CC

Soap MUST TO KNOW IN BACTERIOLOGY
L. monocytogenes
Germicidal
Chlamydia Catalase (+)
3 C’s: Chicken, Coleslaw, Cheese
BAP When delayed: 4’C
Freezing: -20’C
M. gordonae
DNase test Phenotypic
LOA test Gram (+) colonies: Dry, white, sometimes gray
Gram (-) colonies: Gray and moist
Na hypochlorite Destroyed by chlorine
Inoculating needles
Utilizes 1N HCl
Wire loop For Gram (+)
For nonfermentative
50k CFU/mL For Enterobacteriaceae
Pregnant Inactivates HBV (10mins) and HIV (2mins)

Cotton swab Nichrome = F(+) on oxidase test
Not longer than 5cm
Charcoal
Phenotype 2mm diameter
0.001mL urine
Stool
PCR Significant for UTI
Somatic antigen C. albicans
Anton van Leeuwenhoek Lactobacillus
Carrier state
Robert Koch Lawn a culture
Louis Pasteur Toxic to Neisseria
Ehrlich Good for virus
Bacteria
Removes the toxin inoculated by cotton
Cell wall
Basis of identifying organisms
Gram (+) Gram stain and colonies
Not Gram stained
Gram (-)
Most definitive method of identification
Plasma membrane Basis of serotyping

Nucleoid 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 Carries the antibiotic-resistance gene
Drug-resistance
Metachromatic granules Chromosome and plasmid-mediated
Ribosomes Food reserves
Common pili
Sex pili Prokaryotic: 70S
ESBL Eukaryotic: 80S
Endospores
Flagella Bacterial adherence
Gene transfer
Lactobacillus
Autotrophs/Lithotrophs By Gram (-) bacteria
Heterotrophs/ Calcium dipicolinate
Organotrophs Bacillus, Clostridium
Temperature requirements
Monotrichous: one only
pH requirement Amphitrichous: one at both ends
Lophotrichous: tuft at one end
Moisture Peritrichous: all around bacteria (most common)
Salt concentration
Respiration (Aerobic) Aerotolerant anaerobes
Inorganic compound as source of carbon (CO2)
Oxidation (Aerobic) Organic compound as source of carbone (Glucose)
Fermentation (Anaerobic) Pathogenic bacteria
Lag phase Psychrophilic: 0-20’C (ref)
Log/Exponential phase Mesophilic: 20-40’C (pathogenic)
Stationary/plateau phase Thermophilic: 40-60’C

Death/Decline phase Acidophilic: Lactobacillus acidophilus (Doderlein bacillus)
Staining Neutrophilic: pH 7.2-7.6 (optimal) – pathogenic
Capsule stain Basophilic: Vibrio (Halophilic)

Not Gram stained Humidophilic
Halophilic
Gram Stain (Hucker’s Enterococcus and V. parahaemolyticus
modification 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 (-) Safranin O = 30 secs
Gram (-) becomes (+)
Acid Fast staining methods Over-decolorization
Pappenheim’s Old dying
Baumgarten’s Acidic iodine
Fite Faraco Penicillin: omits iodine
Acid fast organisms
Under-decolorization
Ziehl-Neelsen (Hot method) Thick smear

Kinyoun (Cold method) Smear = 2 x 3cm

Auramine-Rhodamine M. smegmatis vs. M. tuberculosis
(Fluorochrome)
M. leprae vs. M. tuberculosis
AFB
Special stains 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 Flagella = Leifson
Electron microscope Nucleic acid = Feulgen
Polar bodies (ex: Y. pestis) = Wayson
Transmission EM Rickettsia = Gimenez
Scanning EM Spirochetes = Levaditi
Inverted Microscope For study of living unstained organisms
Interference microscope
Non staining method For viruses
Pure culture Light source: Electrons
100,000x magnification
Mixed culture Stains:
Stock culture -Negative stain
Liquid -PTA
Semi-solid -Heavy metals (Gold, Silver)
Solid DNA, RNA, chromosomes
Biphasic
General purpose media Surface structures (cell wall, capsule)

