11.1 Colony Morphology and Rapid Presumptive Tests

11.1 Colony Morphology and Rapid Presumptive Tests

Quick Answer: Read hemolysis, pigment, swarming, mucoid capsules, metallic sheen, and odor (never sniff select-agent-risk plates) as presumptive clues, then follow Gram stain → catalase or oxidase → coagulase, indole, and PYR. Do not perform catalase on blood agar (RBC catalase false-positive) or oxidase with nichrome loops (false-positive).

Colony morphology is the first identification result the M(ASCP) candidate is expected to interpret. After overnight incubation, the plate is a data set: hemolysis on sheep blood agar (BAP), pigment, surface spreading, capsule-related mucoidy, indicator-agar reactions such as the green-black metallic sheen of lactose-fermenting Escherichia coli on eosin-methylene blue (EMB), and characteristic odors. Morphology never replaces biochemical or proteomic identification, but it chooses the next test and flags dangerous look-alikes before they are aerosolized. Outline II.J (Identification Methods — theory, interpretation, and application) begins here: you must know what the colony is telling you, which rapid test is valid on that colony, and which result is an artifact of the medium or the loop.

Hemolysis on sheep blood agar

Hemolysis is read as the effect of bacterial products on intact sheep erythrocytes in the agar, not as a Gram-stain color and not as a pigment in the colony itself.

• Beta hemolysis is complete clearing around the colony. Classic beta-hemolytic Gram-positive cocci include Streptococcus pyogenes (group A; large zone relative to colony size), Streptococcus agalactiae (group B; often a smaller, softer zone), and Staphylococcus aureus (wide zone plus gold or cream colonies). Listeria monocytogenes is weakly beta-hemolytic and can be mistaken for GBS until catalase (positive) and tumbling or umbrella motility are applied.
• Alpha hemolysis is incomplete hemoglobin breakdown that greens the agar (biliverdin). Streptococcus pneumoniae is alpha-hemolytic, often mucoid or cratered (checker/draftsman colonies); viridans-group streptococci are also alpha-hemolytic but usually drier and smaller.
• Gamma hemolysis means no visible hemolysis. Many enterococci and coagulase-negative staphylococci are gamma-hemolytic, though some enterococci show small alpha or beta zones depending on strain and atmosphere.

Exam traps: hemolysis type is medium-dependent (human versus sheep blood; anaerobic versus CO2 incubation can enlarge streptococcal zones). Report what you see on the standard BAP used in the laboratory algorithm, then confirm with catalase, PYR, CAMP, hippurate, or optochin as indicated. Do not call a green zone on chocolate agar "alpha hemolysis" — chocolate agar has already lysed the cells.

Pigment, swarming, mucoidy, and metallic sheen

Pigment is a useful genus clue when it is stable and matched to Gram morphology.

• Pyocyanin is the blue-green phenazine of Pseudomonas aeruginosa. Combined with a grape-like or taco-chip odor, spreading growth, and oxidase positivity, pyocyanin is a high-probability presumptive identification that still needs confirmation and susceptibility testing. Pyoverdin (fluorescein) is the yellow-green fluorescent siderophore of fluorescent pseudomonads, best seen under ultraviolet light on iron-poor media.
• Prodigiosin is the red, temperature-dependent pigment of Serratia marcescens, often stronger at room temperature than at 35°C. A red colony at ambient incubation is not Serratia until oxidase (negative), Gram-negative rod morphology, and biochemical or MALDI identification agree.
• Staphyloxanthin gives many S. aureus isolates their golden-yellow color; absence of gold does not exclude S. aureus.

Swarming of Proteus mirabilis (and P. vulgaris) on BAP appears as concentric waves of motility that can overrun a mixed urine culture. CLED or MacConkey agar restricts swarming so other colonies can be picked. Mucoid colonies of Klebsiella pneumoniae reflect a large polysaccharide capsule and can resemble mucoid pneumococcus on BAP until Gram stain (Gram-negative rods versus Gram-positive diplococci) and catalase/oxidase are applied. The green-black metallic sheen of E. coli on EMB is a lactose-fermentation/indicator phenomenon, not a true pigment; it supports but does not prove species identity.

Odor — and when you must not sniff

Older teaching used odor as a rapid clue: P. aeruginosa (grapes or tortilla), Eikenella corrodens (bleach), Haemophilus influenzae (mousy), Proteus (chocolate cake or burnt chocolate), historical Clostridioides difficile (horse stable), Streptomyces (soil). Those associations still appear in exam stems, but do not sniff plates that could contain select agents or other BSL-3 organisms (Brucella, Francisella tularensis, Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei/mallei). If a slowly growing Gram-negative coccobacillus appears on blood or chocolate from a fever-of-unknown-origin, animal-exposure, or biothreat-rule-out case, stop, cover the plate, and follow the select-agent/LRN workup. Odor is never a reportable identification, and a "quick sniff" is not a rapid test on the outline.

