MUST TO KNOW IN BACTERIOLOGY

Soap Germicidal
L. monocytogenes Catalase (+)
3 C’s: Chicken, Coleslaw, Cheese
Chlamydia When delayed: 4’C
Freezing: -20’C
BAP Phenotypic
Gram (+) colonies: Dry, white, sometimes gray
Gram (-) colonies: Gray and moist
M. gordonae Destroyed by chlorine
DNase test Utilizes 1N HCl
LOA test For Gram (+)
For nonfermentative
For Enterobacteriaceae
Na hypochlorite Inactivates HBV (10mins) and HIV (2mins)
Inoculating needles Nichrome = F(+) on oxidase test
Not longer than 5cm
Wire loop 2mm diameter
0.001mL urine
50k CFU/mL Significant for UTI
Pregnant C. albicans
Lactobacillus
Cotton swab Carrier state
Lawn a culture
Toxic to Neisseria
Good for virus
Charcoal Removes the toxin inoculated by cotton
Phenotype Basis of identifying organisms
Gram stain and colonies
Stool Not Gram stained
PCR Most definitive method of identification
Somatic antigen Basis of serotyping
Anton van Leeuwenhoek Father of microbiology
Microscopist
1st to describe bacteria
Robert Koch Germ theory: relationship of organisms to human disease
Louis Pasteur Father of Modern Microbiology
Ehrlich 1st to use dyes for stain
Bacteria Ave. size: 0.4-2μm
Reproduction: Binary fission (two-fold increase)
Cell wall Peptidoglycan (murein)
Protoplast: wall less G(+)
Spheroplast: wall less G(-)
Gram (+) Thick peptidoglycan
Teichoic acid
Gram (-) Thin peptidoglycan
LPS (Lipid A – exotoxin)
Somatic antigen
Plasma membrane Site for energy synthesis (ATP)
Osmotic/permeability barrier
Nucleoid Chromosome: dsDNA
Plasmid: Extrachromosomal DNA
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Plasmid Carries the antibiotic-resistance gene
Drug-resistance Chromosome and plasmid-mediated
Metachromatic granules Food reserves
Ribosomes Prokaryotic: 70S
Eukaryotic: 80S
Common pili Bacterial adherence
Sex pili Gene transfer
ESBL By Gram (-) bacteria
Endospores Calcium dipicolinate
Bacillus, Clostridium
Flagella Monotrichous: one only
Amphitrichous: one at both ends
Lophotrichous: tuft at one end
Peritrichous: all around bacteria (most common)
Lactobacillus Aerotolerant anaerobes
Autotrophs/Lithotrophs Inorganic compound as source of carbon (CO2)
Heterotrophs/ Organic compound as source of carbone (Glucose)
Organotrophs Pathogenic bacteria
Temperature requirements Psychrophilic: 0-20’C (ref)
Mesophilic: 20-40’C (pathogenic)
Thermophilic: 40-60’C
pH requirement Acidophilic: Lactobacillus acidophilus (Doderlein bacillus)
Neutrophilic: pH 7.2-7.6 (optimal) – pathogenic
Basophilic: Vibrio (Halophilic)
Moisture Humidophilic
Salt concentration Halophilic
Enterococcus and V. parahaemolyticus
Respiration (Aerobic) Glucose  CO2 + H2O
Kreb’s cycle
Electron transport chain
Oxidation (Aerobic) Glucose  Acid
Fermentation (Anaerobic) Glucose  Acid/Alcohol
Embden-Meyerhoff pathway (glycolysis)
Lag phase Adjustment
Log/Exponential phase  in growth rate (cell division)
Susceptible to antimicrobial agents
Stationary/plateau phase No net growth
Death = Live cells
Depletion of nutrients
Accumulation of toxic wastes
Sporulation
Death/Decline phase Death rate
Staining Bacteria stain more by basic stains
Capsule stain India ink
Borris method
Nigrosin method
Not Gram stained Chlamydia and Rickettsia = intracellular
Mycoplasma and Ureaplasma = no cell wall
Spirochetes
Gram Stain (Hucker’s Crystal violet = 1min
modification Gram’s iodine = 1min
Acetone-alcohol or 95% ethanol = 30secs-1min
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 Safranin O = 30 secs
Gram (+) becomes (-) Over-decolorization
Old dying
Acidic iodine
Penicillin: omits iodine
Gram (-) becomes (+) Under-decolorization
Thick smear
Acid Fast staining methods Smear = 2 x 3cm
Pappenheim’s M. smegmatis vs. M. tuberculosis
Baumgarten’s M. leprae vs. M. tuberculosis
Fite Faraco M. leprae
Counterstain: Hematoxylin
Acid fast organisms Mycobacterium
Nocardia = Mod. AFS (1% H2SO4 as decolorizer)
Cryptosporidium
Legionella micdadei
Rhodococcus equi
Ziehl-Neelsen (Hot method) 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
Kinyoun (Cold method) 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
Auramine-Rhodamine Most sensitive
(Fluorochrome) 1. Auramine-rhodamine = 1’stain
2. 0.5% Acid alcohol = Decolorizer
3. 0.5% KMnO4 = Counterstain
Results:
AFO = Yellow fluorescence
NAFO = No fluorescence
AFB Read 300 fields
Special stains Capsule = Negative stain
Spore = Dorner, Wirtz, Conklin
Metachromatic granules
- Albert’s
-Loeffler’s Alkaline Methylene Blue (LAMB)

