FINALS

MLS Lab Finals Reviewer: Enteric Bacteria & Antimicrobial Susceptibility

1. ISOLATION OF ENTERIC BACTERIA (Selective/Differential Media)

Lactose Fermentation — 2 key enzymes:

• β-galactoside permease (lactose permease) — transports lactose into cell

• β-galactosidase — breaks down lactose

MacConkey Agar (MAC)

• Contains bile salts → inhibits gram-positive/non-enteric bacteria

• Dyes: neutral red + crystal violet (crystal violet inhibits GP growth)

• LF: red/pink colonies (E. coli = brick red; Enterobacter/Klebsiella = pink-red, mucoid)

• NLF: colorless, transparent colonies

Levine EMB Agar

• Differential; also inhibits gram-positive growth

• E. coli: blue-black with green metallic sheen

• Enterobacter aerogenes: convex, mucoid, dark center, pink/creamy border

• NLF: colorless

2. CARBOHYDRATE FERMENTATION

Phenol Red Broth

• Contains peptone, phenol red indicator, Durham tube, 1 CHO (glucose/lactose/sucrose)

• (+): yellow ± gas in Durham tube

• (-): stays red, no gas

pH Indicators Summary

3. TRIPLE SUGAR IRON (TSI) AGAR — HIGH YIELD

Composition: Glucose (0.1%/1 part), Lactose (1%/10 parts), Sucrose (1%/10 parts), peptone (2%), phenol red, sodium thiosulfate + ferrous sulfate (H₂S detection)

• KIA = same but no sucrose (only glucose + lactose)

• Slant = aerobic chamber; Butt = anaerobic chamber

• Inoculation: stab + streak

Color codes: Yellow = acid; Red = alkaline; Black precipitate = H₂S+; bubbles/cracks = gas+

TSI Reaction Patterns (MEMORIZE)

TSI Quality Control:

• A/A Gas+ → E. coli

• K/A ±Gas, H₂S+ → Salmonella enterica (Typhi)

• K/NC or K/K → Pseudomonas aeruginosa

• K/A H₂S+ → Proteus mirabilis

• K/A → Shigella flexneri

4. ONPG TEST

• Detects late/slow lactose fermenters

• ONPG = structurally similar to lactose, enters cell without permease

• β-galactosidase converts colorless ONPG → galactose + yellow O-nitrophenyl

5. IMViC SERIES (Indole, Methyl Red, Voges-Proskauer, Citrate)

Indole Test

• Tryptophanase breaks down tryptophan → indole + pyruvic acid + NH₃

Methyl Red (MR) Test

• Tests mixed acid fermentation pathway

• Medium: MR-VP broth (Clark & Lubb's dextrose broth)

• (+) Red, pH ≤ 4.5 (lots of acid produced)

• (–) Yellow, pH > 4.5

Voges-Proskauer (VP) Test

• Tests butylene glycol pathway (neutral pathway)

• Glucose → acetoin (acetylmethylcarbinol) → diacetyl (in alkaline pH) + guanidine compounds → red complex

• Reagents: 5% alpha-naphthol (95% ethanol), 40% KOH (alkalinity), 0.5% creatine (prevents false negatives)

• (+) Pink/red; (–) no color change

Citrate Test

• Organism uses citrate as sole carbon source → alkaline byproducts

• Medium: Simmons Citrate Agar (indicator: bromthymol blue)

• pH 6 acid = yellow; pH 6.9 uninoculated = green; pH 7.6 alkaline = blue

• (+) Growth + Prussian blue color (e.g., Enterobacter, Klebsiella)

• (–) No growth, stays green (e.g., E. coli)

Classic IMViC Patterns

6. UREASE TEST

• Urea --(urease)--> CO₂ + NH₃ → (NH₄)₂CO₃ → ↑pH

• Medium: Christensen's Urea Agar / Stuart Urea broth (original /light orange)

• Indicator: phenol red

• (+) Yellow → Pink/magenta

• Rapid producers (PPM): Proteus, Providencia, Morganella (2-4 hrs)

• Weak producers (CKEYS): Citrobacter, Klebsiella, Enterobacter, Yersinia, Serratia (within 4 hrs)

7. SIM / MOTILITY TEST

• Medium: SIM (Sulfide-Indole-Motility) — stab inoculation (¾ depth)

• (+) Motile: spreads from stab line

• (–) Nonmotile: growth only along stab line

• Nonmotile @ 37°C: Shigella, Klebsiella, some Yersinia

• Also tests H₂S (blackening) and Indole (add Kovac's → red = +)

