Free ASCP MLS Exam Practice Questions Mock Test: Part 16 – Microbiology – Analytic Procedures for Bacteriology

1. Neisseria gonorrhoeae is the classic cause of urethral discharge with:

   • Gram-negative intracellular diplococci: Kidney-shaped pairs within neutrophils on Gram stain.

   • Clinical context: Purulent discharge, dysuria; may progress to PID or disseminated infection.

2. Why Not the Others?

   • (a) Acinetobacter baumannii: Gram-negative coccobacilli (environmental, not sexually transmitted).

   • (c) Haemophilus ducreyi: Gram-negative rods/chains (causes chancroid, a genital ulcer disease).

   • (d) Escherichia coli: Gram-negative rods (UTIs, not urethritis with diplococci).

• The lysine decarboxylase test (and similar tests for ornithine or arginine) detects bacterial enzymes that remove carboxyl groups (–COOH) from amino acids, producing amines (e.g., cadaverine from lysine) and CO₂.

  • Positive result: The medium turns alkaline (purple) due to amine production, even after initial acidic fermentation (yellow).

  • Negative result: The medium remains yellow (no decarboxylation).

Key Uses:

• Differentiates Enterobacteriaceae:

  • Lysine +: Salmonella (most), Klebsiella.

  • Lysine –: E. coli (most), Proteus.

Why Not the Other Options?

• b) Citrate test: Detects citrate utilization (e.g., Klebsiella [+], E. coli [–]).

• c) Gelatin hydrolysis: Tests for gelatinase (e.g., Serratia, Pseudomonas).

• d) Oxidase test: Identifies cytochrome c oxidase (e.g., Pseudomonas, Neisseria).

Blood agar is the medium specifically used to detect hemolytic patterns of bacteria, as it contains 5% sheep or horse blood, allowing observation of:

• Beta-hemolysis: Complete lysis of RBCs → clear zones around colonies (e.g., Streptococcus pyogenes).

• Alpha-hemolysis: Partial lysis → greenish discoloration (e.g., Streptococcus pneumoniae).

• Gamma-hemolysis: No hemolysis (e.g., Enterococcus faecalis).

Why Not Other Options?

• (a) MacConkey agar: Selects for Gram-negatives and differentiates lactose fermenters (pink colonies) vs. non-fermenters.

• (c) EMB agar: Selective for Gram-negatives; identifies E. coli (metallic green sheen).

• (d) Citrate agar: Tests citrate utilization (e.g., Klebsiella vs. E. coli).

Selective media contain specific components (e.g., antibiotics, salts, or dyes) that:

• Suppress unwanted bacteria (e.g., Gram-positives, contaminants).

• Promote the growth of target organisms (e.g., pathogens from mixed samples).

Key Examples:

1. MacConkey agar:

   • Selective for Gram-negative bacteria (bile salts inhibit Gram-positives).

   • Also differential (lactose fermenters vs. non-fermenters).

2. Mannitol Salt Agar (MSA):

   • Selective for Staphylococcus (7.5% NaCl inhibits most other bacteria).

Why Not the Others?

• a) Motility: Requires motility agar (stab inoculation), not selective media.

• c) Hemolysis: Detected with blood agar (differential, not selective).

• d) Antimicrobial resistance: Tested via Kirby-Bauer disk diffusion or MIC assays, not selective media.

• The PYR (L-pyrrolidonyl-β-naphthylamide) test is a rapid biochemical test used to identify Enterococcus species (e.g., E. faecalis, E. faecium) and Streptococcus pyogenes (Group A strep).

  • Positive result: Bright red color after adding PYR reagent (due to pyrrolidonyl arylamidase enzyme activity).

  • Negative result: No color change (most other Gram-positive cocci, like staphylococci and non-group A streptococci, are PYR-negative).

Why Not the Other Options?

• b) Coagulase test: Differentiates Staphylococcus aureus (coagulase-positive) from other staphylococci.

• c) Citrate test: Used for Gram-negative bacteria (e.g., Klebsiella, Enterobacter).

• d) DNase test: Helps identify S. aureus (DNase-positive) and some Serratia species.

The gelatin hydrolysis test detects a bacterium’s ability to produce gelatinase, an enzyme that degrades gelatin (a protein) into amino acids.

• Positive result: Liquefaction of the gelatin medium (solid → liquid) even after refrigeration.

• Negative result: Gelatin remains solid when chilled.

Other options:

• (a) DNase test → Detects breakdown of DNA (clearing around colonies on DNase agar).

• (c) Urease test → Detects urea hydrolysis (turns pink if positive, e.g., Proteus).

• (d) Catalase test → Identifies catalase enzyme (bubbles with H₂O₂, e.g., Staphylococcus vs. Streptococcus).

1. Black colonies on Bacteroides Bile Esculin (BBE) agar and catalase-positive are hallmark features of Bacteroides fragilis, the most clinically significant anaerobe in this genus.

   • BBE agar: Selects for bile-resistant anaerobes; blackening occurs due to esculin hydrolysis.

   • Catalase-positive: Differentiates Bacteroides from other anaerobes (e.g., Prevotella, Porphyromonas are catalase-negative).

2. Why Not the Others?

   • (a) Acidaminococcus: Non-pigmented, catalase-negative, and does not grow on BBE agar.

   • (c) Porphyromonas: Forms black-pigmented colonies but is catalase-negative and bile-sensitive (no growth on BBE).

   • (d) Prevotella: May produce black pigment but is catalase-negative and often bile-sensitive.

3. Clinical Relevance:

   • Bacteroides fragilis is a major pathogen in intra-abdominal abscesses (e.g., post-surgery, perforated appendicitis).

   • Resistance: Often produces β-lactamase (treat with metronidazole, carbapenems, or β-lactam/β-lactamase inhibitors).

• The phenylalanine deaminase (PDA) test detects the enzyme phenylalanine deaminase, which converts phenylalanine → phenylpyruvic acid.

• After incubation, 10% ferric chloride is added, forming a green-colored complex with phenylpyruvic acid (positive result).

Key Organisms Identified:

✔ Proteus, Morganella, and Providencia (rapid PDA-positive).
✖ Escherichia coli, Klebsiella, and Salmonella (PDA-negative).

Why Not the Other Tests?

• b) Citrate test → Checks if bacteria use citrate as a carbon source.

• c) Voges-Proskauer (VP) test → Detects acetoin production (e.g., Enterobacter).

• d) Indole test → Identifies tryptophanase activity (e.g., E. coli).

1. lostridium perfringens is an anaerobic, Gram-positive bacillus with:

   • “Box-car” morphology: Short, blunt-ended rods.

   • Beta-hemolysis: Double zone of hemolysis on blood agar (“target hemolysis”).

   • Rapid growth: Often visible within 6–12 hours.

2. Why Not the Others?

   • (a) Actinomyces israelii: Gram-positive, filamentous (not box-car), and non-hemolytic (sulfur granules in pus).

   • (c) Bacillus subtilis: Aerobic, spore-forming, and environmental (not a wound pathogen).

   • (d) Eggerthella lenta: Non-spore-forming, non-hemolytic anaerobe (gut flora, rare in wounds).

• Detects the ability of bacteria to hydrolyze esculin into esculetin and glucose in the presence of bile salts.

• Esculetin reacts with ferric citrate (in the medium) to form a black precipitate, indicating a positive result.

Key Uses:

• Differentiates Enterococcus (positive) from most Streptococcus species (negative).

• Also helps identify group D streptococci and some Listeria species.

Why Not the Other Options?

• a) PYR test → Detects pyrrolidonyl arylamidase (used for Enterococcus and Streptococcus pyogenes).

• c) Citrate utilization test → Determines if bacteria can use citrate as a carbon source (e.g., Klebsiella, Enterobacter).

• d) Indole test → Checks for tryptophanase activity (e.g., E. coli is indole-positive).

• The oxidase test detects the presence of cytochrome c oxidase, an enzyme in the electron transport chain of certain bacteria.

• Neisseria spp. (e.g., N. gonorrhoeae, N. meningitidis) and Pseudomonas spp. (e.g., P. aeruginosa) are oxidase-positive.

• Many other aerobic and facultative anaerobic bacteria (like Vibrio, Campylobacter, and Aeromonas) also test positive.

Why Not the Other Options?

• a) Enterobacteriaceae (e.g., E. coli, Salmonella, Klebsiella) are oxidase-negative.

• c) Staphylococcus species and d) Streptococcus species are also oxidase-negative (they use alternative enzymes for respiration).

The coagulase test is the key test to differentiate Staphylococcus aureus (pathogenic) from other coagulase-negative staphylococci (e.g., S. epidermidis, S. saprophyticus).

