Microbial Metabolism Tests and Reactions

Hydrolysis of Starch

Medium: Starch Agar | Substrate: Starch | Enzyme: Amylase (exoenzyme) | Reagent: Iodine | End Product: Maltose (glucose + glucose) | Positive: Amber zone | Negative: Black color

Hydrolysis of Gelatin

Medium: Gelatin | Substrate: Gelatin | Enzyme: Gelatinase (exoenzyme) | End Product: Amino acids | Positive: Liquid medium | Negative: Solid medium

Fermentation of Carbohydrates

Medium: Phenol broth + protein | Substrates: Glucose, Sucrose, Lactose, Mannitol | Pathway: Glycolysis | Reagent: Phenol red | End Products: Organic acids, ATP, ± CO₂ | Positive: Yellow (A), Yellow with gas (A/G) | Negative: Red or magenta

Litmus Milk - Fermentation

Substrate: Lactose | Pathway: Fermentation | End Products: Organic acids, ATP, ± CO₂ | Positive: Pink milk, ± hard curd

Litmus Milk - Reduction

Substrate: Litmus | Pathway: ETC | End Products: Leucolitmus (white), ATP | Positive: White bottom, apple juice appearance

Litmus Milk - Alkalinization

Substrate: Casein | Enzyme: Proteases | End Products: Small peptides + ammonia | Positive: Blueberry milk

Litmus Milk - Peptonization

Substrate: Casein + lactalbumin | Enzyme: Proteases | End Products: Free amino acids + ammonia | Positive: Grape/apple juice, ± soft curd

Litmus Milk - Curd Formation

Substrate: Casein via fermentation or peptonization | End Product: Curd | Positive: Hard or soft curd | Negative: No curd

Nitrate Reduction

Medium: Nitrate broth | Substrate: KNO₃ | Pathway: ETC | Reagents: DAN + sulfanilic acid | End Products: KNO₂ + ATP | Positive: Magenta | Negative: Peach or no color

Catalase Test

Medium: TSA plate | Substrate: H₂O₂ | Enzyme: Catalase | Reagent: Hydrogen peroxide | End Products: O₂ + H₂O | Positive: Bubbles | Negative: No bubbles

Decarboxylase Production

Medium: Decarboxylase broth | Substrates: Arginine, Lysine, Ornithine | Enzyme: Decarboxylase | Reagent: Brom cresol purple | End Products: Amine + CO₂ | Positive: Violet | Negative: Yellow or control

Urease Test

Medium: Urea broth | Substrate: Urea | Enzyme: Urease | Reagent: Phenol red | End Products: CO₂ + NH₃ | Positive: Hot pink | Negative: Yellow to gold

SIM - Sulfide

Substrate: Cysteine + FeSO₄ | Pathway: ETC | End Products: H₂S → FeS (black ppt), H₂SO₄ | Positive: Black precipitate

SIM - Indole

Substrate: Tryptophan | Reagent: Kovac's | End Products: Indole, pyruvic acid, NH₃ | Positive: Red ring

SIM - Motility

Test: Motility line | Positive: Diffuse growth from stab | Negative: Growth only at stab line

MR Test

Medium: MR broth | Substrate: Glucose | Pathway: Glycolysis | Reagent: Methyl red | End Products: Stable acids | Positive: Red | Negative: Yellow or no change

VP Test

Medium: VP broth | Substrate: Glucose | Pathway: Butylene glycol | Reagents: D.

Napthol + KOH + creatine

End Products: Acetoin (AMC), alcohols | Positive: Red ring | Negative: No ring

Unusual Nitrogen Source

Medium: Ammonium phosphate broth | Substrate: NH₄H₂PO₄ | Reagent: Brom cresol purple | End Products: NH₃ + H₃PO₄ | Positive: Yellow | Negative: Burgundy/purple

Citrate Utilization (Broth)

Medium: Sodium citrate broth | Substrate: Sodium citrate | Enzyme: Citratase | End Products: Acetate + oxaloacetate | Positive: Turbidity | Negative: Clear

Citrate Utilization (Agar)

Medium: Simmons citrate agar | Substrate: Citrate + ammonium | Enzyme: Citrate-permease | Indicator: Bromthymol blue | End Products: Pyruvate + NH₃ | Positive: Blue | Negative: Green

Oxygen Requirements Table

Obligate aerobe: Top growth only | Obligate anaerobe: No growth in air | Facultative: Growth throughout | Microaerophile: Bottom/intermediate growth

MSA (Mannitol Salt Agar)

Medium: MSA | Substrate: Mannitol | Pathway: Glycolysis | Reagent: Phenol red | End Products: Organic acids + ATP | Positive: Yellow | Negative: Red

Coagulase Test

Medium: Rabbit plasma + sodium citrate | Substrate: Sodium citrate | Enzyme: Coagulase | End Product: Clotted plasma | Positive: Solid (clot) | Negative: Liquid

Alpha Hemolysis

Medium: Blood agar | Substrate: RBCs | Pathway: Incomplete | Positive: Green zone around colonies

Beta Hemolysis

Medium: Blood agar | Substrate: RBCs + hemoglobin | Pathway: Complete | Positive: Clear zone

Gamma Hemolysis

Medium: Blood agar | Substrate: Plasma proteins | Pathway: None | Positive: No change (red)

Latex Agglutination

Medium: Latex beads w/ anti-coagulase antibodies | Positive: Clumps | Negative: No clumps | S. aureus: + | S. epi/Strep spp: -

Water Analysis

Test: Lactose broth and EMB | Positive: Acid + Gas (broth); Green colonies (EMB) | Negative: No gas or other colored colonies

UV Light Effects

Shorter exposure: Less kill | Longer exposure: More kill | Lid blocks UV | UV = surface disinfectant

Temperature Effects

Higher temps = more killing | B. subtilis survives heat (spores) | E. coli = more sensitive

Selective Action of GCV

Effective at low conc. | Gram+ more sensitive | S. aureus: Yes | E. coli: Minimal/no effect

Zone of Inhibition

Bacdown (triclosan) = most effective | Pinesol = least | Broad: Bacdown | Narrow: Pinesol

Antibiotic Evaluation

Penicillin: S. aureus & M. luteus sensitive, E. coli resistant | Tetracycline: Broad-spectrum | Erythromycin: Varies

Skin Cleanliness

More washes = fewer transient, more resident flora | Antiseptics remove more bacteria