Rapid tests named on the outline

Coagulase: slide (bound) versus tube (free), plus latex

Bound coagulase (clumping factor) converts fibrinogen on the bacterial surface; the slide coagulase test detects it as visible clumping in plasma (with a saline autoagglutination control). Free coagulase is an extracellular enzyme that reacts with coagulase-reacting factor in plasma to form a fibrin clot; the tube coagulase test (typically rabbit plasma, read at 4 hours and held up to 24 hours if negative) is the classic reference method for S. aureus. Latex agglutination kits detect clumping factor and/or protein A and are faster screening tests used at the bench.

Interpretation rules that show up on exams:

• A positive tube coagulase supports S. aureus (or the animal-associated S. intermedius/S. pseudintermedius group in veterinary or bite-wound contexts).
• Slide or latex positives that are weak or discordant with colony morphology must be confirmed with the tube test.
• S. lugdunensis may be slide-positive (clumping factor) but is tube-coagulase-negative and often PYR-positive — a frequent trap versus S. aureus.
• Autoagglutination in the saline control invalidates the slide test; proceed to tube coagulase or a different method.

Catalase: staphylococci versus streptococci; enterococci are catalase-negative

Catalase (2 H2O2 → 2 H2O + O2) distinguishes catalase-positive Staphylococcus and Micrococcus from catalase-negative Streptococcus and Enterococcus. Use 3% hydrogen peroxide on a colony taken from a non-blood medium (chocolate or nutrient agar), or lift the colony so that agar is not mixed into the reaction. Sheep erythrocytes contain catalase; testing a colony scraped from BAP is a classic false-positive trap. Enterococci are catalase-negative; a weak bubble from peroxidase should not be called positive. Bacillus and most coryneforms are catalase-positive, which is why Gram morphology is the first branch of the algorithm, not catalase alone.

Oxidase: cytochrome c

The oxidase test detects cytochrome c oxidase using tetramethyl-p-phenylenediamine (Kovacs oxidase reagent) that turns dark purple when oxidized. Enterobacterales are oxidase-negative. Pseudomonas, most Neisseria, Moraxella catarrhalis, Campylobacter, Vibrio, and Aeromonas are in the oxidase-positive differential. Use a platinum or plastic loop or a wooden stick; nichrome (iron-containing) loops can give a false-positive oxidase reaction. Do not test from media containing fermentable carbohydrates or dyes that obscure the color change (for example MacConkey) if an alternative colony is available. The reagent must be fresh; a delayed weak color after the manufacturer's read window is not a positive.

Indole: Kovacs and spot

Indole detects tryptophanase. In a tryptophan broth or SIM tube, Kovacs reagent (p-dimethylaminobenzaldehyde in amyl alcohol) yields a red surface ring. Spot indole (often DMACA, which is more sensitive than Kovacs on filter paper) is performed on colonies grown on a tryptophan-containing medium such as blood or chocolate, not MacConkey. E. coli is the prototype indole-positive lactose fermenter; Klebsiella pneumoniae is indole-negative, whereas K. oxytoca is indole-positive — a high-yield species split. Proteus vulgaris is indole-positive; P. mirabilis is indole-negative, which is how you split the two common swarming species at the bench before kits or MALDI.

PYR

PYR hydrolyzes L-pyrrolidonyl-β-naphthylamide; a cinnamaldehyde reagent produces a red color. Group A streptococci (S. pyogenes) and Enterococcus species are PYR-positive. Group B streptococci are PYR-negative. PYR also helps separate S. lugdunensis (usually PYR-positive) from S. aureus (PYR-negative). Do not use PYR as a stand-alone S. pyogenes report from a throat culture without confirming Gram-positive catalase-negative morphology and, in many laboratories, antigen or molecular backup per protocol. A PYR-positive catalase-negative coccus from urine is more likely enterococcus than GAS.

Algorithm: Gram stain → catalase/oxidase → rapid tests

Work from cellular morphology first. Gram-positive cocci in clusters go to catalase (expect positive), then coagulase/latex and, if needed, PYR. Gram-positive cocci in chains or pairs go to catalase (expect negative), then hemolysis-directed tests: PYR and bacitracin for beta-hemolytic colonies, CAMP/hippurate for suspected GBS, optochin/bile solubility for alpha-hemolytic lancet-shaped diplococci. Gram-negative rods go to oxidase: oxidase-negative isolates enter the Enterobacterales biochemical or MALDI path (spot indole is a useful 30-second branch for E. coli); oxidase-positive isolates enter the non-fermenter/Pseudomonas/oxidase-positive fermenter path. This algorithm is theory, interpretation, and application of outline II.J — morphology and rapid tests are presumptive, and discordant results are a stop sign, not a reason to force an identification.