Soap L. monocytogenes Chlamydia BAP M. gordonae DNase test LOA test Na hypochlorite Inoculating needles Wire loop 50k CFU/mL Pregnant Cotton swab
Charcoal Phenotype Stool PCR Somatic antigen Anton van Leeuwenhoek Robert Koch Louis Pasteur Ehrlich Bacteria Cell wall Gram (+) Gram (-) Plasma membrane Nucleoid
Yes MUST TO KNOW IN BACTERIOLOGY Germicidal Catalase (+) 3 C’s: Chicken, Coleslaw, Cheese When delayed: 4’C Freezing: -20’C Phenotypic Gram (+) colonies: Dry, white, sometimes gray Gram (-) colonies: Gray and moist Destroyed by chlorine Utilizes 1N HCl For Gram (+) For nonfermentative For Enterobacteriaceae Inactivates HBV (10mins) and HIV (2mins) Nichrome = F(+) on oxidase test Not longer than 5cm 2mm diameter 0.001mL urine Significant for UTI C. albicans Lactobacillus Carrier state Lawn a culture Toxic to Neisseria Good for virus Removes the toxin inoculated by cotton Basis of identifying organisms Gram stain and colonies Not Gram stained Most definitive method of identification Basis of serotyping Father of microbiology Microscopist 1st to describe bacteria Germ theory: relationship of organisms to human disease Father of Modern Microbiology 1st to use dyes for stain Ave. size: 0.4-2μm Reproduction: Binary fission (two-fold increase) Peptidoglycan (murein) Protoplast: wall less G(+) Spheroplast: wall less G(-) Thick peptidoglycan Teichoic acid Thin peptidoglycan LPS (Lipid A – exotoxin)jsjsb Somatic antigen Site for energy synthesis (ATP) Osmotic/permeability barrier Chromosome: dsDNA Plasmid: Extrachromosomal DNA lec.mt 04 |Page | 55
Plasmid Drug-resistance Metachromatic granules Ribosomes Common pili Sex pili ESBL Endospores Flagella
Lactobacillus Autotrophs/Lithotrophs Heterotrophs/ Organotrophs Temperature requirements pH requirement Moisture Salt concentration Respiration (Aerobic) Oxidation (Aerobic) Fermentation (Anaerobic) Lag phase Log/Exponential phase Stationary/plateau phase
Death/Decline phase Staining Capsule stain Not Gram stained Gram Stain (Hucker’s modification
Carries the antibiotic-resistance gene Chromosome and plasmid-mediated Food reserves Prokaryotic: 70S Eukaryotic: 80S Bacterial adherence Gene transfer By Gram (-) bacteria Calcium dipicolinate Bacillus, Clostridium Monotrichous: one only Amphitrichous: one at both ends Lophotrichous: tuft at one end Peritrichous: all around bacteria (most common) Aerotolerant anaerobes Inorganic compound as source of carbon (CO2) Organic compound as source of carbone (Glucose) Pathogenic bacteria Psychrophilic: 0-20’C (ref) Mesophilic: 20-40’C (pathogenic) Thermophilic: 40-60’C Acidophilic: Lactobacillus acidophilus (Doderlein bacillus) Neutrophilic: pH 7.2-7.6 (optimal) – pathogenic Basophilic: Vibrio (Halophilic) Humidophilic Halophilic Enterococcus and V. parahaemolyticus Glucose CO2 + H2O Kreb’s cycle Electron transport chain Glucose Acid Glucose Acid/Alcohol Embden-Meyerhoff pathway (glycolysis) Adjustment in growth rate (cell division) Susceptible to antimicrobial agents No net growth Death = Live cells Depletion of nutrients Accumulation of toxic wastes Sporulation Death rate Bacteria stain more by basic stains India ink Borris method Nigrosin method Chlamydia and Rickettsia = intracellular Mycoplasma and Ureaplasma = no cell wall Spirochetes Crystal violet = 1min Gram’s iodine = 1min Acetone-alcohol or 95% ethanol = 30secs-1min lec.mt 04 |Page | 56
Gram (+) becomes (-)
Gram (-) becomes (+) Acid Fast staining methods Pappenheim’s Baumgarten’s Fite Faraco Acid fast organisms
Ziehl-Neelsen (Hot method)
Kinyoun (Cold method)
Auramine-Rhodamine (Fluorochrome)
AFB Special stains
Safranin O = 30 secs Over-decolorization Old dying Acidic iodine Penicillin: omits iodine Under-decolorization Thick smear Smear = 2 x 3cm M. smegmatis vs. M. tuberculosis M. leprae vs. M. tuberculosis M. leprae Counterstain: Hematoxylin Mycobacterium Nocardia = Mod. AFS (1% H2SO4 as decolorizer) Cryptosporidium Legionella micdadei Rhodococcus equi Best AFS C-A-M 1. Carbolfuchsin = 1’ stain -Start timing: Vapor (10mins) -Heat = Mordant 2. 3% Acid alcohol = Decolorizer -HCl + 95% etOH -Until no more stain (Max: 3mins) 3. Methylene blue = counterstain -30secs to 1min Results: AFO = Red NAFO = Blue Not used C-A-M 1. Carbolfuchsin = 1’ stain -Phenol, Tergitol = Mordant 2. 3% Acid alcohol = Decolorizer 3. Malachite Green = Counterstain Results: AFO = Red NAFO = Green Most sensitive 1. Auramine-rhodamine = 1’stain 2. 0.5% Acid alcohol = Decolorizer 3. 0.5% KMnO4 = Counterstain Results: AFO = Yellow fluorescence NAFO = No fluorescence Read 300 fields Capsule = Negative stain Spore = Dorner, Wirtz, Conklin Metachromatic granules - Albert’s -Loeffler’s Alkaline Methylene Blue (LAMB) lec.mt 04 |Page | 57
Phase contrast microscope Electron microscope
Transmission EM Scanning EM Inverted Microscope Interference microscope Non staining method Pure culture
Mixed culture Stock culture Liquid Semi-solid Solid Biphasic General purpose media
Enriched media Enrichment media
Differential media
Selective media
Flagella = Leifson Nucleic acid = Feulgen Polar bodies (ex: Y. pestis) = Wayson Rickettsia = Gimenez Spirochetes = Levaditi For study of living unstained organisms For viruses Light source: Electrons 100,000x magnification Stains: -Negative stain -PTA -Heavy metals (Gold, Silver) DNA, RNA, chromosomes Surface structures (cell wall, capsule) For tissue culture Dual light source String’s test (3% KOH) Streak plate = overlap method Pour plate = Water and milk bacteriology Selective medium Animal inoculation = for virus, Chlamydia, Rickettsia 2 or more organisms Stored at refrigeratior or freezer (long term) Broth 0.5-1% agar 2-3% agar Both liquid and solid Ex. Castañeda = Brucella Nonfastidious organisms 1. Sheep BAP = Hemolysis 2. Horse BAP = Haemophilus -Heat-stable, provides X-factor 3. Nutrient agar Solid Fastidous organisms 1. CAP = Heat-labile, provides X & V factor Liquid 1. Selenite F 2. Alkaline peptone water 3. Thioglycollate broth 1. BAP = hemolysis 2. MAC 3. EMB 4. XLD 5. HEA Inhibitory media 1. TCBS 2. SSA 3. TMA 4. CBAP lec.mt 04 |Page | 58
