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
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)
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
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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