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Microbiology
| Question | Answer |
|---|---|
| Bacterial growth requirements | • Temperature • pH • Osmotic pressure • Oxygen • Other atmospheric requirements |
| Bacterial growth requirements: temperature | • Psychrophiles- cold loving; 15°C • Mesophiles- moderate temp; 37° (most pathogenic organisms) • Thermophiles- heat loving; 50-60° |
| Bacterial growth requirements pH | • Bacteria 6.5-7.5 • Fungi 5.0-6.0 • Phosphate salts used to buffer media |
| Bacterial growth requirements osmotic pressure | • Isotonic • Halophiles- (Vibrio) –prefer higher salt concertation |
| Bacterial growth requirements oxygen | • Aerobes- requires O2 • Facultative anaerobes- can grow with or without O2 • Obligate anaerobes- harmed by O2 • Formation of superoxide radicals, toxic; neutralized by catalase and superoxide dismutase (possessed by aerobes and facultative) |
| Bacterial growth requirements other atmospheric requirements | • Microaerophilic- prefer lower O2 than in air • Capnophiles- prefer higher CO2 than in air |
| Culture media: agar | • Polysaccharide derived from marine algae Melts at 100°C Solidifies at approximately 45°C |
| Culture media: complex media | Most common; made of peptones and extracts |
| Culture media: anaerobic media | • Contains reducing agents which bind with dissolved O2 (thioglycolate, cysteine) • Broth tubes should be heated prior to use to drive out O2 •Gas pak envelopes |
| Culture media: anaerobic media gas pak envelopes | • Contain Na2CO3 & sodium borohydride • Add water- produces H2 & CO2 (aids in growth) • Palladium pellets catalyze the reaction • Some require hemin, vitamin K, and yeast extract |
| Incubation | • 5-10% CO2 (incubator or candle jar) • 35-37°C • 50-70% humidity |
| Growth Curve | • Lag phase- enzyme synthesis and cell elongation • Long phase- active reproduction • Stationary phase- exhaustion of nutrients, ↑ waste, ↓ pH, less viability • Death phase- dead cells exceed new cells |
| Sterilization/Inhibition Techniques: heat- denatures protein | • Moist- autoclave (steam under pressure) 15lbs pressure/sq in 121°C, 15mins QC- Bacillus stearothermophilus and B. subtilis • Dry heat Flame, incinerator, hot air oven 170°C, 2hrs • Pasteurization 72°C, 15seconds NOT sterilization |
| Sterilization/Inhibition Techniques: filtration | • Pore size 0.22u-0.45u • Used for sugar solutions, urea media, vaccines |
| Sterilization/Inhibition Techniques: refrigeration | Slows growth |
| Sterilization/Inhibition Techniques: dessication | No multiplication, but organisms remain viable (lyphilization) |
| Sterilization/Inhibition Techniques: osmotic pressure hypertonic | • Causes plasmolysis • “Cured” meat, fruit preserves |
| Sterilization/Inhibition Techniques: radiation | • Forms hydroxyl radicals • Damages DNA |
| Sterilization/Inhibition Techniques: Disinfection | • Phenol- damages cytoplasmic membrane, denatures protein • Halogens (iodine and chlorine) – oxidizers |
| Antimicrobial Therapy: narrow spectrum | Only certain groups covered (Penicillin- gram pos) |
| Antimicrobial Therapy: broad spectrum | Gram pos and gram neg coverage |
| Antimicrobial Therapy: selective toxicity | Action against only microbial structures (70s ribosome, cell wall, etc.) |
| Antimicrobial Therapy: bactericidal action | Kills bacteria without host immune help |
| Antimicrobial Therapy: bacteriostatic action | Reversible inhibition (ultimate destruction depends on host defenses) |
| Antimicrobial Therapy: 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 | • Mueller-Hinton agar • Depth = 4mm • pH = 7.2-7.4 • Physiologic concertation of Ca++ and Mg++ • 35° ambient air • 10^8th organisms (McFarland 0.5) • QC weekly and with each new lot of agar or discs (E. coli, S. aureus, P. aeruginosa) |
| Susceptibility Testing: Broth methods Minimum inhibitory concentration (MIC) | • MIC (minimum inhibitory concentration) Lowest concentration of drug that prevents in vitro growth First dilution tube with no visible growth |
| Susceptibility Testing: Broth methods Minimum bacteriostatic concentration (MBC) | • MBC (minimum bacteriostatic concentration) Lowest concertation that results in >99.9% killing Subculture tubes near MIC to find first plate with no growth |
| Susceptibility Testing: Broth methods Schlichter test | • Serum bactericidal assay • Test patient serum (containing antibiotic) against the infection organism |
| Antibiotics and their actions: B-lactams | Ex: Penicillin's, cephalosporins, carbapenems (Imipenam), Monobactams (Azotreonam), B-lactamase inhibiting combinations (Augmentin, etc.) Action: Inhibits cell wall synthesis Notes: Oxacillin, nafcillin, methicillin for penicillin-resistant Staph |
| Antibiotics and their actions: Glycopeptides | Ex: Vancomycin Action: Inhibits cell wall synthesis Notes: Drug of choice for Clostridium difficile and for MRSA |
| Antibiotics and their actions: Aminoglycosides | Ex: Gentamicin, tobramycin, amikacin Action: Inhibits protein synthesis Notes: Acts on 30S subunit; not active against anaerobes; used with a penicillin for Enterococcus |
| Antibiotics and their actions: Tetracycline | Ex: Tetracycline, doxycycline Action: Inhibits protein synthesis Notes: Acts on 30S subunit; effects bone and teeth in children; may lead to superinfection of yeast |
| Antibiotics and their actions: Chloramphenicol | Ex: Chloramphenicol Action: Inhibits protein synthesis Notes: Acts on 50S subunit; can cause aplastic anemia |
| Antibiotics and their actions: Macrolides | Ex: Erythromycin, clindamycin Action: Inhibits protein synthesis Notes: Acts on 50S subunit; clindamycin for gram + and gram - anaerobes |
| Antibiotics and their actions: Quinolines | Ex: Ciprofloxacin, norfloxacin Action: Inhibits nucleic acid synthesis Notes: For Pseudomonas aeruginosa and other aerobes |