Inhibitory agents PEA Columbia CNA Gonococci Agar (GCA) Gentamicin BAP Bacitracin CAP Cystine Tellurite Blood Agar Cystine Blood Glucose Agar Cystine Trypticase Agar Charcoal Cephalexin Blood agar Bordet-Gengou Agar (Potato Blood Glycerol Agar) BCYE McCoy TSB Sterile specimen Nonsterile specimen Calcium alginate swab Needle aspiration Catheterization Intubation Delay in processing
Transport medium
Biologic safety cabinet BSC Class I
BSC Class II
Antibiotics Dyes, bile salts = inhibit Gram (+) Alcohol (PEA) = inhibit Gram (-) Gram (+) bacteria Gram (+) bacteria Gram (-) cocci S. pneumoniae H. influenzae C. diphtheriae F. tularensis Confirm: Neisseria B. pertussis B. pertusis L. pneumophila Cl. trachomatis Brucella (-) normal flora (+) normal flora Toxic to virus Good for Neisseria Anaerobic and aerobic cultures Needle and syringe for collection Gastritis Vomitus Gastric washing (aerobic culture only) Refrigerate except: 1. CSF = Room temp. or 35’C 2. Blood 3. Swab of N. gonorrhoeae (sensitive to cold) 4. Urine = Boric acid 5. Rectal swab = Cary-Blair 1. Cary Blair = for stool pathogen 2. Stuart’s 3. Amies = Respiratory specimen 4. Transgrow = Neisseria 5. JEMBEC = Neisseria 6. Todd-Hewitt = Vaginal carriage (S. agalactiae) HEPA filter: filters air Negative pressure Environment and MT protected Air velocity = 75 linear ft/min Exhaust air thru HEPA filter Product contaminant Vertical laminar airflow MT, environment and product are protected Air velocity = 75-100 linear ft/min Recommended for hospitals lec.mt 04 |Page | 59
BSC Class III N95 Mask Stool Transposons Fusiform BSL I BSL II BSL III
BSL IV
Blood culture bottle
Disadvantages of SPS
1% gelatin Indications of growth (Blood culture) Subculture (Blood culture) If blood culture = negative Urine culture
CSF culture
Wound specimen Stool specimen
Supply and exhaust air thru HEPA filter Maximum protection Contains HEPA filter For Mycobacterium No direct exam in Microbiology Resistant gene Mobile or jumping F. nucleatum Capnocytophaga No risk Moderate risk High risk Treatment available Inhalation of aerosols Ex. Mycobacteria (BSC Class II) High risk No treatment available Inhalation of aerosols Ex. Small pox BHIB + 0.25% SPS Dilution = 1:10 (1mL blood, 9mL broth) Anti-complementary, anticoagulant, antiphagocytic Neutralizes aminoglycosides Inhibits: -Neisseria -G. vaginalis -S. moniliformis -P. anaerobius Counteract SPS to allow the growth of organisms Hemolysis Turbidity Pellicle BAP MAC = no CO2 CAP 7 days = Bacteremia (Typhoid) 21 days = Brucellosis, SBE Specimen: Catheterized, Midstream, Suprapubic Quantitative: BAP, MAC ->100,000 CFU/mL (or >50,000 CFU/mL) = significant for UTI -<10,000 CFU/mL = not significant (contaminants) DO NOT refrigerate Agents: Neisseria, Haemophilus (Meningitis) Media: BAP, MAC, CAP, BHI C. neoformans: -India ink method -Latex agglutination Gram stain Media: BAP, MAC, Thioglycollate broth Media: MAC, BAP+Ampicillin, CBAP, SSA, Selenite F, TCBS, APW, HEA Oxidase test Biochemical tests lec.mt 04 |Page | 60
Respiratory specimen
Throat swab Vaginal, Urethral swab TB culture
GenPro Moist heat sterilization
Dy heat sterilization
Serologic typing Sputum, NPS TB = 3 sputum specimen Media: BAP, MAC, GBAP, BCAP, Amies, Gram stain and Acid fast stain Sore throat 2 specimen Media: BAP, MTM, Thioglycollate broth Media: CAP, MTM Gram stain NALC-NaOH = Gold standard -NALC = digestion, lyse the mucus -NaOH = decontamination 6% Oxalic acid = Pseudomonas Centrifuge (4’C) for 15 mins at 3000g Media: LJ, Middlebrook 7H11, 7H10 (AST) Incubate at 37’C for 8 weeks ----(NG)----> Report as (-) If (+), after 2-3 weeks: growth is seen Genetic Pro DNA test Result 2 hrs 1. Autoclave (sporicidal) -121’C at 15 lbs/psi for 15 mins -Culture media, bandages, gauze -QC: B. stearothermophilus 2. Inspissation (sporicidal) -75-80’C for 2 hrs on 3 days -Disinfect and solidify protein containing medium (LJ, Loeffler’s) -Water is heated from below and slanting surface gets heated 3. Tyndallization (sporicidal) -100’C for 30mins on 3 days 4. Boiling (Nonsporicidal, disinfectant) -100’C for 30mins -Kills vegetative cells only 5. Pasteurization (Nonsporicidal, disinfectant) -Milk -63’C for 30mins -72’C for 15secs -Phosphatase: to determine if pasteurization is successful. (+): Not pasteurized 1. Hot air oven (Sporicidal) -170-180’C for 2 hrs -Glasswares, cottonswabs, metallic instruments, oils, powders -QC: B. subtilis 2. Incineration (Sporicidal) -Waste disposal -Not recommended 3. Cremation (Sporicidal) -Prevents communicable disease 4. Flaming (Sporicidal) -Needles 5. Gas: Ethylene oxide (sporicidal) -Heat-labile machine instruments lec.mt 04 |Page | 61
Other sterilization methods
Seitz filter Membrane filter Sodium hypochlorite (Clorox) Iodine/Iodophor 70% ethyl alcohol H2 O 2 1% AgNO3 Formaldehyde Glutaraldehyde Phenol (Carbolic acid) Lysol (Cresol) Dyes Zephiran (Benzalkonium chloride) Iatrogenic Antagonistic Synergistic XDR-TB H. influenzae ESBL
Amp C
1. Cold temperature/Freezing (Bacteriostatic) -Preservation 2. Lyophilization (Powderized) -Freeze drying -Best to preserve culture 3. Osmotic pressure (Bacteriostatic) -Preservation 4. Dessication = removal of water 5. UV light = produce pyrimidine dimer to DNA mutation -Reduces airborne infection 6. Ionizing radiation -For disposable materials (gloves, syringe) 7. Filtration -Air: HEPA filter -H2O: cellulose membrane/ membrane filter Filter heat-labile filter Made up of cellulose nitrate, cellulose diacetate, polycarbonate or polyester New: cellulose diacetate w/ a pore diameter of 0.015 to 12 microns Best filter used Spillage disinfectant Sporicidal Iodine + Detergent = Betadine (Best antiseptic) Iodine alone = toxic to skin Nonsporicidal Cleansing of wound Crede’s prophylaxis (New: Erythromycin eye droplets) Prevents ophthalmia neonatorum Sporicidal Sterilant Standard disinfectant Multipurpose Inhibit Gram (+) For decontaminating sputum Instrument caused 1 antibiotic > 2 antibiotics 2 antibiotics > 1 antibiotic Extensively Drug Resistant Tuberculosis Quinolone resistant No treatment at all QC for beta-lactamase Extended spectrum beta-lactamase Produced by Gram (-) = E. coli, Klebsiella Plasmid mediated Test: Beta-lactamase = Keyhole effect (overlapping zones) -Clavulanic acid and cephalosporin Chromosome mediated Produced by Gram (+) and (-) bacteria Test: Beta-lactamase = D zone -(+) to MRSA -Imipenem and cefotixin lec.mt 04 |Page | 62
