Bacteriology PDF

BACTERIOLOGY
Pioneer notes from Ms. A.Aldave and some added notes

Biosafety
Biosafety
Prevents laboratory acquired infection (F.tularensis and C.immitis)
Use of Biosafety Cabinet (BSC Class IIA)
BSC uses HEPA filter (0.3um pore size) and negative pressure
Biosafety safety of medical technologist
Biosecurity safety of biologicals (microorganisms)
Types
BSC Class I
o air velocity: 75 linear feet/min
o 1 HEPA filter: exhaust air thru HEPA filter
o product (culture) contaminant
o process non-pathogens (BSL-1)
BSC Class II
o air velocity: 75-100 linear feet/min
o 2 HEPA filter: exhaust and recirculated air thru HEPA filter
o no product contamination
o vertical laminar flow
o must for laboratory/hospitals (BSC Class IIA)
o process bacterial and fungal pathogens (BSL-2 and BSL-3)
IIA Exhausts air inside the room, Sef-contained, 70% recirculated air
IIB Exhausts air outside the building (radioisotopes, chemicals, carcinogen)
BSC Class III
o supply and exhaust air thru HEPA filter
o close cabinet sealed glove ports
o process viral pathogens (BSL-4)
Classification of Biologic agents (Biosafety Level) Bioterrorism categories

BSL-1 No risk M.gordonae Category A Highly infectious Smallpox virus
B.subtilis Francisella
BSL-2 Moderate risk Y.pestis B.anthracis
Treatment B.anthracis Category B Moderate Ricketssia
Enterics morbidity B.pseudomallei
BSL-3 High risk Mycobacteria Low mortality Coxiella
Treatment Brucella Category C Emerging Yellow fever
Francisella pathogens Dengue fever
Molds Hemorrhagic fever
BSL-4 High risk Virus Ebola
No treatment MERSCOV
Chain of infection: Source, Transmission, Host
Bacterial Characteristics
1. Prokaryotic
Nuclear body: no nuclear membrane, nucleoid region of the cytosol
Cell division: binary fission
Cell wall: with peptidoglycan except Mycoplasma and Ureaplasma
Cytoplasmic membrane: fluid phospholipid bilayer with carbohydrate and sterol
Cell organelles: absent
Site of energy production: cytoplasmic membrance
Site of protein synthesis: free ribosome
2. Has both DNA and RNA
3. 4 morphology cocci, bacilli, spiral, comma
4. Measured in micrometer (um) average size: 0.4-2um
5. Biofilms property of bacteria to attach on solid surface
NUCLEOID
No nuclear membrane
Chromosome dsDNA for reproduction
Plasmid extrachromosomal DNA that carries the antibiotic resistant genes; transfer DNA
CELL WALL
Defines the shape of the bacteria
Pathogenicity:
M protein: Streptococcus pyogenes
Mycolic acid: Mycobacterium spp.
Peptidoglycan (murein layer) consists of glycan chains of alternating N-acetyl-d-glucosamine (NAG) and
N-acetyl-d-muramic acid (NAM)
Mycoplasma and Ureaplasma lack cell walls, only contains sterol
Gram positive and gram negative cells can lose their cell walls and grow as L-forms in media supplemented
with serum or sugar to prevent osmotic rupture of the cell membrane
Gram positive impermeable to alcohol, thick peptidoglycan, teichoic acid, exotoxin
Gram negative Permeable to alcohol, Thin peptidoglycan, LPS, Outer membrance, Periplasm, Lipid A,
Exotoxin and endotoxin, Somatic (O) Ag serotyping
CYTOPLASMIC MEMBRANE
Selectively permeable
Site of energy production (ATP site)
Osmotic/permeability barrier
Regulate transport of nutrients in and out of cell
MESOSOMES
Point of attachment for chromosome
INCLUSIONS
Much granules MTB
Babes-Ernst/metachromatic/volutin granules as food reserve C.diptheriae
Bipolar bodies Yersinia pestis
RIBOSOMES
Bacteria: 70s; Fungi: 80s
For protein synthesis
Viruses are acellular
ENDOSPORE
Resting cell, highly resistant to dessication, heat and chemical agents
Composition: Calcium dipicolinate/Dipicolinic Acid
Bacterial genera with spores: Bacillus and Clostridium
Target of sterilization
Non-reproductive
CAPSULE
Prevents phagocytosis
Antigenic; on the basis of serotyping by Quellung reaction
o Neufeld-Quellung capsular Ag
o (+) capsular swelling due to Ag-Ab
o Ex. Strep.pneumoniae, N.meningitides, H. influenza
o Serotyping:
Somatic O Ag heat stable
Vi Ag and K Ag heat labile
Demonstration
o Animal tissues and fluids
o Media containing milk or serum
Colonies often slimy/mucoid
Stains: HISS, India ink/Nigrosin
PILI
Synonymous to fimbriae
Common/Ordinary pili adherence of bacteria to host cell; virulence factor for Neisseria
Sex pili bacterial conjugation, gene transfer
FLAGELLA Tumbling Listeria
Atrichous no flagellum
Gliding- Capnocytophaga
Monotrichous flagellum on one pole
Amphitrichous single flagellum on each pole Darting - Campylobacter
Lophotrichous tuft of flagella at one or both poles Cork screw Spirochetes (Leptospira,
Peritrichous flagella all over the organism Treponema, Borrelia)
Periplasmic flagella endoflagella/axial filaments Twitching - Kingella
Motility best seen at 37C
Tests for motility semisolid medium and stains
AXIAL FILAMENTS
Spirochete with cork screw motility
Bacterial Virulence Factors
Pathogenicity ability of a microbe to produce disease in a susceptible individual
Virulence relative ability of a microorganism to cause disease or the degree of pathogenicity; usually
measured by the numbers of microorganisms necessary to cause infection in the host
1. Adherence factors pili/fimbriae