| Antibiotics and their actions: Sulfa drugs (Sulfonomides) | Ex: Sulfamethoxazole Action: Analogue of PABA (intermediate in folic acid synthesis) Notes: For UTI, enteric infections; used with trimethoprim (Bactrim, etc.) |
| Routine Media and its purpose : Blood agar (BA, BAP) | Most fastidious bacteria; determines hemolytic reactions |
| Routine Media and its purpose : Chocolate agar | Haemophilus and Neisseria; enriched with hemoglobin or isovitelex |
| Routine Media and its purpose : Phenylethyl Alcohol agar (PEA) | Gram positive Cocci; selects for gram negative anaerobic Bacilli |
| Routine Media and its purpose : Columbia Colistin-Nalidixic Acid (CAN) | Selects for gram positive Cocci |
| Routine Media and its purpose ; Thayer-Martin agar | N. gonorrhoeae and N. meningitides |
| Routine Media and its purpose : CAMPY Blood agar | Campylobacter |
| Routine Media and its purpose : Hektoen Enetric agar (HE) | Salmonella and Shigella |
| Routine Media and its purpose : MaConkey agar (MAC) | Gram negative |
| Routine Media and its purpose : Sabouraud dextrose agar | Fungi and yeast |
| Routine Media and its purpose : Thioglycolate Broth | ‘Back up’ for anaerobes |
| Routine Media and its purpose ; Lowestein-Jensen agar | Mycobacterium sp. |
| Routine Media and its purpose ; Middlebrook 7H10 agar | Mycobacterium sp. |
| Routine Media and its purpose : Petragnani agar | Mycobacterium |
| Anaerobic Media and its purpose: Bacteroides Bile Esculin agar (BBE) | Selects for B. fragilis Group (black colonies) |
| Anaerobic Media and its purpose: Kanamycin-Vancomycin Leaked Blood (KVLB) | Bacteroides sp. (enhances pigment production) |
| Anaerobic Media and its purpose: Cycloserine-Cefoxitin Fructose agar (CCFA) | C. difficile |
| Anaerobic Media and its purpose: CDC Anaerobic Blood agar | Anaerobes (enriched with Hemin, Cystine, and Vitamin K) |
| Anaerobic Media and its purpose: Cooked or chopped meat medium | Anaerobes |
| Special Media and its purpose: Bordet-Gengou Agar | B. pertussis |
| Special Media and its purpose: Buffered Charcoal Yeast Extract (BCYE) | Legionella sp. |
| Special Media and its purpose: Cystine-Glucose Agar | P. tularensis |
| Special Media and its purpose: Fletcher’s Medium | Leptospira |
| Special Media and its purpose: Kelly’s Medium | Borrelia burgdorferi |
| Special Media and its purpose: Skirrow Agar | Helicobacter pylori |
| Special Media and its purpose: Thiosulfate Citrate-Bile Salts (TCBS) | Vibrio sp. |
| Special Media and its purpose: Vaginalis Agar (V-Agar) (human blood) | Gardnerella vaginalis |
| Special Media and its purpose: Cystine-Tellurite Blood (tinsdale) | C. diphtheria (black colonies) |
| Special Media and its purpose: Loeffler’s Medium | C. diphtheria (enhances grouping and Metachromatic granules) |
| Schick test | Corynebacterium diphtheria |
| Dick test | Susceptibility to Scarlet Fever (Streptococcus pyogenes) |
| Schlichter test | Serum bactericidal level |
| Schultz-Charlton test | Antiserum injected into rash; blanches if Scarlet fever |
| Elek test | Confirms toxin production for Corynebacterium diphtheria (in vitro) |
| Specimen Collection and Handling | • Material from infection site • Optimal time • Sufficient quantity • Appropriate collection devices •NEVER refrigerate spinal fluids, anaerobic, or GC specimens • Collect prior to antibiotic therapy • Set up within 2 hours of collection |
| Criteria for Rejection | • Preservatives used • Insufficient quantity • Dry swab • Leaky containers- contaminated specimen as well as biohazard |
| Blood Culture Collection | • Prep skin properly • Best time just prior to fever spike • Draw at least 2 sets BUT NO more than 3 in 24hrs • May use antibiotic removal device (ARD) patient on antibiotics • Best for fungi & acid fast •1:10 blood broth; adult at least 10mL |
| Darkfield microcopy for | Spirochaetes reflected light |
| Culture conditions: plates conditions | • Plates incubated at 35-37°C Campylobacter- 42°C Yersinia- 25-30°C 5-10% CO2 (Campy-microaerophilic) 50-70% humidity |
| Culture conditions: anaerobic conditions | • Anaerobic conditions Broths with thioglycolate or cysteine Pre-reduced media Gas pak jars or anaerobic chamber Environment: 10% H2, 5% CO2, 85% N2, palladium crystals |
| Non-selective media | • Supports most organisms • Blood agar, chocolate agar, tryptcase agar |
| Selective agar | • Contains chemicals, dyes, antibiotics to inhibit certain organisms (EMB, MAC, CAN, Campy-blood) • May also be differential (HE, SS, XLD, EMB, MAC) |
| Inoculation | • Platinum or disposable loops • Calibrated 0.01mL or 0.001mL (0.001mL for urine colony count plates) • Number of colonies X 100 (0.01 loop) or number of colonies X 1000 (0.001 loop) • Read and report after 18-24hrs |
| What color is gram positive | Purple |
| What color is gram negaitve | Pink |
| What is used for the gram stain | 1. Primary stain is Crystal violet 2. Decolorizer is alcohol/acetone 3. Counterstain is Safranin |
| Principle of the gram stain | • Iodine mordant • Methanol or heat fix • Alcohol/acetone for decolorizer • Violet dye and iodine form complex in cell, washes out of gram neg cells |
| Gram Positive Cocci: Staphylococcus | Grape like clusters |
| Staphylococcus aureus | • Coagulase positive • Most common pathogen of genus |
| S. aureus common infections | • Furncles (boils) & carbuncles • Bullous impetigo (listers) • Paronychia (nails) • Post surgical wounds & bacteremia |
| S. aureus intoxications | • Scalded skin syndrome (exfoliation-neonates) • Toxic shock syndrome (TSSST-T) women ages 12-52 • Food poisoning (enterotoxin)- symptoms in 1-5hrs after ingestions (cream dishes like potato salad) |
| S. aureus exotoxins | • Hemolysins • Leukocidins, coagulase, and hyaluronidase (spreading factor) • Nuclease, protease, and lipase |
| S. aureus resistance/sensitivity | • Most resistant to penicillin due to plasmid mediated B-lactamase • Many sensitive to penicillinase-resistant penicillins (PRP’s) (methicillin, oxacillin, etc.) ; if methicillin-resistant S. aureus (MRSA), vancomycin is drug of choice |