Enriched media For tissue culture

Enrichment media Dual light source
String’s test (3% KOH)
Differential media
Streak plate = overlap method
Selective media 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 Antibiotics
Dyes, bile salts = inhibit Gram (+)
PEA Alcohol (PEA) = inhibit Gram (-)
Columbia CNA Gram (+) bacteria
Gonococci Agar (GCA) Gram (+) bacteria
Gentamicin BAP Gram (-) cocci
Bacitracin CAP S. pneumoniae
Cystine Tellurite Blood H. influenzae
Agar C. diphtheriae
Cystine Blood Glucose Agar
Cystine Trypticase Agar F. tularensis
Charcoal Cephalexin Blood Confirm: Neisseria
agar B. pertussis
Bordet-Gengou Agar
(Potato Blood Glycerol B. pertusis
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
Delay in processing Gastric washing (aerobic culture only)
Refrigerate except:
Transport medium 1. CSF = Room temp. or 35’C
2. Blood
Biologic safety cabinet 3. Swab of N. gonorrhoeae (sensitive to cold)
4. Urine = Boric acid
BSC Class I 5. Rectal swab = Cary-Blair
1. Cary Blair = for stool pathogen
BSC Class II 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 Supply and exhaust air thru HEPA filter
N95 Mask Maximum protection
Stool Contains HEPA filter
Transposons For Mycobacterium
Fusiform No direct exam in Microbiology
BSL I Resistant gene
BSL II Mobile or jumping
BSL III F. nucleatum
Capnocytophaga
BSL IV No risk
Moderate risk
Blood culture bottle High risk
Treatment available
Disadvantages of SPS Inhalation of aerosols
Ex. Mycobacteria (BSC Class II)
1% gelatin High risk
Indications of growth No treatment available
(Blood culture) Inhalation of aerosols
Subculture (Blood culture) Ex. Small pox
BHIB + 0.25% SPS
If blood culture = negative Dilution = 1:10 (1mL blood, 9mL broth)
Urine culture Anti-complementary, anticoagulant, antiphagocytic
Neutralizes aminoglycosides
CSF culture Inhibits:
-Neisseria
Wound specimen -G. vaginalis
Stool specimen -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 Serologic typing
Throat swab Sputum, NPS
Vaginal, Urethral swab TB = 3 sputum specimen
TB culture Media: BAP, MAC, GBAP, BCAP, Amies,
Gram stain and Acid fast stain
GenPro Sore throat
Moist heat sterilization 2 specimen
Media: BAP, MTM, Thioglycollate broth
Dy heat sterilization 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 1. Cold temperature/Freezing (Bacteriostatic)
-Preservation
Seitz filter 2. Lyophilization (Powderized)
Membrane filter -Freeze drying
-Best to preserve culture
Sodium hypochlorite 3. Osmotic pressure (Bacteriostatic)
(Clorox) -Preservation
Iodine/Iodophor 4. Dessication = removal of water
5. UV light = produce pyrimidine dimer to DNA  mutation
70% ethyl alcohol -Reduces airborne infection
H2O2 6. Ionizing radiation
1% AgNO3 -For disposable materials (gloves, syringe)
Formaldehyde 7. Filtration
Glutaraldehyde -Air: HEPA filter
Phenol (Carbolic acid) -H2O: cellulose membrane/ membrane filter
Lysol (Cresol) Filter heat-labile filter
Dyes Made up of cellulose nitrate, cellulose diacetate, polycarbonate or polyester
Zephiran (Benzalkonium New: cellulose diacetate w/ a pore diameter of 0.015 to 12 microns
chloride) Best filter used
Iatrogenic Spillage disinfectant
Antagonistic
Synergistic Sporicidal
XDR-TB Iodine + Detergent = Betadine (Best antiseptic)
Iodine alone = toxic to skin
H. influenzae Nonsporicidal
ESBL Cleansing of wound
Crede’s prophylaxis (New: Erythromycin eye droplets)
Amp C 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 Penicillin
Cephalosporin
Cell membrane inhibitors Vancomycin = Tx: MRSA
Broad spectrum:
Ribosome (Protein) Bacitracin
inhibitors Cycloserine
Carbapenems/Imipenem
Nucleic acid (DNA) Penicilinase-resistant: Methicillin, Cloxacillin, Nafcillin
inhibitors Colistin = against Gram (-)
Anti-TB Polymixin = against Gram (-)
Amphotericin B = drug of choice for systemic fungi
Micro/Macrobroth dilution Nystatin = antifungal
Agar dilution Aminoglycosides (30S)
Disk diffusion -False-resistant = P. aeruginosa (Mg2+ and Ca2+)
E test (Epsilometer) Tetracycline (30S)
Kirby-Bauer Disk Diffusion Chloramphenicol (50S)
Erythromycin/Macrolide (50S)
Petroff-Hausser counting -Discovered by Bernardo Aguilar
chamber -For penicillin allergic patients
15mm Clindamycin (50S)
15mins Mitomycin
w/in 15mins Quinolones
False resistant 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 Delay in disc application
If double zone of inhibition Ca2+ and Mg2+ = Aminoglycoside (vs. P. aeruginosa)
If there are colonies inside Thymine-Thymidine = SXT (vs. Enterococcus)
the zone of inhibition pH = tetracycline
AST media pH = aminoglycoside, erythromycin
Expired discs
QC Light inoculums
Thin medium
QA Measure the outer zone
Daily QC Ignore swarming
Gram stain the colonies
Each use (QC)
Weekly QC 1. MHA = std. media
Semi-annually 2. MHA + 2% NaCl = MRSA
ATCC (American Type 3. MHA + 5% Sheep blood = S. pneumoniae (w/ CO2)
Culture Collection) 4. Haemophilus test medium:
ATCC-1234 -MHA + Yeast extract + Hemin + NAD + CO2
5. GC agar = Neisseria (w/ CO2)
Catalase test 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 Rgt: Rabbit EDTA plasma
(+) Clot formation after 4hrs
Mannitol fermentation F (+): Citrate
DNase test F (-): Reading result after 6 hrs (Staphylokinase)
Novobiocin test 1. Slide test (Screening) = detects clumping factor/bound coagulase
Modified oxidase test 2. Tube test (Confirmatory) = detects free/unbound coagulase
Staphylococcus Medium: MSA (7.5% NaCl)
Micrococcus Indicator: Phenol Red
Stomatococcus (+) Yellow
S. aureus (-) Red
1. Toluidine blue (pink zone)/ Methyl green (clear zone)
S. lugdunensis 2. HCl precipitation: no pptn. after adding 1N HCl when DNase (+) = pink
S. intermedius Amt.: 5μg
Lab. Diagnosis (R): <16mm
(Staphylococcus) (S): >16mm
Rgt: tetramethyl-p-phenylenediamine dihydrochloride in dimethylsulfoxide
S. epidermidis (+) 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