2. Anti-phagocytic facrors capsule and self-component of cell wall
3. Enzymes
Coagulase S.aureus
Fibrinolysin spreading and clotting
Hyaluronidase/Duran Renal Factor spreading
4. Toxins: exotoxin and endotoxin
Exotoxin/Enterotoxin Endotoxin
Source Gram positive bacteria/Gram neg Gram negative bacteria
Release Released by living bacteria Released when gram-negative
Do not require bacterial death for bacterial cell is destroyed
release
Composition Peptide and protein Lipopolysaccharide portion of cell
envelope
Heat Stability Heat labile except Staphylococcus Heat stable
enterotoxin
Immunologic Converted to toxoid Not converted to toxoid
Easily neutralized with antitoxin Not easily neutralized
Effect -Kill host cells and help spread -Disruption of clotting, causing clots
bacteria in tissues to form throughout the body (DIC)
-Destroy or interfere with specific -Fever
intracellular activities -Activation of complement and
immune systems
-Circulatory changes that lead to
hypotension, shock and death
Toxicity High Low
Lethal dose Smaller dose Higher dose
Tetanus, Lock jaw UTI, Typhoid
E.coli, S.aureus
Most potent: Botulinum toxin
Bacterial Growth Factors

1. Nutritional Requirement
Carbon
o Litotroph/Autotroph inorganic compound as carbon source (Ex. CO2)
o Heterotroph/Organotroph organic compound as carbon source (Ex. Glucose, pathogens)
Nitrogen
Minerals sulfur and magnesium
Salt
2. Oxygen and Carbon dioxide availability
Aerobes - 21% O2 and small amount of CO2 e.g., Bacillus cereus
Obligate/strict aerobe - Pseudomonas aeruginosa, Mycobacterium tuberculosis, Brucella
Anaerobes - Metabolism is a fermentative type, cannot grow in the presence of O2
Obligate/strict anaerobe - Clostridium perfringens, Clostridium botulinum, Veilonella, Actinomyces
Facultative anaerobe - e.g., Enterobacteriaceae group, Staphylococcus aureus etc.
Aerotolerant anaerobe bacteria not killed by Anaerobic (Gas Pak Jar)
exposure to oxygen eg. Lactobacillus
5% CO2, 10% H2, 85% N2
Microaerophilic - Campylobacter jejuni, Helicobacter
pylori Microaerophilic/Campy Gas (Candle jar)
Capnophilic 5-10% CO2 Haemophilus influenzae, 5% O2, 10% CO2, 85% N2
Neisseria gonorrhoeae etc.
3. Temperature
Psychrophilic/cryophilic = 0-2 C, (refrigerator)

1. Listeria coleslaw food poisoning, milk- BB contam at 4C BB contam at RT
borne diseases Yersinia enterocolitica Staph. epidermidis
2. Y.enterocolitica blood bag contamination Pseudomonas fluorescens Bacillus cereus
(presence of bubbles) Serratia liquifacien
Mesophilic - 20-40C, (pathogenic)
Thermophilic - 40-60C, (Thermus aquaticus PCR enzyme, source of Taq polymerase)
4. pH
pH: 6.5-7.5
a. Acidophilic - Lactobacillus, Fungi
b. Neutrophilic - pathogenic bacteria (pH7.2-7.4)
c. Basophilic - Vibrio (Alkaline Peptone Water)
5. Moisture humidophilic
6. Salt concentration
a. 7.5% NaCl S.aureus (MSA contains 7.5% NaCl)
b. 6.5% NaCl E.faecalis ( UTI and wound infection, index of fecal contamination of marine water)
c. 0,3,5,8,10% NaCl Vibrio (bacteria seen on marine water, V.cholerae contaminated drinking water)
Bacterial Growth Curve

