Microbiology Quiz: Campylobacter and Vibrio spp.
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Questions and Answers

Which characteristic is NOT associated with Campylobacter spp.?

  • Motile with one polar flagellum
  • Fermentative (correct)
  • Microaerophilic
  • Oxidase positive

    • What is the primary growth atmosphere required for isolating Campylobacter spp.?

  • Anaerobic conditions
  • High oxygen levels
  • Standard atmospheric conditions
  • 5% O2, 10% CO2, and nitrogen (correct)

    • Which Campylobacter species is commonly associated with causing enteritis, especially in children?

  • Campylobacter jejuni (correct)
  • Campylobacter mucosalis
  • Campylobacter coli
  • Campylobacter fetus

    • Which of the following biochemical reactions is positive for Campylobacter jejuni?

    <p>Hippurate hydrolysis</p> Signup and view all the answers

    What is the typical colony morphology of Campylobacter jejuni?

    <p>Moist, runny looking, and spreading</p> Signup and view all the answers

    Which characteristic is NOT associated with Vibrio spp.?

    <p>Catalase positive</p> Signup and view all the answers

    Which Vibrio species is typically associated with causing voluminous ‘rice water’ diarrhea?

    <p>Vibrio cholerae</p> Signup and view all the answers

    What is the preferred culture medium for isolating Vibrio spp.?

    <p>Thiosulfate citrate bile salts (TCBS) agar</p> Signup and view all the answers

    Which of the following tests is used to identify Vibrio cholerae?

    <p>String test</p> Signup and view all the answers

    Which Vibrio species is characterized by a positive reaction to Nitrate to Nitrite conversion?

    <p>Vibrio fluvialis</p> Signup and view all the answers

    What is the optimal salt concentration for growing most Vibrio spp. in the lab?

    <p>0.5%</p> Signup and view all the answers

    Which test can yield a positive pink color indicating the presence of V. cholerae?

    <p>Cholera red reaction</p> Signup and view all the answers

    Vibrio spp. typically exhibit which type of lactose fermentation characteristic?

    <p>Lactose negative</p> Signup and view all the answers

    Which species is recognized as the most common human isolate within the Aeromonas genus?

    <p>A. hydrophila</p> Signup and view all the answers

    What characteristic is NOT true for Aeromonas spp.?

    <p>They are oxidase negative.</p> Signup and view all the answers

    Which selective culture medium would you use to grow Aeromonas species for identification?

    <p>MAC Agar</p> Signup and view all the answers

    What is the expected result of the oxidase test for Aeromonas spp.?

    <p>Positive</p> Signup and view all the answers

    Which Vibrio species is known to exhibit variable growth in 6% NaCl?

    <p>V. mimicus</p> Signup and view all the answers

    Which of the following is a characteristic of Campylobacter spp.?

    <p>Microaerophilic</p> Signup and view all the answers

    Which of the following Vibrio species is characterized by producing yellow colonies on TCBS?

    <p>V. cholerae</p> Signup and view all the answers

    Which of the following agent is Aeromonas spp. resistant to?

    <p>O/129</p> Signup and view all the answers

    What distinctive morphological characteristic is associated with Mycobacterium tuberculosis colonies?

    <p>Rough, wrinkled, dry, elevated, buff-colored</p> Signup and view all the answers

    Which biochemical test result is true for Mycobacterium tuberculosis?

    <p>Nitrate positive</p> Signup and view all the answers

    Which of the following mycobacterial species is known to be associated with Buruli ulcers?

    <p>Mycobacterium ulcerans</p> Signup and view all the answers

    What is a common characteristic of the Mycobacterium avium complex?

    <p>Common in HIV/AIDS patients with low CD4 count</p> Signup and view all the answers

    How long does it typically take for Mycobacterium ulcerans to show visible growth during incubation?

    <p>6-9 weeks</p> Signup and view all the answers

    Which culture medium would best support the growth of Listeria monocytogenes?