S. saprophyticus UTI: sexually active women

ID: Streptococcus

Lancefield Taxo A CAMP Hippurate SXT BEM Bile PYR 6.5% Taxo P
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

Viridans Streptococci Most common cause of Otitis media
Lobar pneumonia: Rusty sputum
Nutritionally Variant Lab. Diagnosis:
1. Neufeld Quellung (pptn. test, capsular swelling)
Vancomycin Resistant 2. Bile solubility
Neisseria -BAP: 10% Na desoxycholate
-Tube: 2% Na desoxycholate
ID: Neissera & Moraxella 3. Francis test: skin test
N. gonorrhoeae 4. Mouse virulence test: (+) death
N. meningitidis
N. lactamica S. mitis (mitior)
N. sicca S. salivarius
N. subflava S. uberis
N. cinerea S. constellatus
N. flavescens S. intermedius
N. elongata S. mutans = dental plaques/caries
M. catarrhalis S. sanguis = SBE
N. gonorrhoeae
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

Glucose Maltose Lactose Sucrose DNase
-
+- - - -
-
++ - - -
-
+++ - -
-
++ - + -
+
++ -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)

lec.mt 04 |Page | 67

N. meningitidis = MTM (V-C-N-Trimethoprim lactate)
= MLA (V-C-Anisomycin-T)
M. catarrhalis = NYCA (V-C-Amphotericin B-T)
N. sicca
N. lactamica Carrier: Nasopharynx
Superoxol catalase test Virulence factors:
Beta-lactamase test -Capsule
Mycobacteria -Endotoxin
-Pili
M. tuberculosis -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 2. Decontamination-Digestion = Best
Agar based media -Na citrate: remove metallic compound
Egg based media 3. AFS
(Sterilization: Inspissation) 4. Culture
Liquid media
Niacin test Bone TB