| Coagulase negative Staphylococcus | Opportunist in immunocompromised host and patients with prosthetic valves and devices |
| Staphylococcus aureus lab diagnosis | • BAP- soft, opaque, regular colonies 2-3mm in diam; some are beta hemolytic, and dome have pale golden color • Growth in 7.5% NaCl and ferment mannitol • Catalase positive • Phage typing and susceptibility profile for epidemiologic studies |
| Catalase test | • Reagent: 3$ H2O2 • Add one drop to colony on slide • If catalase present, H2O2 is broken down to water and O2 which bubbles off • Positive: Staph • Negative: Strep |
| Coagulase test | • Reagent-EDTA rabbit plasma • Bound coagulase-clumping on slide (plasma & colony) • Free coagulase-gels in tube test (0.5mL plasma and colony; 35-37°C 4-12hrs) • Agglutination tests-detects coagulase & protein A • Pos: S. aureus • Neg: other Staph |
| S. epidermidis | • Coagulase neg • Infections: endocarditis, prosthetic device infections • Slime capsule • Most methicillin resistant, sensitive to novobiocin |
| S. saprophyticus | • Coagulase neg • Infections: UTI in young women • Resistant to novobiocin |
| Streptococcus | • Spherical or oval; chains or pairs • Lancefield grouping based on C carbohydrate |
| Streptococcus pyogenes (Group A) | • Beta hemolytic • Streptolysin S- stable in O2; non-antigenic • Streptolysin O- oxygen labile; antigenic • Erythrogenic toxin- rash of scarlet fever • Highly sensitive to penicillin |
| S. pyogenes (Group A) infections | • Pharyngitis (Strep- most common cause) • Impetigo • Erysipelas • Wounds, burns • Rheumatic fever (autoimmune sequalae to infection with Streptococcus group A) |
| S. pyogenes (Group A) infections lab diagnosis | • Sensitive to 0.04 units bacitracin disc • Typing |
| S. agalactiae (Group B) | • Narrow zone of beta hemolysis • Neonatal sepsis and meningitis, UTI, vaginal infections |
| S. agalactiae (Group B) lab diagnosis | • Serotyping • CAMP reactions (with S. aureus) • Na hippurate positive |
| Streptococcus (Group D) | • Enterococcus • Growth in 40% bile and 6.5% salt • Alpha, beta or gamma hemolysis • Non-enterococcal- no growth in 6.5% salt • Lab diagnosis- bile esculin positive |
| S. pneumoniae | • Alpha hemolytic crater-like colonies or mucoid, “water drop” colonies • Lancet-shaped diplococci • Check sensitivity to penicillin using OX (oxacillin) disk (>20mm=sensitive) |
| S. pneumoniae causes | • Primary labor pneumonia (rusty sputum) • Meningitis • Bacteremia • Otitis media |
| S. pneumoniae lab diagnosis | • Typical colony morphology • Quelling reaction • Sensitive to optochin • Bile soluble |
| Other alpha Streptococcus (viridians groups) | Subacute bacterial endocarditis |
| Remember Strep | • Pairs or chains • Catalase Neg. Catalase Pos= S. aureus • Growth in 6.5% NaCl (Enterococcus) |
| Remember Staph | • Cluster (grape like) • Catalase Pos • Growth in 7.5% NaCl |
| Streptococci (Catalase neg): S. pyogenes (group A) | • Hemolysis: Beta hemolysis= complete clearing around colony • Bacitracin: Key reactions • Na Hippurate: Neg • Optochin: Neg • Bile Solubility: Neg • Bile Esculin: Neg • 6.5% NaCL: Neg • Infections: Pharyngitis. wound, scarlet fever, impetigo |
| Streptococci (Catalase neg): S. agalactiae (group B) | • Hemolysis: Beta (complete clearing) and no hemolysis • Bacitracin: Resistant • Na Hippurate: Pos • Optochin: Neg • Bile Solubility: Neg • Bile Esculin: Neg • 6.5% NaCL: Neg • Infections : Neonatal septicemia and meningitis, UTI (CAMP +) |
| Streptococci (Catalase neg): Entercoccus (group D) | • Hemolysis: Alpha= greening around colony, Beta (clearing), and no hemolysis • Bacitracin: Resistant • Na Hippurate: Neg • Optochin: Neg • Bile Solubility: Neg • Bile Esculin: Pos • 6.5% NaCL: Pos • Infections: UIT, endocarditis |
| Streptococci (Catalase neg): Non-enterococcus (group D) | • Hemolysis: Alpha (greening) and no hemolysis • Bacitracin: Resistant • Na Hippurate: Neg • Optochin: Neg • Bile Solubility: Neg • Bile Esculin: Pos • 6.5% NaCL; Neg • Infections: Endocarditis (rare) |
| Streptococci (Catalase neg): S. pneumonia | • Hemolysis: Alpha (greening) "water drop"/crater colonies • Bacitracin: Resistant • Na Hippurate: Neg • Optochin: Pos • Bile Solubility: Pos • Bile Esculin: Neg • 6.5% NaCL: Neg • Infections: Pneumonia, meningitis. bacteremia |
| Streptococci (Catalase neg): S. viridans | • Hemolysis: Alpha (greening) and no hemolysis • Bacitracin: Resistant • Na Hippurate: Neg • Optochin: Neg • Bile Solubility: Neg • Bile Esculin: Neg • 6.5% NaCL: Neg • Infections: Endocarditis (rare) |
| Gram Negative Cocci: Neisseria and Moraxella key characteristic | • Diplococci (kidney bean shape) • Oxidase positive |
| Neisseria gonorrhoeae | • Grows on chocolate and Thayer-Martin (contains vancomycin, colistin, nystatin, hemoglobin) • Requires 5-10% CP2; may take 48hrs for growth • Ferments glucose |
| Gonorrhea (cotton swab=toxic) | • STI • Can be asymptomatic; may be mixed with Chlamydia • Gram stain sensitive males but NOT females • Do NOT refrigerate prior to culture • Penicillin or spectinomycin sensitive; perform beta lactamase test to determine penicillin sensitivity |
| Neisseria meningitis | • Grows on blood agar, chocolate, and Thayer-Martin • 5-10% CO2 enhances growth • Ferments glucose and maltose • Transmitted by respiratory droplets and requires close contact |
| Meningitis | • Seen mostly in children under 3 • Waterhouse- Friderichsen syndrome (scattered petechiae) = meningococcemia • Mainly caused by types A, B, C, Y, and W |
| Moraxella (Branhamella) catarrhalis | • Respiratory infections • Grows well on chocolate and BAP but not MacConkey’s • Colony hard and ‘moves over” asaccharolytic • Usually beta lactamase positive |
| Bacterial meningitis | • ↑neutrophils • ↓ glucose • ↑ protein |
| Remember differentiating Neisseria | Glucose Maltose Lactose • N. gonorrhoeae + - - • N. meningitidis + + - • N. lactamase + + + All ferment glucose, Gonorrhoeae only Glucose, Meningitis= glucose & Maltose, Lactamase= glucose & Lactose |
