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Microbiology Quick Reference

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Microbiology Agars

Agar Enrichment Selective Differential Features Use
Blood 5-10% sheep blood Shows hemolysis General growth medium
Chocolate 5-10% lysed sheep blood

2% hemoglobin + supplements

Growth of fastidious organisms
CNA 5-10% sheep blood Gram positives grow
  • Colistin
  • Naladixic acid
 Shows hemolysis Growth of gram positives and yeast
MacConkey Gram negatives grow
  • Bile salts
  • Crystal violet
 Shows lactose fermentation Grow of gram negatives
Agar Features Use
Meuller Hinton
MH + Sheep's blood
  • Enrichment: sheep blood
 AST for specific organisms
  • Streptococcus pneumoniae
Thioglycollate broth
  • Enrichment: hemin & vitamin K
  • Differential: O2 use based on location of growth in tube
 General growth medium, allows presumption of atmospheric growth conditions
Enrichment Media
  • Usually broth
  • Grows fastidious or a specific organism from mixed specimens
  • Contain differential reagents to suppress normal flora for a short period of time
    • Requires subculture within ~ 12-18h
  • Selenite broth
  • Tetrathionate broth
  • Brain-heart Infusion broth
  • Cooked meat broth
Sorbitol MacConkey
  • Selective: for gram negatives
  • Differential: sorbitol fermentation
 Enterohemorrhagic E. coli (non-sorbitol fermenters) vs other E. coli (sorbitol fermenters)
Salmonella-Shigella
  • Selective: bile salts, sodium citrate, brilliant green
    • Selects for gram negative enteric pathogens
  • Differential: lactose fermentation + H2S production
 Isolate enteric pathogens
Hektoen Agar
  • Selective: bile salts
    • Selects for gram negative, mostly enteric pathogens
  • Differential: lactose, sucrose, and salicin fermentation + H2S production
 Isolate enteric pathogens
Cefsulodin Irgasan Novobiocin (CIN)
  • Selective: cefsulodin, irgasan, novobiocin, sodium desoxycholate, crystal violet
    • Inhibit gram + and normal stool flora
  • Differential: mannitol fermentation
 Isolate Yersinia enterocolitica
Campylobacter agar
  • Enrichment: 10% sheep blood
  • Selective:
    • vancomycin (inhibit gram +)
    • amphotericin B + polymyxin B (inhibit yeast/fungus)
    • trimethoprim (prevent Proteus swarming)
    • cefoperazone (inhibit gram -)
    • Sodium bisulfite (creates microaerophillic environment)
 Isolate Campylobacter
Mannitol salt agar
  • Selective: 7.5% salt
    • Selects for halophiles
  • Differential: mannitol fermentation
 Screen for Staphylococcus aureus
New York City agar
  • Enrichment: lysed blood, yeast dialysate, and plasma
  • Selective: vancomycin, colistin, amphotericin B, and trimethoprim

Yeasts

  • Sabourand agar
  • Cornmeal agar (especially for Candida spp.)

Chromogenic Agars

Staphlyococcus aureus

  • MRSA Select agar
  • Denim blue agar

Streptococcus agalactiae (Group B Strep)

  • Brilliance GBS agar

Candida

  • CandiSelect
  • Brilliance Candida

Corynebacterium diptheriae

  • Hoyle's meagar
  • Loeffler's
  • Tinsdale agar

Specimen Collection

Stool O&P

  • Unfixed
  • 10% formalin (general-purpose)
  • PVA polyvinyl alcohol (PCR and staining)
  • SAF sodium acetate-acetic acid-formalin (concentration and staining)

Stool

  • Cary-Blair media

GBS Screen

  • Vaginal-rectal swab
  • Todd-Hewitt broth with gentamicin and nalidixic acid to suppress other flora

Stains

  • Gram stain
  • Ziehl-Neelsen (acid fast)
    • Uses heat for uptake of carbolfuchsin
  • Kinyoun (acid fast)
    • Higher phenol concentration for uptake of carbolfuchsin
  • Auramine/Auramine-Rhodamine (acid fast)
    • fluorescent dye binds to mycolic acids
  • Acridine Orange (organisms without cell wall)
    • fluorescent dye binds to nucleic acid in cells
    • useful for organisms without cell wall (e.g., Mycoplasma)
  • Calcofluor white (fungi)
    • Fluorescent dye binds to cellulose and chitin

Kinyoun = cold, Acridine = nucleic acids, Calcofluor = chitin

Biochemical Tests

Test Purpose Mechanism Results
Hugh-Leifson Oxidation/Fermentation Test Determines if organism can metabolize carbohydrates Incubate tubes with 1% glucose + peptones

Check for pH change in aerobic and anaerobic tubes

  • Fermenter: both +
  • Oxidizer: aerobic + only
  • Non-fermenter/oxidizer: both -
 Positive: yellow

Negative: green

ONPG Differentiate late lactose fermenters from non-lactose fermenters
  • LLF: have β-galactosidase but not permease to allow cell uptake (may mutate or turn gene on after exposure to lactose)
  • NLF: no β-galactosidase (can't utilized lactose)
 Use ONPG disk, which is similar to lactose but small enough to diffuse without permease Positive LLF: any yellow