    <p>McBride Listeria medium with sheep blood</p> Signup and view all the answers

    What differentiates Erysipelothrix rhusiopathiae from Listeria monocytogenes?

    <p>Production of H2S</p> Signup and view all the answers

    What characteristic distinguishes Mycobacterium from gram-negative organisms?

    <p>Gram-positive rods</p> Signup and view all the answers

    What is the primary purpose of incubating Mycobacterium culture media in 5-10% CO2?

    <p>To simulate lung environment for Mycobacterium tuberculosis</p> Signup and view all the answers

    What is the result of the Niacin test for Mycobacterium tuberculosis?

    <p>Positive</p> Signup and view all the answers

    Which of the following statements regarding Erysipelothrix rhusiopathiae is incorrect?

    <p>It always forms filaments</p> Signup and view all the answers

    Why is the Nitrate reduction test used for differentiating Mycobacterium species?

    <p>It distinguishes between Mycobacterium tuberculosis and Mycobacterium bovis</p> Signup and view all the answers

    What characteristic of Mycobacterium colonies can be observed on Middlebrook 7H10 agar?

    <p>Early colony visibility</p> Signup and view all the answers

    Which Vibrio species is characterized by a positive result for Arginine Dihydrolase?

    <p>Vibrio harveyi</p> Signup and view all the answers

    Which species is identified by producing gas from glucose fermentation?

    <p>Vibrio fumissii</p> Signup and view all the answers

    Which Vibrio species shows a negative result for Indole?

    <p>Vibrio metschnikovii</p> Signup and view all the answers

    Which species can be identified by a green coloration on TCBS agar?

    <p>Vibrio harveyi</p> Signup and view all the answers

    Which Vibrio species does not grow at 0% NaCl concentration?

    <p>Vibrio parahaemolyticus</p> Signup and view all the answers

    Which of the following species is a positive lactose fermenter?

    <p>Vibrio vulnificus</p> Signup and view all the answers

    Which of these Vibrio species can reduce nitrate to nitrite?

    <p>Vibrio parahaemolyticus</p> Signup and view all the answers

    Which Vibrio species is indicated as positive for sucrose fermentation?

    <p>Vibrio metschnikovii</p> Signup and view all the answers

    Which characteristic is unique to Campylobacter jejuni compared to Campylobacter coli?

    <p>Hippurate positive</p> Signup and view all the answers

    What is the capacity of Campylobacter spp. to grow in specific environmental conditions?

    <p>Optimal growth in a microaerophilic atmosphere</p> Signup and view all the answers

    Which of the following statements about the motility of Campylobacter is accurate?

    <p>Exhibits motility under phase contrast microscopy</p> Signup and view all the answers

    Which biochemical reaction is associated with Campylobacter jejuni?

    <p>Catalase positive</p> Signup and view all the answers

    What is the expected appearance of colonies of Campylobacter jejuni on culture media?

    <p>Moist and runny-looking</p> Signup and view all the answers

    Which species is least likely to exhibit growth in the presence of 6% NaCl?

    <p>V.fluvialis</p> Signup and view all the answers

    Which organism is characterized by being a non-Inositol fermenter?

    <p>A.hydrophila</p> Signup and view all the answers

    Which selective culture medium primarily supports the identification of Aeromonas spp.?

    <p>MAC Agar</p> Signup and view all the answers

    Which of the following species is recognized for producing yellow colonies on TCBS agar?

    <p>V.fumissii</p> Signup and view all the answers

    Which characteristic is true for V.cholerae?

    <p>It is motile.</p> Signup and view all the answers

    Which Aeromonas species is noted for having unusual susceptibility to ampicillin?

    <p>A.trota</p> Signup and view all the answers

    Which biochemical test gives a positive result for Aeromonas spp.?

    <p>Oxidase test</p> Signup and view all the answers

    Which factor does NOT contribute to isolating Campylobacter spp. in the laboratory?

