11.2 Conventional Biochemicals and Commercial Kits

11.2 Conventional Biochemicals and Commercial Kits

Quick Answer: H. influenzae requires both X (hemin) and V (NAD) and satellites around a staphylococcal streak. N. gonorrhoeae utilizes glucose only on CTA sugars; N. meningitidis utilizes glucose and maltose. Conventional tests (TSI, urea, citrate, decarboxylases, motility, bile esculin, 6.5% NaCl, optochin, bacitracin, CAMP, hippurate) still resolve kit and MALDI gaps. Do not inoculate suspected select agents onto open automated systems.

Conventional biochemicals remain on the M(ASCP) outline because they explain kit reactions, rescue identifications when commercial databases fail, and are the only appropriate tools when an isolate is too dangerous or too unusual for Vitek or MicroScan. Interpretation is as important as setup: inoculum density, atmosphere, and incubation time change the color. Outline II.J expects you to know the theory of each reaction, how to read it, and when a commercial card is the wrong next step.

X and V factors and satellitism

Haemophilus influenzae is a fastidious Gram-negative coccobacillus that cannot grow on unsupplemented sheep blood agar. It requires X factor (hemin) and V factor (NAD). Chocolate agar supplies both because heat lysis of erythrocytes releases hemin and inactivates NADases in the blood. On plain BAP, H. influenzae shows satellitism: tiny colonies grow only in the hemolytic zone around a streak of Staphylococcus aureus, which supplies NAD and, through hemolysis, hemin.

Factor disks or a quad plate separate species in the Haemophilus group:

• H. influenzae: growth only where X and V are both present (XV disk or chocolate agar).
• H. parainfluenzae and other para- species: V factor only.
• H. ducreyi: X factor only (and is rarely recovered on routine genital media).

Exam application: a tiny Gram-negative coccobacillus from sputum, ear, or CSF that grows on chocolate but not BAP, and that satellites around staph, is H. influenzae until biochemical or MALDI confirmation. Do not confuse satellitism with Abiotrophia/Granulicatella (nutritionally variant streptococci), which satellite as Gram-positive cocci around staph because they need thiol or pyridoxal, not X and V. A satellite colony that is a Gram-negative coccobacillus is a Haemophilus problem; a satellite colony that is a Gram-positive coccus is a nutritionally variant streptococcus problem.

Neisseria carbohydrate utilization (CTA)

Cystine trypticase agar (CTA) sugars test acid production from carbohydrates under low-agar conditions that favor Neisseria. Read a yellow (acid) change in the medium, not growth alone. Incubate according to the package insert; CO2 can acidify CTA and produce false-positive sugar reactions if the protocol specifies a non-CO2 atmosphere.

N. gonorrhoeae utilizes glucose only. N. meningitidis utilizes glucose plus maltose. N. lactamica (often recovered from children and not the same pathogen as meningococcus) ferments lactose and will be miscalled N. meningitidis if the lactose tube is omitted. Moraxella catarrhalis is asaccharolytic on CTA; confirm with tributyrin or butyrate esterase and DNase. Superoxol (30% hydrogen peroxide) is strongly positive for gonococci but is not a stand-alone identification. Gram-negative diplococci from a genital specimen still need the full identification algorithm and, in current practice, are often identified by NAAT from the specimen — but when a colony exists, CTA (or a commercial Neisseria panel) remains the conventional biochemical standard on this exam.

Core conventional biochemicals the exam still uses

Triple sugar iron (TSI) agar contains glucose (0.1%), lactose (1%), sucrose (1%), iron salts, and thiosulfate. Interpret slant/butt after 18–24 hours of incubation with a loose cap so that oxidative reactions on the slant can occur:

• K/K (alkaline/alkaline, red/red): no carbohydrate fermentation — typical non-fermenter such as Pseudomonas.
• K/A (red/yellow): glucose fermented, lactose and sucrose not fermented — Shigella, many Salmonella, Proteus, and some Citrobacter.
• A/A (yellow/yellow): glucose plus lactose and/or sucrose — E. coli, Klebsiella, Enterobacter, sucrose-positive Yersinia.
• H2S blackens the butt (Salmonella, Proteus, Citrobacter freundii complex).
• Gas splits or bubbles the agar (E. coli, Klebsiella, many Salmonella; Shigella is typically anaerogenic).

TSI is not a species identification. It is a gate: an oxidase-negative Gram-negative rod that is K/A without gas or H2S belongs in the Shigella/inert E. coli differential and needs serology or molecular confirmation. Reporting "E. coli" from a metallic sheen alone, without TSI or equivalent biochemicals when the isolate is from stool and looks inert, is how Shigella is missed.