Cell wall inhibitors
Cell membrane inhibitors
Ribosome (Protein) inhibitors
Nucleic acid (DNA) inhibitors Anti-TB
Micro/Macrobroth dilution Agar dilution Disk diffusion E test (Epsilometer) Kirby-Bauer Disk Diffusion
Petroff-Hausser counting chamber 15mm 15mins w/in 15mins False resistant
Penicillin Cephalosporin Vancomycin = Tx: MRSA Broad spectrum: Bacitracin Cycloserine Carbapenems/Imipenem Penicilinase-resistant: Methicillin, Cloxacillin, Nafcillin Colistin = against Gram (-) Polymixin = against Gram (-) Amphotericin B = drug of choice for systemic fungi Nystatin = antifungal Aminoglycosides (30S) -False-resistant = P. aeruginosa (Mg2+ and Ca2+) Tetracycline (30S) Chloramphenicol (50S) Erythromycin/Macrolide (50S) -Discovered by Bernardo Aguilar -For penicillin allergic patients Clindamycin (50S) Mitomycin Quinolones Metronidazole (Flagyl) Trimethoprim-Sulfamethoxazole (SXT/Bactrim) = inh. folate synth., synergistic Pyrazinamide Rifampin Isoniazid Streptomycin Ethambutol Reference method (AST) Det. MIC/MBC Many organisms vs. single drug Pure culture vs. many drugs Agar gradient diffusion Antibiotic strip diffusion MIC test MIC = Ellipse zone at intersection Std. Inoculum: 1.5 x 108 Medium: MHA pH: 7.2-7.4 Depth: 4mm Condition: Aerobic, No CO2 Temp: 35-37’C (MRSA: 35’C) Incub. time: 16-18 hrs Std: 0.5 McFarland (1% H2SO4 + 1.175% BaCl2) Antibiotic disc: 6mm (refrigerated/frozen) For bacterial count Distance of antibiotic disc to each other Time for the medium to absorb the bacteria after inoculation Inoculation of discs Incubation Heavy inoculums Thick medium lec.mt 04 |Page | 63
False sensitive If double zone of inhibition If there are colonies inside the zone of inhibition AST media
QC
QA Daily QC
Each use (QC) Weekly QC Semi-annually ATCC (American Type Culture Collection) ATCC-1234
Catalase test
Delay in disc application Ca2+ and Mg2+ = Aminoglycoside (vs. P. aeruginosa) Thymine-Thymidine = SXT (vs. Enterococcus) pH = tetracycline pH = aminoglycoside, erythromycin Expired discs Light inoculums Thin medium Measure the outer zone Ignore swarming Gram stain the colonies 1. MHA = std. media 2. MHA + 2% NaCl = MRSA 3. MHA + 5% Sheep blood = S. pneumoniae (w/ CO2) 4. Haemophilus test medium: -MHA + Yeast extract + Hemin + NAD + CO2 5. GC agar = Neisseria (w/ CO2) 6. Middlebrook 7H10 = Mycobacteria (w/ CO2) Specific Regular basis Checking media and reagents w/ specific organisms to check expected results Set by CLSI (formerly NCCLS) General Snap shot Total process whereby the quality of lab. reports can be guaranteed Oxidase Catalase Incubator Gram stain Refrigerator/Freezer Water bath GasPak Jar ONPG Antibiotic (Newly opened: 30 days QC weekly) Autoclave Biochemical tests Safety hood For AST Stock culture: -20 or -70’C Working culture: 2-8’C Beta-lactamase producers: -S. aureus -N. gonorrhoeae -H. influenzae -Enterococcus -E. coli -P. aeruginosa Rgt: 3% H2O2 (+) Gas bubbles F (+): BAP lec.mt 04 |Page | 64
Coagulase test
Mannitol fermentation
DNase test Novobiocin test Modified oxidase test Staphylococcus
Micrococcus Stomatococcus S. aureus
S. lugdunensis S. intermedius Lab. Diagnosis (Staphylococcus)
S. epidermidis
Rgt: Rabbit EDTA plasma (+) Clot formation after 4hrs F (+): Citrate F (-): Reading result after 6 hrs (Staphylokinase) 1. Slide test (Screening) = detects clumping factor/bound coagulase 2. Tube test (Confirmatory) = detects free/unbound coagulase Medium: MSA (7.5% NaCl) Indicator: Phenol Red (+) Yellow (-) Red 1. Toluidine blue (pink zone)/ Methyl green (clear zone) 2. HCl precipitation: no pptn. after adding 1N HCl when DNase (+) = pink Amt.: 5μg (R): <16mm (S): >16mm Rgt: tetramethyl-p-phenylenediamine dihydrochloride in dimethylsulfoxide (+) Purple Pinhead colonies Mod. oxidase (-) Lysostaphin and Furazolidone (S) Ferments sugar Mod. oxidase (+) Lysostaphin and Furazolidone (R) Oxidizes sugar Mod. oxidase (-) Lysostaphin and Furazolidone (R) Virulence factors: -Protein A (cell wall) -Leukocidin (Panton-Valentine) -Exfoliatin (SSS/Ritter’s disease) -TSST-1 (Tampons) Identification: -Staphyloxanthin (Lipochrome): Yellow-orange colony -(+) Phosphatase, ONPG, Arginine, NO3, VP, Gelatin -(-) PYR Infections: -Carbuncles, furuncles, folliculitis, cellulitis, impetigo, bacteremia, endocarditis, osteomyelitis Slide coagulase (+) PYR (+) Slide coagulase (+) VP (-) Nasal swab: carrier of S. aureus Culture: -Vogel-Johnson: Black colonies -Chapman: Black colonies -Tellurite Glycine: Black colonies -P agar -PEA: selective -Columbia CNA: selective #1 skin flora Blood culture contaminant lec.mt 04 |Page | 65
Biofilm/slime production: Prosthetic heart valve Endocarditis, bacteremia UTI: catheterized UTI: sexually active women
S. saprophyticus ID: Streptococcus Lancefield Group
Taxo A (0.04 U)
A B C, F, G D (Enterococcus) D (non-Enterococcus) S. pneumoniae Streptococcus
S R R R R R
Smith and Brown’s classification
S. pyogenes (Group A) (Beta-hemolytic)
S. agalactiae (Group B) (Beta-hemolytic) Group C (Beta-hemolytic) Group F (Beta-hemolytic) Group D Enterococcus (Alpha, beta or gammahemolytic)
Group D non-Enterococcus (Alpha, beta or gammahemolytic) S. pneumoniae (Alpha-hemolytic)
CAMP
Hippurate
SXT
BEM
Bile Solubility
PYR
6.5% NaCl
R + + + R S R + + + R + R Pinpoint colonies Capnophilic: 5-10% CO2 SBA: Medium of choice PEA: Selective medium Hemolysis: 1. Alpha = incomplete (green) 2. Beta = complete (clear) 3. Gamma = no zone 4. Alpha prime = alpha (around colonies) + beta (around alpha) Universally susceptible to antibiotics Virulence factors: -SLO = O2-labile, subsurface hemolysis, immunogenic -SLS = O2-stable, surface hemolysis, non-immunogenic -Erythrogenic toxin (Scarlet fever) Diseases: -Pharyngitis, AGN, RHF, erysipelas, impetigo -Scarlet fever: a. Dick’s test (red): Skin test b. Schultz-Charlton (rash fade/blanching): Immunity test Vaginal and URT flora #1 neonatal meningitis
Taxo P (5μg)
R R R R R S (>14mm)