Negative NLF: colourless

TSI (Triple Sugar Iron) Detect fermentation of glucose, lactose, and/or sucrose

Detect production of gases and H2S

0.1% glucose is used up first, then either:
  • Non-fermenters use peptones (alkaline in slant)
  • Fermenters use lactose and/or sucrose (acid in both butt and slant)
 Glucose only: NA/A (pink slant, yellow butt)

Multiple carbohydrates: A/A (yellow slant and butt) Aerobic organism (no fermentation): NA/NC (red slant and butt) May also produce gas and/or H2S

MRVP Used to determine type of pyruvate fermentation pathway used
  • MR uses mixed-acid pathway
  • VP uses 2,3-butanediol pathway
 Mixed acid fermentation produces acid end-products (lactic acid and acetic acid), causing pH change

2,3-butanediol pathway produces acetoin

Decarboxylation
Arginine Dihydrolase
Phenylalanine Deaminase (PPD) Used to test for phenylalanine deaminase
  • Present in gram negative bacilli
 Positive: green

Negative: yellow

Simmon's Citrate Positive: blue or green with growth

Negative: green, no growth

Gelatin test
Indole
Sulfide-Indole-Motility (SIM)
Nitrate Test Determines whether organism has nitrate reductase to produce O2 anaerobically Nitrate reductase converts nitrate -> nitrite (NO2)

Some organisms further break down nitrite to nitrogen gas

If red at first tube = nitrites positive

If not red, then but add zinc

  • Red = negative (original nitrates present)
  • No colour = positive (nitrite reduced to N2)
Urea Test

Organism Testing

Test Positive QC Negative QC Uses
Catalase Staphylococcus spp. Streptococcus spp.
Coagulase S. aureus Other Staphylococcus
Staphaureux (Latex agglutination)
PYR
  • S. lugdunensis
  • Enterococcus
  • S. aureus
  • Strep. bovis

Staphylococcus species

Staphylococcus aureus

  • GPC clusters
  • Catalase +
  • Tube Coagulase + (forms clot in plasma due to Staphylocoagulase)
  • Staphaureux +

If coagulase/Staphaureux negative, then CoNS:

  • S. lugdunensis: PYR +, ORN +
    • Has clumping factor that can cause weak false positives!
  • S. saprophyticus (females 12-60): Novobiocin resistant (≤16 mm)
  • Others: S. epidermidis, S. hominis, other CoNS

Related Species (Micrococcus, Stomatococcus, Planococcus)

  • Often normal flora
  • Similar test results to Staphylococcus spp.

Streptococcus species

  • GPC pairs & chains
  • Catalase -

Lancefield grouping mainly used for ID

Lancefield Grouping Organism Features Clinical Relevance
Group A S. pyogenes
  • Beta-heme (large zone)
  • PYR +
  • Bacitracin S
  • Hippurate hydrolysis neg
 iGAS
Group B S. agalactiae
  • Beta-heme (narrow zone)
  • Hippurate +
  • Bacitracin R
  • CAMP test + (enhanced hemolysis when near beta-heme S. aureus)
 Neonatal infections
Group D S. bovis
  • Non-heme
  • PYR -
  • Bile esculin +
  • Sepsis, sub-acute endocarditis, meningitis
  • UTIs
  • Association with GI carcinomas
Enterococcus
  • E. faecalis (most common)
  • E. faecium (majority of VREs)
  • E. gallinarum
  • E. casseliflavus
  • Non-heme, possibly very small beta-heme on some media
  • PYR +
  • Bile esculin +
Group C Streptococcus dysgalactiae subsp equisimilis Beta-heme (large zone) Normally commensal

Possibly cause pharyngitis

Group G
Non-groupable S. pneumoniae
  • Alpha-heme
  • "Checkerboard" colonial appearance
  • Bile soluble
  • Optochin sensitive (≥14mm)
  • Otitis media
  • Sinusitis
  • Pneumonia
  • Meningitis
  • Etc.
Various/non-groupable Viridans Streptococci
  • S. mitis, S. mutans, S. salivarius, S. bovis, S. anginosus
  • Most alpha-heme, but also beta- and non-heme

Enterococcus sp.