Urea (Christensen urea agar): rapid positives include Proteus, Morganella, and many Providencia; K. pneumoniae is urease-positive more slowly; Helicobacter pylori is rapidly urease-positive in gastric testing. A positive urease is a pink alkaline change.

Simmons citrate: growth and a blue color mean the isolate can use citrate as sole carbon source. Klebsiella and Enterobacter are typically positive; E. coli and Shigella are negative. Use a light inoculum from a non-carbohydrate plate — heavy carbohydrate carryover can cause a false-positive blue color.

Decarboxylases (Moeller lysine, ornithine, arginine) require an oil overlay and a control tube without amino acid. A positive is alkaline (purple) versus a fermented, acidic (yellow) control. If the control is not yellow, the test is invalid. Lysine decarboxylase helps separate Salmonella (usually positive) from many Citrobacter (often negative) and Klebsiella pneumoniae (lysine positive, ornithine negative) from Enterobacter cloacae complex (ornithine positive, lysine usually negative).

Motility medium or hanging drop: Proteus and most E. coli and Salmonella are motile; Klebsiella and Shigella are nonmotile. Listeria monocytogenes shows umbrella motility at 25°C but not at 37°C — a classic Gram-positive rod clue that also pairs with catalase positivity and bile-esculin positivity.

Bile esculin and 6.5% NaCl separate enterococci from other catalase-negative Gram-positive cocci. Enterococci hydrolyze esculin in 40% bile (black agar) and grow in 6.5% NaCl. Streptococcus gallolyticus (group D streptococcus, non-enterococcus) is bile-esculin-positive but usually fails 6.5% NaCl. Listeria is bile-esculin-positive and catalase-positive, so Gram morphology and catalase prevent calling it an enterococcus.

Optochin (P disk): S. pneumoniae is susceptible (zone diameter interpreted with the current disk size and package-insert cutoff, classically ≥14 mm with a 6 mm disk); viridans-group streptococci are resistant. Confirm borderline zones with bile solubility (sodium desoxycholate): pneumococcus lyses, mitis-group organisms do not.

Bacitracin (A disk): S. pyogenes is susceptible; GBS and most other beta-hemolytic streptococci are resistant. PYR has largely replaced bacitracin as a more specific rapid test, but both appear on exams and both are applied only after catalase-negative Gram-positive cocci in chains are documented.

CAMP test: a perpendicular streak to a beta-lysin-producing S. aureus strain yields an arrowhead of enhanced hemolysis with GBS. Listeria can produce a rectangular (block) CAMP reaction. S. pyogenes is CAMP-negative. Do not read a positive CAMP from a catalase-positive rod and call it GBS.

Hippurate hydrolysis: GBS hydrolyzes hippurate to glycine, detected with ninhydrin as a purple color. S. pyogenes is hippurate-negative. Campylobacter jejuni is hippurate-positive, a species-level conventional clue versus C. coli.

Commercial kits: API, Vitek cards, MicroScan panels

API strip galleries (API 20E for Enterobacterales, 20NE for non-fermenters, Staph, NH for Neisseria/Haemophilus, and anaerobic galleries) are inoculated with a standardized suspension, incubated, and converted to a numerical profile that is looked up in a database. Vitek cards and MicroScan panels automate many of the same biochemical reactions and often combine them with susceptibility wells on the same instrument run.

Three operational rules decide whether the profile is trustworthy:

1. Inoculum must match the manufacturer's McFarland standard. Too heavy a suspension yields false-positive reactions; too light yields false-negatives and "unidentified" or low-probability profiles. Picking several colony types into one suspension produces a chimeric code that matches nothing real.
2. Incubation time, temperature, and atmosphere are kit-specific. Reading an API 20E at 6 hours when the biocode is designed for 18–24 hours, or incubating a Neisseria card in the wrong CO2 condition, produces identifications that should not be reported.
3. Databases contain only the taxa the manufacturer validated. Unusual environmental Gram-negatives, newly named species, and select agents may be absent, misidentified, or identified as a near neighbor (Bacillus anthracis reported as B. cereus group; Brucella reported as Ochrobactrum or another oxidase-positive Gram-negative on some older phenotypic systems).

Do not place suspected bioterrorism (select) agents on open automated identification systems. The cards, pipettes, and instrument loaders generate aerosols, contaminate expensive equipment that is difficult to decontaminate, delay public-health referral, and may print a dangerously wrong name. Stop work, secure the isolate, and follow LRN/select-agent protocol. That limitation is an identification-method application item on outline II.J, not only a safety item from laboratory operations.

When a commercial profile disagrees with Gram stain, oxidase, catalase, or colony morphology, believe the smear and the conventional rapid tests. Repeat the kit only after you have verified purity and inoculum; then reflex to MALDI-TOF, additional biochemicals, or molecular methods as in the next section. A kit is an application of biochemical theory — it is not more authoritative than a wrong Gram stain.