• Coagulase-positive (S. aureus):

  • Produces coagulase, an enzyme that clots plasma (via fibrinogen conversion to fibrin).

  • Associated with serious infections (abscesses, sepsis, endocarditis).

• Coagulase-negative staphylococci:

  • Do not produce coagulase (e.g., S. epidermidis—opportunistic in implants/catheters).

Why Not the Others?

• a) Catalase test:

  • All Staphylococcus species are catalase-positive (differentiates Staphylococcus from Streptococcus, which is catalase-negative).

• c) Oxidase test:

  • Staphylococci are oxidase-negative (used to differentiate from Pseudomonas, which is oxidase-positive).

• d) Indole test:

  • Used for Gram-negative bacteria (e.g., E. coli is indole-positive; Klebsiella is indole-negative).

1. Sorbitol-MacConkey agar (SMAC) is the standard medium to screen for E. coli O157:H7 because:

   • E. coli O157:H7 is sorbitol non-fermenting (forms colorless colonies).

   • Most other E. coli strains ferment sorbitol (pink colonies).

2. Why Not the Others?

   • (a) Indole: A biochemical test (post-culture), not a component of MacConkey agar.

   • (b) Citrate: Used in Simmons citrate agar (irrelevant here).

   • (d) Lactose: Standard MacConkey contains lactose, but E. coli O157:H7 ferments lactose (pink colonies), making it indistinguishable from normal flora without sorbitol differentiation.

The CAMP test (Christie–Atkins–Munch-Petersen test) is a specific biochemical assay used to identify Group B Streptococcus (S. agalactiae).

How It Works:

1. S. agalactiae produces CAMP factor, a protein that synergizes with Staphylococcus aureus beta-hemolysin.

2. When streaked perpendicular to S. aureus on blood agar, it creates an arrowhead-shaped zone of enhanced hemolysis (positive result).

Clinical Importance:

• Group B strep is a leading cause of neonatal sepsis/meningitis and postpartum infections.

• The CAMP test is rapid and reliable for screening pregnant women (vaginal/rectal swabs) to prevent transmission during delivery.

Why Not the Others?

• a) S. pyogenes (Group A strep): Identified by bacitracin sensitivity or Pyr test.

• c) S. aureus: Detected via coagulase test or Mannitol Salt Agar fermentation.

• d) N. meningitidis: Diagnosed via oxidase test, glucose/maltose fermentation, or PCR.

The described organism is a gram-negative bacillus that is:

• Indole-positive → Rules out Proteus mirabilis (indole-negative).

• Urease-positive → Consistent with Proteus, Morganella, and some Providencia species.

• >100,000 CFU/mL in urine → Suggests a true urinary tract infection (UTI) pathogen.

Key differentiating factors:

• Proteus vulgaris: Indole positive, urease positive.

• Proteus mirabilis: Indole negative, urease positive.

• Morganella morganii: Indole positive, urease variable (often positive).

• Providencia stuartii: Indole positive, urease negative.

• The catalase test detects the enzyme catalase, which breaks down hydrogen peroxide (H₂O₂) into water (H₂O) and oxygen gas (O₂).

• Positive result: Immediate bubbling or effervescence (due to O₂ release).

• Negative result: No bubbles (lack of catalase).

Key Uses:

• Differentiates:

  • Staphylococcus (catalase +) from Streptococcus/Enterococcus (catalase –).

  • Bacillus (catalase +) from Clostridium (catalase –).

Why Not the Other Options?

• b) CO₂ production is detected in carbohydrate fermentation tests (e.g., Durham tube).

• c) Indole formation is tested via Kovac’s reagent (tryptophanase activity).

• d) Nitrate reductase is detected in the nitrate reduction test.

• The X and V factor requirement test is used to identify Haemophilus species, which are fastidious bacteria that need specific growth factors:

  • X factor (hemin) – required for cytochrome synthesis.

  • V factor (NAD or NADP) – required for metabolic processes.

Key Points:

• Haemophilus influenzae requires both X and V factors.

• Some species (e.g., Haemophilus parainfluenzae) require only V factor.

• The test helps differentiate Haemophilus from other bacteria that may grow on blood agar but do not need these factors.

Why Not the Others?

• Neisseria – Identified by oxidase test and sugar fermentation.

• Corynebacterium – Identified by tellurite agar and biochemical tests.

• Streptococcus – Identified by hemolysis patterns and Lancefield grouping.

• The CAMP test identifies Group B Streptococcus (Streptococcus agalactiae) by detecting the CAMP factor, a protein that synergistically enhances the beta-hemolysis caused by Staphylococcus aureus‘s sphingomyelinase (beta-hemolysin).

• When S. agalactiae is streaked perpendicular to S. aureus on sheep blood agar, the CAMP factor intensifies hemolysis, forming a distinct arrowhead-shaped zone at the intersection.

Key Points:

✔ CAMP factor does not cause hemolysis alone—it amplifies S. aureus‘s beta-hemolysin activity.
✔ S. aureus must be streaked first, and the test organism (S. agalactiae) streaked perpendicularly toward it.

Why Not the Other Options?

• a) Streptococcus pyogenes → Produces its own hemolysins (streptolysins) but does not interact with the CAMP factor.

• c) Enterococcus faecalis → Non-hemolytic or gamma-hemolytic; irrelevant to the CAMP test.

• d) Bacillus cereus → Produces hemolysins but does not synergize with the CAMP factor.

The citrate utilization test determines if a bacterium can use citrate as its sole carbon source and ammonium salts as its nitrogen source, producing alkaline byproducts.

• Positive result: Blue color change (due to pH rise from citrate metabolism, e.g., Klebsiella, Enterobacter).

• Negative result: Green/no change (e.g., E. coli).

Why Not Other Options?

• (a) Methyl red test: Detects mixed-acid fermentation (e.g., E. coli vs. Enterobacter).

• (c) Voges-Proskauer test: Identifies acetoin production (e.g., Enterobacter).

• (d) Indole test: Detects tryptophan hydrolysis (e.g., E. coli vs. Klebsiella).

1. Definition: Clue cells are vaginal epithelial cells that appear stippled or granular under microscopy due to being coated with bacteria (primarily Gardnerella vaginalis).

2. Diagnostic Significance:

   • They are the hallmark of bacterial vaginosis (BV), the most common vaginal infection in women of reproductive age.

   • BV is a polymicrobial infection involving G. vaginalis, Mobiluncus spp., and anaerobes (Prevotella, Bacteroides).

Why Not the Others?

• a) Candida albicans: Causes vulvovaginal candidiasis (yeast infection), diagnosed by budding yeast/hyphae on KOH prep (no clue cells).

• c) Trichomonas vaginalis: Causes trichomoniasis, diagnosed by motile flagellated protozoa on wet mount (no clue cells).

• d) Neisseria gonorrhoeae: Diagnosed via NAAT or culture (intracellular gram-negative diplococci); unrelated to clue cells.

Nutritionally variant streptococci (NVS) are fastidious bacteria that require pyridoxal (vitamin B6) or cysteine for growth. They were reclassified into two genera:

1. Abiotrophia

2. Granulicatella

These organisms:

• Cause culture-negative endocarditis (may not grow on standard blood agar).

• Exhibit satellitism (growth only near Staphylococcus colonies providing nutrients).

Why Not Other Options?

• (a) Enterococci: Grow on standard media (e.g., Enterococcus faecalis).

• (b) Group D enterococci: Part of Enterococcus genus (not nutritionally fastidious).

• (c) Beta-hemolytic streptococci: Grow readily on blood agar (e.g., S. pyogenes).

• Streptococcus pyogenes (Group A β-hemolytic Streptococcus) is susceptible to bacitracin, meaning it shows a zone of inhibition around a bacitracin disk.

• Other β-hemolytic streptococci (e.g., Streptococcus agalactiae – Group B) are typically resistant to bacitracin.

Why Not the Other Options?

• b) Coagulase test: Used to differentiate Staphylococcus aureus (coagulase-positive) from other staphylococci.

• c) Oxidase test: Helps identify Pseudomonas or Neisseria, but streptococci are oxidase-negative.

• d) Indole test: Used for gram-negative bacteria (e.g., E. coli).

1. Mucoid colonies:

   • Klebsiella pneumoniae produces thick polysaccharide capsules, creating shiny, mucoid colonies on agar.

   • This capsule contributes to its virulence and is associated with pneumonia and nosocomial infections.

2. Lactose fermentation:

   • K. pneumoniae is a lactose fermenter, forming pink colonies on MacConkey agar (due to acid production).

   • This differentiates it from non-lactose fermenters like Pseudomonas (a) and Salmonella (d).

Why Not the Others?

• a) Pseudomonas aeruginosa:

  • Non-lactose fermenter (colorless on MacConkey agar).

