Bacteriology Analytic Procedures

The Bench Triage Every MLS Candidate Must Know

Bacteriology items reward fast, correct triage from a Gram stain plus a handful of confirmatory tests. The reliable opening fork: gram-positive cocci in clusters point to Staphylococcus; in chains or pairs point to Streptococcus/Enterococcus; gram-negative rods point to Enterobacterales or non-fermenters; gram-negative diplococci point to Neisseria/Moraxella.

First-Tier Confirmatory Tests

Three tests resolve most gram-positive and many gram-negative puzzles:

• Catalase (3% H2O2 -> bubbles): positive separates Staphylococcus (positive) from Streptococcus/Enterococcus (negative).
• Coagulase (clots rabbit plasma): positive identifies Staphylococcus aureus; negative leaves the coagulase-negative staph group (S. epidermidis, S. saprophyticus).
• Oxidase (cytochrome c oxidase): positive flags Pseudomonas, Neisseria, Aeromonas, Vibrio, Campylobacter; Enterobacterales are oxidase-negative -- a single oxidase result rules a gram-negative rod in or out of the family.

Selective and Differential Media

Media choice is a recurring item type. Know what each plate selects for and how it differentiates:

A worked example: a clear, non-lactose-fermenting colony on MacConkey that is oxidase-positive, produces a grape-like odor and blue-green pigment (pyocyanin), and grows at 42 deg C is Pseudomonas aeruginosa. Contrast that with a clear MAC colony that is oxidase-negative and produces H2S on TSI/Hektoen -- that points to Salmonella.

Gram-Positive Cocci Algorithms

For catalase-negative chains, hemolysis plus a disk test closes the identification:

• Beta-hemolytic, bacitracin (A disk) susceptible, PYR positive = group A Streptococcus pyogenes.
• Beta-hemolytic, CAMP positive, hippurate hydrolysis positive = group B Streptococcus agalactiae (screened in pregnancy).
• Alpha-hemolytic, optochin susceptible, bile soluble = Streptococcus pneumoniae.
• Alpha-hemolytic, optochin resistant, bile insoluble = viridans group strep.
• Gamma/variable, bile-esculin positive, growth in 6.5% NaCl = Enterococcus.

For catalase-positive clusters, coagulase splits S. aureus from the rest, and novobiocin resistance identifies S. saprophyticus (a common cause of UTI in young women). The classic trap is choosing optochin for a staphylococcus question or bacitracin for an enterococcus question -- match the disk test to the catalase result first, then to hemolysis, then choose the best answer.

Gram-Negative Identification and Fastidious Organisms

Gram-negative rods are sorted first by oxidase, then by lactose fermentation, then by TSI (triple sugar iron) and the IMViC panel. TSI is a frequent item: an alkaline slant over an acid butt with gas and no H2S, plus indole-positive, points to E. coli; an alkaline slant over acid butt with H2S blackening points to Salmonella; Shigella gives an acid butt with no gas and no H2S. Urease distinguishes the swarming, urease-positive Proteus from the non-motile, mucoid, indole-negative Klebsiella. Citrate-positive, indole-negative organisms (Citrobacter, Enterobacter, Klebsiella) separate from the citrate-negative E. coli.

Fastidious and Curved Organisms

Some organisms will not grow on routine media and need special conditions the exam highlights:

The satellite phenomenon is a classic best-answer: small colonies of Haemophilus growing only adjacent to a Staphylococcus aureus streak because the staph supplies V factor. Recognizing it instantly identifies H. influenzae without a full biochemical panel.

Building the Answer Stepwise

A worked example: a sputum from a child grows oxidase-negative, lactose-fermenting (pink) colonies on MacConkey that are indole-positive, methyl red-positive, Voges-Proskauer-negative, and citrate-negative -- the IMViC ++-- pattern of E. coli. Change one reaction (citrate-positive, VP-positive, indole-negative) and the answer shifts to the Klebsiella-Enterobacter group. The exam rewards candidates who let every listed reaction constrain the choice rather than locking onto the first recognizable clue.

Finally, do not ignore colony morphology and odor clues that the exam deliberately plants: the grape-like odor and metallic sheen of Pseudomonas, the swarming film of Proteus, the mousy odor of E. coli, and the dry, wrinkled colonies of some mycobacteria-adjacent organisms. These sensory descriptors are legitimate bench findings and are fair game as the deciding detail in a one-best-answer item, so treat them as data, not decoration.

Two additional discriminators close many gram-negative items. Motility separates the swarming, actively motile Proteus from the consistently non-motile Klebsiella and Shigella. The spot indole and rapid urease tests give a one-step answer on suspect colonies: a swarming, urease-positive, indole-variable rod is Proteus, whereas a mucoid, urease-positive but non-swarming rod is Klebsiella. When colony appearance, biochemical reactions, and a quick spot test all agree, you have the best answer; when they conflict, repeat or suspect a mixed culture rather than forcing a single name.