MLS Study Guide: High-Yield Microbiology and Clinical Chemistry Topics for the ASCP Board Exam

The ASCP MLS exam rewards people who understand patterns and lab logic, not just lists. This guide focuses on high-yield Microbiology and Clinical Chemistry topics you will see across questions. You will find what to recognize, what to calculate, why it matters, and how to avoid common traps.

How the exam tests Microbiology and Clinical Chemistry

• Expect integrated vignettes: a patient scenario, a lab figure (Gram stain, growth pattern, troponin series), and a question asking for the next step, the best interpretation, or the likely organism.
• “Best answer” questions depend on understanding pre-analytical variables, test limitations, and clinical context.
• Microbiology often tests you on specimen quality, colony morphology, rapid tests, and resistance patterns.
• Chemistry questions often hinge on calculations, method interferences, critical values, and pattern recognition (e.g., cholestatic vs hepatocellular liver injury).

Microbiology: specimen basics and Gram stain logic

• Specimen quality drives accuracy. Why: contamination alters culture results and antibiotic choices.
  • Sputum: Reject if many squamous epithelial cells; accept if many PMNs. Poor quality suggests oral contamination.
  • Blood cultures: Draw from two separate sites; skin antisepsis is critical. One-of-two bottles positive with skin flora suggests contamination.
  • Urine: Midstream clean-catch, process promptly or refrigerate. Delays let bacteria multiply and skew counts.
• Gram stain = first fork in the road. Why: It narrows media, identification tests, and empiric therapy.
  • Gram-positive cocci in clusters → think Staphylococcus.
  • Gram-positive cocci in chains/pairs → think Streptococcus/Enterococcus.
  • Gram-negative diplococci → think Neisseria/Moraxella.
  • Gram-negative rods → split by lactose fermenters (pink on MAC) vs non-fermenters.
  • Pleomorphic coccobacilli in respiratory sources → consider Haemophilus.

Culture media and colony clues you must know

• Blood agar (BAP): Hemolysis patterns help.
  • Beta (clear zone): Strep pyogenes (A), Strep agalactiae (B), some Staph aureus.
  • Alpha (green): Strep pneumoniae, viridans group.
  • Gamma (none): Enterococcus, some Strep.
• MacConkey (MAC): Selects Gram-negatives; lactose fermenters are pink/purple.
  • LF: E. coli (often dry pink with bile precipitate), Klebsiella, Enterobacter.
  • NLF: Pseudomonas, Proteus, Salmonella, Shigella, Acinetobacter.
• Chocolate agar: For fastidious organisms (e.g., Haemophilus, Neisseria).
• Selective media tips:
  • HEK/SS/XLD for stool pathogens. Black centers = H2S producers (often Salmonella).
  • Campy agar at 42°C microaerophilic for Campylobacter.
  • Thayer–Martin for pathogenic Neisseria.
  • BCYE for Legionella.

Gram-positive cocci: fast algorithms

• Staphylococcus:
  • S. aureus: Catalase +, coagulase +, beta-hemolytic, often yellow pigment. MRSA = mecA (PBP2a) detection is definitive. Why: penicillin-binding protein alteration drives resistance.
  • Coagulase-negative Staph (CoNS): Often contaminants in blood cultures; true pathogen in prosthetic devices and catheters.
• Streptococcus/Enterococcus:
  • Group A Strep (S. pyogenes): Beta-hemolytic, bacitracin susceptible, PYR +. Causes pharyngitis, skin infections, and post-strep complications.
  • Group B Strep (S. agalactiae): Beta-hemolytic, CAMP +, hippurate +. Neonatal sepsis/meningitis risk in colonized mothers.
  • Strep pneumoniae: Alpha-hemolytic, bile soluble, optochin susceptible; lancet diplococci. Why: capsule drives virulence.
  • Viridans group: Alpha-hemolytic, optochin resistant; dental flora; endocarditis.
  • Enterococcus (E. faecalis, E. faecium): PYR +, grow in 6.5% NaCl, often vancomycin-resistant (vanA/vanB). Why: altered cell wall targets reduce glycopeptide binding.