  • Produces green pigments (pyocyanin) and has a grape-like odor.

• c) Proteus mirabilis:

  • Non-lactose fermenter and forms swarming motility on agar (not mucoid).

• d) Salmonella enterica:

  • Non-lactose fermenter (colorless on MacConkey agar) and is typically associated with gastroenteritis, not sputum.

• The CAMP test detects the CAMP factor, produced by Group B Streptococcus (Streptococcus agalactiae).

• This factor enhances hemolysis caused by the β-hemolysin (a hemolysin) produced by Staphylococcus aureus.

• When both organisms are streaked perpendicular to each other on blood agar, a characteristic arrowhead-shaped zone of enhanced hemolysis appears at the junction — a positive CAMP test.

Other options:

• a) Streptolysin O – Hemolysin produced by Streptococcus pyogenes (Group A), but not involved in CAMP test.

• b) Beta-lactamase – Enzyme that breaks down β-lactam antibiotics, unrelated to hemolysis.

• d) Urease – Breaks down urea; not involved in hemolysis.

Viral meningitis typically shows the following CSF findings:

• Cell count: Lymphocytic pleocytosis (WBCs 50–500/μL, predominantly lymphocytes).

• Glucose: Normal (≈2/3 of serum glucose).

• Protein: Mildly elevated (50–100 mg/dL).

• Lactate: Normal (unlike bacterial meningitis, where it’s elevated).

Why Not Other Options?

• (a) Decreased protein: Rare in meningitis (protein is usually normal/mildly elevated in viral cases).

• (b) Increased glucose: Nonspecific; low glucose suggests bacterial/TB/fungal meningitis.

• (c) Increased lactate: Suggests bacterial meningitis (due to anaerobic glycolysis).

1. Legionella pneumophila is a fastidious Gram-negative bacillus that requires:

   • Buffered charcoal yeast extract (BCYE) agar, enriched with L-cysteine and iron (essential for growth).

   • pH 6.9 (optimized with ACES buffer).

   • Incubation: 3–5 days in 5% CO₂ at 35°C.

2. Why Not the Others?

   • (a) Thiosulfate citrate bile salts (TCBS) agar: Selective for Vibrio spp. (e.g., V. cholerae).

   • (c) MacConkey agar: Does not support Legionella (no cysteine/iron).

   • (d) Blood agar: Lacks cysteine/iron; Legionella may grow poorly or not at all.

The bile solubility test is the key test to differentiate:

• Streptococcus pneumoniae: Bile-soluble (colonies lyse within minutes when bile is added).

• Viridans streptococci: Bile-insoluble (no lysis).

Why Not Other Options?

• (b) Coagulase test: Used for Staphylococcus aureus (irrelevant for streptococci).

• (c) Catalase test: All streptococci are catalase-negative (rules out Staphylococcus only).

• (d) Oxidase test: Streptococci are oxidase-negative (irrelevant).

Supportive Tests for S. pneumoniae:

• Optochin sensitivity (zone of inhibition around P disk).

• Alpha-hemolysis on blood agar (though some viridans streptococci also show this).

• The satellite phenomenon occurs when Haemophilus influenzae (a fastidious bacterium requiring factor V [NAD] and factor X [hemin] for growth) forms small colonies around streaks of Staphylococcus aureus.

  • S. aureus secretes NAD (factor V) during growth, which diffuses into the surrounding agar, allowing H. influenzae to grow as “satellite colonies” near the streak.

• This phenomenon is a classic diagnostic clue for identifying H. influenzae in cultures.

Why Not the Other Options?

• b) Neisseria meningitidis: Requires cystine and iron, not NAD/hemin; no satellite growth.

• c) Proteus mirabilis: Swarms on agar but does not depend on S. aureus for growth factors.

• d) Escherichia coli: Non-fastidious; grows independently without S. aureus.

The PYR (L-pyrrolidonyl-β-naphthylamide) test is used to differentiate Enterococcus species (PYR-positive) from non-enterococcal Group D streptococci (PYR-negative).

• Bile esculin hydrolysis (b) helps identify Group D streptococci (including both Enterococcus and non-enterococcal species).

• Growth in 6.5% NaCl (c) is a characteristic of Enterococcus but not all Group D streptococci.

• Oxidase test (d) is irrelevant for differentiating these Gram-positive cocci.

The oxidase test detects the presence of cytochrome c oxidase, a key enzyme in the electron transport chain (ETC) of aerobic respiration.

• Positive result (e.g., Pseudomonas, Neisseria):

  • Turns dark blue/purple (using reagents like tetramethyl-p-phenylenediamine).

  • Indicates the bacterium uses cytochrome oxidase for oxygen metabolism.

• Negative result (e.g., Enterobacteriaceae like E. coli):

  • No color change.

Why Not the Others?

• b) Catalase: Detected via the catalase test (bubbles with H₂O₂).

• c) Urease: Detected in the urease test (e.g., Helicobacter pylori turns urea broth pink).

• d) Nitrate reductase: Detected via the nitrate reduction test (red color with zinc dust).

• The CAMP test (Christie-Atkins-Munch-Petersen test) is used to identify Group B Streptococcus (Streptococcus agalactiae).

• Principle:

  • S. agalactiae produces a CAMP factor (protein) that synergizes with Staphylococcus aureus‘s beta-hemolysin to enhance hemolysis on sheep blood agar.

  • This creates a characteristic arrowhead-shaped zone of hemolysis at the intersection of the two organisms.

Why Not the Others?

• a) Staphylococcus aureus – Used in the CAMP test as the helper strain, but not identified by it.

• c) Pseudomonas aeruginosa – Identified by oxidase, pigment production, and growth at 42°C, not CAMP.

• d) Clostridium perfringens – Identified by double zone hemolysis and Nagler reaction, not CAMP.

The Triple Sugar Iron (TSI) agar test is a key biochemical test used to differentiate members of the Enterobacteriaceae family (e.g., E. coli, Salmonella, Shigella, Proteus). It assesses:

1. Sugar fermentation (glucose, lactose, sucrose) → Acid production (yellow).

2. Gas production → Cracks or bubbles in agar.

3. H₂S production → Black precipitate (e.g., Salmonella, Proteus).

Why Not the Others?

• a) Oxidase test: Enterobacteriaceae are oxidase-negative, so this test doesn’t differentiate within the family.

• c) Catalase test: Most Enterobacteriaceae are catalase-positive (useful to distinguish from Streptococcus, but not within the group).

• d) Spore staining: Enterobacteriaceae do not form spores (only relevant for Bacillus or Clostridium).

• The rapid antigen detection test (RADT) is specifically designed for quick identification of Group A Streptococcus (GAS; Streptococcus pyogenes) directly from throat swabs.

• It detects Group A-specific carbohydrate antigens in minutes, allowing for prompt diagnosis and treatment of strep throat.

Key Features:

✔ Fast results (5–15 minutes).
✔ High specificity (low false positives).
✔ Used in clinics for immediate decision-making (e.g., prescribing antibiotics).

Why Not the Other Tests?

• a) Latex agglutination → Used for confirming Group A Strep in culture, not rapid throat swabs.

• b) PYR test → Helps differentiate S. pyogenes (PYR-positive) but requires culture isolation first.

• d) Coagulase test → Used for Staphylococcus aureus, not streptococci.

1. Purpose: The optochin (ethylhydrocupreine) disk test differentiates:

   • S. pneumoniae (sensitive to optochin, zone of inhibition ≥14 mm)

   • Viridans streptococci (resistant, no zone or <14 mm)

2. Mechanism:

   • Optochin disrupts the cell membrane of S. pneumoniae but not other alpha-hemolytic streptococci.

   • Test is performed on blood agar with a 6-mm P disk.

3. Clinical Utility:

   • Rapid identification of S. pneumoniae (a leading cause of pneumonia/meningitis) vs. commensal viridans strep.

   • Confirms alpha-hemolytic colonies that are bile-soluble (another S. pneumoniae marker).

Why Not the Others?

• a) Staphylococcus vs. Streptococcus: Differentiated by catalase test (Staph = catalase+, Strep = catalase-).

• c) E. coli vs. Klebsiella: Distinguished by indole test (E. coli = indole+, Klebsiella = indole-).

• d) Pseudomonas vs. Acinetobacter: Differentiated by oxidase test (Pseudomonas = oxidase+, Acinetobacter = oxidase-).

1. Gram-positive bacillus:

   • Listeria monocytogenes appears as short, non-branching rods on Gram stain.

2. Tumbling motility at 25°C:

   • Listeria exhibits characteristic tumbling motility when cultured at room temperature (20–25°C) due to its flagella.

   • Motility is lost at 37°C (unlike most bacteria, which are more motile at body temperature).