S. equisimilis S. equi S. zoopedemicus S. dysagalactiae S. anginosus E. faecalis E. faecium E. durans E. avium Cause UTI Drug-resistant: VRE S. bovis S. equinus Cause UTI Lancet-shaped, diplococci Colonies: Mexican hat/ Dome-shaped Encapsulated #1 Adult bacterial meningitis lec.mt 04 |Page | 66
Viridans Streptococci
Nutritionally Variant
Vancomycin Resistant Neisseria
Most common cause of Otitis media Lobar pneumonia: Rusty sputum Lab. Diagnosis: 1. Neufeld Quellung (pptn. test, capsular swelling) 2. Bile solubility -BAP: 10% Na desoxycholate -Tube: 2% Na desoxycholate 3. Francis test: skin test 4. Mouse virulence test: (+) death S. mitis (mitior) S. salivarius S. uberis S. constellatus S. intermedius S. mutans = dental plaques/caries S. sanguis = SBE Abiotrophia Granulicatella Require Vit. B6 (pyridoxine) (+) Staph. Streak test Leuconostoc = LAP (-) Pediococcus = LAP (+) Aerobic Gram (-) diplococci Oxidase (Taxo N): Presumptive test (+) CTA: Confirmatory test Capnophilic: 5-10% CO2
ID: Neissera & Moraxella N. gonorrhoeae N. meningitidis N. lactamica N. sicca N. subflava N. cinerea N. flavescens N. elongata M. catarrhalis N. gonorrhoeae
Glucose Maltose Lactose Sucrose + + + + + + + + + + + V Pili: Adherence Diseases: -Gonorrhea (“Clap”) -Ophthalmia neonatorum (Tx: Erythromycin eye drops) -Salphingitis -Epididymitis Lab. Diagnosis: -Culture: a. Sterile: = CAP: (+) Growth = BAP: (-) Growth (Fastidious) b. Nonsterile: = GC agar: AST media = TMA (Vancomycin-Colistin-Nystatin)
DNase +
lec.mt 04 |Page | 67
N. meningitidis
M. catarrhalis
N. sicca N. lactamica Superoxol catalase test Beta-lactamase test
Mycobacteria
M. tuberculosis
= MTM (V-C-N-Trimethoprim lactate) = MLA (V-C-Anisomycin-T) = NYCA (V-C-Amphotericin B-T) Carrier: Nasopharynx Virulence factors: -Capsule -Endotoxin -Pili -IgA protease Diseases: -Meningitis -Meningococcemia -Waterhouse-Friderichsen syndrome (Adrenal gland hemorrhage) -DIC Lab. Diagnosis: -Culture: a. BAP = (+) Growth b. CAP = (+) Growth -Serotypes: A, B, C, Y, W135 (Capsular Antigens) Commensal of URT (+) NO3 NO2 (+) Butyrate disk (+) Tributyrin hydrolysis (G) Nutrient Agar Colony: Hockey Puck 3rd cause of Otitis media Breadcrumb/wrinkled colony (+) ONPG 30% H2O2 (+) N. gonorrhoeae 1. Chromogenic cephalosporin test or Nitrocefin/Cefinase disk test -(+) Pink/red color 2. Acidimetric -Phenol red (+) Colorless 3. Acidimetric -I2 (+) Yellow AFB (Mycolic acid) Much granules = metachromatic Aerobic nonsporeformer 3 Groups: 1. M. tuberculosis complex = cause TB a. M. tuberculosis = pulmonary TB b. M. bovis = intestinal TB, bovine TB (BCG) c. M. africanum = pulmonary TB in Africa 2. MOTT (New: NTM) 3. M. leprae “Koch bacillus” Capnophilic Virulence: Cord Factor and Sulfatides Lab Dx: 1. GS = qualify specimen > 10 epithelial cells = saliva < 10 epithelial cells (>25 pus cells) = sputum lec.mt 04 |Page | 68
Pott’s disease Agar based media Egg based media (Sterilization: Inspissation) Liquid media Niacin test
Heat stable catalase test
Nitrate reduction test Tween 80 hydrolysis test
Arylsulfatase test
TCH Susceptibility test Old Skin Test (TB)
PPD
2. Decontamination-Digestion = Best -Na citrate: remove metallic compound 3. AFS 4. Culture Bone TB Duboi’s oleic acid albumin medium Mitchison’s medium Middlebrook 7H10 and 7H11 (AST) Petragnani medium Löwenstein Jensen (Slant) medium American Thoracic Society medium Dorset egg medium BACTEC 12B Septi-Chek Middlebrook 7H9 Principle: Niacin ---(enzyme)---> Niacin ribonucleotide + Cyanogen bromide + aniline dye (No yellow color) M. tuberculosis: Niacin ---(no enzyme)---> Niacin + C.bromide + A.dye = (+) Yellow color Temp: 68’C Medium: Tween 80 Rgt: 30% H2O2 Principle: Tween 80 + Mycobacterium + 30% H2O2 ----(Heat at 68’C)----> (+) Gas bubbles (+) M. kansasii (-) M. tuberculosis Principle: Na Nitrate ---(Nitroreductase)---> Nitrite Nitrite + HCl + Sulfanilamide + N-1-naphthylethylenediamine --> (+) Red/Pink Tween 80: Polyoxyethylene sorbitan mono-oleate Principle: Tween 80 ---(Tween 80 lipase)---> (+) Oleic acid (Red) (+) M. kansasii and Differentiates M. gordonae (+) from (-) M. scrofulaceum For rapid growers Principle: Tripotassium + --------(Arylsulfatase)---------> (+) Free phenolphthalein (Red) Phenolphthalein disulfide/sulfate (+) M. fortuitum-chelonae TCH: Thiopene-2-carboxylic acid hydrazide (S): M. bovis (R): M. tuberculosis 1. Mantoux = intracutaneous (most sensitive) 2. Von Pirquet = scratch on skin 3. Vollmer’s patch test = piece of cloth w/ PPD skin 4. Moro percutaneous test 5. Tuberculin time test = multiple puncture technique 0.1 mL (5 TU) (+): Induration (≥10mm) Doubtful: 5-<10mm/(-): <5mm lec.mt 04 |Page | 69
Photochromogens (Group I)
Scotochromogens (Group II)
Non-photochromogens (Group III)
Rapid Growers (Group IV)
1. M. marinum (“Of the sea”) -(+) pyrazinamidase -(+) urease 2. M. asiaticum 3. M. simiae -1st: Macaca rhesus monkey 4. M. kansasii (“Yellow bacillus”) -(+) Heat-stable catalase -(+) Tween 80 hydrolysis -(+) NO3 reduction -Pulmonary infection -#2 isolate 1. M. scrofulaceum -(+) Urease -Scrofula: cervical lymphadenitis 2. M. szulgai -Photochromogen at 25’C -Scotochromogen at 35-37’C 3. M. gordonae (“Tap water bacillus”) 4. M. flavescens 5. M. xenopi -Hot and cold H2O taps -Cornmeal agar: “Bird’s nest” -Optimal growth at 42’C -(G) 37’C -(NG) 25’C -(-) Tellurite reduction -(-) Catalase 6. M. thermoresistible -(G) 52’C Colony: Cream/buff 1. M. terrae-triviale -(+) Catalase a. M. terrae (“Raddish bacillus”) -(NG) NaCl b. M. triviale -(G) NaCl 2. M. avium-intracellulare (“Battey bacillus”) -(+) Tellurite reduction 3. M. gastri -(+) Urease 4. M. haemophilum = require hemin (CAP) 5. M. ulcerans (Buruli) 6. M. malmoense 7. M. tuberculosis 1. M. fortuitum-chelonae -(+) Arylsulfatase -(G) MAC a. M. fortuitum -(+) NO3 reduction -(+) 5% NaCl -(+) Iron uptake lec.mt 04 |Page | 70
Growth: 10-21 days Growth: 3-7 days
b. M. chelonei -(-) NO3 reduction -(-) 5% NaCl -(-) Iron uptake 2. M. smegmatis 3. M. phlei (“Hay bacillus”) Groups I, II, III (Runyon’s Classification) Group IV (Rapid growers)
