CLBACT - Gram Negative Cocci Test Summary

Gram Stain (Neisseria)

Principle: differential stain based on cell wall structure. Gram-negative organisms lose the crystal violet-iodine complex during decolorization and take up the safranin counterstain.

Gram Stain (Neisseria)

Positive: pink/red, kidney-bean-shaped diplococci, often seen intracellularly within PMNs. Negative: absence of typical gram-negative diplococcus morphology.

Gram Stain (Neisseria)

Intracellular GNDC on a direct smear from a symptomatic male urethral discharge is presumptive evidence of gonorrhea; far less reliable from female or extragenital specimens — culture or NAAT is required for confirmation.

Oxidase Test (Neisseria)

Principle: detects cytochrome c oxidase, which oxidizes the reagent (tetramethyl-p-phenylenediamine) to a colored indophenol end-product.

Oxidase Test (Neisseria)

Positive: colony turns deep purple to blue-black within 10–30 seconds. Negative: no color change. Mandatory screening step before species-level testing; the reagent is bactericidal, so subculture before applying if the isolate is needed further.

Superoxol (Catalase) Test

Principle: detects catalase activity using strong (30%) hydrogen peroxide; pathogenic Neisseria give a markedly more vigorous reaction than most other catalase-positive organisms.

Superoxol (Catalase) Test

Positive: immediate, vigorous, sustained effervescence — strongly supports N. gonorrhoeae. Negative: weak, delayed, or absent bubbling.

Superoxol (Catalase) Test

Distinguishes the very strong catalase reaction of N. gonorrhoeae from the more moderate reaction of N. meningitidis. Use fresh 30% H₂O₂ — it degrades quickly and gives false-weak reactions if old.

CTA Sugar Utilization

Principle: detects acid production from oxidative (not fermentative) carbohydrate breakdown in cystine trypticase agar (CTA) sugar medium, read via a pH indicator (glucose, maltose, lactose, sucrose tubes).

CTA Sugar Utilization

Positive: yellow color change (acid) in the corresponding sugar tube. Negative: no color change — medium remains red/orange. Weak oxidizers — use a heavy inoculum and allow up to 72 hours before calling a tube negative.

CTA sugar pattern — N. gonorrhoeae

Glucose only.

CTA sugar pattern — N. meningitidis

Glucose + maltose.

CTA sugar pattern — N. lactamica

Glucose + maltose + lactose.

Chromogenic Cephalosporin Test

Principle: detects beta-lactamase enzyme, which hydrolyzes the beta-lactam ring of a chromogenic cephalosporin substrate (nitrocefin), producing a visible color shift.

Chromogenic Cephalosporin Test

Positive: disk changes from yellow to pink/red within 15 minutes — penicillinase-producing (PPNG) strain. Negative: no color change.

Chromogenic Cephalosporin Test

A negative result does NOT rule out penicillin resistance — chromosomally mediated resistance mechanisms are not detected by this test.

Selective Media (MTM / Martin-Lewis)

Principle: enriched chocolate-agar base selective for pathogenic Neisseria; incorporated antibiotics (vancomycin, colistin, nystatin/anisomycin, trimethoprim) suppress normal flora while permitting fastidious Neisseria growth.

Selective Media (MTM / Martin-Lewis)

Positive: small, gray-white, glistening, convex colonies after 24–48 hours (5% CO₂, 35–37°C). Negative: no growth. Plates must go into a CO₂-enriched, humid incubator promptly; never refrigerate specimens or plates.

NAAT (Neisseria panel)

Principle: amplifies and detects species-specific nucleic acid sequences directly from clinical specimens without requiring a viable organism.

NAAT (Neisseria panel)

Positive: detectable amplified target signal. Negative: no signal detected. CDC-preferred method for N. gonorrhoeae/C. trachomatis screening; provides no isolate for susceptibility testing — culture is still required for resistance surveillance or medicolegal cases.

Serogroup Latex Agglutination

Principle: detects capsular polysaccharide serogroup-specific antigens using antibody-coated latex particles (serogroups A, B, C, Y, W135).

Serogroup Latex Agglutination

Positive: visible agglutination with the corresponding group antiserum. Negative: none. Used for rapid serogroup determination during outbreak investigation and vaccine-strain surveillance; culture confirmation is still required.