3. Other Features:

   • Catalase-positive (rules out Streptococcus and Enterococcus).

   • Beta-hemolytic on blood agar.

   • Cold enrichment improves detection from food/clinical samples.

Why Not the Others?

• a) Bacillus anthracis: Non-motile, forms spores, and is catalase-positive (but no tumbling motility).

• b) Corynebacterium diphtheriae: Non-motile, forms metachromatic granules, and has a “picket fence” arrangement.

• d) Clostridium botulinum: Anaerobic, forms spores, and produces neurotoxins (no motility at 25°C).

• The urease test detects the enzyme urease, which hydrolyzes urea into ammonia and CO₂, raising the pH and turning the medium pink/red (due to the pH indicator phenol red).

• Proteus species (e.g., P. mirabilis) are rapid urease-positive, while Escherichia coli is urease-negative.

Why Not the Other Options?

• a) Klebsiella pneumoniae → Some strains are urease-positive, but this is not the primary differentiating test from E. coli.

• c) Pseudomonas aeruginosa → Urease-negative (like E. coli), so not useful for differentiation.

• d) Neisseria meningitidis → Does not produce urease and is not relevant in this context.

1. Purpose: The string test is a rapid, simple test used to identify Vibrio cholerae and some other Vibrio species.

2. Procedure:

   • A loop is mixed with bacterial colonies in saline.

   • If positive, the mixture forms a viscous “string” (>2 mm) when the loop is pulled away, due to the organism’s capsular polysaccharide or extracellular mucin.

3. Specificity: While other organisms (e.g., Aeromonas) may occasionally give a weak string reaction, it is most strongly associated with V. cholerae.

Why Not the Others?

• b) Campylobacter jejuni – No string test; identified by oxidase+, motility, and microaerophilic growth.

• c) Helicobacter pylori – Diagnosed via urease test, not string test.

• d) Aeromonas hydrophila – Rarely weak string-positive, but not diagnostic (more reliably identified by oxidase+ and resistance to O/129).

• The satellite phenomenon (or satellite test) is used to identify Haemophilus influenzae, which requires V factor (NAD or NADP) for growth.

• When cultured near Staphylococcus aureus (which secretes V factor), H. influenzae forms small colonies “satelliting” around the S. aureus streaks.

• This occurs because H. influenzae cannot grow without V factor unless it is provided by nearby bacteria.

Why Not the Other Options?

• b) Neisseria gonorrhoeae → Identified by oxidase test and selective media (e.g., Thayer-Martin agar).

• c) Escherichia coli → Grows independently and does not require V factor.

• d) Streptococcus pyogenes → Identified by beta-hemolysis and bacitracin sensitivity.

Clostridium septicum is an anaerobic, Gram-positive bacillus that forms subterminal spores and is associated with:

• Peritoneal abscesses (often secondary to bowel pathology, e.g., colon cancer).

• Gas gangrene (myonecrosis) in traumatic or spontaneous infections.

Why Not Other Options?

• (a) Bacillus cereus: Aerobic, spore-forming, foodborne pathogen (not anaerobic).

• (c) Eggerthella lenta: Anaerobic Gram-positive non-spore-forming rod (part of gut flora).

• (d) Bifidobacterium dentium: Anaerobic Gram-positive branching rod (commensal, no spores).

• The Indole test determines if a bacterium can break down the amino acid tryptophan into indole using the enzyme tryptophanase.

• Kovac’s reagent (containing *p*-dimethylaminobenzaldehyde) is added to the culture.

  • Positive result: A red/pink ring forms at the top (indole reacts with Kovac’s reagent).

  • Negative result: No color change (yellow reagent remains).

Key Uses:

• Helps differentiate:

  • Escherichia coli (indole +) from Klebsiella pneumoniae (indole –).

  • Proteus vulgaris (indole +) from Proteus mirabilis (indole –).

Why Not the Other Options?

• a) Methyl Red (MR) test: Detects mixed-acid fermentation (e.g., E. coli vs. Enterobacter).

• c) Urease test: Identifies bacteria that hydrolyze urea (e.g., Proteus, Helicobacter pylori).

• d) Citrate test: Tests for citrate utilization (e.g., Klebsiella + vs. E. coli –).

1. Streptococcus pyogenes (Group A Streptococcus, GAS) is the most common bacterial cause of acute exudative pharyngitis (strep throat).

   • Clinical features: Sudden-onset sore throat, fever, tonsillar exudate, and tender cervical lymphadenopathy.

   • Diagnosis: Rapid antigen detection test (RADT) or throat culture.

2. Why Not the Others?

   • (a) Staphylococcus aureus: Causes skin infections (e.g., abscesses) or toxic shock syndrome, not classic pharyngitis.

   • (b) Streptococcus pneumoniae: A pathogen of pneumonia/meningitis/otitis media, not pharyngitis.

   • (c) Streptococcus agalactiae (Group B Streptococcus): Neonatal sepsis/meningitis; no role in pharyngitis.

The beta-lactamase test detects the enzyme penicillinase (a type of beta-lactamase), which breaks down the beta-lactam ring in penicillin antibiotics, conferring resistance.

• Positive result: Rapid degradation of penicillin (e.g., color change in nitrocefin test).

• Clinically relevant for Staphylococcus aureus, Haemophilus influenzae, and Neisseria gonorrhoeae.

Why Not Other Options?

• (a) Urease: Hydrolyzes urea (e.g., Proteus, H. pylori).

• (c) Oxidase: Detects cytochrome c oxidase (Pseudomonas, Neisseria).

• (d) Coagulase: Clots plasma (S. aureus vs. S. epidermidis).

• Simmons citrate agar contains bromothymol blue, a pH indicator that is green at neutral pH and turns blue in alkaline conditions.

• Bacteria that utilize citrate (e.g., Klebsiella, Enterobacter) metabolize it into carbonates and bicarbonates, raising the pH.

• A positive result shows blue coloration (alkaline pH), while a negative result remains green.

Why Not the Other Options?

• a) Red to yellow → Seen in urea hydrolysis (e.g., Proteus).

• c) Yellow to pink → Occurs in MacConkey agar (lactose fermentation).

• d) Colorless to black → Indicates H₂S production (e.g., Salmonella in TSI agar).

In Triple Sugar Iron (TSI) agar:

• Yellow butt (acidic) + Red slant (alkaline) = Glucose fermentation only.

  • The small amount of glucose (0.1%) is fermented first, acidifying the entire medium (yellow butt).

  • Once glucose is depleted, oxidative metabolism resumes in the slant (aerobic region), reverting to red due to protein catabolism (alkaline byproducts).

  • Lactose/sucrose (1% each) are not fermented (no acid in slant).

Other TSI Interpretations:

• (b) Lactose/sucrose fermentation: Yellow butt + yellow slant (both sugars fermented).

• (c) No fermentation: Red butt + red slant (inert organism, e.g., Pseudomonas).

• Gas/H₂S production: Cracks (gas) or black precipitate (H₂S) may accompany results.

The phenol red lactose broth test detects:

1. Lactose fermentation: Acid production turns the medium yellow (phenol red indicator).

2. Gas production: Visible as bubbles in the Durham tube.

• Example: E. coli (ferments lactose with gas) vs. Shigella (no fermentation).

Why Not Other Options?

• (a) SIM test: Detects sulfide production (black precipitate), indole (red ring with Kovac’s), and motility (cloudy spread).

• (c) Citrate test: Identifies citrate utilization (blue color change, e.g., Klebsiella).

• (d) Urease test: Detects urea hydrolysis (pink color, e.g., Proteus).

• The indole test detects the ability of bacteria (e.g., E. coli) to produce indole from the amino acid tryptophan using the enzyme tryptophanase.

• Kovac’s reagent (containing p-dimethylaminobenzaldehyde) is added to the culture.

• A positive result forms a red/pink ring at the top of the medium due to the reaction between indole and Kovac’s reagent.

Why Not the Other Options?

• b) Blue-green color → Seen in oxidase test (e.g., Pseudomonas).

• c) Black precipitate → Occurs in H₂S production tests (e.g., SIM agar).

• d) Gas bubble release → Indicates fermentation (e.g., in Durham tube tests).

Prosthetic valve endocarditis (PVE) occurring within 2–3 months post-surgery is most commonly caused by coagulase-negative staphylococci (CoNS), particularly Staphylococcus epidermidis. These organisms:

• Form biofilms on prosthetic material.

• Enter via contamination during surgery or postoperative bacteremia.

Why Not Other Options?

• (a) Streptococcus pneumoniae: Rarely causes PVE; more associated with native valve endocarditis.

• (b) Streptococcus pyogenes: Causes acute rheumatic fever or pharyngitis, not early PVE.