AFB Grading (National Standard) CDC Method to Report AFB 0 No AFB/300 fields 0 0 AFB/field +/- 1-9 AFB/100 fields +/- 1-2 AFB/300 fields 1+ 10-99 AFB/100 fields 1+ 1-9 AFB/100 fields 2+ 1-10 AFB/field in at least 50 fields 2+ 1-9 AFB/10 fields 3+ >10 AFB/field in at least 20 fields 3+ 1-9 AFB/field -------------------------------------------------------------- 4+ >9 AFB/field BACTEC 460 Middlebrook Principle: RIA 14C Palmitic acid + organisms ----------> 14CO 7H12 2 (+): >10 growth index Mycobacteria Growth Fluorometric based Indicator test (MGIT) O2 consumption = Fluorescence BACTEC 12B + NAP Rgt: p-nitroacetylamino-beta-hydroxypropiophenone (NAP) (Growth inhibition test) (NG) = (+) M. tuberculosis M. leprae Cigarette-packet/picket-fence Not cultivable in agar Can hydrolyze DOPA Tropism to peripheral nerves Tx: Dapsone Lepromatous Leonine face (-) Lepromine AFB Tuberculoid (+) Lepromine AFB Lab. Diagnosis (Leprosy) Specimen: Ear lobe, nasal scraping Culture: Foot pads of Armadillo Stain: Fite Faraco M. genavensi Disseminated infection in AIDS BACTEC (+) M. paratuberculosis Crohn’s disease Rhodococcus equi Pleomorphic (rod-cocci) (+) pink colonies Nocardia Partially acid fast (Mod. AFS: H2SO4) (+) Urease Cause pneumonia N. asteroides = casein hydrolysis (-) N. brasiliensis = casein hydrolysis (+) Corynebacteria Pleomorphic Club shape: X, Y, V, L (Chinese characters) Palisade appearance: diphtheroids Nonmotile, nonsporeforming, nonencapsulated (+) catalase and oxidase lec.mt 04 |Page | 71
C. diphtheriae
Biotypes of C. diphtheriae C. pseudodiphtheriticum C. xerosis C. minutissimum Transduction B. anthracis
B. cereus B. subtilis Clostridium
C. perfringens (Old: C. welchii)
“Klebs-Loeffler” bacillus Virulence: Exotoxin (A and B) – heat labile Diphtheria: pseudomembrane of the pharynx Lab. Diagnosis: -Elek test: definitive -Roemer test: incubates the bacteria in pig -Schick’s test: skin test (Ab’s) -(+) DNase -(-) Urease -Ferments dextrose 1. Gravis = gray, large, beta-hemolytic, starch/glycogen fermentation (+) 2. Mitis = black, medium-size, beta-hemolytic, starch/glycogen fermentation (-) 3. Intermedius = black, small, nonhemolytic “Hoffman’s bacillus” Ferments glucose, maltose and sucrose Agent of erythrasma Coral red fluorescence on Wood’s lamp (porphyrin) Lysogeny (no lysis) Aerobic, catalase (+) Zoonotic Virulence: -Exotoxin: edema and lethal -Capsule: D-glutamate Diseases: -Malignant pustule (Black eschar) -Woolsorter’s disease (Respiratory) -Gastroenteritis (Bloody diarrhea) Lab. Diagnosis: -PLET: sensitive medium -Colony: medusa-head, inverted pine tree growth -(+) catalase -String of pearls on BAP + 0.05 U of penicillin -Gamma phage susceptible -Ascoli test “Fried rice bacillus” Exotoxin (cholera-like): cAMP = H2O, Na+, K+ = dehydration (-) capsule Common lab. contaminant Causes eye infection in heroin addicts Anaerobic, catalase (-) Saccharolytic except: C. tetani, C. septicum 1. Neurotoxic: C. tetani (spastic paralysis), C. botulinum (flaccid paralysis) 2. Histotoxic: C. perfringens, C. septicum 3. Enteric: C. difficile Encapsulated, nonmotile Double zone of hemolysis: -Inner (complete): due to theta toxin -Outer (incomplete hemolysis): due to alpha toxin Lab. Diagnosis: -Chopped meat: growth + gas (anaerobic growth) -Nagler test: lecithinase test (alpha toxin) = Medium: McClung or Neomycin egg yolk lec.mt 04 |Page | 72
= (+) Opalescence on agar w/o antitoxin = (-) No opalescence on agar w/ antitoxin C. botulinum “Canned-good bacillus” Virulence: -Heat labile toxin: block release of acetylcholine (muscle) flaccid paralysis -Botulinum toxin: Most potent toxin -Not cultured Diseases: -Wound botulism -Infant botulism: grow in gut and honeybee (SIDS) C. tetani “Tack head bacillus” Tennis head, drumstick Oval, terminal spore Swarms on BAP Virulence: Exotoxin -Tetanolysin -Tetanospasmin: binds to ganglioside receptors and inhibit neurons in CNS spastic paralysis Disease: -Lockjaw -Risus sardonicus: Sardonic smile -Opisthotonus C. difficile Normal flora of the colon Clindamycin-associated pseudomembranous colitis Lab. Diagnosis: -Cytotoxin assay -Culture: CCFA (Yellow colonies w/ horse manure odor) Anaerobic bacteriology Collection: Needle aspiration Reduced media: -Anaerobic BAP -Schaedler medium -Bacteroides bile medium -Laked Kanamycin-Vancomycin BAP -Anaerobic PEA -Egg yolk agar -Chopped meat -Peptone yeast extract glucose -Thioglycollate broth -Lombard Dowell Agar Methods to promote 1. GasPak Jar or Mcintosh Fildesh, Brewer, Torbal or any anaerobic jar anaerobiosis -CO2, N2, no O2 -H + O2 = H2O -Candle jar = not to be used 2. PRAS = roll tube of Hungate Anaerobic indicators: a. Resazurin = pink b. Methylene blue = colorless ID: Kanamycin Vancomycin Colistin Kanamycin Vancomycin Colistin B. fragilis R R R Fusobacterium S R S B. ureolyticus S R S lec.mt 04 |Page | 73
Veillonella Clostridium Gram (+) cocci Porphyromonas P. anaerobius Prevotella Brick red fluorescence Red fluorescence Pitting of agar Swarming Molar tooth colony, sulfur granules Breadcrumb colony Gram (+) Anaerobic Bacilli
Gram (-) Anaerobic Bacilli
Gram (+) Anaerobic Cocci Gram (-) Anaerobic Cocci A. bovis P. acnes L. acidophilus Mobiluncus Anaerobic infections B. fragilis F. necrophorum (F. moriferum) P. anaerobius P. asaccharolyticus P. niger H2S indicators Phenol red
S S Kanamycin S R R R Prevotella Porphyromonas Veillonella B. ureolyticus C. tetani C. septicum A. israelii
R S Vancomycin S S S R
S R Colistin R R R S
Fusobacterium Actinomyces Bifidobacterium Eubacterium Propionebacterium Lactobacillus Mobiluncus Bacteroides Porphyromonas Prevotella Fusobacterium Peptostreptococcus Peptococcus Veillonella Megasphera Acidaaminococcus Lumpy jaw Acne vulgaris (+) Catalase, Indole Tomato juice agar (-) Catalase Vaginitis (G. vaginalis) (-) Catalase Foul odor Needs 20% bile Vincent’s angina (B. vincenti) SPS sensitive (Neut: 1% gelatin) (-) Indole, Catalase (-) Catalase Staphylococcus-like Black TSI = Ferrous sulfate LIA = Ferric ammonium citrate SIM = Lead acetate Acid = Yellow Alkaline = Red lec.mt 04 |Page | 74
Methyl red Neutral red Bromthymol blue Enterobacteriaceae
VP (+)
H2S (+)
Lactose Fermenters
Deaminase (+)
Urease (+)