Heat stable catalase test Duboi’s oleic acid albumin medium
Mitchison’s medium
Nitrate reduction test Middlebrook 7H10 and 7H11 (AST)
Tween 80 hydrolysis test
Petragnani medium
Arylsulfatase test Löwenstein Jensen (Slant) medium
American Thoracic Society medium
TCH Susceptibility test Dorset egg medium
Old Skin Test (TB)
BACTEC 12B
PPD 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
Scotochromogens 2. M. asiaticum
(Group II) 3. M. simiae
-1st: Macaca rhesus monkey
Non-photochromogens 4. M. kansasii (“Yellow bacillus”)
(Group III) -(+) Heat-stable catalase
-(+) Tween 80 hydrolysis
Rapid Growers -(+) NO3 reduction
(Group IV) -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 b. M. chelonei
Growth: 3-7 days -(-) 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
7H12 14C Palmitic acid + organisms ----------> 14CO2

(+): >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
Biotypes of C. diphtheriae Diphtheria: pseudomembrane of the pharynx
C. pseudodiphtheriticum Lab. Diagnosis:
C. xerosis -Elek test: definitive
C. minutissimum -Roemer test: incubates the bacteria in pig
Transduction -Schick’s test: skin test (Ab’s)
B. anthracis -(+) DNase
-(-) Urease
B. cereus -Ferments dextrose
B. subtilis 1. Gravis = gray, large, beta-hemolytic, starch/glycogen fermentation (+)
Clostridium 2. Mitis = black, medium-size, beta-hemolytic, starch/glycogen fermentation (-)
3. Intermedius = black, small, nonhemolytic
C. perfringens “Hoffman’s bacillus”
(Old: C. welchii) 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 RRR

Fusobacterium SRS

B. ureolyticus SRS

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 lec.mt 04 |Page | 74
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
Gram (-) Anaerobic Bacilli Bifidobacterium
Eubacterium
Gram (+) Anaerobic Cocci Propionebacterium
Lactobacillus
Gram (-) Anaerobic Cocci Mobiluncus

A. bovis Bacteroides
P. acnes Porphyromonas
Prevotella
L. acidophilus Fusobacterium

Mobiluncus Peptostreptococcus
Peptococcus
Anaerobic infections
B. fragilis Veillonella
F. necrophorum Megasphera
(F. moriferum) Acidaaminococcus
P. anaerobius
Lumpy jaw
P. asaccharolyticus
P. niger Acne vulgaris
(+) Catalase, Indole
H2S indicators
Tomato juice agar
Phenol red (-) 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

Methyl red Acid = Red
Neutral red Alkaline = Yellow
Bromthymol blue
Enterobacteriaceae Acid = Pink/red
Alkaline/neutral = Colorless
VP (+)
H2S (+) Acid = Yellow
Lactose Fermenters Neutral = Green
Alkaline = Blue
Deaminase (+)
Urease (+) 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

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
Colorless
EMB Eosin Y Lactose Eosin Y Red purple
Methylene blue Colorless
MAC Methylene blue (E. coli: GMS)
Bile salts Colorless
SSA Crystal violet Lactose Neutral red Red/pink Colorless
DCA
TCBS Bile salts Lactose Neutral red Red Green
Lactose Neutral red Red/pink
Bile salts Sucrose Bromthymol blue
Yellow
Bile salts