1. LAG phase/Adjustment phase
increase in cell size NOT in number
little or no multiplication
adaptation phase
2. LOG phase/exponential phase
increase in growth rate (cell division)
susceptible to antimicrobial agents
organisms grow at maximum rate (exponential rate)
3. Stationary/Plateau phase
NO net growth (death = live cells)
Growth ceases because nutrients are exhausted or toxic metabolic products have accumulated
4. Death phase/Period of decline
viable count decreases
Bacterial Gene Transfer

1. Conjugation 2. Transduction
plasmid mediated bacteriophage (virus) mediated
sex pili Tox gene of C.diptheriae
transposon mobile, jumping genes 3. Transformation
gram negative bacteria (ESBL naked DNA
positive) Strep. pneumonia (virulent-avirulent)
Bacterial Metabolism
Respiration (Aerobic process)
Krebs cycle aerobil process
Electron transport chain aerobic process
Glucose CO2 and H2O
Oxidation (Aerobic process)
Glucose acid
non-fermentative organism (Pseudomonas)
Fermentation (Anaerobic process)
Glycolysis (EMP): Glucose acid/alcohol
Ex. E.coli and S.aureus
Culture Media
I. According to physical state/consistency:

Preparation of culture media
a. Liquid (broth) rapid culture, without agar
Water deionized or distilled
b. Semisolid 0.5%-1% agar, for motility pH 7.2-7.4
Salmonella (+), Shigella (-) Sterile autoclave
Listeria (+), Corynebacterium (-) Dissolved clear and no particles
c. Solid 2-3% agar Steps weigh, dissolve, sterilize, dispense
d. Biphasic both liquid and solid (Castaneda for
Brucella blood culture medium)
II. According to composition: synthetic/chemically-defined, complex/non-synthetic, tissue
III. Dispensing/Distribution: plated or tubed
IV. Function and use
a. Simple/Basal/Supportive/General Isolation/General Purpose Media
Non-fastidious bacteria
Nutrient agar/broth, trypticase soy agar/broth general media for g(+) and g(-)
BAP hemolysis study, general bacterial media
Sheep BAP Streptococcus
Horse BAP Haemophilus spp.
Human BAP Gardnerella vaginalis
b. Enriched Media
Fastidious bacteria
CAP
o With X (hemin) and V (NAD)
o 0% chocolate, heated BAP
o Horse blood
o Base medium for TMA
c. Enrichment Media
Enhance growth of organism
Increase lag phase of normal flora, decrease lag phase of pathogen
Selenite broth and tetrathionate broth: for Salmonella and Shigella
Alkaline peptone water: Vibrio
d. Selective Media
Inhibitory agents:
o Antibiotics eg.CNA, inhibits g(-), growth of g(+)
o dyes, bile salt, alcohol (PEA, inhibits gram negative)
Inhibitors for gram positive bacteria: crystal violet, bile salts
Inhibitors for gram negative bacteria: potassium tellurite, sodium azide
TCBS, SSA, TMA, CBAP enteric media, recommended for non-sterile samples
Examples:
Lowenstein Jensen Media MTB