Acid = Red Alkaline = Yellow Acid = Pink/red Alkaline/neutral = Colorless Acid = Yellow Neutral = Green Alkaline = Blue All ferment glucose All are motile except: -Shigella -Klebsiella -Y. pestis -S. gallinarum -S. pullorum All are catalase (+) All reduce NO3 NO2 All are oxidase (-) except P. shigelloides (+) Antigens: -K = Capsular (for serotyping) -O = Somatic (cell wall, heat-stable) -H = Flagellar (heat-labile) “KESH” Klebsiella Enterobacter Serratia Hafnia “SPACEd” Salmonella Proteus Arizona Citrobacter Edwardsiella “ACH” (Slow) “EKE” (Rapid) Arizona Citrobacter Hafnia E. coli Klebsiella Enterobacter ----------------------------------------------------Y. enterocolitica = (+) ONPG, TSI: A/A “PMP” Proteus Morganella Providencia “PMP” (except P. alcalifaciens) “K.pn/ox” “E” “Y.ps/en” Proteus Morganella Providencia K. pneumoniae lec.mt 04 |Page | 75
Aerogenic
SSA DCA TCBS
Inhibitory Eosin Y Methylene blue Bile salts Crystal violet Bile salts Bile salts Bile salts
HEA
Bile salts
XLD
Bile salts
BSA
Brilliant green (Old: Wilson-Blair)
EMB MAC
Escherichia coli Enterobacter aerogenes Enterobacter gergoviae Edwardsiella tarda Arizona Serratia Hafnia Salmonella enteritidis Salmonella choleraesuis Enterobacter cloacae Enterobacter sakazakii Enterobacter taylorae Salmonella typhi Klebsiella pneumoniae Klebsiella oxytoca Klebsiella ozaenae E. coli
K. oxytoca E. gergoviae Y. pseudotuberculosis Y. enterocolitica Klebsiella E. coli Salmonella (except S. typhi, S. gallinarum) Proteus Arizona Citrobacter Enterobacter Enteric Media CHO Indicator LF Lactose Eosin Y Red purple Methylene blue (E. coli: GMS) Lactose Neutral red Red/pink
NLF Colorless Colorless
Lactose Lactose Sucrose
Neutral red Neutral red Bromthymol blue
Red Red/pink Yellow
Colorless Colorless Green
Salicin Lactose Sucrose Xylose Lactose Sucrose Glucose
Bromthymol blue
Yellow
Green
Phenol Red
Yellow
Red
Bismuth sulfite
S. typhi = black colonies
Lysine-Ornithine-Arginine Reactions “(LO)9(OA)3(L)4(O)6(N)6” - Enterobacteriaceae L O A + + Shigella sonnei + + Proteus mirabilis + + Morganella morganii + + Citrobacter + + Yersinia enterocolitica + + Salmonella paratyphi + + Providencia + + Proteus vulgaris + + Klebsiella rhinoscleromatis + + Pantoea agglomerans + + Yersinia pseudotuberculosis + + Yersinia pestis + Plesiomonas shigelloides + Vibrio + Aeromonas hydrophila + Pseudomonas aeruginosa “Colon Bacillus” #1 UTI #2 Neonatal meningitis (K1 Ag) MUG (+) except E. coli O157:H7
L + + + -
O + + + + + + + + -
A + + +
lec.mt 04 |Page | 76
-MUG (+): Electric blue fluorescence ETEC
EPEC EIEC
EHEC/VTEC E. coli O157:H7 EAEC Other Escherichia spp
Enterobacter K. pneumoniae
K. rhinoscleromatis PMP Group (Proteeae) Proteus
Salmonella
S. typhi
Traveler’s diarrhea Montezuma revenge Turista Belly Delhi Cholera like, watery diarrhea (SL, LT) Infantile diarrhea Pili: pathogenicity island Dysentery (Shigella)-like diarrhea Invasin: Pathogenicity Stool: bloody w/ mucus Sereny test HUS Hemorrhagic colitis Verotoxin: Shigella-like toxin EHEC/VTEC Sorbitol MAC (-) Acute and chronic diarrhea Aggregative adhesion fimbriae E. hermanni = yellow colonies E. vulneris = yellow colonies Alkalescens dispar (Anaerogenic E. coli/E. coli inactive) -Nonmotile, (-) H2S, NLF, PG (-), Vi (-) UTI, wound, septicemia E. sakazakii = yellow pigment E. (Pantoea) agglomerans = yellow pigment “Friedlander’s bacillus” (+)Capsule, ESBL Diseases: -Pneumonia: “Currant jelly-like sputum” -Wound infections -Meningitis -UTI Biochemically inert All are indole (+) except P. mirabilis (-) Swarm on BAP but not on MAC #2 UTI Renal stone association Odor: Burnt gun powder/Burnt chocolate P. mirabilis: Rapid urease producer Indole (-) BSA & SSA: Black colonies Gold standard: Culture Kaufmann White schema -Vi Ag = heat-labile, masks the O Ag -O Ag -H ag Tx: Chloramphenicol Typhoid fever Meningitis Osteomyelitis lec.mt 04 |Page | 77
Complication: Psychosis
S. paratyphi C (S. choleraesuis) S. enteritidis Shigella
Shigella Subgroups (O Ag)
Serratia marcescens
Serratia odorifera Y. pestis
Y. enterocolitica Y. pseudotuberculosis Vibrio
V. cholerae
Polymyxin Susceptibility
Lab Diagnosis: -1st week: Blood -2nd week: Urine and stool (carrier) Septicemia “Gardner’s Bacillus” Biochemically inert SSA: colorless Related to E. coli: -E. coli: Acetate (+) -Shigella: Acetate (-) Intestinal pathogen: Dysentery Lab. Diagnosis: -BEST: Culture of fresh stool w/ mucous flecks -Rectal swab of ulcer A = S. dysenteriae (“Shiga Bacillus”) B = S. flexneri (“Strong Bacillus”) C = S. boydii D = S. sonnei (Cross reacts w Plesiomonas) Red pigment (Prodigiosin) (+) DNase, Gelatinase, LPS Red milk Resistant to many antibiotics Rancid, potato-like odor “Plague Bacillus” (+) Stalactite on broth medium V and W antigens MOT: Rat flea bite Infections: -Bubonic plague: buboes, lymphadenopathy -Pneumonic plague -Septicemic plague -Black death Motile at RT’ Appendicitis-like infection (+) ONPG Motile at RT’ Mesenteric lymphadenitis, septicemia “Comma Bacillus” Comma-shaped Darting motility Oxidase (+) except V. mitschnikovii (-) Halophilic except: -V. cholerae -V. mimicus (+) Indole (+) String test (0.5% Na desoxycholate) Cholera: Ricewater stool “Fish in the stream” appearance Classical Biotype S
El Tor Biotype R lec.mt 04 |Page | 78
Lysis by bacteriophage Chicken RBC agglutination Hemolysis of sRBC VP test Anti-Ogawa Anti-Inaba Lab. Diagnosis (V. cholerae)
V. parahaemolyticus
V. vulnificus
V. alginolyticus Aeromonas
Plesiomonas
Campylobacter
H. pylori
OF test
P. aeruginosa
+ + Classical Biotype El Tor Biotype + + Ogawa Serotype Inaba Serotype Hikojima Serotype + + + + Culture: -TCBS: Sucrose fermenter -TTGA: Tellurite Taurocholate Gelatin Agar -APW: 6-8 hrs Subculture (TCBS) -Cary Blair: Transport medium (stool) -Rectal swab: Carrier O129 Susceptibility test = (S) Halophilic (8% NaCl) (+) Indole #1 Gastroenteritis in Japan (seafood) Nonsucrose fermenter (TCBS) Kanagawa (+) = Beta hemolysis on Wagatsuma agar Stool pathogen (+) Blood culture (+) Lactose fermentation, ONPG Nonsucrose fermenter (TCBS) Sucrose fermenter (TCBS) Motile (monotrichous) (+) Oxidase Beta-hemolytic (R) O129 (+) DNase (+) Bile esculin hydrolysis (+) Oxidase Non-hemolytic (V) O129 (-) DNase (-) Bile esculin hydrolysis (G) 42’C Curved, S-shaped (Wings of Seagull) (+) Catalase (+) Oxidase Darting motility C. jejuni: (+) Hippurate (+) Catalase (+) Oxidase (+) Urease Test: Urease breath test Fermentative (close) = O/F = +/+ = Y/Y Oxidative (open) = O/F = +/- = Y/G Nonutilizer = O/F = -/- = G/G Medium: Hugh and Leifson (1% glucose, 1% agar) Indicator: Bromthymol blue Old: P. pyocyanea lec.mt 04 |Page | 79