| Gram Positive Rods: Corynebacterium Diphtheria key characteristics | • Small pleomorphic rods w/ clubbed ends • Palisade “Chinese letter” • Metachromatic granules (stain redpurple w/ methylene blue) • Tinsdale agar-black colonys from tellurite hydrolysis • Elek test-determines toxin production • White/gray nonspore |
| Corynebacterium Diphtheria Loeffler’s | Inhibits normal flora and enhances palisade arrangement and metachromatic granules |
| Corynebacterium Diphtheria | • Exotoxin productions by only lysogenic organisms carrying a B phage • Produces pseudomembrane on tonsils, uvula, or soft palate • Causes diphtheria |
| Gram Positive Rods: Listeria Monocytogenes key characteristics | • Small colonies with narrow zone of beta hemolysis • Catalase positive • Tumbling motility; “umbrella” motility in SIM @RT but NOT 37°C • Bile esculin positive • (non-spore) |
| Listeria Monocytogenes causes | Neonatal meningitis and sepsis; sepsis in immunocompromised hosts |
| Gram Positive Rods: Erysipelothrix key characteristics | • Non-motile • Catalase negative • “test tube brush” growth in gelatin; H2S positive in TSI • (non-spore) |
| Erysipelothrix is seen in occupations in | Occupational infection for fishermen, butchers, veterinarians, rose growers |
| Gram Positive Rods; Bacillus Sp. (Spore formers) key characteristics | • Larger ground glass colonies • Beta hemolysis (except B. anthracis) • Catalase positive • Large gram positive to variable rods in chains with spores |
| B. anthracis | • Very long chains (bamboo shoots) • “Medusa head” colonies • Non-motile, non-hemolytic • Anthrax- cutaneous, pulmonary, or gastrointestinal |
| B. cereus | Food poisoning due to preformed toxin- “fried rice” |
| Corynebacterium | • Catalase: Pos • Esculin: Neg • H2S/TSI: Neg • Beta hemolysis: +/- • Chinese letter arrangement, metachromatic granules (Loeffler's slants), Tellurite hydrolysis (Tinsdale agar), Elek test determines toxin production |
| Listeria | • Catalase: Pos • Esculin: Pos • H2S/TSI: Neg • Beta hemolysis: Pos • Tumbling motility at 25C but Not 37C, cold enrichment, neonatal meningitis and sepsis. sepsis in immunocompromised host |
| Erysipelothrix | • Catalase: Neg • Esculin: Neg • H2S/TSI: Pos • Beta hemolysis: Neg • "Test tube brush" growth in gelatin, infection in fishermen, butchers and veterinarians |
| Bacillus | • Catalase: Pos • Esculin: Varriable • H2S/TSI: Neg • Beta hemolysis: -/+ • "Ground glass" hemolytic colonies, • B. anthracis (Non-hemolytic, non-motile, "Medusa" head colonies), long bamboo shoots. • B. cereus (food poising, enterotoxin) B hemolytic |
| Gram Negative Rods: Enterobacteriaceae | • Peritrichous flagella when motile • Ferment glucose • Reduce NO3 to NO2 • Oxidase negative • Antigens use in typing |
| Enterobacteriaceae: antigens use in typing | • Flagella = H Ag • Envelope = K Ag • Cell wall LPS (lipopolysaccharide) = O Ag • All possess LPS endotoxin; some produce exotoxins |
| Gram Negative Rods: Escherichia coli | • Key characteristics Indole and lactose positive IMViC = +±- • Most common cause of UTI in females • Intestinal infections • K1 strains can cause neonatal meningitis |
| Escherichia coli intestinal infections: Enterotoxigenic E. coli | LT toxin (heat labile) & ST toxin (heat stable) |
| Escherichia coli intestinal infections: Enteroinvasive E. coli | Penetrate epithelial cells in large intestine |
| Escherichia coli intestinal infections: Enterohemorrhagic E. coli- strain 0157:H7 | • Shigella-like toxin • Food poisoning associated with undercooked meat • Hemolytic uremic syndrome |
| Gram Negative Rods: Shigella key characteristics | • Lactose negative • Non-motile • Anaerogenic |
| Shigella | • Bacillary dysentery- penetrate epithelial cells in small intestine (fecal oral) • <200 organisms needed for disease |
| Shigella groups and what is most sever and most common | • S. dysenteria (Group A) – most sever • S. flexneri (Group B) • S, boydii (Group C) • S. sonnei (Group D) – most common |
| Remember Shigella: S. dysenteria | Group A (longest letters) |
| Remember Shigella: S. flexneri | Group B (2nd longest letters) |
| Remember Shigella: S. boydii | Group C (3rd longest letters but "b" comes before "s" |
| Remember Shigella: S. sonnei | Group D (3rd longest letters but "s" comes after "b" |
| Gram Negative Rods: Klebiellae | • Opportunist; UTI, pneumonia; ampicillin-resistant • Non-motile • Has capsule • Urea positive • Ornithine negative • Can cause labor pneumonia |
| Klebiellae Enterobacter | • Motile • Ornithine positive |
| Klebiellae Serratia | • May produce red pigment • DNase positive |
| Gram Negative Rods: Salmonella | Large number needed for infection (>10,00) |
| Salmonella biochemical reactions | • H2S positive • Lysine positive • Indole negative • Urea negative |
| Salmonella cholerasuis | May cause septicemia |
| Salmonella typhi | • Typhoid fever • Blood positive early- 1st week • Stool positive in 2nd and 3rd week |
| Salmonella Arizona | ONPG pos (others neg) |
| Gram Negative Rods: Cirtobacter | • Opportunist • Lysine negative • Similar to Salmonella biochemically |
| Gram Negative Rods: Proteus | • Key characteristics Urea positive Deaminase positive • P. mirabilis Most sensitive to penicillins Indole negative • P. vulgaris Indole positive H2S positive |
| Gram Negative Rods: Yersinia | • Not motile • Y. enterocolitica Optimal growth =RT, cold enrichment Invasive and toxigenic • Y. pseudotuberculosis- acute mesenteric lymphadenitis and “pseudotubercules” • Y. pestis- bubonic plague |
| Common Gram Neg selective media | • MAC( MacConkey) • EMB (Eosin Methylene Blue) • HE (Hektoan-Enteric) • SS (Salmonella-Shigella) • XLD (Xylose-Lysine Deoxycholate) |
| Common Gram Neg selective media: MAC( MacConkey) | • Differentiating agent: Lactose • Selective agent: Crystal violet, Bile salts • H2S indicator: None • Lactose pos: Red • Lactose neg: Transparent |
