Microbiology

Bacterial Growth Requirements

• Temperature

  • Psychrophiles - cold loving; optimum temperature = 15C

  • Mesophiles - moderate temperature; optimum temperature = 37C (most pathogenic organisms)

  • Thermophiles - heat loving; optimum temperature = 50-60C

• pH optimized for most

  • Bacteria 6.5-7.5

  • Fungi 5.0-6.0

• Oxygen

  • Aerobes - require O₂

  • Facultative anaerobes - can grow with or without O₂

  • Obligate anaerobes - harmed by O₂

  • Formation of superoxide radicals, toxic; neutralized by catalase, peroxidase and superoxide dismutase (possessed by aerobes and facultatives)

• Other atmospheric requirements

  • Microaerophiles - prefer lower O₂ than in air

  • Capnophiles - prefer higher CO₂ than in air

Psychrophiles

Cold loving; optimum temperature = 15C

Mesophiles

Moderate temperature; optimum temperature = 37C (most pathogenic organisms)

Thermophiles

Heat loving; optimum temperature = 50-60C

pH for Bacteria? For Fungi?

Bacteria 6.5-7.5

Fungi 5.0-6.0

Aerobes

Require O₂

Facultative anaerobes

Can grow with or without O₂

Obligate anaerobes

Harmed by O₂

Formation of superoxide radicals, toxic; neutralized by…

catalase, peroxidase and superoxide dismutase

Microaerophiles

Prefer lower O₂ than in air

Capnophiles

Prefer higher CO₂ than in air

Culture Media Requirements

• Must meet growth requirements

• Agar - polysaccharide derived from marine algae

  • Melts at 100C

  • Solidifies at approx. 45C

• Complex media - most common, made of peptones and extracts

• Anaerobic media - contain reducing agents which bind with dissolved O₂ (thioglycolate, cysteine)

• Broth tubes should be heated prior to use to drive out O2

• Gas pak envelopes

  • Contain Na₂CO₃ and sodium borohydride

  • Add water - produces H₂ and CO₂ (aids in growth)

  • Palladium pellets catalyze the reaction

  • Some require hemin, Vitamin K, and yeast extract

• Typical incubation

  • 5-10% CO2 (incubator or candle jar)

  • 35-37C

  • 50-70% humidity

• May be selective and/or differential

Sterilization/Inhibition Techniques

• Heat - denatures protein

  • Moist - autoclave (steam under pressure)

    • 15lbs pressure/sq. in., 121C, 15 minutes

    • QC - Bacillus stearothermophilus

  • Dry heat

    • Flame, incinerator

    • Hot air oven 170C, 2 hrs

  • Pasteurization, ultra high temperature

    • 140C, 3 seconds

    • NOT sterilization

• Filtration

  • Pore size 0.22μ - 0.45μ

  • Used for sugar solutions, urea media, vaccines

• Refrigeration - slows growth

• Dessication - no multiplication, but organisms remain viable (lyophilization)

• Osmotic pressure hypertonic solution

  • Causes plasmolysis

  • “Cured” meat, fruit preserves

• Radiation

  • Forms hydroxyl radicals

  • Damages DNA

• Disinfection

  • Phenol - damages cytoplasmic membrane, denatures protein

  • Halogens (halogen and chlorine)

  • Alcohols - denature protein and dissolve lipids

Sterilization vs. Disinfection

• Sterilization - Kills all microorganisms (including spores and viruses)

  • Examples: Autoclave (121C at 15 psi for 15mins), incineration, filtration (physically removes microorganisms)

• Inactivation or inhibition of microorganisms (may not affect spores)

  • Example: Bleach (1:10 Hypochlorite)

β-lactams (examples, action, notes)

• Penicillins, Cephalosporins, Carbapenams (Imipenam), Monobactams (Azotreonam), β-lactamase Inhibiting Combinations (Augmentin, etc.)

• Inhibits cell wall synthesis

• Oxacillin, nafcillin, methicillin for penicillin-resistant Staph

Glycopeptides (examples, action, notes)

• Vancomycin

• Inhibits cell wall synthesis

• Drug of choice for Clostridium difficile and MRSA

Aminoglycosides (examples, action, notes)

• Gentamicin, Tobramycin, Amikacin

• Inhibits protein synthesis

• Acts on 30S subunit; not active against anaerobes; used with a penicillin for Enterococcus

Tetracyclines (examples, action, notes)

• Tetracycline, Doxycycline

• Inhibits protein synthesis

• Acts on 30S subunit; effects bone and teeth in children; may lead to superinfection of yeast

Chloramphenicol (examples, action, notes)

• Chloramphenicol

• Inhibits protein synthesis

• Acts on 50S subunit; can cause aplastic anemia

Macrolides (examples, action, notes)

• Erythromycin, Clindamycin

• Inhibits protein synthesis

• Acts on 50S subunit; clindamycin for gram pos and gram neg anaerobes

Quinolines (examples, action, notes)

• Ciprofloxacin, Norfloxacin

• Inhibits nucleic acid synthesis

• For Pseudomonas aeruginosa and other anaerobes

Sulfa Drugs (Sulfonomides) (examples, action, notes)

• Sulfamethoxazole

• Analogue of PABA (intermediate in folic acid synthesis)

• For UTI, enteric infections; used with trimethoprim (Bactrim, etc.)

Streptogramins (examples, action, notes)

• Quinupristin/dalfopristin

• Inhibits protein sythesis

• For gram pos, especially vancomycin resistant Enterococcus faecium

Oxazdidinones (examples, action, notes)

• Linezolid

• Inhibits protein sythesis

• For gram pos, including those resistant to other antibiotics

Antimicrobial Therapy

• Narrow spectrum - only certain groups covered

• Broad spectrum - gram pos and gram neg coverage

• Selective toxicity - action against only microbial structures (70s ribosome, cell wall, etc.)