B. cepacia
B. pseudomallei
B. mallei P. stutzeri
S. maltophilia
P. syncyanea Shewanella putrefaciens Acinetobacter
Pyocyanus: Blue pus agent (+) Oxidase Motile (monotrichous) Capsule: Slimy layer Grapelike odor (aminoacetaphenone) (G) 42’C Cetrimide medium: selective Pigments: -Pyocyanin: Best -Pyoverdin/Fluorescein: (+) P. putida/P. fluorescens Infections: -#2 Burns (#1: S. aureus) -Wound: Ecthyma gangrenosum -Swimmer’s ear (Otitis externa) -Dermatitis: whirlpool baths -Contact lens infection -#1 Cystic fibrosis (+) Oxidase Motile (lophotrichous) #2 Cystic fibrosis (+) Lactose, ONPG “Whitmore’s Bacillus” Vietnamese timebomb Melioidosis/Glanders-like disease Ashdown medium: Wrinkled colony (G) 42’C Motile (lophotrichous) (+) Lactose Long incubation period: 10-20 years Nonmotile Glander’s disease (Horses) Brown (buff colored) wrinkled colony (+) 6.5% NaCl (+) NO2 N2 (-) Lactose Found in stagnant water (-) Oxidase (+) DNase (+) Maltose and Glucose Motile (lophotrichous) Colony: Lavender green Common w/ the use of catheter Blue milk TSI: K/K + H2S (+) Oxidase (-) Oxidase (+) Catalase Nonmotile MAC: purple colonies Mistaken as Neisseria – (+) oxidase Causes UTI lec.mt 04 |Page | 80
A. anitratus (A. baumanii) A. lwoffi Alcaligenes faecalis
M. lacunata
Flavobacterium (Chryseobacterium) meningosepticum
E. corrodens
Kingella spp Haemophilus
H. influenzae H. aegyptius H. haemolyticus H. parainfluenzae H. parahaemolyticus H. paraphrophilus H. ducreyi H. aphrophilus Porphyrin test H. influenzae
Oxidizer Herella vaginocola Nonoxidizer Mima polymorpha (+) Oxidase (+) Catalase Motile (peritrichous) Asaccharolytic “Fruity” odor (+) Pitting of agar (+) Oxidase (+) Catalase Assacharolytic (NG) MAC Infection: Blepharoconjunctivitis Mistaken as Neisseria Flavin = Yellow pigment (+) Oxidase (+) DNase (+) Indole (+) Gelatin hydrolysis Nonmotile Causes neonatal meningitis Twitching motility MOT: Human bite “Clenched fist” Corrodes agar Odor: Bleach-like (-) Catalase (+) Oxidase Cause SBE (HACEK) Pits agar Require X factor (hemin) and V factor (NAD) (+) Satellitism (+) Oxidase HCAP: medium of choice (5% CO2) X factor V factor Porphyrin + + + + + + + + + + + + + + X factor D-ALA (+) Protoporphyrin (Red) “Pfeiffer’s Bacillus” Serotype polysaccharide: -Hib = Haemophilus type B: capsular antigen type B (polyribose ribitol PO4) Infections: -Epiglotitis (Major) lec.mt 04 |Page | 81
H. ducreyi
H. aegyptius B. pertussis
B. pertussis B. parapertussis B. bronchiseptica B. bronchiseptica Brucella
-#3 Meningitis (#1: S. pneumoniae/#2: N. meningitidis) -Otitis media, CF, conjunctivitis, pneumonia, URTI, sepsis Lab. Diagnosis: -Culture: Grayish, dew drop colony w/ Mousy odor a. CAP b. Levinthal c. Fildes “Chancroid Bacillus” (G) CAP + Vancomycin (33’C) Soft chancre (painful) School of red fish “Koch Week’s Bacillus” Pink eye conjunctivitis Brazilian purpuric fever “Whooping cough Bacillus” Strictly aerobic Encapsulated, nonmotile Require cysteine and methionine for growth Toluidine blue: Bipolar granules Stages: Whooping cough 1. Catarrhal: mild 2. Paroxysmal: successive cough 3. Convalescence: recovery Lab. Diagnosis: -NPS: Carrier -Culture media: a. BG (PBGA) = mercury droplet colonies (pearl-like) b. RL (Charcoal horse blood) c. Jones Kendrich (Charcoal, yeast extract) d. CCBA e. Stainer and Scholte f. Casamino broth Motility Urease Oxidase + + + + + “Kennel cough Bacillus” (-) Capsule Obligate aerobe Nonmotile Facultatively intracellular (phagocytes) Erythritol: enhances growth Zoonotic: found in animal placenta Infections: -Undulant fever -Malta fever -Aboriton in cattle -Endocarditis Lab. Diagnosis: -Castañeda broth: Best medium -TSB: Best medium -W (Wisconsin) medium: selective
MAC/BAP + +
lec.mt 04 |Page | 82
B. abortus B. abortus B. melitensis B. suis B. canis F. tularensis
P. multocida
Treponema Leptospira Borrelia T. pallidum Syphilis
Neurosyphilis
-(+) Rose Bengal & 2-ME agglutination “Bang’s Bacillus” Urease CO2 Thionine (Dye Inhibition) Fuchsin (Dye Inhibition) + + - (NG) + (G) + + (G) + (G) + + (G) - (NG) + + (G) - (NG) (+) Capsule Nonmotile Facultatively intracellular Obligate aerobe Lab. acquired infection (BSL II) Tularemia: -Lymphadenopathy -Deerfly, lemming, rabbit, water rat trapper’s disease Lab. Diagnosis: 1. GCBA: Glucose cysteine blood agar 2. PCA: Peptone cysteine agar 3. CHA: Cysteine heart agar 4. Biochem: -(+) Catalase -(-) Oxidase -(-) Urease -(-) MAC Multocida: Multiple killing (+) Capsule Nonmotile (+) Oxidase (+) Catalase (+) Glucose (+) Ornithine (+) Indole (+) Urease (G) BAP (NG) MAC Infections: -Animal (cat) bite wound infection -Shipping fever (-) Catalase Dx: Serology (+) Catalase Dx: Culture (-) Catalase Dx: Giemsa (blood smear), Serology Noncultivable on agar Obligate intracellular (Rabbit’s testicle) 1’ = Hard chancre (Painless) 2’ = Condylomata lata (HPV: Condylomata acuminata) Latent: Asymptomatic, Dx: Serology 3’: Gummas, neurosyphilis Congenital syphilis: Hutchinsonian triad, abortion, still birth Tabes dorsalis lec.mt 04 |Page | 83
Penicillin T. pertenue T. carateum T. endemicum T. cuniculi Lab. Diagnosis (Treponemes)
L. interrogans icterohemorrhagiae
Borrelia
H. aphrophilus A. actinomycetemcomitans C. hominis E. corrodens K. kingae A. actinomycetemcomitans C. hominis E. corrodens Chlamydia (Old: Bedsonia)
Treatment for syphilis (Jarisch-Herxheimer reaction) Yaws, Framboesia Pinta Bejel Rabbit syphilis 1. Darkfield microscopy: -Corkscrew motility -1’ and 2’ syphilis 2. Stain: Levaditi silver impregnation 3. Serology: -Latent, 3’ syphilis -Reagin test (VDRL, RPR, TRUST) -Trep. Antibody test (FTA-ABS [confirm], MHA-TP, HATTS) Obligate aerobes Spiral w/ hooked ends Weil’s disease (Zoonotic): -1st week: blood, CSF -2nd week: urine (chronic) Animal serum: 30’C for 6 weeks Culture media: -Fletcher’s -Noguchi -EMJH Blood spirochete 1. Relapsing fever: a. B. recurrentis: louse-borne b. B. anserine: tick-borne c. B. turicatae: tick-borne d. B. parkeri: tick-borne Diagnosis: Wright’s/Giemsa (blood/BM) 2. Lyme disease: B. burgdorferi -MOT: Tick bite (I. dammini) a. 1’ stage: Erythema chronicum migrans (Bull’s eye rash) b. 2’ stage: neurological (meningitis), cardiac c. 3’ stage: Arthritis (joint fluid) Diagnosis: Culture and Serology -Culture on Barber Stoenner Kelly medium = 33’C for 6 weeks Differential: HACEK Catalase Oxidase MAC + + + + + Star-like colony Dots and dashes of Morse code (+) Indole Teardrop shape Rosette formation Asaccharolytic Obligate intracellular