8. DECARBOXYLASE TEST

• Tests ability to decarboxylate amino acids (removes carboxyl group → amine) → alkaline pH

• Moeller broths: 1% Lysine, 1% Ornithine, 1% Arginine + glucose

• Indicator: Bromcresol purple → (+) Purple/alkaline; (–) Yellow/acid

• Triple (+): Plesiomonas shigelloides; Triple (–): Pantoea agglomerans

9. LYSINE IRON AGAR (LIA)

• Tests lysine decarboxylation (slant/butt both) and deamination, plus H₂S

• Medium: glucose + lysine; H₂S indicator = ferric ammonium citrate; pH indicator = bromcresol purple

• Inoculation: double stab + streak

Interpretation:

LIA Quality Control:

• Alkaline slant/butt, H₂S+ → Citrobacter freundii, Salmonella typhimurium

• Alkaline slant/butt (no H₂S) → E. coli

• Red slant, acid butt → Proteus mirabilis

10. INOCULATION TECHNIQUE SUMMARY (Enterobacteriaceae)

11. SPOT OXIDASE TEST

• Reagent: 1% tetramethyl-p-phenylenediamine dihydrochloride on filter paper

• Rub colony on moistened paper

• (+) Color change to blue/purple within 10 seconds

• Note: If TSI shows non-fermentative GNR → do oxidase test; if oxidase+ → do oxidative-fermentative (O-F) test

12. ANTIBIOTICS & ANTIMICROBIAL SUSCEPTIBILITY TESTING (AST)

Key Definitions

• Antibiotics: natural/semi-synthetic/synthetic molecules to treat/prevent disease

• Antimicrobial agents: broader term — antibacterials, antifungals, antiseptics, disinfectants, sterilants, preservatives

• Antimicrobial resistance: natural consequence of drug exposure/misuse

Narrow vs Broad Spectrum

Resistance Mechanisms (6)

1. Enzymatic inactivation (β-lactamases)

2. Target modification (PBPs, DNA gyrase/topoisomerase IV mutations)

3. Reduced permeability (porin loss/mutation)

4. Efflux pumps (→ MDR)

5. Target bypass/alternative pathways (e.g., sulfonamide resistance)

6. Biofilm formation

Kirby-Bauer Disk Diffusion Method

1. Standardize inoculum to 0.5 McFarland (~1.5 × 10⁸ CFU/mL)

2. Swab onto Mueller-Hinton agar using "3+1" lawn technique (streak 3x, rotate 60°, +1 edge swab)

3. Dry 5-10 min, apply antibiotic disks (press firmly, don't move once placed)

4. Incubate 16-18 hrs at 35°C

5. Measure zone of inhibition diameter (nearest mm) → compare to CLSI/EUCAST chart

Standard organisms used: S. aureus (ATCC 25923) and E. coli (ATCC 11229)

Interpretation Categories

MIC vs MBC

• MIC (Minimum Inhibitory Concentration): lowest concentration that prevents visible growth

• MBC (Minimum Bactericidal Concentration): lowest concentration that kills ≥99.9% of original population

  • Determined by subculturing from MIC tubes (no growth) onto antibiotic-free agar

AST Methods

Factors Influencing AST Results

1. pH of medium

2. Agar thickness (~4mm standard)

3. Growth rate of microbe

4. Inoculum density (0.5 McFarland)

5. Medium suitability (Mueller-Hinton standard)

6. Antibiotic diffusibility (size/solubility)

7. Inoculum/incubation technique consistency

Major Antibiotic Classes (by mechanism)

Why Combination Antibiotic Therapy?

1. Prevent resistance — reduces chance of single mutation conferring survival

2. Synergistic effect — drugs enhance each other (e.g., Ampicillin + Gentamicin for Enterococcus)

3. Broaden spectrum — useful in empiric therapy

4. Target mixed infections — covers multiple pathogens

Combination effects:

• Synergy: combined > sum of individual effects

• Indifference: combined = sum (no benefit/harm)

• Antagonism: combined < sum (e.g., chloramphenicol + penicillin — chloramphenicol slows growth, reducing penicillin's effect on dividing cells)

Important Special Cases

• mecA gene → oxacillin resistance in staph; cefoxitin disk is a better inducer than oxacillin disk for detecting it

• VRE detected via vancomycin screen agar or vanA/vanB gene assays

• β-lactam-resistant staph = resistant to all β-lactams regardless of individual test results

• ESBL-producers = resistant to all cephalosporins, penicillins, aztreonam (common in Enterobacterales)

• Anaerobe testing reference method = agar dilution (CLSI)