| Common Gram Neg selective media: EMB (Eosin Methylene Blue) | • Differentiating agent: Lactose, Sucrose • Selective agent: Eosin Y Methylene Blue • H2S indicator: None • Lactose pos: Green sheen, purplish and brownish amber • Lactose neg: Transparent |
| Common Gram Neg selective media: HE (Hektoan-Enteric) | • Differentiating agent: Lactose, Sucrose, Salicin • Selective agent: Bile salts • H2S indicator: Sodium Thiosulfate • Lactose pos: Salmon/yellow • Lactose neg: Green to blue (H2S+=Black) |
| Common Gram Neg selective media: SS (Salmonella-Shigella) | • Differentiating agent: Lactose • Selective agent: Brilliant green, Bile salts • H2S indicator: Sodium Thiosulfate • Lactose pos: Red • Lactose neg: Transparent (H2S+=Black center) |
| Common Gram Neg selective media: XLD (Xylose-Lysine Deoxycholate) | • Differentiating agent: Lactose, Sucrose, Xylose • Selective agent: Bile salts • H2S indicator: Sodium Thiosulfate • Lactose pos: Yellow • Lactose neg: Transparent on Red medium (H2S+=Black) |
| Oxidase test | • Reagent tetramethy p-phyenylenediamine dihydrochloride • Positive = blue |
| Nitrate test | • Reagent- α-naphthylamine, sulfanilic acid • Positive = pink (use zinc powder to confirm) |
| ONPG test | • Detects B-d-galactosidase • Reagent- O-nitrophenyl-B-d-galactopyranoside • Positive = yellow |
| TSI (triple sugar iron agar) slant | • 0.1% glucose, 1% sucrose, 1% lactose • Yellow butt- glucose fermented • Yellow slant- lactose or sucrose fermented • Red slant- neither lactose nor sucrose fermented • Black butt- H2S produced |
| KIA test | Same as TSI but with only glucose and lactose and NO sucrose |
| Citrate test | • Media green • Positive = blue |
| Decarboxylase test measures | • Measures ability to decarboxylate amino acids • Lysine -> Cadaverine • Ornithine -> Putrescine • Arginine -> Putrescine • Indicator dye = bromcresol purple |
| Decarboxylase test: Lysine iron agar (LIA) | • Has H2S indicator • 0.1% glucose and 1% lysine • Positive = purple butt • Slant of LIA turns red for lysine deaminase |
| Decarboxylase test: Motility-indole-ornithine (MIO) | • 0.1% glucose and 1% ornithine • Positive = purple butt • Also test for motility and indole (Kovac’s) |
| Indole test | • Indole split from trypophan • Reagent (Kovac’s) – p-dimethyl-aminobezaldehyde • Positive = pink |
| Urease test | • Urea hydrolyzed to ammonia and CO2 • Phenol red indicator turns pink if positive |
| Voges-Proskauer (VP | • Detects acetylmethylcarbinol (acetoin) • Reagents = KOH and α-naphthol • Positive = pink |
| Phenylalanine deaminase | • Reagent = ferric chloride • Positive = green |
| TSI-Biochemical reactions: A/A | • Escherichia (Indole+) • Enterobacter (ODC+, sugars) • Klebsiella (ODC-) • Serratia (sugars) |
| TSI-Biochemical reactions: A/A, H2S+ | • Proteus (urea+, deam+) • Arizona (LDC+) • Citrobacter (LDC-) |
| TSI-Biochemical reactions: K/A, H2S+ | • Salmonella (mal-, ONPG-) • Citrobacter (LDC-) • Edwardsiella (indole+, LDC=) |
| TSI-Biochemical reactions: K/A | • Shigella (citrate-, non-motile) • Providencia (deam+) • Citrobacter (citrate+) • Proteus (urea+, deam+) • Yersinia (small colonies, urea+, deam-) |
| TSI-Biochemical reactions: K/K | Pseudomonas (ox+, blue-green pigment, growth at 42C, growth in cetrimide) |
| Remember Enterobacteriaceae (TSI reactions): picture yourself in a desert. The Yellow sun shining over the hot desert = A/A (SEEK shelter in the hot desert ) | A/A (sun over the desert) SEEK shelter in the hot desert Serratia Escherichia Enterobacter Klebsiella |
| Remember Enterobacteriaceae (TSI reactions): picture yourself in a desert. The moon shining over the desert = K/A (a CASE of fire crackers going off smells like sulphur (H2S+) ) | A/A, H2S+ (moon over the desert) a CASE of fire crackers going off smells like sulphur (H2S+) Citrobacter Arizona Salmonella Edwardsiella |
| Remember Enterobacteriaceae (TSI reactions): picture yourself in a desert. The moon over the mountain =K/K | K/K (moon over the moutain) Pseudomonas (Pseudo "moon"as) |
| Remember Enterobacteriaceae (TSI reactions): picture yourself in a desert. The Yellow sun shining over the desert =A/A, H2S+ (Wear a cap to protect yourself from the geyser (H2S+) ) | A/A, H2S+ (sun over the desert) (Wear a cap to protect yourself from the geyser (H2S+) Citrobacter Arizona Proteus |
| Remember Enterobacteriaceae (TSI reactions): picture yourself in a desert. The moon over the desert = K/A (Sk(c)ippy Coyote howls at the moon) | K/A (moon over the desert) (Sk(c)ippy Coyote howls at the moon) Shigella Citrobacter Providencia Proteus Yersinia |
| Enterobacteriaceae primary differentiating test: Citrobacter | • TSI: K(A)/A, Gas • Ornithine: Variable • VP: Neg • Urease: Neg • Lysine: Neg • Motility: Pos • H2S: V • Deaminase: Neg • DNAse: Neg |
| Enterobacteriaceae primary differentiating test: Enterobacter | • TSI: A/A, Gas • Ornithine: Pos • VP: Pos • Urease: Neg • Lysine: Variable • Motility: Pos • H2S: Neg • Deaminase: Neg • DNAse: Neg |
| Enterobacteriaceae primary differentiating test: Escherichia | • TSI: A(K)/A, Gas • Ornithine: Variable • VP: Neg • Urease: Neg • Lysine: Pos • Motility: Variable • H2S: Neg • Deaminase: Neg • DNAse: Neg |
| Enterobacteriaceae primary differentiating test: Klebsiella | • TSI: A/A, Gas • Ornithine: Neg • VP: Pos • Urease: Variable • Lysine: Pos • Motility: Neg • H2S: Neg • Deaminase: Neg • DNAse: Neg |
| Enterobacteriaceae primary differentiating test: Morganella | • TSI: K/A, Gas • Ornithine: Pos • VP: Neg • Urease: Pos • Lysine: Neg • Motility: Pos • H2S: Neg • Deaminase: Pos • DNAse: Neg |
| Enterobacteriaceae primary differentiating test: Proteus | • TSI: K/A, Gas • Ornithine: Variable • VP: Neg • Urease: Pos • Lysine: Neg • Motility: Pos • H2S: Pos • Deaminase: Pos • DNAse: Neg |
| Enterobacteriaceae primary differentiating test: Providencia | • TSI: K/A, Gas • Ornithine: Neg • VP: Neg • Urease: Variable • Lysine: Neg • Motility: Pos • H2S: Varible • Deaminase: Pos • DNAse: Neg |