CO2 + + + + +
lec.mt 04 |Page | 84
C. trachomatis
C. psittaci
C. pneumoniae
Rickettsia
Ehrlichia
R. rickettsii R. akari R. typhi R. prowazekii O. tsutsugamushi R. quintana E. chaffeensis
Energy (ATP) parasites Inclusion body: diagnostic (stained by Giemsa), not Gram stained a. Elementary body = infectious b. Reticulate body = reproductive Immunofluorescence: det. Chlamydia antigen (N. gonorrhoeae) Contains glycogen ---(I2)---> Brown TRIC agent: Trachoma Inclusion Conjunctivitis Diseases: -LGV (Frei’s test) -PID (pelvic inflammatory disease) -#1 STD in US -#1 NGU (S) Sulfonamide Lab. Diagnosis: 1. Inclusion body (Halberstadter Prowazeik) -Stain (Glycogen): Iodine (brown)/Giemsa (purple) -Elementary body: Round-shaped 2. Fitz-Hugh Curtis 3. McCoy (shell vial) = Best medium (cell culture) 4. Direct IF = Ag 5. DNA amplification = most sensitive 6. Transport = 4’C/-70’C (long term) Parrot fever/Psittacosis/Ornithosis Man: Pneumonia Inclusion body (Levinthal Cole Lillie) -Elementary body: Round-shaped -(-) glycogen (R) Sulfonamide TWAR agent Pneumonia: Human-to-human Growth on: -Human lines -Hep-2 cell Guillain-Barré syndrome: ascending paralysis Obligate intracellular (endothelial cells) except Coxiella (extracellular) Arthropod borne except Coxiella (inhalation) Lab. Diagnosis: 1. Weil-Felix test: cross react w/ Proteus (det. Rickettsial Ab) 2. Special stain: Gimenez, Macchiavelo 3. Culture: Embryonated egg, cell culture Intracellular (WBCs) Morulae: diagnostic form Tick-transmitted Cause sennetsu fever Vector Disease Tick RMSF Mite Rickettsialpox Rat flea Murine/endemic typhus Human louse Epidemic typhus/Brill Zinsser dis. Chigger Scrub typhus Human louse Trench fever Tick Monocytic Ehrlichiosis lec.mt 04 |Page | 85
E. equi C. burnettii Mycoplasma
M. pneumoniae
M. hominis
U. urealyticum
G. vaginalis
C. granulomatis
S. moniliformis
Tick Granulocytic Ehrlichiosis Inhalation Q fever No cell wall (pleomorphic) Fried egg/mulberry Cell membrane: Sterol (bilipid layer) Require sterol for growth (R) Penicillin Diene’s stain: stain for Mycoplasma colonies (blue) Eaton agent PPLO: Pleuropneumonia-like organism PAP: Primary atypical pneumonia Walking pneumonia Lab. Diagnosis: 1. Media: PPLO agar 2. Confirm: Hemadsorption test (not a serological test), RBCs Colonies 3. Screening: Cold agglutination 4. Confirmatory: IF Tx: Tetracyclin/Erythromycin Large fried egg colony Infections: Post abortal, post partum fever, PID Media: -A7/A8: selective -NYCA -SP4: arginine T strain = tiny fried egg (+) Urease = brown Cause NGU Media: -A7/A8 -SP4: urea -NYCA Old: Haemophilus v., Corynebacterium v. (-) Catalase (-) Oxidase (S) SPS (Counteracted by 1% gelatin) Whiff’s test: 3% KOH Fishy amine-like odor Media: HBTA (selective), V (vaginalis) agar, Columbia CNA Gram (V) (+) Capsule Safety pin MOT: Sexual contact Causes granuloma inguinale (Donovanosis) Giemsa stain: Donovan bodies (macrophage w/ Gram [-] bacilli) Not cultured String of beads Broth: fluff balls HI: fried egg S. moniliformis (w/ cell wall) ---(Penicillin)---> L forms (no cell wall) (S) SPS Infections: -Rat bite fever -Haverhill disease lec.mt 04 |Page | 86
S. minus (S. minor) C. violaceum
Capnocytophaga
B. henselae
B. bacilli formis
L. pneumophila
L. monocytogenes
E. rhusiopathiae
Spiral organism Causes rat bite fever Cannot be cultivated Old: Achromobacterium Violacein: violet colored MAC: NLF NH4 cyanide Fusiform Gliding motility Periodontal disease (oral flora) = periodontitis Large spreading colonies (-) Oxidase (-) Catalase (-) Indole (+) Esculin hydrolysis, NO3 reduction Infections: Cat scratch disease Bacillary angiomatosis Peliosis Hepatitis (NG) MAC Vector: Sandfly Infections: -Carrion’s disease -Verruga peruana: skin eruptions -Oroya fever: anemia Aerobic Facultatively intracellular Require cysteine and iron for growth (NG) MAC = no cysteine Isolated from airconditioners and water-cooling towers Infections: -Broadstreet pneumonia -Pontiac fever (+) Catalase (+) Oxidase (+) DNase (+) Beta-lactamase Lab. Diagnosis: 1. DF Ab test: Ag (IF: Ab) 2. BCYE: Blue green cut glass colony 3. Safranin: 10 mins 4. Transport: 4’C or frozen Tumbling motility: head over heels/end-to-end motility Listeriolysin O Infections: -Granulomatosis infantiseptica -Meningitis Lab. Diagnosis: Anton’s test (Skin test) Growth: Test tube brush Erysipeloid: Butcher’s cut L. monocytogenes E. rhusiopathiae lec.mt 04 |Page | 87
Catalase Motility (25’C) Hemolysis VP H2 S Bile esculin Hippurate Gluconate Media Water Bacteriology Sodium thiosulfate 1. Presumptive test 2. Confirmatory test 3. Completed test MPN (Most Probable Number) Reference laboratories (Water Bacteriology) Milk Bacteriology Streptococcus lactis Coliform bacteria Bacillus subtilis Clostridium butyricum Clostridium perfringens Pseudomonas syncyanea Serratia marcescens Pseudomonas aeruginosa Flavobacterium synxanthum Alcaligenes viscosus
+ + Beta Alpha L. monocytogenes E. rhusiopathiae + + + + + Mc Bride, Cold enrichment BAP E. coli: Index of fecal contamination Det. coliforms Neutralizes chlorine Lactose broth/ + H2O ----------(35’C for 24 hrs)----> (+) Gas Lauryl tryptose broth (-) No gas after 48 hrs EMB/Endo agar + Inoculum -----(24 hrs)-----------> (+) Colony Lactose broth fermentation tube ---(35’C for 24-48 hrs)---> (+) Acid + Gas Most common method for H2 analysis Estimation of coliform group density # of positive coliforms from multiple decimal dilution EAMC FDA Acid forming, causes normal souring of milk Gas-forming, hydrolyze the milk protein casein Hay bacteria, proteolytic action on coagulated milk Stormy fermentation of milk Blue milk Red milk Blue green milk Yellow milk Slimy or ropy milk (capsule formation)
lec.mt 04 |Page | 88