    <p>High salt concentration</p> Signup and view all the answers

    Which Vibrio species exhibits a positive reaction for both Lysine Decarboxylase and Arginine Dihydrolase?

    <p>V. cincinnatiensis</p> Signup and view all the answers

    Which of the following species is characterized by a negative result for Ornithine Decarboxylase?

    <p>G. hollisae</p> Signup and view all the answers

    Which Vibrio species shows variability in its reaction to Lysine Decarboxylase testing?

    <p>V. harveyi</p> Signup and view all the answers

    Which species shows a positive growth reaction in 0% NaCl conditions?

    <p>V. cholerae</p> Signup and view all the answers

    Which Vibrio species is recognized for showing a positive reaction to Lysine Decarboxylase but does not reduce nitrate?

    <p>V. cholerae</p> Signup and view all the answers

    Which species among the following shows a negative reaction for Arginine Dihydrolase?

    <p>V. harveyi</p> Signup and view all the answers

    Which Vibrio specie is classified as variable in its Ornithine Decarboxylase testing?

    <p>V. mimicus</p> Signup and view all the answers

    Which of these Vibrio species is NOT known to exhibit a positive reaction for Lysine Decarboxylase?

    <p>V. parahaemolyticus</p> Signup and view all the answers

    What is a unique morphological characteristic of Mycobacterium tuberculosis colonies?

    <p>Rough, wrinkled, dry, elevated, buff-colored colonies</p> Signup and view all the answers

    Which biochemical test shows a positive result in Mycobacterium tuberculosis?

    <p>Niacin production</p> Signup and view all the answers

    What is the incubation period for Mycobacterium ulcerans to show visible growth?

    <p>6-9 weeks</p> Signup and view all the answers

    Which of the following characteristics is NOT associated with the Mycobacterium avium complex?

    <p>Fast growth and visible within a few days</p> Signup and view all the answers

    Which of the following is true about the biochemical characteristics of Mycobacterium tuberculosis?

    <p>Catalase positive at 68°C</p> Signup and view all the answers

    Which is the optimal culture medium for isolating Vibrio spp. to promote growth?

    <p>Thiosulfate citrate bile salts (TCBS) agar</p> Signup and view all the answers

    Which of the following Vibrio species does NOT produce gas from glucose?

    <p>Vibrio fumissii</p> Signup and view all the answers

    What does a positive Cholera red reaction indicate when testing for Vibrio cholerae?

    <p>Formation of a pink color</p> Signup and view all the answers

    Which of the following biochemical tests shows a positive result for all Vibrio species listed?

    <p>Oxidase</p> Signup and view all the answers

    Which Vibrio species exhibits a positive result for Ornithine Decarboxylase?

    <p>Vibrio cholerae</p> Signup and view all the answers

    What is the characteristic colony appearance of Vibrio species on Sheep Blood Agar (SBA)?

    <p>Large, smooth, and iridescent colonies</p> Signup and view all the answers

    Which Vibrio species is characterized by producing a green coloration on TCBS agar?

    <p>Vibrio hollisae</p> Signup and view all the answers

    In laboratory diagnostics, which test is primarily used for the identification of Vibrio cholerae?

    <p>String test with sodium deoxycholate</p> Signup and view all the answers

    Which culture medium is commonly used to detect Listeria monocytogenes beta hemolysis?

    <p>Trypticase agar</p> Signup and view all the answers

    Which characteristic is unique to Erysipelothrix rhusiopathiae in comparison to Listeria monocytogenes?

    <p>Produces H2S</p> Signup and view all the answers

    What feature distinguishes Mycobacterium cultures when using Lowenstein-Jensen (L-J) medium?

    <p>Green color and opaque appearance</p> Signup and view all the answers

    In which test is Mycobacterium tuberculosis identified by a positive Niacin reaction?