| Enterobacteriaceae primary differentiating test: Salmonella | • TSI: K/A, Gas • Ornithine: Pos • VP: Neg • Urease: Neg • Lysine: Pos • Motility: Pos • H2S: Pos • Deaminase: Neg • DNAse: Neg |
| Enterobacteriaceae primary differentiating test: Serratia | • TSI: K(A)/A • Ornithine: Pos • VP: Pos • Urease: Neg • Lysine: Pos • Motility: Pos • H2S: Neg • Deaminase: Neg • DNAse: Pos |
| Enterobacteriaceae primary differentiating test: Shigella | • TSI: K/A • Ornithine: Variable • VP: Neg • Urease: Neg • Lysine: Neg • Motility: Neg • H2S: Neg • Deaminase: Neg • DNAse: Neg |
| Gram Negative Non-Fermenters | • Opportunists • Glucose NOT fermented (TSI=alklalk); lower acid production (OF media = lower peptide content) • Oxidase positive (some exceptions) • May not grow on MacConkey’s |
| Gram Negative Non-Fermenters -Pseudomonads | • Oxidase positive (except Burkholderia cepacia and Stenotrophomonas) • Motile by polar flagella |
| Pseudomonas aeruginosa | • Grape/corn smell • Most common • Lactose neg on MAC • Mucoid strain found in cystic fibrosis pt • Treated with aminoglycosides, 3rd generation cephalosporins and extended spectrum penicillins • Infections-burns, pneumonia, swim ear, eye in, UTI |
| Pseudomonas aeruginosa | • May produce: Pyocyanin (blue-green) Pyorubin (rust) Pyoverdin (blue-white under UV) |
| Gram Negative Oxidase Positive Fermenters- Vibrio characteristic | • Curved rods with polar flagella • TCBS selective for Vibrio • BAP- hemolytic colonies • Lactose negative (differs from enterics) • NO3 positive (NO3 to NO2) |
| Vibrio cholera (serogroup 01) | • Most serious • Gastroenteritis- rapid onset 3-10hrs; profuse diarrhea • Need high concentration of organism unless hypochorohydric • Stools contain mucus flecks (described as “rice water” stools) • Yellow on TCBS |
| Vibrio parahemolyticus | • Green on TCBS • Enteritis |
| Vibrio vulnificus | • 2nd worst • Green on TCBS |
| Vibrio septicemia can kill | Immunocompromised or diabetics |
| Campylobacter | Gram Negative Oxidase Positive Fermenters |
| Campylobacter jejuni | • Small curved rods (seagull appearance) (light staining) • Microaerophilic 2-4 days for growth • Growth on CAMPY agar • Found in raw poultry and contaminated water (like Salmonella) • Erythromycin or tetracycline for treatment |
| Campylobacter jejuni Biochemical reactions | • Catalase positive • Oxidase positive • Hippurate positive |
| Helicobacter pylori | • Associated with gastric and duodenal ulcers • Produces large amounts of urease |
| Aeromonas | • Motile by polar flagella • Most are indole positive • Growth on MAC • Cellulitis, wound infections, and diarrhea |
| Chromobacterium violaceum | • Grows on MAC • Motile by single polar and lateral flagellum • Produces violet color (seen best at room temp) |
| Zoonotic diseases (acquired directly or indirectly from animals)- Brucella | • Gram neg coccobacillus • Disease is Brucellosis • Blood culture Pos in first 2 weeks (hold 21 days) |
| Brucella abortus (Cows) | • CO2 + • H2S + • Thionine +/- • Basic fuschin + |
| Brucella sus (Pigs) | • CO2 - • H2S +/- • Thionine + • Basic fuschin - |
| Brucella melentensis (Goats) | • CO2 - • H2S - • Thionine + • Basic fuschin + |
| Remember: Brucella | • Cows eat Basic Fuschin • Pigs do NOT eat Basic Fuschin • Goats eat anything |
| Francisella tularensis | • Gram Neg coccobacillus • Pinpoint colonies ("mercury drop") • Cystine-Glucose media • H2S + with lead acetate • Disease: Tularemia "rabbit fever" • Infected by tick bite, high risk to lab personnel (wear gloves and work under hood) |
| Yersinia pestis | • Gram Neg • Bio-polar staining (resembles safety pin) • Disease: plague • Transmitted by fleas, rats, other mammal reservoirs |
| Pasturella multicida | • Gram Neg rod • Bio-polar staining • Oxidase and Indole Pos • Ferments glucose and sucrose • Contracted from cat and dog bites • Mousy odor/mushrooms |
| Streptobacillus monilliformis | • Long filamentous Gram Neg rod with swellings (pleomorphic); "puffball" or "string of pears" colonies in Thioglycolate broth • "Rat bite fever" Haverhill fever • Acitic fluid sample needed; SPS inhibits |
| Fastidious Gram Negative Rods | • Source- mouth flora • Pathogenic in immunocompromised host • Grow slowly and requires 5-10% CO2; NO growth on MAC |
| Fastidious Gram Negative Rods Pathogenic in immunocompromised host: | • Periodontal and jaw abscesses (Eikenella, Actinobacillus, Capnocytophaga) • Bacterial vaginosis (Gardnerella) • Bacteremia (Capnocytophaga, Cardiobacterium) |
| Gardnerella Vaginalis | • Bacterial vaginosis • “Clue cells” • 10% KOH added to discharge causes “fish” odor • Tiny colonies at 48hrs on BAP and chocolate • Catalase and oxidase negative; hippurate and starch positive |
| Clue cells | High number of squamous epithelial cells colonized with gram variable rods |
| Bordetella Pertussis | • Gram negative coccobacilli; causes “whooping cough” • Requires special media • Old method for collection- cough plate; better to collect NP swab and plate directly; fluorescent antibody technique more rapid |
| Bordetella Pertussis special media | • Classic = Bordet-Gengon (potato infusion with glycerol and 20% SRB’s) and penicillin • Regan and Lowe (oxoid charcoal agar, 10% horse blood, cephalexin); longer shelf life |
| Bordetella Pertussis colony | • Incubate 72-96hrs @35°C • Pinpoint “mercury droplet” colonies |
| Haemophilus | • Small, non-motile gram negative rod • Requires growth factors • Non-gonococcal urethritis, post partem bacteremia, neonatal meningitis (rare) • Conjunctivitis (pink eye) contagious Caused by H. aegyptius – probably a strain of H. influenza |
| Haemophilus characteristics part 1 | • Grow only on chocolate, NOT blood agar plate (unless mixed with S. aureus) • Use X & V disks on Mueller-Hinton agar to detect growth requirements |