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 Flagella = Leifson
Nucleic acid = Feulgen
Polar bodies (ex: Y. pestis) = Wayson
Rickettsia = Gimenez
Spirochetes = Levaditi
Phase contrast microscope For study of living unstained organisms
Electron microscope For viruses
Light source: Electrons
100,000x magnification
Stains:
-Negative stain
-PTA
-Heavy metals (Gold, Silver)
Transmission EM DNA, RNA, chromosomes
Scanning EM Surface structures (cell wall, capsule)
Inverted Microscope For tissue culture
Interference microscope Dual light source
Non staining method String’s test (3% KOH)
Pure culture Streak plate = overlap method
Pour plate = Water and milk bacteriology
Selective medium
Animal inoculation = for virus, Chlamydia, Rickettsia
Mixed culture 2 or more organisms
Stock culture Stored at refrigeratior or freezer (long term)
Liquid Broth
Semi-solid 0.5-1% agar
Solid 2-3% agar
Biphasic Both liquid and solid
Ex. Castañeda = Brucella
General purpose media Nonfastidious organisms
1. Sheep BAP = Hemolysis
2. Horse BAP = Haemophilus
-Heat-stable, provides X-factor
3. Nutrient agar
Enriched media Solid
Fastidous organisms
1. CAP = Heat-labile, provides X & V factor
Enrichment media Liquid
1. Selenite F
2. Alkaline peptone water
3. Thioglycollate broth
Differential media 1. BAP = hemolysis
2. MAC
3. EMB
4. XLD
5. HEA
Selective media Inhibitory media
1. TCBS
2. SSA
3. TMA
4. CBAP

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Inhibitory agents Antibiotics
Dyes, bile salts = inhibit Gram (+)
Alcohol (PEA) = inhibit Gram (-)
PEA Gram (+) bacteria
Columbia CNA Gram (+) bacteria
Gonococci Agar (GCA) Gram (-) cocci
Gentamicin BAP S. pneumoniae
Bacitracin CAP H. influenzae
Cystine Tellurite Blood C. diphtheriae
Agar
Cystine Blood Glucose Agar F. tularensis
Cystine Trypticase Agar Confirm: Neisseria
Charcoal Cephalexin Blood B. pertussis
agar
Bordet-Gengou Agar B. pertusis
(Potato Blood Glycerol
Agar)
BCYE L. pneumophila
McCoy Cl. trachomatis
TSB Brucella
Sterile specimen (-) normal flora
Nonsterile specimen (+) normal flora
Calcium alginate swab Toxic to virus
Good for Neisseria
Needle aspiration Anaerobic and aerobic cultures
Catheterization Needle and syringe for collection
Intubation Gastritis
Vomitus
Gastric washing (aerobic culture only)
Delay in processing 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
Transport medium 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)
Biologic safety cabinet HEPA filter: filters air
Negative pressure
BSC Class I Environment and MT protected
Air velocity = 75 linear ft/min
Exhaust air thru HEPA filter
Product contaminant
BSC Class II Vertical laminar airflow
MT, environment and product are protected
Air velocity = 75-100 linear ft/min
Recommended for hospitals

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BSC Class III Supply and exhaust air thru HEPA filter
Maximum protection
N95 Mask Contains HEPA filter
For Mycobacterium
Stool No direct exam in Microbiology
Transposons Resistant gene
Mobile or jumping
Fusiform F. nucleatum
Capnocytophaga
BSL I No risk
BSL II Moderate risk
BSL III High risk
Treatment available
Inhalation of aerosols
Ex. Mycobacteria (BSC Class II)
BSL IV High risk
No treatment available
Inhalation of aerosols
Ex. Small pox
Blood culture bottle BHIB + 0.25% SPS
Dilution = 1:10 (1mL blood, 9mL broth)
Anti-complementary, anticoagulant, antiphagocytic
Neutralizes aminoglycosides
Disadvantages of SPS Inhibits:
-Neisseria
-G. vaginalis
-S. moniliformis
-P. anaerobius
1% gelatin Counteract SPS to allow the growth of organisms
Indications of growth Hemolysis
(Blood culture) Turbidity
Pellicle
Subculture (Blood culture) BAP
MAC = no CO2
CAP
If blood culture = negative 7 days = Bacteremia (Typhoid)
21 days = Brucellosis, SBE
Urine culture 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)
CSF culture DO NOT refrigerate
Agents: Neisseria, Haemophilus (Meningitis)
Media: BAP, MAC, CAP, BHI
C. neoformans:
-India ink method
-Latex agglutination
Wound specimen Gram stain
Media: BAP, MAC, Thioglycollate broth
Stool specimen Media: MAC, BAP+Ampicillin, CBAP, SSA, Selenite F, TCBS, APW, HEA
Oxidase test
Biochemical tests