• Bactericidal action - kills bacteria without host immune help

• Bacteriostatic action - reversible inhibition (ultimate destruction depends on host defenses)

• Drug combination

  • Synergism - combined better than the sum 1 + 2 = 4

  • Antagonism - one decreases activity of other: 1 + 2 = 1

Susceptibility Testing

• Kirby-Bauer Method

• Broth methods

• Modified methods for testing slow-growing or fastidious bacteria

• Extended spectrum beta-lactamase (ESBL)

• D-test

• Detection of MRSA

Kirby-Bauer Method

• Mueller-Hinton agar

• Depth = 4mm

• pH = 7.2-7.4

• Psychologic concentration of Ca++ and Mg++

• 35C, ambient air

• 10⁸ organisms (McFarland 0.5)

• QC weekly and with each new lot of agar or discs (E. coli, S. aureus, P. aeruginosa)

Broth methods

• MIC (minimum inhibitory concentration)

  • Lowest concentration of drug that prevents in vitro growth

  • First dilution tube with no visible growth

• MBC (minimum bacteriostatic concentration)

  • Lowest concentration that results in >99.9% killing

  • Subculture tubes near MIC to find first plate with no growth

Modified methods for testing slow-growing or fastidious bacteria

• Haemophilus test medium

• Supplemented MH for S. pneumoniae

• Supplemented GC agar for N. gonorrhoeae

Extended spectrum beta-lactamase (ESBL)

• Enzymes for resistance to extended-spectrum (third generation) of cephalosporins and monobactams but do no affect cephamycins

• Enzyme activity may vary

• If an ESBL is detected, all penicillins, cephalosporins, and aztreonam should be reported as resistant

• Especially consider Escherichia and Klebsiella as potential ESBL-producing organisms

D-test

• Used for detection on inducible clindamycin resistance

• Clindamycin 2μg disks and erythromycin 15μg disks used

• Inducible strains form a “D”- shaped zone of inhibition

Detection of MRSA

• Zone of ≤ 10mm with no oxacillin 1μg/ml disk on Mueller-Hinton

• Molecular tests for mecA gene

Probable Cause of Tetracycline Zone too large and Clindamycin too small with E.coli or S. aureus controls

pH of agar too low

Probable Cause of Tetracycline Zone too small and Clindamycin too large with E.coli or S. aureus controls

pH of agar too high

Probable Cause of Aminoglycoside Zone too small with P. aeruginosa control

Ca⁺⁺ and/or Mg⁺⁺ too high in agar

Probable Cause of Aminoglycoside Zone too large with P. aeruginosa control

Ca⁺⁺ and/or Mg⁺⁺ too low in agar

Probable Cause of Zones Universally too large on Control Plates

• Inoculum too light

• Nutritionally poor medium

• Slow-growing organism (not seen with controls)

• Agar depth too thin

Probable Cause of Zones Universally too small on Control Plates

• Inoculum too heavy

• Agar depth too thick

Probable Cause of Methicillin Zone Indeterminate in Disk Test

Methicillin being degraded by strong β-lactamase producing Staphylococci

Probable Cause of Methicillin Zone Decreasing over Days or Weeks with Control Organisms

Methicillin degrading during refrigerator storage

Probable Cause of Colonies within Zone of Inhibition

• Mixed culture

• Resistant mutants within zone

Probable Cause of “Zone within a Zone” Phenomenon

• A swarming Proteus

• Feather edges of zones around penicillin or ampicillin disks usually occur with β-lactamase neg. strains of S. aureus

• β-lactamase pos.

  • H. influenzae with penicillin or ampicillin

Routine Medias and their Purpose

• Blood Agar (BA, BAP) - Most bacteria; determines hemolytic reactions

• Chocolate Agar - Haemophilus and Neisseria sp.; enriched with Hemoglobin or IsoVitalleX

• Phenylethyl Alcohol Agar (PEA) - Selects for gram pos cocci and anaerobic gram neg bacilli

• Columbia Colistin-Nalidixic Acid (CNA) - Selects for gram pos cocci

• Thayer-Martin Agar - N. gonorrhoeae and N. meningitidis

• CAMPY-Blood Agar - Campylobacter sp.

• Thioglycolate Broth - “Back-up” for Anaerobes

• Lowenstein-Jensen Agar, Middlebrook 7H10 Agar, and Petragnani Agar - Mycobacterium sp.

Anaerobic Media and their Purpose

• Bacteroides Bile Esculin Agar (BBE) - Selects for B. fragilis Group (Black Colonies)

• Kanamycin-Vancomycin Laked Blood (KVLB) - Bacteroides sp. (Enhances Pigment Production

• Cycloserine-Cefoxitin Fructose Agar (CCFA) - C. difficile

• CDC Anaerobic Blood Agar - Anaerobes (enriched with Hemin, Cystine, and Vitamin K)

• Cooked or Chopped Meat Medium - Anaerobes

Special Media and their Purpose

• Bordet-Gengou Agar - B. pertussis

• Buffered Charcoal Yeast Extract (BYCE) - Legionella sp.

• Cystine-Glucose Agar - F. tularensis

• Fletcher’s Medium - Leptospira

• Skirrow Agar - Helicobacter pylori

• Thiosulfate Citrate-Bile Salts (TCBS) - Vibrio sp.

• Vaginalis Agar (V-Agar) (human blood) - Gardnerella vaginalis

• Cystine-Tellurite Blood (Tinsdale) - C. diphtheriae (Black Colonies)

• Loeffler’s Medium - C. diphtheriae (Enhances Grouping and Metachromatic Granules)