Penicillin Disk Elongation Test

Principle: subinhibitory concentrations of penicillin cause bacillary elongation in Moraxella spp. and several nonpathogenic Neisseria-like coccobacilli, whereas true cocci retain their round shape.

Penicillin Disk Elongation Test

Positive: elongated bacillary forms on Gram stain taken from the edge of the zone of inhibition. Negative: cells remain coccoid. The key test distinguishing true cocci (pathogenic Neisseria, M. catarrhalis) from bacillary mimics.

Growth on MacConkey Agar

Principle: MacConkey agar is selective for gram-negative organisms (bile salts and crystal violet inhibit gram-positives); a pH indicator differentiates lactose fermenters from non-fermenters.

Growth on MacConkey Agar

Positive: growth present, typically as non–lactose-fermenting colonies. Negative: no growth. Most true Neisseria spp. and M. lacunata/M. lincolnii fail to grow here; growth points toward M. catarrhalis, M. canis, or select N. elongata subspecies.

Nitrate Reduction Test

Principle: detects nitrate reductase, which reduces nitrate to nitrite; nitrite is revealed by a diazotization reaction (sulfanilic acid + alpha-naphthylamine) producing a red dye.

Nitrate Reduction Test

Positive: red color develops immediately after reagents are added. Negative: no color change; add zinc dust — if still no red color, nitrate was reduced past nitrite (true reduction).

Nitrate Reduction Test

Differentiates among Moraxella spp.: M. lacunata and M. nonliquefaciens are typically positive; M. lincolnii and M. osloensis are variable to negative.

Nitrite Reduction Test

Principle: detects nitrite reductase, which reduces nitrite further to nitrogen gas or ammonia. Performed only when the nitrate test is negative.

Nitrite Reduction Test

Positive: no red color develops after reagents/zinc are added — nitrite has been reduced past the point of detection. Negative: red color develops — unreduced nitrite still present.

DNase Test (differentiation)

Principle: detects extracellular deoxyribonuclease; hydrolyzed DNA no longer precipitates with acid or indicator dye.

DNase Test (differentiation)

Positive: clear zone (or pink halo with toluidine blue) around growth. Negative: no clearing. Contributes to the differential panel separating Moraxella atlantae and related low-virulence species.

Sodium Acetate Utilization

Principle: tests the ability to use acetate as a sole carbon source; utilization causes an alkaline pH shift detected by a bromothymol blue indicator.

Sodium Acetate Utilization

Positive: color change to blue (alkaline shift) — acetate utilized. Negative: no color change — medium remains green.

Sodium Acetate Utilization

The definitive test separating the two most commonly isolated Moraxella species: M. osloensis is acetate-positive, M. nonliquefaciens is acetate-negative.

Loeffler's Serum Slant

Principle: detects proteolytic enzyme activity capable of liquefying coagulated serum protein on the slant surface.

Loeffler's Serum Slant

Positive: visible depressions, pitting, or liquefaction of the slant surface. Negative: slant surface remains intact. Distinctive for M. lacunata, which also pits and forms dark halos on blood and chocolate agar.

Catalase Test (differentiation)

Principle: same peroxide-based principle used throughout the gram-negative and gram-positive cocci panels; helps separate genera within this group.

Catalase Test (differentiation)

Positive: bubbling — typical of Moraxella spp. Negative: no bubbling — typical of most Neisseria spp.

Catalase Test (differentiation)

Exceptions exist: N. elongata subsp. nitroreducens and some N. bacilliformis strains are catalase-positive. Oxidase alone cannot separate the two genera — all members of this group are oxidase-positive.

Pathogenic Neisseria key concept

Oxidase-positive, gram-negative diplococci growing on selective media (MTM/Martin-Lewis) are presumptively Neisseria; CTA sugar utilization is the pivotal test separating N. gonorrhoeae, N. meningitidis, and N. lactamica from one another.

Neisseria/Moraxella key concept

The penicillin elongation test is the gateway step: true cocci (pathogenic Neisseria, M. catarrhalis) stay coccoid, while Moraxella spp. and several commensal Neisseria-like organisms elongate into bacillary forms — only then proceed to nitrate/nitrite, DNase, acetate, and serum liquefaction testing to speciate.