Mueller Tellurite Agar C.diphtheriae
PEA (phenyletyl-alcohol) for gram positive
inhibits gram negative
Thayer Martin, Modified Thayer Martin, Martin Lewis, New York City Agar N.gonorrhoeae
Mannitol Salt Agar Staphylococcus
Thiosulfate Citrate Bile Salt Agar Vibrio
Columbia CAN (Colistin-Nalidixic) Agar Gram positive bacteria
Gonoccoci Agar Gram negative cocci
Neisseria
Gentamicin BAP Streptococcus pneumonia
Bacitracin CAP H.influenzae
Cystine Blood Glucose Agar Francisella
Cystine Tellurite Blood Agar C.diphteriae
Tinsdale with Potassium Tellurite
Cystine Trypticase Agar Confirmatory for Neisseria
Charcoal Cephalexin Blood Agar B. pertussis (more preferred than
Potato blood agar)
Buffered Charcoal Yeast Extract Legionella
McCoy C.trachomatis
TSB Brucella spp
Potato Blood Glycerol Agar/Bordet Gengou Agar B.pertussis
Cysteine Lactose Electrolyte Deficient Urinary tract bacteria
Inhibits proteus swarm
Bile Esculin Agar Group D streptococci
Granada Group B streptococci (red)
Cetrimide Agar P.aeruginosa
Lysogeny broth Shigella and E.coli
Leifson agar, SSA, Onoz agar, Hektoen, XLD Salmonella and Shigella
Nutrient Agar Pigment production
SMAC E.coli 0157:H7 colorless colony,
negative for sorbitol
e. Differential Media
BAP, MAC, EMB, XLD, HEA (Presumptive ID)
Enterobacteriaceae: Rapid Lactose Fermenters, Late Lactose Fermenters, Non-lactose
Fermenters
Selective Differential Media for gram negative EMB, MAC, HEA, SSA
f. Medium for susceptibility testing: Mueller-Hinton Agar
g. Media for biochemical testing: TSI, LIA
h. Types of Culture
Pure culture one organism, ID/AST
o To indicate pure colony: same appearance and ID can be made
o Streak plate (best), Pour plate, Selective medium, Animal inoculation
Mixed culture more than 2 organism
Stock culture for quality control (ATCC American Type Culture Collection)
Equipment, Supplies, and Instrumentation

1. Incubator
Set at 35-37C
18-24 hours: aerobic culture
24-48 hours: anaerobic culture
Bacteria: 35C, Virus: 37C, Fungi: 25-30C
2. Inoculating needles
Nichrome, platinum
No longer than 5cm
Nichrome false positive in oxidase because it contains iron
3. Typing sera serotyping of bacteria
4. Pasteur pipette transfer liquid
5. Durham tube
water bacteriology
Gas detector but not for H2S (black)
6. Cotton swab carrier state, 2 samples (for culture and gram stain)
Nasopharyngeal swab
N.meningitidis, H.influenza, B.pertusis
Nasal swab: S.aureus
Toxic to Neisseria: add charcoal on culture media to detoxify
Good for viruses
7. Tuberculin syringe: Mantoux skin test for TB, test for cellular immunity
8. Commercial ID system
Rapid: API (Analytical Profile Index)
Enterotube
Crystal ID
Requirement: Decrease substitute, increase inoculum, unique substrate
Collection, transport, processing and staining of specimens

Type of Specimen
1. Sterile without normal flora

2. Non-sterile use selective medium
Rules
1. Collect before antibiotic theraphy

2. Aseptic collection
Betadine/iodophor blood culture
Bottle #2 - CSF
3. Acute stage
Leptospirosis 1st to be positive in blood and CSF
Typhoid fever 1sts to be positive in blood and bone marrow
4. Quantify sufficient, prompt delivery, proper sterile containers
Collection
1. Swab (aerobic)s
Cotton - toxic to Neisseria, good for virus
Calcium alginate toxic to virus, good for Neisseria
2. Needle aspiration: aerobic and anaerobic
3. Catheterization: aspirate from catheter tube
Foley Catheter urine
IV catheter - blood
Storage
1-2 hours delay

Refrigerated: stool, sputum, urine, swab except genital (because Neisseria is cold sensitive)
Room temperature (not ref): CSF, blood, body fluids,

Swab
genital swab of N.gonorrhea
Dacron and Rayon good for bacteria and
Transport Medium viruses
Cary Blair: tool pathogens, Vibrio
Swab recommended for:
Stuarts: bacteria and virus
Amies: respiratory Virus: Dacron
Transgrow: Neisseria Urogenital specimen: cotton swab with
JEMBEC: Neisseria charcoal
Swab not for fungi and anaerobes
Todd Hewitt: S.agalactiae (vaginal swab)
LIM: modified Todd-Hewitt Transport Temperature
Note: Transport Medium CSF transport (RT), storage (35-37C)

