Micrococcus from typically negative streptococci and enterococci. A positive tube coagulase supports Staphylococcus aureus. Oxidase helps distinguish organisms such as Pseudomonas aeruginosa (usually positive) from most Enterobacterales, including E. coli (negative). These tests guide identification; none alone proves a species or group.

What does an IMViC result of ++−− mean?

Indole positive, methyl red positive, Voges-Proskauer negative and citrate negative. That is the classic pattern for Escherichia coli. The reverse pattern, −−++, is typical of Klebsiella pneumoniae, K. aerogenes and Enterobacter cloacae.

What should I do if my unknown does not fit any branch of the key?

Check that the culture is pure and fresh (usually 18 to 24 hours for these organisms), repeat the doubtful test with known positive and negative controls, and recheck how long each tube was incubated. If it still does not fit, use the key your lab provides and ask your instructor, because strains vary and lab keys differ.