• (c) Staphylococcus aureus: More common in late PVE (>12 months) or IV drug use-related endocarditis.

The hippurate hydrolysis test identifies Group B Streptococcus (GBS) by detecting the enzyme hippuricase, which breaks down sodium hippurate into glycine and benzoic acid.

• Positive result: Formation of glycine is detected with ninhydrin reagent, turning deep purple (indicating S. agalactiae).

• Negative result: No color change (other streptococci).

Why Not Other Options?

• (a) S. pyogenes (Group A): Identified by bacitracin sensitivity or PYR test.

• (c) S. pneumoniae: Identified by optochin sensitivity and bile solubility.

• (d) E. faecalis: Identified by PYR test and growth in 6.5% NaCl.

The nitrate reduction test is used to determine if bacteria can reduce nitrate (NO₃⁻) to nitrite (NO₂⁻) or further to nitrogen gas (N₂) or other nitrogenous compounds. This test helps differentiate bacteria based on their nitrate reductase enzyme activity.

The other options are unrelated:

• a) Acid production from lactose is detected by tests like the MacConkey agar or phenol red broth.

• c) Hydrogen sulfide (H₂S) formation is detected using SIM agar or TSI agar.

• d) Beta-lactamase activity is tested using assays like the nitrocefin test or penicillin disk methods.

• Staphylococcus saprophyticus is resistant to novobiocin, while Staphylococcus epidermidis is susceptible.

• The test involves placing a novobiocin disk on a culture plate:

  • Zone of inhibition ≤16 mm → S. saprophyticus (resistant).

  • Zone >16 mm → S. epidermidis (susceptible).

Why Not the Other Tests?

• a) Coagulase test: Both S. saprophyticus and S. epidermidis are coagulase-negative (this test only identifies S. aureus).

• c) Catalase test: Both are catalase-positive (does not differentiate them).

• d) DNase test: Not routinely used for this purpose (though S. saprophyticus is usually DNase-negative, while S. epidermidis may be variable).

The coagulase test (a) is the most commonly used test to differentiate Staphylococcus aureus (coagulase-positive) from other Staphylococcus species and Micrococcus (both coagulase-negative).

Why not the others?

• Oxidase test (b): Micrococcus is oxidase-positive, but Staphylococcus is usually negative (though some strains may vary).

• Bacitracin test (c): Micrococcus is often susceptible, while Staphylococcus is resistant, but this is less reliable.

• Catalase test (d): Both are catalase-positive, so it doesn’t differentiate.

• The ONPG (o-nitrophenyl-β-D-galactopyranoside) test detects the enzyme β-galactosidase, which hydrolyzes ONPG into o-nitrophenol (yellow) and galactose.

• A yellow color indicates a positive result, confirming β-galactosidase activity.

Key Uses:

✔ Differentiates late lactose fermenters (e.g., Citrobacter, Shigella sonnei) from non-lactose fermenters (e.g., Salmonella).
✔ Helps identify E. coli (β-galactosidase-positive) vs. Shigella (usually negative).

Why Not the Other Tests?

• b) Indole test → Detects tryptophanase (e.g., E. coli).

• c) Urease test → Identifies urease enzyme (e.g., Proteus).

• d) Catalase test → Checks for catalase (e.g., Staphylococcus vs. Streptococcus).

• The β-lactamase test detects the enzyme beta-lactamase, which hydrolyzes the β-lactam ring in penicillin and related antibiotics (e.g., ampicillin, amoxicillin), conferring resistance.

• A positive result (e.g., color change in nitrocefin-based tests) indicates the bacterium can inactivate penicillin.

Key Uses:

✔ Rapidly identifies penicillin-resistant strains (e.g., Staphylococcus aureus, Haemophilus influenzae, Neisseria gonorrhoeae).
✔ Guides antibiotic therapy (e.g., avoiding penicillin for β-lactamase-producing bacteria).

Why Not the Other Options?

• a) Cytochrome oxidase → Detected by the oxidase test (e.g., Pseudomonas).

• c) Nitrate reductase → Involved in nitrate reduction tests (e.g., E. coli).

• d) DNA polymerase → Irrelevant to antibiotic resistance testing.

1. Purpose: The urease breath test (UBT) is a non-invasive diagnostic tool specifically designed to detect active Helicobacter pylori infections, a bacterium linked to gastritis, peptic ulcers, and gastric cancer .

2. Mechanism:

   • H. pylori produces urease, an enzyme that breaks down urea into ammonia and carbon dioxide (CO₂).

   • During the test, the patient ingests urea labeled with a carbon isotope (¹³C or ¹⁴C). If H. pylori is present, urease converts the labeled urea into labeled CO₂, which is exhaled and measured .

3. Clinical Utility:

   • Used for initial diagnosis and post-treatment verification of eradication .

   • Preferred over serology (which cannot distinguish active vs. past infections) and stool antigen tests (less convenient for some patients) .

Why Not the Others?

• a) Salmonella typhi: Diagnosed via blood/stool culture or Widal test, not urease breath testing.

• c) Shigella dysenteriae: Detected through stool culture or PCR; no urease-based breath test exists.

• d) Clostridioides difficile: Diagnosed via stool toxin assays or PCR; unrelated to urease activity.

1. Gram-negative diplococci – This morphology is classic for Neisseria meningitidis (and N. gonorrhoeae, but the latter rarely causes meningitis).

   • Rules out:

     • Streptococcus pneumoniae (a): Gram-positive lancet-shaped diplococci.

     • Haemophilus influenzae (b): Gram-negative coccobacilli (not diplococci).

     • Listeria monocytogenes (d): Gram-positive rods.

2. Clinical Context (CSF):

   • N. meningitidis is a leading cause of bacterial meningitis, especially in young adults and outbreaks.

   • H. influenzae (b) is now rare due to vaccination (Hib vaccine).

Confirmatory Tests:

• Oxidase-positive + glucose/maltose fermentation (maltose+ distinguishes N. meningitidis from N. gonorrhoeae).

• Growth on Thayer-Martin agar in 5% CO₂.

1. Purpose:

   • The D-test detects inducible macrolide-lincosamide-streptogramin B (iMLSB) resistance in Staphylococcus aureus and other Gram-positive bacteria.

   • Specifically, it identifies strains that appear susceptible to clindamycin but harbor erm genes that can induce resistance during treatment.

2. Mechanism:

   • Erythromycin (a macrolide) induces resistance to clindamycin (a lincosamide) in iMLSB-resistant strains.

   • The test uses:

     • An erythromycin disk (15 µg) placed 15–20 mm from a clindamycin disk (2 µg).

     • Positive result: Flattening of the clindamycin zone near the erythromycin disk (D-shaped zone), indicating inducible resistance.

3. Clinical Impact:

   • If the D-test is positive, clindamycin should not be used (risk of treatment failure).

   • If negative, clindamycin remains a treatment option.

Why Not the Others?

• a) Vancomycin: Resistance is detected via vanA/vanB genes or MIC testing (e.g., VRE).

• c) Penicillin: Resistance in S. aureus is detected via beta-lactamase or mecA gene (for MRSA).

• d) Ciprofloxacin: Resistance is chromosomal or plasmid-mediated (no D-test equivalent).

1. Gram-negative bacillus – All options (a-d) fit, so further differentiation is needed.

2. Indole-positive – Strongly suggests E. coli (b), as most strains are indole-positive.

   • Klebsiella pneumoniae (a) is indole-negative.

   • Proteus mirabilis (c) is indole-negative (P. vulgaris is indole-positive, but less common in UTIs).

   • Serratia marcescens (d) is typically indole-negative.

3. Motile – E. coli is motile, further supporting the answer.

Why Not the Others?

• a) Klebsiella pneumoniae – Indole-negative, non-motile.

• c) Proteus mirabilis – Indole-negative (though motile and common in UTIs).

• d) Serratia marcescens – Usually indole-negative and less common in uncomplicated UTIs.

Gram staining classifies bacteria based on fundamental differences in their cell wall structure, which directly affects their staining properties:

1. Gram-positive bacteria:

   • Thick peptidoglycan layer (60–90% of cell wall).

   • Retains crystal violet dye (purple color) after alcohol decolorization.

   • Examples: Staphylococcus, Streptococcus.

2. Gram-negative bacteria:

   • Thin peptidoglycan layer (10–20%) + outer lipid membrane.

   • Loses crystal violet during decolorization and takes up safranin counterstain (pink/red).

   • Examples: E. coli, Pseudomonas.

Why Not the Others?

• a) Enzyme activity: Requires biochemical tests (e.g., catalase/oxidase).

• c) Oxygen requirement: Determined by culture conditions (aerobic/anaerobic), not staining.

• d) Growth rate: Observed on culture media, unrelated to Gram classification.