Enterobacterales and key stool pathogens

• Screen by lactose and oxidase:
  • E. coli: Lactose fermenter (LF), indole +, motile; common UTI; EHEC O157:H7 is sorbitol-negative on SMAC and linked to HUS (avoid antibiotics).
  • Klebsiella: LF, urease +, non-motile; mucoid colonies from capsule.
  • Proteus: NLF, swarming, urease +, H2S +; struvite stones via urease alkalinization.
  • Salmonella: NLF, H2S +, motile; poultry/eggs; invades M cells.
  • Shigella: NLF, non-motile, H2S −; low infectious dose.
• ESBL and CRE:
  • ESBL hydrolyze 3rd-gen cephalosporins. Why it matters: ceftriaxone failure risk; choose carbapenem if severe.
  • Carbapenemases (KPC, NDM, OXA-48) cause broad resistance; confirm with molecular or phenotypic tests and use strict infection control.

Nonfermenters and fastidious Gram-negatives

• Pseudomonas aeruginosa: NLF, oxidase +, blue-green pigment, grape odor; grows at 42°C; intrinsic resistance via efflux and porins.
• Acinetobacter: NLF, oxidase −, often multi-drug resistant; coccobacillary.
• Haemophilus influenzae: Requires X (hemin) and V (NAD) factors; satellitism near S. aureus on BAP; epiglottitis, otitis, COPD exacerbations.
• Neisseria gonorrhoeae: GN diplococci; oxidase +; grows on Thayer–Martin; glucose + only. N. meningitidis uses glucose + maltose +.
• Moraxella catarrhalis: GN diplococci; DNase +; beta-lactamase commonly; otitis/sinusitis in kids, COPD flares in adults.
• Campylobacter jejuni: Curved GN rods; microaerophilic at 42°C; oxidase +; bloody diarrhea; post-infectious Guillain–Barré risk.
• Legionella pneumophila: Requires BCYE; urinary antigen detects serogroup 1; think water systems and severe pneumonia with hyponatremia.

Anaerobes and other special bacteria

• Bacteroides fragilis group: Anaerobic GN rods; beta-lactamase production; intra-abdominal abscesses.
• Clostridioides difficile: Toxin-mediated colitis. Best diagnosis is algorithmic: GDH + toxin EIA +/− confirm by NAAT. Why: NAAT alone detects colonization, not toxin activity.
• Actinomyces israelii: Anaerobic, branching GPR; “sulfur granules”; jaw/ILO infections after dental procedures.
• Listeria monocytogenes: GPR with tumbling motility, cold growth; neonatal sepsis/meningitis; risk foods (soft cheeses, deli meats).

Mycobacteria and fungi: high-yield essentials

• Mycobacterium tuberculosis: Acid-fast bacilli (AFB) on Ziehl–Neelsen/Kinyoun; slow growth on Lowenstein–Jensen; NAAT accelerates detection.
• Rapid growers (M. fortuitum, M. chelonae): grow in 7 days; device infections.
• Fungi:
  • Yeast: Candida albicans germ tube +; Candida auris is often multidrug-resistant.
  • Mold: Aspergillus septate hyphae with acute-angle branching; Mucorales broad, non-septate, right-angle branching in DKA/immunocompromised.

Antimicrobial susceptibility: what the exam expects

• Kirby–Bauer disk diffusion and MIC testing compare to breakpoints to call S/I/R. Why: Predicts clinical success at standard dosing.
• Inducible clindamycin resistance (D-test): Flattened clindamycin zone next to erythromycin disk = inducible MLSB resistance; report clinda as resistant.
• MRSA: Detect mecA or PBP2a; oxacillin/cefoxitin screen.
• VRE: vanA/vanB; be alert to high-level aminoglycoside resistance (HLAR) in Enterococcus for synergy decisions.
• ESBL/CRE: Use confirmatory tests; consider infection control flags and cascade reporting.

Blood cultures: pathogen vs contaminant

• Likely contaminants when recovered from a single set: CoNS, Cutibacterium acnes, Corynebacterium spp., Bacillus (non-anthracis).
• Likely true pathogens: S. aureus, beta-hemolytic streptococci, Enterobacterales, P. aeruginosa, Candida, anaerobes.
• Why it matters: Prevents overtreatment and guides source control workup.

Clinical Chemistry quality and pre-analytical traps

• Specimen handling:
  • Hemolysis falsely increases K, LD, AST; red color interferes with spectrophotometry.
  • Lipemia/icterus cause turbidity/absorbance errors; use ultracentrifugation or blanking.
  • Glycolysis lowers glucose by ~5–7%/hour at room temp; use fluoride/oxalate or prompt separation.
• Delta checks catch ID errors or acute changes; follow up with repeat draw if results conflict with prior data or physiology.
• Westgard rules (e.g., 1_2s warning; 1_3s, 2_2s, R_4s, 4_1s, 10_x rejection) protect from bias and imprecision. Why: QC violations signal analytical error before patient harm.