    <p>Enzymatic hydrolysis of Tween 80</p> Signup and view all the answers

    Which culture method is used for identifying Erysipelothrix rhusiopathiae?

    <p>Phenylethylalcohol agar</p> Signup and view all the answers

    Which of the following is true regarding Mycobacterium species and their culture conditions?

    <p>Prefer incubation in 5-10% CO2</p> Signup and view all the answers

    What biochemical reaction differentiates Mycobacterium tuberculosis from Mycobacterium bovis?

    <p>Nitrate reduction</p> Signup and view all the answers

    Which of the following characteristics is NOT true for Mycobacterium species?

    <p>Completely gram-negative</p> Signup and view all the answers

    Study Notes

    Non-Enteric Gastrointestinal Pathogens

    • Vibrio spp. are oxidase positive, except V. metschnikovii
    • Vibrio spp. produce gas from glucose, except V. fumissii, which causes voluminous "rice water" diarrhea.
    • Vibrio spp. are best cultured in alkaline peptone broth (pH 8.4)
    • Thiosulfate citrate bile salts (TCBS) agar is the best medium for isolating Vibrio.
    • Vibrio spp. are gram-negative, comma-shaped, curved, or straight bacilli.
    • Vibrio spp. are facultative anaerobes.
    • Vibrio spp. are catalase negative and oxidase-positive, reducing nitrate to nitrite except V. metschnikovii.

    Vibrio spp. Other Reactions of V. cholerae

    • V. cholerae: MR is negative.
    • V. cholerae: VP is positive/variable.
    • V. cholerae: Citrate is positive.
    • V. cholerae: Urease is negative.
    • V. cholerae: TSI shows A/A, gas is negative, and H2S is negative.
    • String test (emulsify organism in 0.5% sodium deoxycholate on a slide)
    • Cholera red reaction (add concentrated sulphuric acid to 24hrs peptone water culture with V. cholerae results in positive pink color)

    Vibrio spp. Salt Concentration (0.5%)

    • Nutrient agar or sheep blood agar (SBA) are commonly used laboratory media.
    • Colonies on SBA or chocolate agar (CHOC) appear smooth, opaque, and iridescent with a greenish hue (medium to large colonies).
    • Cefsulodin-Irgasan-Novobiocin (CIN) agar is a selective differential medium for Vibrio spp.
    • MAC agar is a selective differential medium.

    Differentiation of Pathogenic Vibrio Species

    • Table detailing the differentiation based on TCBS, Lact. Ferm., Cholera Red React., String Test, and Kanagawa Pheno for different pathogenic Vibrio species.

    Summary for Vibrio spp.

    • Summarized traits specific to different Vibrio species: positive oxidase with different sugars, lysines, decarboxylase, salt concentrations, color on TCBS, etc

    Indole for Vibrio spp.

    • Table of Vibrio spp. categorized by positive and negative Indole.

    Glucose for Vibrio spp.

    • Table of Vibrio spp. categorized by positive and negative Glucose.

    Sucrose for Vibrio spp.

    • Table of Vibrio spp. categorized by positive and negative Sucrose.

    Lysine Decarboxylase for Vibrio spp.

    • Table of Vibrio spp. categorized by positive and negative Lysine Decarboxylase.

    Arginine Dihydrolase for Vibrio spp.

    • Table of Vibrio spp. categorized by positive and negative Arginine Dihydrolase.

    Ornithine Decarboxylase for Vibrio spp.

    • Table of Vibrio spp. categorized by positive and negative Ornithine Decarboxylase.

    Growth in 0% NaCl for Vibrio spp.

    • Table of Vibrio spp. categorized by positive and negative growth in 0% NaCl.

    Growth in 6% NaCl for Vibrio spp.

    • Table of Vibrio spp. categorized by positive and negative growth in 6% NaCl.
    • Colony color and characteristics on TCBS agar.