HEA Bile salts Salicin Bromthymol blue Yellow Green
Lactose Phenol Red
XLD Bile salts Sucrose Yellow Red
Bismuth sulfite
BSA Brilliant green Xylose S. typhi = black colonies
(Old: Wilson-Blair) Lactose
Sucrose
Escherichia coli
Enterobacter aerogenes Glucose
Enterobacter gergoviae
Edwardsiella tarda Lysine-Ornithine-Arginine Reactions LO A
Arizona “(LO)9(OA)3(L)4(O)6(N)6” - Enterobacteriaceae -+ -
Serratia -+ -
Hafnia LOA -+ -
Salmonella enteritidis -+ -
Salmonella choleraesuis + + - Shigella sonnei -+ -
Enterobacter cloacae + + - Proteus mirabilis -+ -
Enterobacter sakazakii -- -
Enterobacter taylorae + + - Morganella morganii -- -
Salmonella typhi + + - Citrobacter -- -
Klebsiella pneumoniae -- -
Klebsiella oxytoca + + - Yersinia enterocolitica -- -
Klebsiella ozaenae + + - Salmonella paratyphi -- -
E. coli ++ +
+ + - 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

lec.mt 04 |Page | 76

ETEC -MUG (+): Electric blue fluorescence

EPEC Traveler’s diarrhea
EIEC Montezuma revenge
EHEC/VTEC Turista
E. coli O157:H7 Belly Delhi
EAEC Cholera like, watery diarrhea (SL, LT)
Other Escherichia spp Infantile diarrhea
Enterobacter Pili: pathogenicity island
K. pneumoniae Dysentery (Shigella)-like diarrhea
Invasin: Pathogenicity
K. rhinoscleromatis Stool: bloody w/ mucus
PMP Group (Proteeae) Sereny test
Proteus HUS
Hemorrhagic colitis
Salmonella Verotoxin: Shigella-like toxin
EHEC/VTEC
S. typhi 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) Lab Diagnosis: El Tor Biotype
S. enteritidis -1st week: Blood R
Shigella -2nd week: Urine and stool (carrier)
lec.mt 04 |Page | 78
Shigella Subgroups (O Ag) Septicemia
Serratia marcescens
Serratia odorifera “Gardner’s Bacillus”
Y. pestis
Biochemically inert
Y. enterocolitica SSA: colorless
Y. pseudotuberculosis Related to E. coli:
Vibrio -E. coli: Acetate (+)
-Shigella: Acetate (-)
V. cholerae Intestinal pathogen: Dysentery
Lab. Diagnosis:
Polymyxin Susceptibility -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

Lysis by bacteriophage +-
Chicken RBC agglutination
Hemolysis of sRBC -+
VP test
Anti-Ogawa Classical Biotype El Tor Biotype
Anti-Inaba
Lab. Diagnosis (V. cholerae) -+

V. parahaemolyticus -+

V. vulnificus Ogawa Serotype Inaba Serotype Hikojima Serotype

V. alginolyticus +-+
Aeromonas
- ++
Plesiomonas
Culture:
Campylobacter
-TCBS: Sucrose fermenter
H. pylori -TTGA: Tellurite Taurocholate Gelatin Agar
-APW: 6-8 hrs  Subculture (TCBS)
OF test
-Cary Blair: Transport medium (stool)
P. aeruginosa -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 Pyocyanus: Blue pus agent
(+) Oxidase
B. pseudomallei Motile (monotrichous)
Capsule: Slimy layer
B. mallei Grapelike odor (aminoacetaphenone)
P. stutzeri (G) 42’C
Cetrimide medium: selective
S. maltophilia Pigments:
-Pyocyanin: Best
P. syncyanea -Pyoverdin/Fluorescein: (+) P. putida/P. fluorescens
Shewanella putrefaciens Infections:
Acinetobacter -#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) Oxidizer
A. lwoffi
Alcaligenes faecalis Herella vaginocola

M. lacunata Nonoxidizer
Mima polymorpha
Flavobacterium
(Chryseobacterium) (+) Oxidase
meningosepticum (+) Catalase

E. corrodens Motile (peritrichous)
Asaccharolytic
Kingella spp “Fruity” odor
Haemophilus
(+) Pitting of agar
H. influenzae (+) Oxidase
H. aegyptius
H. haemolyticus (+) Catalase
H. parainfluenzae Assacharolytic
H. parahaemolyticus (NG) MAC
H. paraphrophilus
H. ducreyi Infection: Blepharoconjunctivitis
H. aphrophilus Mistaken as Neisseria
Porphyrin test
H. influenzae 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