Organisms of infection control concern

E. faecalis

  • PYR +
  • Bile esculin +
  • Arabinose -
  • MGP -

E. faecium

  • PYR +
  • Bile esculin +
  • Arabinose +
  • MGP -

Other Enterococci (no infection control concern)

E. gallinarum

  • MGP +

E. casseliflavus

Enterobacterales

  • GNB, non-spore formers
  • Oxidase -
  • Reduce nitrates → nitrites
  • Ferment carbohydrates
  • All motile at 37C except Klebsiella, Shigella, Yersinia

E coli

Klebsiella

  • Mucoid (capsule)
  • Non-motile

Proteus

  • Swarming motility
  • PPD +
  • Urea +
  • H2S +

Providencia

  • H2S -

Morganella

  • H2S -

Serratia

  • Some species produce red pigment
  • ONPG + (LLF)
  • DNAse +

Citrobacter

Edwardsiella

Enterobacter

Pantoea

Salmonella

  • NLF
  • H2S +

Shigella

  • NLF
  • H2S -
  • Non-motile
  • Latex group testing (A, B, C, D)

E. coli O157

  • LF usually
  • Non-sorbitol fermenter
  • Otherwise, similar to other E. coli
  • Serology (O157:H7 antisera)

Yersinia

  • NLF
  • Non-motile
  • Bullseye on CIN agar (darker red/pink middle, light pink rim)
  • Grows at RT and 4C

Antibiotic Susceptibility Testing

MRSA

  • Penicillins bind to PBP (penicillin binding protein) = inhibits cell wall formation
  • MRSAs have mecA gene encoding PBP2a
    • PBP2a resistant to ALL beta-lactams
  • Test using oxacillin screen and Kirby-Bauer (cefoxitin)

BORSA (Borderline Oxacillin Resistant Staphylococcus aureus)

  • NOT due to mecA (therefore, have altered PBP but NOT PBP2a)
  • Due to superproduction of beta-lactamase
  • MIC above oxacillin breakpoints (>4 μg/mL), but don't grow on oxacillin screen plates

VISA

  • Vancomycin MIC breakpoint 4-8 μg/mL
  • Possibly due to thickened cell wall, trapping antibiotic
  • Test using vancomycin screen

VRSA

  • Vancomycin MIC breakpoint >8 μg/mL
  • vanA resistance from plasmids or transposons (e.g., from VREs)
  • Test using vancomycin screen

VRE

  • vanA or vanB associated with outbreaks (E. faecalis and E. faecium)
  • vanC intrinsic - not associated with outbreaks (E. gallinarum and E. casseliflavus)

D-Test

  • Test for inducible clindamycin resistance
    • Presence of erm gene
    • Causes flattening of zone

Mycology

  • Malassezia furfur = Tinea versicolour = budding yeast and hyphae in 10% KOH ("spaghetti and meatballs")
  • Candida albicans = polymorphic, chlamydospore +, germ tube +, urea -
  • Candida auris = germ tube -
  • Cryptococcus = germ tube -, india ink +, urea +

Parasitology

  • Definitive/true host = where parasite develops into sexually mature (adult) form
  • Intermediate/secondary host = in-between host where parasite is sexually immature (e.g., larvae)
  • Entamoeba histolytica = spoke-like central karyosome(s)
  • Trichomonas vaginalis = one nuclei (often oval and to one side), flagella
  • Balantidium coli = large, ciliated
  • Schistosoma = egg with prominent spine/spike
  • Enterobius vermicularis = pinworm, egg is elongated oval with one side flattened
  • Strongyloides = larvae migrating through agar

Polymerase Chain Reaction

  • Taq polymerase used along with dNTPs (nucleotides for elongation), and primers (DNA or RNA sequence of interest)
  • Buffer pH at 8.0-9.5, include salts MgCl2 and KCl
  • Amplified through multiple cycles (usually 25-30)
  • 95C denature - 60C anneal - 72C extend
  • qPCR provides amplification and detection together
    • detect products as they're produced
    • use fluorescent dye
      • SYBR green or Taqman probe
    • SYBR green binds to any double-stranded DNA and fluoresces (more DNA produced = more fluorescence)
    • Taqman is more specific, and binds to a specific target section
      • 5' fluorescent dye and 3' quencher
      • No fluorescence when dye and quencher are close together
      • During PCR, the extension of polymerase will cut the Taqman probe with an exonuclease, releasing the fluorescent probe
      • As the probe is cut away from the quencher, a signal is released
      • As amplification continues, more probes are separated from the quencher resulting in a larger signal
    • Get amplification curve over ~ 40 cycles
      • Baseline phase (little fluorescent signal)
      • Exponential phase as PCR product amplifies significantly ~ cycles 16-25
      • Stationary phase once components are used up and no further amplification occurs
    • Threshold line is the point where there is a certain level of fluorescence above the background signal (set per instrument)
    • Cycle threshold (Ct) is the amount of cycles required to reach the threshold line
      • If there's less target DNA/RNA present, it will take more cycles to amplify to this level = higher Ct
      • If there's more target DNA/RNA present, it will take fewer cycles to reach this level = lower Ct
      • Can use known standards to get absolute quantitation of target present by comparing the curves

MALDI-ToF

Matrix-assisted laser desorption ionization time-of-flight

  • Laser vapourizes complex molecules into ionized protein molecules
    • Desorption removes molecules from sample
    • Ionization produces positively charged proteins that move through mass spec tube
  • Matrix (cinnamic acid) absorbs energy and protects sample
  • Measure mass-to-charge (m/z) ratio of molecules
  • Generates mass spectrum based on the flight time (speed) of the molecules = mass of ions
    • Smaller ions move faster
  • Compare results to database of known organisms
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