microlab final exam

Key sources of error for Kirby Bauer Disk

• Incorrect lawn density (too heavy/light inoculum) → zones too small/large.

• Wrong medium or pH → alters antibiotic activity.

• Old/expired disks or improper storage → decreased potency.

• Plates dried out or incubated incorrectly → altered diffusion/growth.

• Reading after an incorrect incubation period → misleading zone sizes.

• Measuring over confluent growth (not a uniform lawn) or reading fuzzy edges incorrectly.

Kirby–Bauer Disk Diffusion

• Look for a clear zone of inhibition (area where no bacterial growth) around the antibiotic disk.

Kirby–Bauer Disk Diffusion

Measure the diameter of that zone across the center (include the disk) and compare to the standard interpretive chart (resistant/intermediate/sensitive)

E-test

• An E-test strip has a gradient of antibiotic. After incubation you get an ellipse of inhibition. The MIC is read where the ellipse edge intersects the scale on the strip (the lowest drug concentration that inhibits growth).

E-test key sources of error

• Same factors as Kirby–Bauer (inoculum, medium, disk/strip handling, incubation).

• Poor contact between strip and agar (gaps) → distorted ellipse.

• Misreading the scale if there’s trailing growth or pinpoint colonies inside the ellipse.

Catalase test

catalase enzyme breaks hydrogen peroxide into water + oxygen → visible bubbling if positive.

Catalase test errors

• Using organisms that produce weak catalase or aged cultures → weak/no bubbles.

• Contamination with blood (has catalase) or using colonies from blood agar can give misleading bubbles.

• Too much reagent or too large a colony can appear exaggerated.

Staph Latex Agglutination

latex beads coated with antibodies bind bacterial surface factors (protein A/coagulase) → visible clumping (agglutination) indicates Staphylococcus aureus.

Staph Latex Agglutination errors

• Over-reading background clumping (interpret negative control first).

• Using old/poorly stored reagents → weak reaction.

• Highly mucoid strains or heavy inoculum can give nonspecific clumping.

Oxidase test

detects cytochrome c oxidase activity via a reagent that turns purple/blue when oxidized. Positive = rapid color change.

Oxidase test errors

• Slow or late color development may be misread — read at the specified short window.

• Using culture older than recommended or colonies from certain media can give false negatives/positives.

• Metal loops or contamination with oxidizing agents can produce false color.

Indole test

tests for tryptophanase — organisms that convert tryptophan to indole will produce an indole reagent color (typically red in reagent layer).

Indole test error

• Using the wrong reagent or letting reagent mix too long can change the color.

• Very weak producers may give a faint color that’s hard to interpret.

Bile Esculin (BEA)

why it turns black: certain organisms (e.g., Enterococcus) hydrolyze esculin to esculetin; esculetin reacts with ferric ions in the medium → dark brown/black complex.

Bile Esculin (BEA) error tips

non-Enterococcus that can sometimes hydrolyze esculin or overgrowth can confuse interpretation; check growth control.

Optochin (P disk)

used to identify Streptococcus pneumoniae (opt-sensitive). A clear zone around the disk suggests sensitivity (S. pneumoniae); no zone = resistant (other alpha-hemolytic strep).

Optochin (P disk) error

inoculum density and media age affect zone size; incubation conditions matter.

Bacitracin (A disk)

differentiates Streptococcus pyogenes (bacitracin sensitive) from other beta-hemolytic streptococci. Zone of inhibition = sensitive.

Bacitracin (A disk) error

ambiguous small zones need confirmation by other tests.

Catalase

differentiates staphylococci (catalase positive) from streptococci/enterococci (catalase negative).

Staph Latex Agglutination

identifies S. aureus via clumping (protein A/coagulase).

Mannitol Salt Agar (MSA)

why it turns color: contains mannitol and a pH indicator; organisms that ferment mannitol (e.g., S. aureus) produce acid → indicator turns yellow around colonies. Non-fermenters leave it red/pink.

Mannitol Salt Agar (MSA) errors

Some organisms tolerate salt but don’t ferment mannitol; interpret growth (salt tolerance) separately from fermentation (color change).