| Haemophilus characteristics part 2 | • Susceptibility zone interpretations differ from conventional K-B zones (use Haemophilus test media (HTM), supplemented Mueller-Hinton agar and incubated in 5-10% CO2) |
| Haemophilus characteristics part 3 | • Perform beta lactamase to determine sensitivity to ampicillin; cefuroxime, ceftriaxone, cefotaxime (meningitis) |
| Haemophilus growth factors | • X factor = hemin • V factor = NAD • Both factors found in blood but nee to heat to break down RBC and release factors to chocolate used |
| Haemophilus satellitism | • S. aureus produces V factor and releases X factor by hemolyzing blood • Haemophilus will grow in the hemolytic zone surrounding staph as satellite colonies on blood agar plate • Need 5% horse or rabbit blood to hemolysis |
| Haemophilus infections | • Meningitis- H. influenza 1 months to 2 years Preceded by nasopharyngeal colonization and bacteremia |
| Haemophilus epiglottitis | • Ages 2-4, do NOT collect throat culture (blood culture best) • May required intubation |
| Chancroid- Haemophilus ducreyi | • Painful gential ulcers or soft chancres • Gram stain of drainage shows tiny gram negative coccobacilli with a “school of fish” arrangement |
| Mycoplasma and Ureaplasma | • Smallest free-living microorganisms • Lack cell wall (bound by single triple layered membrane) • Does NOT stain with Gram’s stain • Center of colony grows into special media (contains sterols) giving appearance of inverted “fried egg” |
| Mycoplasma pneumoniae | • Primary atypical pneumonia or “walking” pneumonia • Causes positive cold agglutinin titer (>1:32); false positive RPR • Treat with erythromycin or tetracycline |
| Ureaplasma urealyticum | • Non-gonococcal, non-chlamydial urethritis, especially in males • Produces urease • Treat with tetracycline or spectrnomycin |
| Mycoplasma hominis | • May colonize GU tract; postpartum fever • Tetracycline; resistant to erythromycin (all other Mycoplasma are sensitive) |
| Actinobacillus | • Oxidase +/- • Catalase + • Nitrate + • Indole - • Periodontal and jaw abscesses, high number in plaque; center of colony has 4-6 pointed star |
| Eikenella | • Oxidase + • Catalase - • Nitrate + • Indole - • "Bleachy" odor, pits agar, 3 zones of growth, periodontal and jaw abscesses |
| Capnocytophaga | • Oxidase - • Catalase - • Nitrate +/- • Indole - • Needs 5-10% CO2 or anaerobic conditions, gliding motility, fusiform shapr, bacteremia |
| Haemophilus aphrophilus | • Oxidase -/weak + • Catalase - • Nitrate + • Indole - • Colonies similar to Actinobacillus, endocarditis |
| Cardiobacterium | • Oxidase + • Catalase - • Nitrate - • Indole = • Can cause endocarditis, can give false pos gam reactions |
| Gardnerella vaginalis | • Tiny colonies at 48hrs on blood and chocolate, catalase Neg, Oxidase Neg, hippurate Pos, starch pos • Disease: bacteral vaginosis • "Clue cells" 10% KOH added to discharge=fishy odor |
| Bordetella pertussis | • Gram Neg coccobacillus, pinpoint, "Mercury droplet" colonies • Disease: Pertussis (Whooping cough) • Bordet-Gengou media; NP swab and plate directly |
| Haemophlius | • Small non-motile Gram Neg rods, H. ducreyi-school of fish • Disease: H. influenzae-causes influenza/meningitis/epiglottitis. H. ducreyi-causes genital ulcers • Requires X and V factors |
| Legionella pneumophillia | • Growth on BCYE • Disease: Legionnaires' disease • No growth routine media |
| Mycoplasma/Ureaplasma | • Colony appears as inverted fried egg • Disease: M. pneumoniae-causes primary atypical pneumonia (increased cold agglutinin titer) • Dienes stain NOT gram stain |
| Clues to anaerobic infection part 1 | • Foul odor • Location in close proximity to mucosal surface • Animal/human bit • Gas in specimen • Previous therapy with aminoglycosides |
| Clues to anaerobic infection part 2 | • Black discoloration of blood containing exudates • Presence of “sulfur granules” • Unique morphology on gram stain • Failure to grow organisms seen on smear aerobically • Growth in anaerobic zone or bubbles in fluid media • slime in UTI |
| Specimen and transport for Anaerobes | • Site containing a resident flora (oral, GI, GU) not appropriate for anaerobic culture • Best to aspirate with syringe and needle and place in a transport vial or tube under reduced conditions (swab samples NOT good) |
| Culture techniques for Anaerobes | • “classic” principle Jar technique (Gas Pak jar) Catalyst- palladium pellets Envelope generates H2 and CO2 when water is added Methylene blue or resazurin- indicators (blue and pink, respectively when oxidized; clear when reduced) |
| Other methods for Anaerobes | • Anaerobic bags • Roll tube technique • Anaerobic chamber |
| Other methods for Anaerobes Roll tube technique | PRAS )pre-reduced anaerobically sterilized media) inoculated under constant flow of O2- free gas |
| Other methods for Anaerobes Anaerobic chamber | • Plates put in chamber through a pass box that is reduced • Incubator in chamber; also contains palladium catalyst |
| Other methods for Anaerobes Anaerobic bags | Clear bag with gas generating ampules; plates can be read without opening bag |
| Examination of primary plate | • Pitting • Large colonies with double zone of hemolysis • Bread crumb or speckled colonies • Molar tooth colonies of gram positive branching rods • BBE • KVLB |
| Examination of primary plate: Pitting | Bacteroides ureolyticus (could be Eikenella) |
| Examination of primary plate: Large colonies with double zone of hemolysis | Clostridium perfringens; set up egg yolk agar for Naegler test |
| Examination of primary plate: Bread crumb or speckled colonies | Gram negative slender fusiforms- Fusobacterium nucleatum |
| Examination of primary plate: Molar tooth colonies of gram positive branching rods | Actinomyces |
| Examination of primary plate BBE | >1mm- Bacteroides fragilis |
| Examination of primary plate KVLB part 1 | • Look for pigment and examine under UV light Perform aero-tolerance test on colonies by sub-culturing each colony type to an anaerobic blood plate and a chocolate plate |