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 Serologic typing
Respiratory specimen Sputum, NPS
TB = 3 sputum specimen
Media: BAP, MAC, GBAP, BCAP, Amies,
Gram stain and Acid fast stain
Throat swab Sore throat
2 specimen
Media: BAP, MTM, Thioglycollate broth
Vaginal, Urethral swab Media: CAP, MTM
Gram stain
TB culture 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
GenPro Genetic Pro
DNA test
Result  2 hrs
Moist heat sterilization 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
Dy heat sterilization 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

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Other sterilization methods 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
Seitz filter Filter heat-labile filter
Membrane 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
Sodium hypochlorite Spillage disinfectant
(Clorox)
Iodine/Iodophor Sporicidal
Iodine + Detergent = Betadine (Best antiseptic)
Iodine alone = toxic to skin
70% ethyl alcohol Nonsporicidal
H2 O 2 Cleansing of wound
1% AgNO3 Crede’s prophylaxis (New: Erythromycin eye droplets)
Prevents ophthalmia neonatorum
Formaldehyde Sporicidal
Glutaraldehyde Sterilant
Phenol (Carbolic acid) Standard disinfectant
Lysol (Cresol) Multipurpose
Dyes Inhibit Gram (+)
Zephiran (Benzalkonium For decontaminating sputum
chloride)
Iatrogenic Instrument caused
Antagonistic 1 antibiotic > 2 antibiotics
Synergistic 2 antibiotics > 1 antibiotic
XDR-TB Extensively Drug Resistant Tuberculosis
Quinolone resistant
No treatment at all
H. influenzae QC for beta-lactamase
ESBL Extended spectrum beta-lactamase
Produced by Gram (-) = E. coli, Klebsiella
Plasmid mediated
Test: Beta-lactamase = Keyhole effect (overlapping zones)
-Clavulanic acid and cephalosporin
Amp C Chromosome mediated
Produced by Gram (+) and (-) bacteria
Test: Beta-lactamase = D zone
-(+) to MRSA
-Imipenem and cefotixin
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Cell wall inhibitors Penicillin
Cephalosporin
Vancomycin = Tx: MRSA
Broad spectrum:
Bacitracin
Cycloserine
Carbapenems/Imipenem
Penicilinase-resistant: Methicillin, Cloxacillin, Nafcillin
Cell membrane inhibitors Colistin = against Gram (-)
Polymixin = against Gram (-)
Amphotericin B = drug of choice for systemic fungi
Nystatin = antifungal
Ribosome (Protein) Aminoglycosides (30S)
inhibitors -False-resistant = P. aeruginosa (Mg2+ and Ca2+)
Tetracycline (30S)
Chloramphenicol (50S)
Erythromycin/Macrolide (50S)
-Discovered by Bernardo Aguilar
-For penicillin allergic patients
Clindamycin (50S)
Nucleic acid (DNA) Mitomycin
inhibitors Quinolones
Metronidazole (Flagyl)
Trimethoprim-Sulfamethoxazole (SXT/Bactrim) = inh. folate synth., synergistic
Anti-TB Pyrazinamide
Rifampin
Isoniazid
Streptomycin
Ethambutol
Micro/Macrobroth dilution Reference method (AST)
Det. MIC/MBC
Agar dilution Many organisms vs. single drug
Disk diffusion Pure culture vs. many drugs
E test (Epsilometer) Agar gradient diffusion
Antibiotic strip diffusion MIC test
MIC = Ellipse zone at intersection
Kirby-Bauer Disk Diffusion 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)
Petroff-Hausser counting For bacterial count
chamber
15mm Distance of antibiotic disc to each other
15mins Time for the medium to absorb the bacteria after inoculation
w/in 15mins Inoculation of discs  Incubation
False resistant Heavy inoculums
Thick medium

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 Delay in disc application
Ca2+ and Mg2+ = Aminoglycoside (vs. P. aeruginosa)
Thymine-Thymidine = SXT (vs. Enterococcus)
pH = tetracycline
pH = aminoglycoside, erythromycin
Expired discs
False sensitive Light inoculums
Thin medium
If double zone of inhibition Measure the outer zone
Ignore swarming
If there are colonies inside Gram stain the colonies
the zone of inhibition
AST media 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)
QC Specific
Regular basis
Checking media and reagents w/ specific organisms to check expected results
Set by CLSI (formerly NCCLS)
QA General
Snap shot
Total process whereby the quality of lab. reports can be guaranteed
Daily QC Oxidase
Catalase
Incubator
Gram stain
Refrigerator/Freezer
Water bath
Each use (QC) GasPak Jar
ONPG
Weekly QC Antibiotic (Newly opened: 30 days QC  weekly)
Autoclave
Biochemical tests
Semi-annually Safety hood
ATCC (American Type For AST
Culture Collection) Stock culture: -20 or -70’C
Working culture: 2-8’C
ATCC-1234 Beta-lactamase producers:
-S. aureus
-N. gonorrhoeae
-H. influenzae
-Enterococcus
-E. coli
-P. aeruginosa
Catalase test Rgt: 3% H2O2
(+) Gas bubbles
F (+): BAP