Electrolytes, osmolality, and acid–base

• Anion gap (AG) = Na − (Cl + HCO3). High AG metabolic acidosis: MUDPILES pattern (classically lactate, ketoacids, toxins). Why: unmeasured anions accumulate.
• Osmolality (calculated) ≈ 2Na + Glucose/18 + BUN/2.8. Osmolal gap = measured − calculated; elevated in toxic alcohols (methanol, ethylene glycol).
• ABG interpretation:
  • Step 1: Acidotic or alkalotic (pH).
  • Step 2: Respiratory (pCO2) vs metabolic (HCO3−) primary.
  • Step 3: Compensation appropriate? Winter’s formula for metabolic acidosis: pCO2 ≈ 1.5×HCO3 + 8 ± 2.
  • Step 4: Anion gap and delta gap to unmask mixed disorders.
• Hyponatremia pitfalls:
  • Pseudohyponatremia from indirect ISE with severe lipemia/proteinemia; direct ISE avoids dilution artifact.
  • Hyperglycemia lowers Na by water shift; correction ~1.6 mEq/L Na per 100 mg/dL glucose above normal.

Renal function: patterns that matter

• Creatinine reflects GFR; BUN is affected by protein intake and volume status.
• BUN:Cr ratio:
  • Prerenal azotemia: ratio >20:1 (urea reabsorbed with water).
  • Intrinsic renal: ratio ~10–15:1.
  • Postrenal: variable; look for obstruction signs.
• eGFR reports stage CKD; know that muscle mass and race adjustments affect equations.

Liver tests: interpret the pattern

• Hepatocellular injury: High AST/ALT relative to ALP. Ischemic or toxic injury can push ALT >1000.
• Cholestatic pattern: High ALP and GGT relative to AST/ALT; think bile duct obstruction or infiltrative disease.
• Bilirubin fractions:
  • Unconjugated (indirect) rises with hemolysis or impaired conjugation (e.g., Gilbert).
  • Conjugated (direct) rises with cholestasis or hepatocellular excretion defects.
• Albumin and PT/INR reflect synthetic function; INR changes fast with acute failure.

Cardiac markers: time matters

• High-sensitivity troponin is the primary marker. Rise and/or fall pattern confirms acute injury. Why: dynamic change distinguishes acute from chronic elevation.
• CK-MB is less specific; may help detect reinfarction if troponin remains elevated.
• BNP/NT-proBNP support heart failure diagnosis; interpret with renal function and age.

Lipids: calculations and caveats

• Friedewald LDL (when TG not very high): LDL ≈ TC − HDL − (TG/5) in mg/dL.
• Do not use Friedewald if TG ≥400 mg/dL; consider direct LDL or alternative formulas.
• Severe hypertriglyceridemia (≥1000 mg/dL) raises pancreatitis risk; treat urgently.

Glucose and diabetes testing

• Fasting plasma glucose, OGTT, and HbA1c diagnose diabetes; know that A1c may be unreliable with hemoglobin variants, anemia, or rapid RBC turnover.
• Sample handling: use glycolysis inhibitors or rapid centrifugation to avoid falsely low glucose.
• Ketones: Nitroprusside detects acetoacetate; may miss beta-hydroxybutyrate in DKA. Why: redox shift favors beta-hydroxybutyrate.

Thyroid and endocrine quick hits

• TSH-first strategy: High TSH + low free T4 = primary hypothyroidism. Low TSH + high free T4/T3 = hyperthyroidism. Why: pituitary is a sensitive sensor.
• Biotin interference can falsely skew immunoassays (often low TSH, high T4). Ask about supplements.
• Calcium/PTH:
  • Primary hyperparathyroidism: high Ca, high/inappropriately normal PTH, low/normal phosphate.
  • Hypocalcemia: check magnesium; low Mg blunts PTH release and action.
• Adrenal:
  • Cortisol peaks in the morning; timing matters.
  • Dexamethasone suppression screens Cushing physiology; non-suppression suggests autonomous cortisol.