The main purpose of a Gram stain is to differentiate bacteria based on their cell wall composition:

• Gram-positive bacteria have a thick peptidoglycan layer (retains crystal violet, appearing purple).

• Gram-negative bacteria have a thin peptidoglycan layer and an outer lipid membrane (loses crystal violet, counterstains pink/red).

This classification is critical because:

• Guides the choice of culture media (e.g., MacConkey for Gram-negatives).

• Informs initial antibiotic selection (e.g., penicillin targets Gram-positive peptidoglycan).

• Directs further biochemical testing.

Why Not the Others?

• a) Spore formation → Detected with specialized stains (e.g., malachite green for endospores).

• c) Motility → Assessed via wet mount microscopy or motility agar.

• d) Oxygen requirements → Determined by culturing in aerobic/anaerobic conditions, not Gram staining.

The phenol red lactose broth test is a carbohydrate fermentation test used to determine if bacteria can ferment lactose, producing:

1. Acid (lowers pH → yellow color change).

2. Gas (trapped in a Durham tube).

How It Works:

• Phenol red (pH indicator):

  • Yellow (pH < 6.8): Lactose fermented → acid production.

  • Red/orange (pH ~7.4): No fermentation.

  • Pink/magenta (pH > 8.4): Degradation of peptones → alkaline byproducts.

• Durham tube: Captures CO₂/H₂ gas (visible as bubbles).

Clinical Utility:

• Differentiates Enterobacteriaceae:

  • Lactose fermenters (e.g., E. coli, Klebsiella → yellow + gas).

  • Non-fermenters (e.g., Salmonella, Shigella → red, no gas).

• Screens for fecal coliforms in water testing.

Why Not the Others?

• a) Spore formation: Detected with malachite green stain (e.g., for Bacillus).

• c) Urease activity: Tested with urea broth (pink = positive).

• d) Gelatin liquefaction: Requires gelatin agar stab (liquefaction at 4°C).

The carbohydrate utilization test (e.g., Cystine Trypticase Agar (CTA) sugars) is a standard rapid method to confirm Neisseria gonorrhoeae by assessing its ability to ferment specific carbohydrates:

• N. gonorrhoeae: Ferments glucose (acid production) but not maltose, lactose, or sucrose.

• N. meningitidis: Ferments glucose and maltose.

Why Not Other Tests?

• (a) Oxidase test: Neisseria species are oxidase-positive, but this only confirms the genus, not the species.

• (c) Latex agglutination: Used for Cryptococcus or Strep pneumoniae (not standard for N. gonorrhoeae).

• (d) Urease test: Used for Proteus or H. pylori (irrelevant here).

The Voges-Proskauer (VP) test detects bacteria that ferment glucose via the butanediol pathway, producing acetoin (a precursor to 2,3-butanediol).

How It Works:

1. Bacteria are cultured in glucose-phosphate broth.

2. Barritt’s reagents (α-naphthol + KOH) are added.

3. Positive result: A red/pink color (acetoin reacts with reagents).

4. Negative result: No color change (yellow/brown).

Why Not the Others?

• a) Mixed acid fermentation: Detected by the methyl red (MR) test (red = positive).

• c) Indole production: Tested via Kovac’s reagent (red ring = positive).

• d) Gas production: Observed in Durham tubes (e.g., lactose broth).

1. Mucoid, dark pink (lactose-fermenting) colonies on MacConkey agar in an alcoholic patient strongly suggest Klebsiella pneumoniae.

   • Mucoid appearance: Due to the organism’s polysaccharide capsule (a virulence factor).

   • Lactose fermentation: Pink colonies from acid production (neutral red indicator).

2. Why Not the Others?

   • (a) Edwardsiella tarda: Typically non-lactose fermenting (colorless colonies on MacConkey).

   • (c) Escherichia coli: Ferments lactose but forms pink, non-mucoid colonies (less viscous).

   • (d) Proteus vulgaris: Non-lactose fermenting and swarms on agar (no pink color).

Group B streptococci (GBS) are the most common cause of neonatal purulent meningitis, particularly in:

• Early-onset disease (≤7 days after birth): Acquired during delivery from maternal vaginal colonization.

• Late-onset disease (7–90 days): Often linked to nosocomial or community exposure.

Why Not Other Options?

• (a) Neisseria meningitidis: More common in older children/adults (rare in neonates).

• (b) Streptococcus pneumoniae: Causes meningitis in all ages but is less frequent in neonates.

• (d) Haemophilus influenzae: Now rare due to Hib vaccination (historically affected infants >1 month).

1. Gram-positive cocci in clusters – Suggests Staphylococcus (streptococci and enterococci form chains or pairs).

2. Catalase-positive – Rules out Streptococcus (b) and Enterococcus (d), which are catalase-negative.

3. Coagulase-positive – Confirms Staphylococcus aureus (c), as it is the primary coagulase-positive Staphylococcus.

   • Staphylococcus epidermidis (a) is coagulase-negative (common contaminant in blood cultures).

Why Not the Others?

• a) Staphylococcus epidermidis – Coagulase-negative (part of normal skin flora, often a contaminant).

• b) Streptococcus agalactiae – Catalase-negative, gram-positive cocci in chains.

• d) Enterococcus faecalis – Catalase-negative, gram-positive cocci in pairs/short chains.

1. Sorbitol-MacConkey agar (SMAC) is the standard medium to screen for E. coli O157:H7 because:

   • E. coli O157:H7 is sorbitol non-fermenting (forms colorless colonies).

   • Most other E. coli strains ferment sorbitol (pink colonies).

2. Why Not the Others?

   • (a) Indole: A biochemical test (post-culture), not a component of MacConkey agar.

   • (b) Citrate: Used in Simmons citrate agar (irrelevant here).

   • (d) Lactose: Standard MacConkey contains lactose, but E. coli O157:H7 ferments lactose (pink colonies), making it indistinguishable from normal flora without sorbitol differentiation.

The SIM agar test (Sulfide-Indole-Motility) is a multitest medium that detects:

1. Motility: Cloudy growth radiating from the stab line (motile bacteria, e.g., Proteus).

2. Indole production: Red ring with Kovac’s reagent (e.g., E. coli).

3. H₂S production: Black precipitate (e.g., Salmonella).

Why Not Other Options?

• (b) Citrate test: Identifies citrate utilization (e.g., Klebsiella).

• (c) Voges-Proskauer test: Detects acetoin (e.g., Enterobacter).

• (d) Oxidase test: Identifies cytochrome c oxidase (e.g., Pseudomonas).

• BCYE agar is the standard medium for culturing Legionella pneumophila because it contains:

  • Charcoal (to detoxify free radicals and absorb inhibitory substances)

  • Yeast extract (provides essential nutrients)

  • L-cysteine & iron (required for Legionella growth)

  • Buffer (to maintain optimal pH)

Why not the others?

• a) MacConkey agar – Used for Gram-negative enteric bacteria (e.g., E. coli), but Legionella does not grow on it.

• c) Thayer-Martin agar – Selective for Neisseria gonorrhoeae (contains antibiotics).

• d) Blood agar – Supports many bacteria, but Legionella requires additional supplements (L-cysteine & iron) not present in standard blood agar.

• Haemophilus influenzae is a fastidious gram-negative rod that requires both X (hemin) and V (NAD) factors for growth, typically cultured on chocolate agar (heated blood agar) or supplemented media like Haemophilus Test Medium (HTM) .

• This distinguishes it from other gram-negative rods:

  • Brucella melitensis (b): Requires CO₂ but not X/V factors; grows on standard blood agar .

  • Bordetella pertussis (c): Needs charcoal-blood agar (Regan-Lowe or Bordet-Gengou media) but not X/V .

  • Legionella pneumophila (d): Grows on buffered charcoal yeast extract (BCYE) agar, no X/V requirement .

• MacConkey agar is a selective and differential medium specifically designed to distinguish between lactose-fermenting and non-lactose-fermenting Gram-negative bacilli.

  • Lactose fermenters (e.g., Escherichia coli, Klebsiella spp.): Produce pink/red colonies due to acid production, which reacts with the neutral red pH indicator.

  • Non-lactose fermenters (e.g., Salmonella, Shigella, Pseudomonas spp.): Form colorless/transparent colonies (no acid produced).

Why Not the Other Options?

• b) Citrate utilization test: Differentiates bacteria based on their ability to use citrate as a carbon source (e.g., Klebsiella [+], E. coli [–]).

• c) PYR test: Identifies Enterococcus and Streptococcus pyogenes (Gram-positive cocci).

• d) Coagulase test: Distinguishes Staphylococcus aureus (coagulase +) from other staphylococci.