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Coagulase test 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
Mannitol fermentation Medium: MSA (7.5% NaCl)
Indicator: Phenol Red
(+) Yellow
(-) Red
DNase test 1. Toluidine blue (pink zone)/ Methyl green (clear zone)
2. HCl precipitation: no pptn. after adding 1N HCl when DNase (+) = pink
Novobiocin test Amt.: 5μg
(R): <16mm
(S): >16mm
Modified oxidase test Rgt: tetramethyl-p-phenylenediamine dihydrochloride in dimethylsulfoxide
(+) Purple
Staphylococcus Pinhead colonies
Mod. oxidase (-)
Lysostaphin and Furazolidone (S)
Ferments sugar
Micrococcus Mod. oxidase (+)
Lysostaphin and Furazolidone (R)
Oxidizes sugar
Stomatococcus Mod. oxidase (-)
Lysostaphin and Furazolidone (R)
S. aureus 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
S. lugdunensis Slide coagulase (+)
PYR (+)
S. intermedius Slide coagulase (+)
VP (-)
Lab. Diagnosis Nasal swab: carrier of S. aureus
(Staphylococcus) Culture:
-Vogel-Johnson: Black colonies
-Chapman: Black colonies
-Tellurite Glycine: Black colonies
-P agar
-PEA: selective
-Columbia CNA: selective
S. epidermidis #1 skin flora
Blood culture contaminant

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 Biofilm/slime production: Prosthetic heart valve  Endocarditis, bacteremia
UTI: catheterized
S. saprophyticus UTI: sexually active women
ID: Streptococcus
Lancefield Taxo A Bile 6.5% Taxo P
CAMP Hippurate SXT BEM PYR
Group (0.04 U) Solubility NaCl (5μg)
A S - - R - - + - R
B R + + R - - - - R
C, F, G R - - S - - - - R
D (Enterococcus) R - - R + - + + R
D (non-Enterococcus) R - - R + - - - R
S. pneumoniae R - - R - - - - S (>14mm)
Streptococcus Pinpoint colonies
Capnophilic: 5-10% CO2
SBA: Medium of choice
PEA: Selective medium
Smith and Brown’s Hemolysis:
classification 1. Alpha = incomplete (green)
2. Beta = complete (clear)
3. Gamma = no zone
4. Alpha prime = alpha (around colonies) + beta (around alpha)
S. pyogenes Universally susceptible to antibiotics
(Group A) Virulence factors:
(Beta-hemolytic) -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
S. agalactiae Vaginal and URT flora
(Group B) #1 neonatal meningitis
(Beta-hemolytic)
Group C S. equisimilis
(Beta-hemolytic) S. equi
S. zoopedemicus
S. dysagalactiae
Group F S. anginosus
(Beta-hemolytic)
Group D Enterococcus E. faecalis
(Alpha, beta or gamma- E. faecium
hemolytic) E. durans
E. avium
Cause UTI
Drug-resistant: VRE
Group D non-Enterococcus S. bovis
(Alpha, beta or gamma- S. equinus
hemolytic) Cause UTI
S. pneumoniae Lancet-shaped, diplococci
(Alpha-hemolytic) Colonies: Mexican hat/ Dome-shaped
Encapsulated
#1 Adult bacterial meningitis
lec.mt 04 |Page | 66
 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
Viridans Streptococci S. mitis (mitior)
S. salivarius
S. uberis
S. constellatus
S. intermedius
S. mutans = dental plaques/caries
S. sanguis = SBE
Nutritionally Variant Abiotrophia
Granulicatella
Require Vit. B6 (pyridoxine)
(+) Staph. Streak test
Vancomycin Resistant Leuconostoc = LAP (-)
Pediococcus = LAP (+)
Neisseria Aerobic
Gram (-) diplococci
Oxidase (Taxo N): Presumptive test (+)
CTA: Confirmatory test
Capnophilic: 5-10% CO2
ID: Neissera & Moraxella
Glucose Maltose Lactose Sucrose DNase
N. gonorrhoeae + - - - -
N. meningitidis + + - - -
N. lactamica + + + - -
N. sicca + + - + -
N. subflava + + - V -
N. cinerea - - - - -
N. flavescens - - - - -
N. elongata - - - - -
M. catarrhalis - - - - +
N. gonorrhoeae 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)