TDM and toxicology

• Aminoglycosides: concentration-dependent killing; monitor peaks (efficacy) and troughs (toxicity).
• Vancomycin: troughs approximate AUC; kidney function guides dosing.
• Digoxin: draw at steady state and at least 6–8 hours post-dose; low K and low Mg increase toxicity.
• Phenytoin: total level misleading in hypoalbuminemia; measure free or adjust using albumin. Why: only unbound drug is active.
• Tox screens:
  • Acetaminophen, salicylate, and ethanol levels are quantitative and time-critical.
  • CO poisoning: carboxyhemoglobin measured by co-oximetry; pulse oximetry is unreliable.
  • Toxic alcohols: high osmolal gap early; anion gap acidosis later as acids accumulate.

Analytical methods and interferences

• Spectrophotometry and Beer–Lambert law (A = εbc): absorbance is proportional to concentration. Deviations arise from turbidity and stray light.
• Turbidimetry/nephelometry: measure immune complexes for proteins (CRP, immunoglobulins). Prozone (antigen excess) can cause underestimation; dilution fixes it.
• Ion-selective electrodes (ISE):
  • Direct ISE (blood gas analyzers) measure undiluted samples; not affected by lipemia/proteinemia.
  • Indirect ISE uses dilution; risk of pseudohyponatremia with high solids fraction.
• Immunoassays: susceptible to heterophile antibodies and biotin. Mismatched clinical picture should trigger confirmation by an orthogonal method.
• Chromatography/Mass spectrometry: high specificity for drugs, steroids, vitamins; reference methods for confirmation.
• Hook effect (very high analyte saturates antibodies) yields falsely low results in sandwich assays (e.g., hCG, prolactin). Dilution reveals the true high value.

Key equations and quick-reference values

• Anion gap = Na − (Cl + HCO3).
• Corrected Na for hyperglycemia ≈ Na + 1.6 × [(glucose − 100) / 100].
• Serum osm (calc) ≈ 2Na + Glucose/18 + BUN/2.8.
• Osmolal gap = measured − calculated osm.
• Winter’s formula for metabolic acidosis: pCO2 ≈ 1.5 × HCO3 + 8 ± 2.
• FENa% = 100 × (U_Na × P_Cr) / (P_Na × U_Cr). Prerenal typically <1%.
• Friedewald LDL = TC − HDL − (TG/5) if TG < 400 mg/dL.
• Corrected Ca ≈ measured Ca + 0.8 × (4 − albumin).

Common exam traps and how to avoid them

• One positive blood culture with CoNS in an asymptomatic patient → likely contaminant; do not overcall bacteremia.
• Sorbitol-negative E. coli on SMAC with bloody diarrhea → think EHEC; avoid antibiotics due to HUS risk.
• Hyponatremia with severe hypertriglyceridemia and normal serum osmolality → pseudohyponatremia from indirect ISE.
• Unexpected low TSH and high T4 in a patient on high-dose biotin → assay interference; hold biotin and repeat.
• Negative urine ketone strip in DKA → beta-hydroxybutyrate predominates; order specific assay.
• Flat clindamycin zone near erythromycin disk (D-test) → report clindamycin resistant.

Study strategy: build reliable instincts

• Start with patterns: Gram stain morphology, lactose fermentation, oxidase reaction, and hemolysis rapidly narrow IDs.
• Practice calculations daily: anion gap, osmolal gap, LDL, corrected calcium, Winter’s formula. Repetition makes them automatic.
• Use “if X then Y” flashcards:
  • If hemolyzed sample → suspect high K, LD, AST.
  • If ALP high and GGT high → cholestasis more likely than bone disease.
  • If high osmolal gap + metabolic acidosis → evaluate for toxic alcohols.
• Review QC failures with Westgard rules until you can pick the correct corrective action on sight.
• Simulate lab decisions: choose appropriate media, rapid tests, and next steps based on the initial Gram stain.

Final takeaways

• Microbiology success comes from linking specimen quality, Gram stain, media, and a few discriminatory tests.
• Chemistry success comes from fast, accurate calculations, recognizing interferences, and reading patterns that match physiology.
• Always ask “does this result make sense?” That one question catches most exam tricks and many real-world errors.

G S Sachin

I am a Registered Pharmacist under the Pharmacy Act, 1948, and the founder of PharmacyFreak.com. I hold a Bachelor of Pharmacy degree from Rungta College of Pharmaceutical Science and Research. With a strong academic foundation and practical knowledge, I am committed to providing accurate, easy-to-understand content to support pharmacy students and professionals. My aim is to make complex pharmaceutical concepts accessible and useful for real-world application.

Mail- Sachin@pharmacyfreak.com