| Examination of primary plate KVLB part 2 and 3 | • Incubate chocolate at 36°C under 5-10% CO2 and the anaerobe BAP at 36°C in a gas jar or anaerobe chamber • If growth on both, organism is facultative; if growth only on anaerobic blood the organism is an anaerobe |
| Spirochetes- Spirillaceae | Rigid, helically curved rods with one or more turns; corkscrew motility by polar flagella; gram negative |
| Spirochetes- Spirillum minor | • “rate bite” fever • Visualize by darkfield or stain with Giemsa • 2-3 spirals and bipolar polytrichous tufts of flagella • No growth on artificial media |
| Spirochetes- Treponema pallidum | • Stain with silver impregnation • Darkfield- slow motility and flexion • No growth on artificial media • Sensitive to penicillin, tetracycline, and erythromycin • Detected through serological test • Syphilis |
| Treponema pallidum- Syphilis | • Primary lesion 2-10 weeks after infec, chancre appears Heals w/o treatment 3-8 weeks; may do darkfield or direct FA on fluid • 2nd lesion Dissemination- skin rash erosion on genitalia • Latent stage 2-20 years later Affects skin, bone, CNS |
| Leptospira | • Spirals w/hooked ends • Animal pathogen passed via H2O contaminated w/animal UA • + darkfield or direct FA • Can grow in Fletcher’s semi-solid media • Incubate 6 weeks @30°C in the dark • Perform darkfield from several centimeters into media |
| Borrelia recurrentis | • “Relapsing fever” from ticks or lice • Looser coils; best seen with Giemsa or Wright’s stain of blood smear • Mutates during disease; relapse due to inability to recognize new antigen • May exhibit cross reaction with Protens OX K on febrile agglutin |
| Borrelia burgdorferi | • Lyme disease • Transmitted by ticks deer/mouse • Chronic migratory erythematous rash, fever, muscle, joint pain; later meningioecephalitis, myocarditis, and arthritis • Culture in Kelly@33°C darkfield weekly 1month • Serological diagnos is faster |
| Chlamydia | • Obligate intracellular parasites • Gram negative cell wall, with no peptidoglycan; possess ribosomes for protein synthesis • Dependent on host for APT • Growth in yolk sac of chick embryo or tissue culture (McCoy cell) |
| Chlamydia- Lab diagnosis part 1 | • Giemsa stain (purple inclusion bodies) or iodine stain (glycogen synthesized in large amounts by C. trachomatis and surrounds elementary body; stains brown with iodine; not produced with other species) |
| Chlamydia- Lab diagnosis part 2 | • Direct FA. Tissue culture, or complement fixation |
| Chlamydia psittaci | • Psittacosis (parrot fever) – occupational hazard for pet bird handlers and poultry workers • Acute lower respiratory infection |
| Chlamydia trachomatis part 1 | •Eye infections Trachoma- leading cause of blindness in underdeveloped countries Inclusion conjunctivitis Adults and newborns Can colonize nasopharynx (-> pneumonia) and genital tract Appears 2-25 days after birth as a purulent eye discharge |
| Chlamydia trachomatis part 2 | • Genital tract infections (sexually transmitted disease) Non-gonococcal urethritis and epididymitis in males Cervicitis and salpingitis (PID) in females Can be passed to newborn as conjunctivitis or pneumonia |
| Rickettsiae | • Small gram negative coccobacilli • Obligate intracellular parasites • Spread by arthropod vector • Seen better with Giemsa • Arthropod bite- causes fever, headache, rash (Q fever- no rash and organism survives outside host) • Weil-Felix test |
| Rickettsiae- Weil-Felix test | • Protens OX-19, OX-2, and OX-K used as antigens to detect Rickettsial antibody • 4-fold rise in titer or 1:160 titer |
| Fungus-like Bacteria- Mycobacteria morphology | • Slim gram variable rods; don’t stain well due to high lipid content in wall |
| Fungus-like Bacteria- Mycobacteria | • Acid fast stain Ziehl-Neilsen- “hot” stain Kinyoun- “cold” stain • Auramine-Rhodamine- fluorescent stain • All stains based on presence of mycolie acid (lipid-waxy) in cell wall • Any number seen on a smear is significant |
| Fungus-like Bacteria- Mycobacteria growth requirements | • Lowestein-Jensen; 60 egg in nutrient base • Tween 80 (aleic acid) – aids in dispersing colonies in liquid media • ↑ CO2 (especially 24hrs) • 3-6 weeks for growth • Most grow at 36°C; some require 30°C |
| Fungus-like Bacteria- Mycobacteria specimen collection | • Sputum (fist morning; on 3 consecutive mornings); bronchial washing, gastrics, urine and tissue • Collect aseptically and place in sterile, tightly capped container • May be refrigerated overnight (neutralize gastrics and urines if holing overnight) |
| Fungus-like Bacteria- Mycobacteria specimen decontamination | • N-acetyl-L-cysteine (NALC) – mucolytic; NaOH decontaminates; time dependent • Stain and report slides within 24hrs of processing • Centrifuge decontaminated specimen for 20min at 300rpm prior to making smears and inoculating media (use sediment) |
| Viral structure | • RNA or DNA- NOT both • Does DON’T contain structural elements required for protein synthesis • Replicates in host cells |
| Virology specimen collection and handing | • Pre and post convalescent sera- ship on dry ice • Specimen for viral culture- similar to transport media for bacteria but contains nutrients (fetal calf serum or albumin) and antibodies |
| Virology lab methods | • EIA- presence of viral antibody or antigen (ex. HbsAg and anti-HBsAb) • Viral culture • Election microscopy • Molecular techniques |
| Virology special procedures- DNA | • DNA probes Molecular cloning of specific DNA sequence If viral unknown “matches” clone, the viral identity is confirmed |
| Virology special procedures- Polymerase chain reaction (PCR) | • Polymerase chain reaction (PCR) Method in which nucleic acid sequences can be amplified in vitro Carried out in cycles, each cycle doubling the amount of desired nucleic acid product |
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