Microbiology Quick Reference

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

Chromogenic Agars

Staphlyococcus aureus

• MRSA Select agar
• Denim blue agar

Streptococcus agalactiae (Group B Strep)

• Brilliance GBS 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)

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.
Coagulase	S. aureus	Other Staphylococcus
Staphaureux (Latex agglutination)
PYR	S. lugdunensis

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

Catalase

Oxidase

PYR

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