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 = MTM (V-C-N-Trimethoprim lactate)
= MLA (V-C-Anisomycin-T)
= NYCA (V-C-Amphotericin B-T)
N. meningitidis 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)
M. catarrhalis Commensal of URT
(+) NO3  NO2
(+) Butyrate disk
(+) Tributyrin hydrolysis
(G) Nutrient Agar
Colony: Hockey Puck
3rd cause of Otitis media
N. sicca Breadcrumb/wrinkled colony
N. lactamica (+) ONPG
Superoxol catalase test 30% H2O2
(+) N. gonorrhoeae
Beta-lactamase test 1. Chromogenic cephalosporin test or Nitrocefin/Cefinase disk test
-(+) Pink/red color
2. Acidimetric
-Phenol red  (+) Colorless
3. Acidimetric
-I2  (+) Yellow
Mycobacteria 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
M. tuberculosis “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 1. M. marinum (“Of the sea”)
(Group I) -(+) 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
Scotochromogens 1. M. scrofulaceum
(Group II) -(+) 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
Non-photochromogens Colony: Cream/buff
(Group III) 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
Rapid Growers 1. M. fortuitum-chelonae
(Group IV) -(+) Arylsulfatase
-(G) MAC
a. M. fortuitum
-(+) NO3 reduction
-(+) 5% NaCl
-(+) Iron uptake

lec.mt 04 |Page | 70
 b. M. chelonei
-(-) NO3 reduction
-(-) 5% NaCl
-(-) Iron uptake
2. M. smegmatis
3. M. phlei (“Hay bacillus”)
Growth: 10-21 days Groups I, II, III (Runyon’s Classification)
Growth: 3-7 days 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
7H12 14C Palmitic acid + organisms ----------> 14CO
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 “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
Biotypes of C. diphtheriae 1. Gravis = gray, large, beta-hemolytic, starch/glycogen fermentation (+)
2. Mitis = black, medium-size, beta-hemolytic, starch/glycogen fermentation (-)
3. Intermedius = black, small, nonhemolytic
C. pseudodiphtheriticum “Hoffman’s bacillus”
C. xerosis Ferments glucose, maltose and sucrose
C. minutissimum Agent of erythrasma
Coral red fluorescence on Wood’s lamp (porphyrin)
Transduction Lysogeny (no lysis)
B. anthracis 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
B. cereus “Fried rice bacillus”
Exotoxin (cholera-like): cAMP = H2O, Na+, K+ = dehydration
(-) capsule
B. subtilis Common lab. contaminant
Causes eye infection in heroin addicts
Clostridium 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
C. perfringens Encapsulated, nonmotile
(Old: C. welchii) 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 S R S
Clostridium S S R
Kanamycin Vancomycin Colistin
Gram (+) cocci S S R
Porphyromonas R S R
P. anaerobius R S R
Prevotella R R S
Brick red fluorescence Prevotella
Porphyromonas
Red fluorescence Veillonella
Pitting of agar B. ureolyticus
Swarming C. tetani
C. septicum
Molar tooth colony, sulfur A. israelii
granules
Breadcrumb colony Fusobacterium
Gram (+) Anaerobic Bacilli Actinomyces
Bifidobacterium
Eubacterium
Propionebacterium
Lactobacillus
Mobiluncus
Gram (-) Anaerobic Bacilli Bacteroides
Porphyromonas
Prevotella
Fusobacterium
Gram (+) Anaerobic Cocci Peptostreptococcus
Peptococcus
Gram (-) Anaerobic Cocci Veillonella
Megasphera
Acidaaminococcus
A. bovis Lumpy jaw
P. acnes Acne vulgaris
(+) Catalase, Indole
L. acidophilus Tomato juice agar
(-) Catalase
Mobiluncus Vaginitis (G. vaginalis)
(-) Catalase
Anaerobic infections Foul odor
B. fragilis Needs 20% bile
F. necrophorum Vincent’s angina (B. vincenti)
(F. moriferum)
P. anaerobius SPS sensitive (Neut: 1% gelatin)
(-) Indole, Catalase
P. asaccharolyticus (-) Catalase
P. niger Staphylococcus-like
Black
H2S indicators TSI = Ferrous sulfate
LIA = Ferric ammonium citrate
SIM = Lead acetate
Phenol red Acid = Yellow
Alkaline = Red
lec.mt 04 |Page | 74
Methyl red Acid = Red
Alkaline = Yellow
Neutral red Acid = Pink/red
Alkaline/neutral = Colorless
Bromthymol blue Acid = Yellow
Neutral = Green
Alkaline = Blue
Enterobacteriaceae 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)
VP (+) “KESH”
Klebsiella
Enterobacter
Serratia
Hafnia
H2S (+) “SPACEd”
Salmonella
Proteus
Arizona
Citrobacter
Edwardsiella
Lactose Fermenters “ACH” (Slow) “EKE” (Rapid)
Arizona
Citrobacter
Hafnia
E. coli
Klebsiella
Enterobacter
-----------------------------------------------------
Y. enterocolitica = (+) ONPG, TSI: A/A
Deaminase (+) “PMP”
Proteus
Morganella
Providencia
Urease (+) “PMP” (except P. alcalifaciens)
“K.pn/ox”
“E”
“Y.ps/en”
Proteus
Morganella
Providencia
K. pneumoniae
lec.mt 04 |Page | 75
 K. oxytoca
E. gergoviae
Y. pseudotuberculosis
Y. enterocolitica
Aerogenic Klebsiella
E. coli
Salmonella (except S. typhi, S. gallinarum)
Proteus
Arizona
Citrobacter
Enterobacter
Enteric Media
Inhibitory CHO Indicator LF NLF
EMB Eosin Y Lactose Eosin Y Red purple Colorless
Methylene blue Methylene blue (E. coli: GMS)
MAC Bile salts Lactose Neutral red Red/pink Colorless
Crystal violet
SSA Bile salts Lactose Neutral red Red Colorless
DCA Bile salts Lactose Neutral red Red/pink Colorless
TCBS Bile salts Sucrose Bromthymol blue Yellow Green

