CLBACT - Gram Positive Cocci Test Summary

Catalase Test

Principle: detects the enzyme catalase, which breaks down hydrogen peroxide into water and oxygen gas. Separates Staphylococcus/Micrococcus (positive) from Streptococcus/Enterococcus (negative).

Catalase Test

Positive: immediate bubbling/effervescence with 3% H₂O₂. Negative: no bubble formation.

Catalase Test

Never pick colonies from blood agar for this test — RBC catalase causes false positives. A few catalase-negative staph strains exist; Enterococcus/Aerococcus/Rothia may show a weak pseudocatalase reaction.

Coagulase Test — Slide (Rapid)

Principle: detects bound coagulase ("clumping factor") on the cell wall, which reacts directly with plasma fibrinogen to cause visible clumping.

Coagulase Test — Slide (Rapid)

Positive: visible clumping/agglutination within 10–20 seconds. Negative: none.

Coagulase Test — Slide (Rapid)

Screening test only. Some S. aureus strains are slide-negative but tube-positive — always confirm a negative slide result with the tube method.

Coagulase Test — Tube (Confirmatory)

Principle: detects free coagulase, which activates a coagulase-reacting factor that converts plasma fibrinogen to fibrin, forming a visible clot.

Coagulase Test — Tube (Confirmatory)

Positive: clot formation of any size, usually within 1–4 hours. Negative: no clot after full 24-hour incubation.

Coagulase Test — Tube (Confirmatory)

Gold standard for S. aureus identification. Read at 4 hours and again at 24 hours — some strains produce fibrinolysin that dissolves the clot if read too late.

DNase Test

Detects extracellular deoxyribonuclease, which depolymerizes DNA in the medium; hydrolyzed DNA no longer precipitates with acid or dye indicator. Used to confirm S. aureus when coagulase results are ambiguous or discordant.

DNase Test

Positive: clear zone (or pink halo with toluidine blue) around growth. Negative: no clearing / no color change.

Mannitol Salt Agar (MSA)

Principle: 7.5% NaCl inhibits most non-staphylococcal organisms; mannitol fermentation lowers pH, turning the phenol red indicator yellow.

Mannitol Salt Agar (MSA)

Positive: yellow halo surrounding colonies (mannitol fermented) — typical of S. aureus. Negative: pink/red colonies, no color change — typical of CoNS (e.g., S. epidermidis).

Mannitol Salt Agar (MSA)

S. saprophyticus also ferments mannitol and can mimic S. aureus on MSA — never use this medium alone for definitive ID.

Novobiocin Susceptibility

Disk diffusion assay (5 µg disk on Mueller-Hinton agar) separating coagulase-negative staphylococci into susceptible vs. resistant groups. Primary use: identifying S. saprophyticus among CoNS from urine.

Novobiocin Susceptibility

Susceptible: zone ≥ 16 mm (e.g., S. epidermidis, S. haemolyticus, S. lugdunensis). Resistant: zone < 16 mm (e.g., S. saprophyticus, S. cohnii, S. xylosus).

Microdase (Modified Oxidase) Test

Detects cytochrome C oxidase activity, present in Micrococcus spp. but absent in Staphylococcus spp. Best interpreted together with bacitracin susceptibility for reliable genus-level separation.

Microdase (Modified Oxidase) Test

Positive: colony turns blue within 2 minutes — Micrococcus spp. Negative: no color change — Staphylococcus spp.

Bacitracin Susceptibility (Staph/Micrococcus)

Disk diffusion test separating Micrococcus from Staphylococcus. Used as a companion test to the microdase test, not a stand-alone identifier.

Bacitracin Susceptibility (Staph/Micrococcus)

Positive: susceptible (zone present) — Micrococcus spp. Negative: resistant (growth to edge of disk) — Staphylococcus spp.

PYR Test (Staph/Micrococcus)

Detects pyrrolidonyl arylamidase, which hydrolyzes L-pyrrolidonyl-β-naphthylamide; the liberated β-naphthylamide reacts with a cinnamaldehyde developer to form a red color.

PYR Test (Staph/Micrococcus)

Positive: bright pink-to-red color within 2 minutes. Negative: no color change (remains colorless/orange).

PYR Test (Staph/Micrococcus)

Positive in S. lugdunensis and S. intermedius, helping distinguish them from other coagulase-negative/variable staphylococci.

Latex Agglutination (CF + Protein A)

Latex particles coated with fibrinogen and IgG simultaneously detect clumping factor and Protein A for rapid presumptive S. aureus screening.

Latex Agglutination (CF + Protein A)

Positive: visible agglutination within 1 minute. Negative: no agglutination (smooth suspension).

Latex Agglutination (CF + Protein A)

Increasing reports of false negatives with MRSA/CA-MRSA strains — do not rely on this test alone; confirm with coagulase testing.

Coagulase (pivotal test, CoNS workup)

Coagulase-positive isolates are presumptively S. aureus; coagulase-negative isolates (CoNS) require novobiocin, PYR, and further biochemical testing to speciate.

Hemolysis on Blood Agar

Detects hemolysins (e.g., streptolysin O/S) that lyse red blood cells on 5% SBA (stab inoculation); the pattern gives a presumptive group. Stabbing enhances detection of oxygen-labile streptolysin O.

Hemolysis on Blood Agar

Positive: beta (complete, clear zone) or alpha (partial, greenish zone). Negative: gamma — no hemolysis/no change to medium.

PYR Test (Strep/Enterococcus)

Same principle as the staph PYR test. Positive: pink/red — S. pyogenes (Group A) and Enterococcus spp. Negative: no color change — S. agalactiae (Group B), viridans streptococci, S. anginosus group.