• Pathogenic Neisseria species (N. gonorrhoeae and N. meningitidis) can be differentiated from nonpathogenic Neisseria species based on their ability to ferment specific carbohydrates:

  • N. gonorrhoeae ferments glucose (but not maltose, lactose, or sucrose).

  • N. meningitidis ferments glucose and maltose.

  • Nonpathogenic Neisseria (e.g., N. sicca, N. lactamica) may ferment additional sugars like lactose or sucrose.

Why Not the Other Tests?

• a) Oxidase test → All Neisseria species are oxidase-positive, so it doesn’t differentiate pathogenic vs. nonpathogenic.

• c) PYR test → Used for identifying Streptococcus pyogenes and Enterococcus, not Neisseria.

• d) Gelatin hydrolysis test → Not a standard test for Neisseria differentiation.

Gram staining is universally performed first in bacterial classification because it:

1. Divides bacteria into two fundamental groups:

   • Gram-positive (thick peptidoglycan, retains crystal violet → purple)

   • Gram-negative (thin peptidoglycan + outer membrane → pink/red)

2. Guides all subsequent testing:

   • Determines choice of culture media (e.g., blood agar vs. MacConkey).

   • Informs antibiotic selection (e.g., vancomycin targets Gram-positives).

   • Directs further biochemical tests (e.g., coagulase for Gram-positive cocci; TSI for Gram-negative rods).

Why Not the Others?

• a) Oxidase test: Used after Gram staining to differentiate Gram-negatives (e.g., Pseudomonas vs. Enterobacteriaceae).

• c) Catalase test: Follows Gram stain to separate Staphylococcus (catalase-positive) from Streptococcus (catalase-negative).

• d) Coagulase test: Specific for Gram-positive cocci (e.g., S. aureus identification).

Purpose of Mannitol Salt Agar (MSA):

• Selective medium: Contains 7.5–10% NaCl, which inhibits most bacteria except Staphylococcus species (salt-tolerant).

• Differential medium: Contains mannitol and a pH indicator (phenol red).

  • Staphylococcus aureus ferments mannitol → produces acid → turns the medium yellow.

  • Other Staphylococcus species (e.g., S. epidermidis) do not ferment mannitol → medium remains pink/red.

Why Not the Other Options?

• a) Lactose fermenters → MacConkey agar or EMB agar are used instead.

• c) Hemolysis detection → Blood agar (e.g., sheep or horse blood) is used.

• d) Motility determination → Motility test medium or SIM agar is used.

The indole test identifies bacteria that produce the enzyme tryptophanase, which hydrolyzes the amino acid tryptophan into:

• Indole (primary metabolite)

• Pyruvic acid and ammonia (byproducts)

How It Works:

1. Bacteria are cultured in tryptone broth (rich in tryptophan).

2. Kovac’s reagent (contains *p*-dimethylaminobenzaldehyde) is added.

3. Positive result: A red/pink layer forms (indole reacts with the reagent).

4. Negative result: No color change (yellow reagent remains).

Clinical Utility:

• Differentiates Escherichia coli (indole-positive) from other Enterobacteriaceae:

  • Klebsiella (indole-negative)

  • Proteus mirabilis (indole-negative) vs. Proteus vulgaris (indole-positive)

Why Not the Others?

• a) Nitrate reduction: Detected via nitrate reductase test (red color with zinc).

• b) Urea hydrolysis: Tested with urease test (pink color from ammonia).

• d) Citrate utilization: Assessed with Simmons’ citrate agar (blue color change).

• Neisseria species are differentiated primarily by their ability to ferment specific carbohydrates in CTA (cystine trypticase agar) or rapid enzymatic tests:

  • Pathogenic species:

    • Neisseria gonorrhoeae ferments glucose only (no maltose/sucrose).

    • Neisseria meningitidis ferments glucose and maltose (no sucrose).

  • Nonpathogenic species:

    • Neisseria lactamica ferments glucose, maltose, and lactose.

    • Neisseria sicca ferments sucrose.

Why Not the Other Options?

• b) Lactose fermentation only → Too limited; lactose helps distinguish N. lactamica but not other Neisseria.

• c) Citrate utilization → Used for Enterobacteriaceae, not Neisseria.

• d) Voges-Proskauer test → Detects acetoin production (e.g., Enterobacter), irrelevant here.

The oxidase test is the most reliable method to differentiate Pseudomonas (oxidase-positive) from Enterobacteriaceae (oxidase-negative).

• Pseudomonas spp. (e.g., P. aeruginosa):

  • Oxidase-positive → Turns dark blue/purple (due to cytochrome c oxidase).

  • Aerobic, opportunistic pathogens (e.g., wound/respiratory infections).

• Enterobacteriaceae (e.g., E. coli, Salmonella, Klebsiella):

  • Oxidase-negative → No color change.

  • Facultative anaerobes, common in gut flora and infections (UTIs, sepsis).

Why Not the Others?

• a) Catalase test: Both groups are catalase-positive (useless for differentiation).

• c) TSI agar test: Used to differentiate within Enterobacteriaceae (e.g., Salmonella vs. E. coli), but Pseudomonas does not ferment sugars in TSI.

• d) Coagulase test: Only relevant for Staphylococcus (e.g., S. aureus is coagulase-positive).

1. Purpose: The PYR (L-pyrrolidonyl-β-naphthylamide) test is a rapid biochemical test used to identify:

   • Group A streptococci (Streptococcus pyogenes)

   • Some enterococci (e.g., Enterococcus faecalis)

2. Mechanism:

   • The test detects the enzyme pyrrolidonyl arylamidase (PYRase)

   • S. pyogenes produces this enzyme, which hydrolyzes the PYR substrate

   • A color change to bright pink/red indicates a positive result

3. Clinical Utility:

   • Rapid differentiation of S. pyogenes (PYR+) from other β-hemolytic streptococci

   • Useful for diagnosing streptococcal pharyngitis and guiding antibiotic therapy

Why Not the Others?

• b) Staphylococcus aureus: PYR-negative (identified by coagulase test)

• c) Proteus vulgaris: PYR-negative (identified by urease and phenylalanine deaminase tests)

• d) Klebsiella pneumoniae: PYR-negative (identified by lactose fermentation and capsule production)

1. ESBL Definition:

   • Extended-spectrum beta-lactamases (ESBLs) hydrolyze penicillins, cephalosporins (e.g., ceftazidime, cefotaxime), and aztreonam, but are inhibited by clavulanic acid.

2. Synergy Test (c):

   • The ceftazidime/clavulanic acid combination disk test is the gold standard for ESBL detection.

   • Positive result: Increased zone of inhibition around ceftazidime+clavulanate vs. ceftazidime alone (≥5 mm difference).

   • Alternative: Cefotaxime/clavulanic acid can also be used.

Why Not the Others?

• a) Oxidase test: Identifies Pseudomonas or Neisseria (not related to resistance).

• b) Cefoxitin disk test: Detects methicillin-resistant Staphylococcus aureus (MRSA) or inducible AmpC beta-lactamases (not ESBLs).

• d) Bacitracin susceptibility: Screens for Streptococcus pyogenes (Group A Strep), not resistance mechanisms.

• The primary purpose of AST is to guide antibiotic selection by identifying which antimicrobial agents are effective against a specific bacterial isolate.

• Common methods include disk diffusion (Kirby-Bauer), E-test, and broth microdilution (MIC determination).

Key Objectives:

✔ Avoid ineffective antibiotics, reducing treatment failure.
✔ Prevent antibiotic resistance by promoting targeted therapy.
✔ Confirm empirical treatment (e.g., adjusting therapy based on lab results).

Why Not the Other Options?

• a) Bacterial morphology → Determined by microscopy (e.g., Gram stain).

• c) Spore formation → Observed via specialized stains (e.g., malachite green).

• d) Gram reaction → Identified through Gram staining, not AST.

1. Gram-negative coccobacilli – Rules out:

   • Streptococcus pneumoniae (Gram-positive cocci, option a)

   • Mycobacterium tuberculosis (acid-fast, not seen on Gram stain, option d)

   • Pseudomonas aeruginosa (Gram-negative rods, not coccobacilli, option c)

2. Grows only on chocolate agar – Haemophilus influenzae requires X (hemin) and V (NAD) factors, which are provided by chocolate agar (heated blood agar). It does not grow on standard blood agar.

3. Clinical context (sputum with neutrophils) – Consistent with H. influenzae respiratory infections (e.g., pneumonia, exacerbations of COPD).

Why Not the Others?

• a) S. pneumoniae – Gram-positive, grows on blood agar (shows alpha-hemolysis).

• c) P. aeruginosa – Gram-negative rod, grows on standard media (e.g., MacConkey agar).

• d) M. tuberculosis – Requires Lowenstein-Jensen medium or specialized mycobacterial culture, not chocolate agar.