    Aeromonas spp. Key Characteristics

    • Gram-negative, straight bacilli
    • Facultative anaerobe
    • Fermentative
    • Indole positive
    • Oxidase positive
    • Motile
    • Resistant to vibriostatic agent O/129 (2,4 diamino-6,7-diisopropylpteridine)
    • Non-inositol fermenter
    • A. hydrophila is the most common human isolate

    Aeromonas spp. Selective Culture Media

    • MAC Agar, ferment lactose (A. caviae), unusual universal susceptibility to ampicillin.
    • CIN Medium, pink-centered colonies from fermentation of mannitol, uneven clear apron resembling Yersinia enterocolitica.
    • Alkaline peptone water.

    Aeromonas spp. Presumptive Identification

    • Oxidase test (positive)
    • Spot indole test on suspicious colonies (especially beta-hemolytic colonies)
    • Ability to grow in the presence of NaCl.

    Campylobacter spp. Key Characteristics

    • Gram-negative, comma, curved, S-shaped, seagull wing-shaped bacilli
    • Microaerophilic (5% O2)
    • Capnophilic
    • Motile (one polar flagellum)
    • Oxidase-positive
    • Non-fermentative
    • Sodium Hippurate Hydrolysis-positive
    • Urease-negative
    • Exhibit characteristic motility (hanging drop preparations/phase contrast microscopy).

    Campylobacter spp. (Campylobacter jejuni and Campylobacter coli)

    • Causes enteritis (esp. children)
    • Leading cause of acute diarrhea worldwide.
    • Sometimes responsible for bacteremia or endocarditis
    • Culture: Incubate in 5% O2, 10% CO2, and nitrogen atmosphere for 48 hours.
    • Isolation media: "Campy-thio" or "Campy-blood" agar.
    • Use antibiotics in the media to make it selective.

    Campylobacter spp. Biochemical reactions of C. jejuni

    • Non-fermentative
    • Oxidase (+), Catalase (+), Nitrate (+), H2S (-)
    • Grows in 1% glycine
    • Hippurate (+). C. coli is negative.
    • C. jejuni and other enteric Campylobacters
    • Moist, runny look; spreading, usually non hemolytic, some round and others flat.
    • C. fetus subsp. fetus
    • Produces smooth, convex, translucent colonies.
    • C. mucosalis and C. hyointestinalis
    • Can produce a dirty yellow pigment.

    Helicobacter pylori

    • Curved, flagellated, motile gram-negative rods
    • "Triple positive": catalase (+), oxidase (+), urease (+)
    • Diagnosis: Biopsy, Urea breath test, Stool antigen

    Aerobic Gram-Positive Bacilli

    • Corynebacterium

    Corynebacterium

    • Non-spore-forming, nonbranching, catalase-positive bacilli.
    • Also called Kleb Coeffler's bacillus
    • Microscopy: Highly pleomorphic gram-positive bacilli.
    • Club-shaped swellings and beaded forms are common.
    • Metachromatic areas (Babes-Ernst granules) stain intensely.
    • Facultative anaerobes.
    • Best growth at 37°C, but can multiply from 15°C to 40°C.
    • Grows well on nutrient agar, serum/blood-containing media (ex. Coeffler Serum or Pai Agar).
    • Cystine-tellurite blood agar (CTBA) - modification of Tinsdale medium, sheep red blood cells, bovine serum, cystine, potassium tellurite. Inhibits non-Coryneform bacteria.
    • Characteristics of C. diphtheriae- non motile, nitrate reduction (+), urease (-), catalase (+), glucose & maltose fermented, sucrose not fermented.