HEA Bile salts Salicin Bromthymol blue Yellow Green

Lactose
Sucrose
XLD Bile salts Xylose Phenol Red Yellow Red
Lactose
Sucrose
BSA Brilliant green Glucose Bismuth sulfite S. typhi = black colonies
(Old: Wilson-Blair)
Lysine-Ornithine-Arginine Reactions
“(LO)9(OA)3(L)4(O)6(N)6” - Enterobacteriaceae
L O A L O A
Escherichia coli + + - Shigella sonnei - + -
Enterobacter aerogenes + + - Proteus mirabilis - + -
Enterobacter gergoviae + + - Morganella morganii - + -
Edwardsiella tarda + + - Citrobacter - + -
Arizona + + - Yersinia enterocolitica - + -
Serratia + + - Salmonella paratyphi - + -
Hafnia + + - Providencia - - -
Salmonella enteritidis + + - Proteus vulgaris - - -
Salmonella choleraesuis + + - Klebsiella rhinoscleromatis - - -
Enterobacter cloacae - + + Pantoea agglomerans - - -
Enterobacter sakazakii - + + Yersinia pseudotuberculosis - - -
Enterobacter taylorae - + + Yersinia pestis - - -
Salmonella typhi + - - Plesiomonas shigelloides + + +
Klebsiella pneumoniae + - - Vibrio + + -
Klebsiella oxytoca + - - Aeromonas hydrophila + - +
Klebsiella ozaenae + - - Pseudomonas aeruginosa - - +
E. coli “Colon Bacillus”
#1 UTI
#2 Neonatal meningitis (K1 Ag)
MUG (+) except E. coli O157:H7
lec.mt 04 |Page | 76
 -MUG (+): Electric blue fluorescence

ETEC Traveler’s diarrhea

Montezuma revenge
Turista
Belly Delhi
Cholera like, watery diarrhea (SL, LT)
EPEC Infantile diarrhea
Pili: pathogenicity island
EIEC Dysentery (Shigella)-like diarrhea
Invasin: Pathogenicity
Stool: bloody w/ mucus
Sereny test
EHEC/VTEC HUS
Hemorrhagic colitis
Verotoxin: Shigella-like toxin
E. coli O157:H7 EHEC/VTEC
Sorbitol MAC (-)
EAEC Acute and chronic diarrhea
Aggregative adhesion fimbriae
Other Escherichia spp E. hermanni = yellow colonies
E. vulneris = yellow colonies
Alkalescens dispar (Anaerogenic E. coli/E. coli inactive)
-Nonmotile, (-) H2S, NLF, PG (-), Vi (-)
Enterobacter UTI, wound, septicemia
E. sakazakii = yellow pigment
E. (Pantoea) agglomerans = yellow pigment
K. pneumoniae “Friedlander’s bacillus”
(+)Capsule, ESBL
Diseases:
-Pneumonia: “Currant jelly-like sputum”
-Wound infections
-Meningitis
-UTI
K. rhinoscleromatis Biochemically inert
PMP Group (Proteeae) All are indole (+) except P. mirabilis (-)
Proteus Swarm on BAP but not on MAC
#2 UTI
Renal stone association
Odor: Burnt gun powder/Burnt chocolate
P. mirabilis: Rapid urease producer
Salmonella 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
S. typhi Typhoid fever
Meningitis
Osteomyelitis

lec.mt 04 |Page | 77

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
Complication: Psychosis
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 El Tor Biotype
S R
lec.mt 04 |Page | 78
Lysis by bacteriophage + -
Chicken RBC agglutination - +
Classical Biotype El Tor Biotype
Hemolysis of sRBC - +
VP test - +
Ogawa Serotype Inaba Serotype Hikojima Serotype
Anti-Ogawa + - +
Anti-Inaba - + +
Lab. Diagnosis (V. cholerae) 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)
V. parahaemolyticus Halophilic (8% NaCl)
(+) Indole
#1 Gastroenteritis in Japan (seafood)
Nonsucrose fermenter (TCBS)
Kanagawa (+) = Beta hemolysis on Wagatsuma agar
V. vulnificus Stool pathogen
(+) Blood culture
(+) Lactose fermentation, ONPG
Nonsucrose fermenter (TCBS)
V. alginolyticus Sucrose fermenter (TCBS)
Aeromonas Motile (monotrichous)
(+) Oxidase
Beta-hemolytic
(R) O129
(+) DNase
(+) Bile esculin hydrolysis
Plesiomonas (+) Oxidase
Non-hemolytic
(V) O129
(-) DNase
(-) Bile esculin hydrolysis
Campylobacter (G) 42’C
Curved, S-shaped (Wings of Seagull)
(+) Catalase
(+) Oxidase
Darting motility
C. jejuni: (+) Hippurate
H. pylori (+) Catalase
(+) Oxidase
(+) Urease
Test: Urease breath test
OF 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
P. aeruginosa Old: P. pyocyanea

lec.mt 04 |Page | 79
 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
B. cepacia (+) Oxidase
Motile (lophotrichous)
#2 Cystic fibrosis
(+) Lactose, ONPG