The slide coagulase test is a rapid method to identify Staphylococcus aureus by detecting clumping factor (a cell wall-bound protein that binds fibrinogen and causes plasma agglutination).

• Positive result: Immediate clumping of bacterial cells when mixed with rabbit plasma.

• Negative result: No clumping (e.g., S. epidermidis or S. saprophyticus).

Why Not Other Options?

• (b) Catalase test: All Staphylococcus species are catalase-positive (only rules out Streptococcus).

• (c) Indole test: Used for Gram-negatives (e.g., E. coli vs. Klebsiella).

• (d) TSI agar: Used for Enterobacteriaceae (e.g., Salmonella, Shigella).

In Triple Sugar Iron (TSI) agar, a black precipitate forms when bacteria produce hydrogen sulfide (H₂S). This occurs due to:

1. Reduction of thiosulfate (in the medium) → H₂S gas.

2. Reaction with ferrous sulfate (indicator) → ferrous sulfide (FeS), a black precipitate.

Key Bacteria That Produce H₂S:

• Salmonella spp. (e.g., S. Typhi → prominent blackening).

• Proteus spp. (e.g., P. mirabilis).

• Some Citrobacter and Edwardsiella strains.

Clinical Relevance:

• Helps differentiate enteric pathogens (e.g., Salmonella vs. Shigella, which is H₂S-negative).

• Supports diagnosis of gastrointestinal infections (e.g., typhoid fever).

Why Not the Others?

• a) Nitrate reduction: Detected via nitrate broth (red color with zinc).

• c) Indole: Tested with Kovac’s reagent (red ring = positive).

• d) Urease activity: Turns urea broth pink (e.g., Proteus, H. pylori).

• Bile solubility test is a key presumptive test for Streptococcus pneumoniae.

  • S. pneumoniae is bile-soluble (lyses in the presence of bile salts like sodium deoxycholate), while other alpha-hemolytic streptococci (e.g., Viridans streptococci) are not.

• Optochin sensitivity (P disk test) is another confirmatory test for S. pneumoniae, but bile solubility is also highly specific.

Why Not the Others?

• b) Catalase production – S. pneumoniae is catalase-negative, but this is a general test to differentiate staphylococci (catalase+) from streptococci (catalase-).

• c) Coagulase test – Used to identify Staphylococcus aureus (coagulase+), not pneumococci.

• d) Urease activity – Used for organisms like Proteus or Helicobacter pylori, not S. pneumoniae.

1. Blue-green pigment:

   • Produces pyocyanin (blue) and pyoverdine (green), which combine to create its distinctive blue-green color in cultures and infected tissues .

   • This pigment is visible on agar plates and in clinical specimens (e.g., wound exudates or sputum) .

2. Grape-like odor:

   • The organism emits a sweet, fruity odor due to the production of 2-aminoacetophenone, often described as resembling grapes or corn tortillas .

   • This odor is a diagnostic clue in clinical settings (e.g., infected burns or respiratory secretions) .

Why Not the Others?

• a) Serratia marcescens: Produces a red pigment (prodigiosin) and lacks the grape-like odor .

• c) Enterobacter cloacae: Non-pigmented or pale yellow; no distinctive odor .

• d) Acinetobacter baumannii: Non-pigmented and odorless; known for antibiotic resistance, not pigment production .

• The oxidase reagent (e.g., tetramethyl-p-phenylenediamine) is a redox indicator that turns purple/blue when oxidized by cytochrome c oxidase in bacteria.

• However, the reagent is light-sensitive and unstable—it auto-oxidizes upon prolonged exposure to air, leading to a color change even without bacterial enzymes, which can cause false-positive results.

• Therefore, results must be read within 10–30 seconds for accuracy.

Why Not the Other Options?

• a) While some reagents can be toxic, this isn’t the primary reason for rapid reading.

• b) Evaporation isn’t a significant issue with oxidase reagents.

• d) The oxidase test detects cytochrome c oxidase, not catalase (catalase activity is tested separately with hydrogen peroxide).

The first step in identifying an unknown bacterial isolate is Gram staining, which categorizes bacteria into:

• Gram-positive (purple)

• Gram-negative (pink/red)

This basic classification guides further testing (e.g., culture media selection, biochemical tests). While other steps (b, c, d) are important, they come after initial Gram staining.

Why not the others?

• b) Culture on selective media: Requires prior knowledge (e.g., Gram type) to choose the right media.

• c) Biochemical testing: Performed after preliminary identification (e.g., Gram reaction).

• d) Antibiotic susceptibility testing: Done after identifying the bacterium.

A positive DNase test indicates the organism produces deoxyribonuclease (DNase), an enzyme that hydrolyzes DNA into smaller fragments.

• Test method: Growth on DNase agar with methyl green (clearing around colonies) or toluidine blue (pink halo).

• Examples:

  • Staphylococcus aureus (DNase-positive vs. S. epidermidis).

  • Serratia marcescens (DNase distinguishes it from other Enterobacteriaceae).

Why Not Other Options?

• (b) Nitrate utilization: Detected by nitrate reduction test.

• (c) H₂S production: Seen in SIM or TSI agar (e.g., Salmonella, Proteus).

• (d) Urea breakdown: Detected by urease test (e.g., Proteus, H. pylori).

The catalase test detects the enzyme catalase, which breaks down hydrogen peroxide (H₂O₂) into water and oxygen. This test is primarily used to differentiate:

• Staphylococcus (catalase-positive → bubbles form with H₂O₂).

• Streptococcus (catalase-negative → no bubbles).

Why Not Other Options?

• (a) Gram-positive cocci vs. Gram-negative rods: Catalase status varies within these groups (e.g., Enterococcus is Gram-positive/catalase-negative).

• (c) Lactose fermenters: Differentiated by MacConkey agar or phenol red broths.

• (d) Motility: Detected via motility agar or microscopic examination.

A positive bile esculin test results in blackening of the medium due to:

• Hydrolysis of esculin into esculetin and glucose.

• Esculetin reacts with ferric citrate (in the medium) to form a black precipitate.

This indicates that the organism can grow in bile and hydrolyze esculin, typical of Group D streptococci (including Enterococcus) and some other bacteria.

Other options:

• (a) Pink color → PYR test (for Enterococcus).

• (c) Gas in Durham tube → Fermentation tests (e.g., lactose fermentation).

• (d) Blue-green → Pseudomonas pigments (irrelevant here).

The TSI test is a fundamental biochemical assay for differentiating Enterobacteriaceae (a large family of Gram-negative rods including E. coli, Salmonella, Shigella, and Proteus). It evaluates three key metabolic traits simultaneously:

1. Sugar fermentation (glucose, lactose, sucrose) → Acid production (yellow).

2. Gas production → Cracks or lifting of the agar.

3. Hydrogen sulfide (H₂S) production → Black precipitate (e.g., Salmonella, Proteus).

Why Not the Others?

• a) Oxidase test: Enterobacteriaceae are uniformly oxidase-negative; this test helps distinguish them from other Gram-negative bacteria (e.g., Pseudomonas).

• c) Catalase test: Most Enterobacteriaceae are catalase-positive; this test is more useful for separating Staphylococcus (catalase-positive) from Streptococcus (catalase-negative).

• d) Bile solubility test: Used to differentiate Streptococcus pneumoniae (soluble in bile) from other alpha-hemolytic streptococci.

Key Clinical Utility:

The TSI test is low-cost, rapid, and highly informative for:

• Identifying enteric pathogens (e.g., Salmonella in stool cultures).

• Distinguishing lactose fermenters (e.g., E. coli, Klebsiella) from non-fermenters (e.g., Shigella, Salmonella).

• The hanging drop method is a microscopy technique used to observe live, unstained bacteria in a suspended droplet to determine motility.

  • Motile bacteria exhibit purposeful, directional movement (e.g., Proteus spp. with flagella).

  • Non-motile bacteria show only Brownian motion (random vibration due to water molecules).

Why Not the Other Options?

• a) Spore formation: Observed via malachite green stain (e.g., Bacillus, Clostridium).

• b) Capsule production: Visualized with India ink or negative staining.

• d) Cell wall structure: Requires Gram staining or electron microscopy.

The optochin sensitivity test uses an ethylhydrocupreine (P disk) to differentiate:

• Streptococcus pneumoniae: Sensitive to optochin (zone of inhibition ≥14 mm).

• Viridans streptococci: Resistant to optochin (no zone or small zone).

Why Not Other Options?

• (a) Group A Streptococcus (S. pyogenes): Identified by bacitracin sensitivity or PYR test.

• (b) Group B Streptococcus (S. agalactiae): Identified by CAMP test or hippurate hydrolysis.

• (d) Enterococcus species: Identified by PYR test and growth in 6.5% NaCl.