    Corynebacterium (continued)

    • Culture from threat, nasopharynx, skin wounds
    • Special culture media: Coeffler's or Tinsdale
    • Black colonies on Tinsdale media
    • Metachromatic (blue/red) granules on Coeffler's media
    • Media to enhance pleomorphism and granule production
    • Coeffler serum agar + poached egg colonies, Pai coagulated egg medium

    Listeria monocytogenes

    • Facultative intracellular Catalase positive
    • Tumbling motility (at room temp) but usually nonmotile at 35-37°C
    • Hippurate hydrolysis-positive
    • Esculin-positive, salicin (+)
    • CAMP positive (hemolysis looks like a shovel, not arrowhead)
    • Grows from 0.5-45°C
    • Cold enrichment may be used

    Listeria monocytogenes (Culture)

    • Sheep blood agar (detect beta-hemolysis, blue-green colonies)
    • McBride Cisteria medium (sheep blood).
    • Phenylethylalcohol agar (inhibits gram-negative organisms).
    • Hold suspect cultures at 4°C up to 6 months, subculture weekly for 1 month, monthly thereafter.
    • Umbrella-shaped pattern.

    Erysipelothrix rhusiopathiae

    • Slender, pleomorphic gram-positive rods that form filaments
    • Alpha hemolytic (on blood agar)
    • Decolorizes easily in gram stain, may appear gram-negative.
    • Catalase, oxidase, indole negative
    • Produces H2S, differentiating it from Listeria monocytogenes.
    • Test tube brush growth.

    Mycobacterium

    • Obligate aerobes
    • Slender, non-spore-forming, acid-fast, gram-positive rods.
    • Do not gram stain well.
    • Culture media (incubate in 5-10% CO2):
    • -Cowenstein-Jensen (C-J) (green color and opaque) - Contains egg glycerol, promotes growth of more species (less drying), good for niacin test. Difficult to prepare, often used for cultures.
    • -Middlebrook 7H10 or 7H11 agar (Clear agar)
    • Good for antimicrobial susceptibility testing, fewer contaminants grow.
    • Contains glycerol. Enhances growth of human strain of M. tuberculosis.

    Mycobacterium (Identification)

    • Pigment production, growth rate, biochemical reactions.
    • Niacin test (best for M. tuberculosis) - enzymatic hydrolysis of Tween 80 releases oleic acid. The indicator (neutral red) changes color depending on the species.
    • Runyon groups I and III positive in 5 days.
    • M. tuberculosis positive in 5 days.

    Mycobacterium (Nitrate reduction)

    • M. tuberculosis is positive
    • M. bovis is negative
    • M. kansasii is a positive control.

    Mycobacterium (Catalase reaction)

    • Almost all Mycobacteria are catalase-positive.
    • M. tuberculosis and M. bovis are catalase-negative after incubation at 69°C for 30 minutes.

    Biochemical tests (NTM identification)

    • Includes tests for Tween-80 hydrolysis, Niacin test, Nitrate reduction test, Tellurite reduction test, Urea hydrolysis test and Citrate utilization test for Mycobacteria other than tuberculosis.

    Mycobacterium tuberculosis

    • Rough, wrinkled, dry, elevated, buff-colored ("cauliflower-like") at 35-37°C
    • Slow growth: 2-3 weeks or longer
    • Innoculate in 2 C-J slants, wrap in aluminum foil.
    • Incubate at 35°C until visible growth occurs in unshielded tube, expose covered tube to light for 1 hour, cover again, and reincubate overnight.
    • Niacin positive/yellow
    • Tween 80 hydrolyzed in 10-20 days (pink color)
    • Nitrate positive
    • Catalase at 68C: negative
    • No growth in MacConkey Medium
    • Arylsulfatase negative

    Mycobacterium ulcerans

    • Associated with skin lesions in tropical and subtropical areas (Buruli ulcers in Africa)
    • Requires several weeks (6-9) of incubation at 32°C for growth to become visible.
    • Negative for niacin, nitrate reduction, Tween 80.