B. pseudomallei “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
B. mallei Nonmotile
Glander’s disease (Horses)
P. stutzeri Brown (buff colored) wrinkled colony
(+) 6.5% NaCl
(+) NO2  N2
(-) Lactose
Found in stagnant water
S. maltophilia (-) Oxidase
(+) DNase
(+) Maltose and Glucose
Motile (lophotrichous)
Colony: Lavender green
Common w/ the use of catheter
P. syncyanea Blue milk
Shewanella putrefaciens TSI: K/K + H2S
(+) Oxidase
Acinetobacter (-) Oxidase
(+) Catalase
Nonmotile
MAC: purple colonies
Mistaken as Neisseria – (+) oxidase
Causes UTI

lec.mt 04 |Page | 80
A. anitratus (A. baumanii) Oxidizer
Herella vaginocola
A. lwoffi Nonoxidizer
Mima polymorpha
Alcaligenes faecalis (+) Oxidase
(+) Catalase
Motile (peritrichous)
Asaccharolytic
“Fruity” odor
M. lacunata (+) Pitting of agar
(+) Oxidase
(+) Catalase
Assacharolytic
(NG) MAC
Infection: Blepharoconjunctivitis
Mistaken as Neisseria
Flavobacterium Flavin = Yellow pigment
(Chryseobacterium) (+) Oxidase
meningosepticum (+) DNase
(+) Indole
(+) Gelatin hydrolysis
Nonmotile
Causes neonatal meningitis
E. corrodens Twitching motility
MOT: Human bite
“Clenched fist”
Corrodes agar
Odor: Bleach-like
(-) Catalase
(+) Oxidase
Kingella spp Cause SBE (HACEK)
Pits agar
Haemophilus Require X factor (hemin) and V factor (NAD)
(+) Satellitism
(+) Oxidase
HCAP: medium of choice (5% CO2)
X factor V factor Porphyrin
H. influenzae + + -
H. aegyptius + + -
H. haemolyticus + + -
H. parainfluenzae - + +
H. parahaemolyticus - + +
H. paraphrophilus - + +
H. ducreyi + - -
H. aphrophilus - - +
Porphyrin test X factor
D-ALA  (+) Protoporphyrin (Red)
H. influenzae “Pfeiffer’s Bacillus”
Serotype polysaccharide:
-Hib = Haemophilus type B: capsular antigen type B (polyribose ribitol PO4)
Infections:
-Epiglotitis (Major)

lec.mt 04 |Page | 81
 -#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
H. ducreyi “Chancroid Bacillus”
(G) CAP + Vancomycin (33’C)
Soft chancre (painful)
School of red fish
H. aegyptius “Koch Week’s Bacillus”
Pink eye conjunctivitis
Brazilian purpuric fever
B. pertussis “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 MAC/BAP
B. pertussis - - + -
B. parapertussis - + - +
B. bronchiseptica + + + +
B. bronchiseptica “Kennel cough Bacillus”
Brucella (-) 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

lec.mt 04 |Page | 82
 -(+) Rose Bengal & 2-ME agglutination
B. abortus “Bang’s Bacillus”
Urease CO2 Thionine (Dye Inhibition) Fuchsin (Dye Inhibition)
B. abortus + + - (NG) + (G)
B. melitensis + - + (G) + (G)
B. suis + - + (G) - (NG)
B. canis + - + (G) - (NG)
F. tularensis (+) 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
P. multocida Multocida: Multiple killing
(+) Capsule
Nonmotile
(+) Oxidase
(+) Catalase
(+) Glucose
(+) Ornithine
(+) Indole
(+) Urease
(G) BAP
(NG) MAC
Infections:
-Animal (cat) bite wound infection
-Shipping fever
Treponema (-) Catalase
Dx: Serology
Leptospira (+) Catalase
Dx: Culture
Borrelia (-) Catalase
Dx: Giemsa (blood smear), Serology
T. pallidum Noncultivable on agar
Obligate intracellular (Rabbit’s testicle)
Syphilis 1’ = Hard chancre (Painless)
2’ = Condylomata lata (HPV: Condylomata acuminata)
Latent: Asymptomatic, Dx: Serology
3’: Gummas, neurosyphilis
Congenital syphilis: Hutchinsonian triad, abortion, still birth
Neurosyphilis Tabes dorsalis