PYR Test (Strep/Enterococcus)

The single most useful rapid test for presumptive Group A strep ID and, combined with LAP-positive/6.5% NaCl growth, for Enterococcus screening.

Bacitracin Susceptibility (Strep)

Disk diffusion test assessing zone of inhibition around a low-dose bacitracin disk on blood agar. Positive: susceptible (any zone) — presumptive Group A strep. Negative: resistant (growth to disk edge).

Bacitracin Susceptibility (Strep)

No longer recommended alone for S. pyogenes ID — Groups C and G strep are also bacitracin-susceptible. PYR testing is preferred.

CAMP Test

Detects the CAMP factor, a diffusible protein produced by S. agalactiae that synergizes with (enhances) the beta-lysin produced by S. aureus. Method: cross-streak against a known S. aureus beta-hemolysin strain.

CAMP Test

Positive: arrowhead-shaped zone of enhanced hemolysis at the intersection of the two streaks — confirms S. agalactiae. Negative: no arrowhead formation.

Hippurate Hydrolysis

Detects hippuricase, which hydrolyzes sodium hippurate to glycine and benzoic acid; glycine is then oxidized by ninhydrin to produce a purple color.

Hippurate Hydrolysis

Positive: deep purple/blue color — S. agalactiae (Group B) and Enterococcus spp. Negative: no color change. Used with the CAMP test to confirm GBS.

Bile Esculin (BE) Test

Detects hydrolysis of esculin in the presence of 40% bile (oxgall); the esculetin end-product reacts with ferric citrate to form a dark brown/black precipitate.

Bile Esculin (BE) Test

Positive: blackening of more than half the slant. Negative: no darkening — medium remains unchanged.

Bile Esculin (BE) Test

Positive for Group D streptococci and enterococci, but also for Leuconostoc, Pediococcus, and Aerococcus viridans — no longer specific for enterococci alone.

6.5% NaCl Tolerance

Tests the ability to grow in a high-salt broth (heart infusion broth, 6.5% NaCl), a hallmark of salt-tolerant enterococcal physiology.

6.5% NaCl Tolerance

Positive: visible turbidity/growth. Negative: no growth — broth remains clear.

6.5% NaCl Tolerance

Used with bile esculin and PYR for presumptive Enterococcus ID; Aerococcus and Pediococcus can also grow here, so never use alone.

Optochin Susceptibility

Disk diffusion using optochin (ethylhydrocupreine hydrochloride), which selectively disrupts the S. pneumoniae cell membrane/ATPase system. SBA lawn, incubated with 5% CO₂.

Optochin Susceptibility

Positive: susceptible, zone ≥ 14 mm — presumptive S. pneumoniae. Negative: resistant, growth to disk edge — viridans streptococci.

Optochin Susceptibility

Presumptive only — occasional S. oralis/S. mitis strains are optochin-sensitive. Always confirm with the bile solubility test.

Bile Solubility Test

Bile salts (2% sodium desoxycholate) activate autolytic amidase enzymes in the S. pneumoniae cell wall, causing rapid lysis. Considered the definitive confirmatory test for S. pneumoniae, superior to optochin alone.

Bile Solubility Test

Positive: colony/suspension clears (lyses) within minutes — confirms S. pneumoniae. Negative: suspension remains turbid — viridans streptococci (incl. S. pseudopneumoniae).

Voges-Proskauer (VP) Test

Detects acetoin (acetylmethylcarbinol), an intermediate of glucose fermentation via the butylene glycol pathway. Reagents: VP broth, Barritt's reagents (α-naphthol and KOH).

Voges-Proskauer (VP) Test

Positive: red/pink color within 15–30 minutes. Negative: no color change (medium remains copper-colored).

Voges-Proskauer (VP) Test

A positive VP with a negative PYR identifies a beta-hemolytic isolate as S. anginosus group.

LAP Test (Leucine Aminopeptidase)

Detects leucine aminopeptidase, which hydrolyzes leucine-β-naphthylamide; the liberated naphthylamide reacts with a color developer to produce a red color.

LAP Test (Leucine Aminopeptidase)

Positive: red/cherry color within 2 minutes. Negative: no color change (colorless/yellow).

LAP Test (Leucine Aminopeptidase)

Enterococci are LAP-positive; Leuconostoc and Pediococcus are LAP-negative — useful when an unexpected vancomycin-resistant, "strep-like" isolate is seen.

Vancomycin Screen (30 µg disk)

Disk diffusion test assessing intrinsic susceptibility patterns among catalase-negative, gram-positive cocci genera. Flags intrinsic resistance — do not mistake for acquired VRE resistance.

Vancomycin Screen (30 µg disk)

Positive: susceptible (any zone) — Streptococcus, Enterococcus, Aerococcus, Gemella, Lactococcus. Negative: resistant (no zone) — Pediococcus, Leuconostoc, most Lactobacillus.

Lancefield Grouping (Latex)

Detects group-specific cell wall carbohydrate antigens using latex particles coated with group-specific antisera (Groups A, B, C, D, F, G). Rapid and definitive from 1–2 colonies; more costly than biochemical screening.

Lancefield Grouping (Latex)

Positive: visible agglutination with the corresponding group antiserum. Negative: no agglutination with any antiserum tested.

Hemolysis pattern (workup path)

Beta-hemolytic isolates proceed to PYR/bacitracin/CAMP/hippurate grouping; alpha-hemolytic isolates are ruled in/out for S. pneumoniae (optochin + bile solubility) before considering viridans streptococci or enterococci (6.5% NaCl + bile esculin + PYR + LAP).