    Mycobacterium leprae

    • Hansen's bacillus
    • Causes Hansen's disease (leprosy)
    • Acid-fast rod in nasal mucosa of affected patients.
    • Smears of tissue juice - acid-fast stain for lepra cells (macrophages containing acid-fast bacilli).
    • Culture: Grows very slowly, not cultured on artificial media or human tissue. Culture can be performed on footpad of mice.
    • Lepromin test - skin test for leprosy using a sterile extract from lepromatous nodules (24-48 hrs = early/Fernandez; 3-4 weeks = late/Mitsuda reaction)

    Nocardia

    • Thin, branching and beaded modified acid-fast positive rods.
    • Key characteristics: obligate aerobe, partially acid-fast, delicate mycelium, catalase (+), urease (+).
    • Culture: growth on any media without antibiotics.
    • Complete biochemical tests needed for identification.

    Anaerobes

    • Found throughout human bodies as part of normal microbiota.
    • Frequently cause polymicrobial infections.
    • Anaerobic culture methods: Enriched agar, selective media:
      • KV (kanamycin-vancomycin) blood agar (anaerobic gram (-) rods).
      • KVL (kanamycin-vancomycin-laked) blood agar (esp. Bacteroides melaninogenicus).
      • Neomycin-vancomycin blood agar (for Fusobacteria, Veilonella).
      • Neomycin blood agar (for clostridia and anaerobic gram-positive cocci).
      • Naegler agar (with egg yolk, neomycin) (for clostridia).
      • Chopped meat glucose broth, supplemented thioglycollate broth. Boil 10 min and cool rapidly to drive off oxygen prior to inoculation.

    Commonly Isolated Anaerobes

    • Clostridium (anaerobic, gram-positive, spore-forming, motile rods; mostly peritrichous flagella)
    • May produce lecithinase and lipase, ferment sugars, digest proteins.
    • Table displaying motility, lecithinase, lipase, glucose and lactose results for different Clostridium species. C. perfringens, C. botulinum, C. tetani, and C. difficile.

    Clostridium tetani

    • Terminal spores ('lollipop').
    • Positive for gelatin hydrolysis, glucose fermentation.
    • Produces neurotoxin (tetanospasmin) causes tetanus (spastic paralysis).

    Clostridium botulinum

    • Causes botulism (floppy baby syndrome in infants)

    Clostridium perfringens

    • Causes gas gangrene (clostridial myonecrosis).
    • Alpha toxin destroys muscle tissue, causes hemolysis.
    • Phospholipase (lecithinase) degrades phospholipids in cell membranes.
    • Double zone of hemolysis around colonies on blood agar.

    Clostridium difficile

    • Causes pseudomembranous colitis in patients undergoing prolonged antibiotic therapy.
    • Cycloserine-cefoxitin-fructose agar (CCFA) used to recover C. difficile from stool cultures.
    • Naegler's test - inhibition by anti-perfringens antitoxin of the precipitate around growth on egg yolk agar.

    Antimicrobial Susceptibility Testing (Kirby-Bauer Disk Diffusion Test)

    • Principle: Inverse linear relationship between zone diameter inhibited growth and antibiotic MIC.
    • Testing parameters:
      • Use Mueller Hinton Agar in a 150x15mm plate.
      • pH: 7.2-7.4
      • Agar depth constant at 4mm (some sources 4-6mm).
      • Inoculum density: equivalent to 0.5 McFarland standard (1.5 x 108 CFU/mL) using 0.5 mL of 1.175% BaCl2 and 99.5 mL of 1% H2SO4
      • Too heavy inoculum = smaller zones, too light = larger zones
    • Incubate plate at 35 ± 2°C for 18 hours.
      • <16 hours = ↑ zone, false susceptible

      • 18 hours = ↓ zone, false resistant

    • Measure zone of inhibition using ruler, caliper, or template (on the opposite of blood agar plate).
    • Resistant, intermediate, or susceptible based on zone inhibition in millimeters.
    • QC strains of E. coli, S. aureus, P. aeruginosa, Enteroccocus faecalis for 20-30 days for frequent testing. Reduce frequency to weekly if results are acceptable.

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