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Penicillin Treatment for syphilis
(Jarisch-Herxheimer reaction)
T. pertenue Yaws, Framboesia
T. carateum Pinta
T. endemicum Bejel
T. cuniculi Rabbit syphilis
Lab. Diagnosis (Treponemes) 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)
L. interrogans Obligate aerobes
icterohemorrhagiae 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
Borrelia 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 CO2
H. aphrophilus - + - +
A. actinomycetemcomitans + - - +
C. hominis - + - +
E. corrodens - + - +
K. kingae - - + +
A. actinomycetemcomitans Star-like colony
Dots and dashes of Morse code
C. hominis (+) Indole
Teardrop shape
Rosette formation
E. corrodens Asaccharolytic
Chlamydia (Old: Bedsonia) Obligate intracellular

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 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)
C. trachomatis 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)
C. psittaci Parrot fever/Psittacosis/Ornithosis
Man: Pneumonia
Inclusion body (Levinthal Cole Lillie)
-Elementary body: Round-shaped
-(-) glycogen
(R) Sulfonamide
C. pneumoniae TWAR agent
Pneumonia: Human-to-human
Growth on:
-Human lines
-Hep-2 cell
Guillain-Barré syndrome: ascending paralysis
Rickettsia 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
Ehrlichia Intracellular (WBCs)
Morulae: diagnostic form
Tick-transmitted
Cause sennetsu fever
Vector Disease
R. rickettsii Tick RMSF
R. akari Mite Rickettsialpox
R. typhi Rat flea Murine/endemic typhus
R. prowazekii Human louse Epidemic typhus/Brill Zinsser dis.
O. tsutsugamushi Chigger Scrub typhus
R. quintana Human louse Trench fever
E. chaffeensis Tick Monocytic Ehrlichiosis

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E. equi Tick Granulocytic Ehrlichiosis
C. burnettii Inhalation Q fever
Mycoplasma 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)
M. pneumoniae 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
M. hominis Large fried egg colony
Infections: Post abortal, post partum fever, PID
Media:
-A7/A8: selective
-NYCA
-SP4: arginine
U. urealyticum T strain = tiny fried egg
(+) Urease = brown
Cause NGU
Media:
-A7/A8
-SP4: urea
-NYCA
G. vaginalis 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)
C. granulomatis (+) Capsule
Safety pin
MOT: Sexual contact
Causes granuloma inguinale (Donovanosis)
Giemsa stain: Donovan bodies (macrophage w/ Gram [-] bacilli)
Not cultured
S. moniliformis 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

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S. minus (S. minor) Spiral organism
Causes rat bite fever
Cannot be cultivated
C. violaceum Old: Achromobacterium
Violacein: violet colored
MAC: NLF
NH4 cyanide
Capnocytophaga Fusiform
Gliding motility
Periodontal disease (oral flora) = periodontitis
Large spreading colonies
(-) Oxidase
(-) Catalase
(-) Indole
(+) Esculin hydrolysis, NO3 reduction
B. henselae Infections:
Cat scratch disease
Bacillary angiomatosis
Peliosis
Hepatitis
(NG) MAC
B. bacilli formis Vector: Sandfly
Infections:
-Carrion’s disease
-Verruga peruana: skin eruptions
-Oroya fever: anemia
L. pneumophila 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
L. monocytogenes Tumbling motility: head over heels/end-to-end motility
Listeriolysin O
Infections:
-Granulomatosis infantiseptica
-Meningitis
Lab. Diagnosis: Anton’s test (Skin test)
E. rhusiopathiae Growth: Test tube brush
Erysipeloid: Butcher’s cut
L. monocytogenes E. rhusiopathiae

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Catalase + -
Motility (25’C) + -
Hemolysis Beta Alpha
L. monocytogenes E. rhusiopathiae
VP + -
H2 S - +
Bile esculin + -
Hippurate + -
Gluconate + -
Media Mc Bride, Cold enrichment BAP
Water Bacteriology E. coli: Index of fecal contamination
Det. coliforms
Sodium thiosulfate Neutralizes chlorine
1. Presumptive test Lactose broth/ + H2O ----------(35’C for 24 hrs)----> (+) Gas
Lauryl tryptose broth (-) No gas after 48 hrs
2. Confirmatory test EMB/Endo agar + Inoculum -----(24 hrs)-----------> (+) Colony
3. Completed test Lactose broth fermentation tube ---(35’C for 24-48 hrs)---> (+) Acid + Gas
MPN (Most Probable Number) Most common method for H2 analysis
Estimation of coliform group density
# of positive coliforms from multiple decimal dilution
Reference laboratories EAMC
(Water Bacteriology) FDA
Milk Bacteriology
Streptococcus lactis Acid forming, causes normal souring of milk
Coliform bacteria Gas-forming, hydrolyze the milk protein casein
Bacillus subtilis Hay bacteria, proteolytic action on coagulated milk
Clostridium butyricum Stormy fermentation of milk
Clostridium perfringens
Pseudomonas syncyanea Blue milk
Serratia marcescens Red milk
Pseudomonas aeruginosa Blue green milk
Flavobacterium synxanthum Yellow milk
Alcaligenes viscosus Slimy or ropy milk (capsule formation)

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