-#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”
H. aegyptius (G) CAP + Vancomycin (33’C)
B. pertussis Soft chancre (painful)

B. pertussis School of red fish
B. parapertussis
B. bronchiseptica “Koch Week’s Bacillus”
B. bronchiseptica
Brucella 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 MAC/BAP
-
- -+ +
+
-+-

+++

“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

lec.mt 04 |Page | 82

-(+) Rose Bengal & 2-ME agglutination

B. abortus “Bang’s Bacillus”
B. abortus
B. melitensis Urease CO2 Thionine (Dye Inhibition) Fuchsin (Dye Inhibition)
B. suis
B. canis ++ - (NG) + (G)
F. tularensis
+- + (G) + (G)
P. multocida
+- + (G) - (NG)
Treponema
Leptospira +- + (G) - (NG)
Borrelia
T. pallidum (+) Capsule
Syphilis Nonmotile
Facultatively intracellular
Neurosyphilis
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

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Penicillin Treatment for syphilis
T. pertenue
T. carateum (Jarisch-Herxheimer reaction)
T. endemicum
T. cuniculi Yaws, Framboesia
Lab. Diagnosis (Treponemes)
Pinta
L. interrogans
icterohemorrhagiae Bejel

Borrelia Rabbit syphilis

H. aphrophilus 1. Darkfield microscopy:
A. actinomycetemcomitans -Corkscrew motility
C. hominis
E. corrodens -1’ and 2’ syphilis
K. kingae 2. Stain: Levaditi silver impregnation
A. actinomycetemcomitans
C. hominis 3. Serology:
E. corrodens -Latent, 3’ syphilis
Chlamydia (Old: Bedsonia) -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 CO2
+
-+- +
+
+- - +
+
-+-

-+-

- -+

Star-like colony

Dots and dashes of Morse code

(+) Indole

Teardrop shape
Rosette formation

Asaccharolytic

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
C. psittaci
Diseases:
C. pneumoniae -LGV (Frei’s test)
-PID (pelvic inflammatory disease)
Rickettsia
-#1 STD in US
Ehrlichia -#1 NGU

R. rickettsii (S) Sulfonamide
R. akari Lab. Diagnosis:
R. typhi 1. Inclusion body (Halberstadter Prowazeik)
R. prowazekii
O. tsutsugamushi -Stain (Glycogen): Iodine (brown)/Giemsa (purple)
R. quintana -Elementary body: Round-shaped
E. chaffeensis 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

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E. equi Tick Granulocytic Ehrlichiosis
C. burnettii
Mycoplasma Inhalation Q fever
M. pneumoniae
No cell wall (pleomorphic)
M. hominis
U. urealyticum Fried egg/mulberry
Cell membrane: Sterol (bilipid layer)
G. vaginalis Require sterol for growth

C. granulomatis (R) Penicillin
S. moniliformis 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) Spiral organism
C. violaceum
Capnocytophaga Causes rat bite fever
B. henselae Cannot be cultivated
B. bacilli formis
L. pneumophila Old: Achromobacterium

L. monocytogenes Violacein: violet colored
E. rhusiopathiae 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

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Catalase +-
Motility (25’C)
Hemolysis +-

VP Beta Alpha
H2S
Bile esculin L. monocytogenes E. rhusiopathiae
Hippurate
Gluconate +-
Media
Water Bacteriology -+

Sodium thiosulfate +-
1. Presumptive test
+-
2. Confirmatory test
3. Completed test +-
MPN (Most Probable Number)
Mc Bride, Cold enrichment BAP
Reference laboratories
(Water Bacteriology) E. coli: Index of fecal contamination
Milk Bacteriology
Streptococcus lactis Det. coliforms
Coliform bacteria
Bacillus subtilis Neutralizes chlorine
Clostridium butyricum
Clostridium perfringens Lactose broth/ + H2O ----------(35’C for 24 hrs)----> (+) Gas
Pseudomonas syncyanea Lauryl tryptose broth (-) No gas after 48 hrs
Serratia marcescens
Pseudomonas aeruginosa EMB/Endo agar + Inoculum -----(24 hrs)-----------> (+) Colony
Flavobacterium synxanthum
Alcaligenes viscosus 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)

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