Urine preservative: boric acid
Nutrients to maintain the growth of organism Urine, stool, swab, viral specimens, sputum,
Buffer to maintain pH foreign devices: 4C
Small amount of agar to maintain moisture Serum for serology: -20C
Tissue specimen for long term storage: -70C
Transport temperature for anaerobes: RT
Clinical Specimen
1. Blood
Iodophore (betadine) and opposite arms
Collect before height of fever (at 37.5 or 37.8 bacteria are dead)
TSB, BHIB with 0.025% SPS, 1% gelatin
o (+) cloudy, gas bubbles, hemolysis, pellicle
Blood to broth rati = 1:10 critical
o Adult = 1:5 to 1:10 (1:5 more preferred)
o Children = 1:10 to 1:20 (1:10 more preferred)
Subculture on BAP, CAP and MAC
o TAT: 7 days
o 21 days Brucellosis, Endocarditis, SBE
Note: 0.025% SPS (liquid) anticoagulant, anti-complementary, anti-phagocytic, neutralizes
aminoglycosides and bactericidal effect on serum
o SPS inhibits: G.vaginalis, Neisseria, S,monoliforms, P.anaerobius
o 1% gelatin: counteracts SPS
Automation: Uses BACTEC 9120
o TAT: 5 days
o Medium: BACTEC broth (1:10 ratio) with SPS and ARD
o (+) Fluorescence Subculture on BAP, CAP, and MAC ID and AST on Vitek
2. Urine
Random, catheterized, midstream, suprapubic (anaerobic)
Centrifuge: collect sediment for culture and GS
Quantitative: use BAP and MAC
o For suspected infection like UTI
(E.coli gram negative; Enterococcus and S.saprophyticus gram positive)
o Colony count in CFU/ml = number of colony x 1000 (if 0.001mL loop)
o >100,000CFU/ml = ID and AST of UTI
o <10,000 CFU/ml ID only
3. CSF
placed on bottle 2, not refrigerated only incubator
Routine test: India ink and gram stain
Isolation of: Neisseria and Haemophilus
Centrifuge: sediment for culture and GS
o BAP, CAP (5-10% CO2)
o BHIB, MAC (incubator No CO2)
o India ink method (capsule): CSF Latex agglutionation (capsular Ag)
4. Wound
BAP, MAC, THIO, GS
Swab collected at edge after NSS aerobic culture
Needle aspiration - anaerobic culture
5. Stool
do not GS
1-2grams stool on sterile vial
Rectal swab on Cary Blair
MAC, BAP, CAP, SSA, TCBS, HEA, XLD
SELENITE F (SSA), APW (TCBS), CBAP (42C for 48 hours)
Presumptive: oxidase test, biochemical test
Confirmatory: Serotyping
6. Respiratory (Sputum, NPS)
Processing done on BSC
Gram stain (>25PMN, <10EC)
Gentamicin BAP S.pneumoniae
Bacitracin CAP H.influenza (5-10% CO2 incubator, MAC 35C incubator)
Do gram stain and AFS
Bartletts Classification
Assess the quality of sputum
Enumerate the number of neutrophils and epithelial cells/LPF
0 score or less saliva (no inflammation)
>1 score inflammation/infection
7. Throat swab
Sore throat
Diptheria
BAP, MTM, Gram stain
8. Vaginal urethral swab CAP, MTM, Gram stain
9. TB culture
1 sputum for GS; 2 sputum for AFS
GOLD STANDARD: NALC-NaOH
NALC (n-acetyl-L-cystine): digestant/mucolytic
2.4% NaOH: decontamination
Oxalic acid Pseudomonas contamination (cystic fibrosis)
Anti-formin: chlorox
Refrigerated centrifuge for 15 mins at 3000xg (4C)
Lowenstein-Jensen, Middlebrook 7H11, 7H10
Reporting:
(-) = 37C for 8 weeks of no growth
(+) = 2-3 weeks growth seen
BACTEC = radiometric method
TAT:
L-J = 8 weeks (-) GX (Gene Expert) = 2-3hours
Bactec = 2 weeks (-)
Methods of Studying Microorganism
I. Living State (Unstained)
1. Wet mount preparation
2. Hanging drop preparation
II. Fixed State (Stained)
Staining Methods
1. Simple 1 dye
2. Differential 2 dyes (GS, AFS)
3. Special bacterial structures
4. Indirect/Reflief/Negative capsule
a. India ink test/Borris method
b. Nigrossin method
GRAM STAIN
Purpose Reagents Gram (+) Gram (-)

Primary V (Crystal violet) Purple Purple
Mordant I (Iodine) basic Purple Purple
Decolorizer-critical A (Acetone-alcohol) or 95% EtOH Purple Colorless
Counterstain S (Safranin) Purple Red
*Examine microscopicallt under an oil immersion lens at 1000x for phagocytes, bacteria and other cellular material
Gram (+) becomes gram (-)

Over-decolorization
Old
dying
use of acidic iodine as mordant
penicillin
omit iodine
Gram (-) becomes gram (+)

Under-decolorization
thick smear
Gram stain general rule
1. All cocci are gram (+) except Neisseria, Veilonella, Moraxella

2. All bacilli are gram (-) except Mycobacteria, Corynebacteria, Clostridia, Bacillus, Lactobacillus, Listeria,
Erysiphilothrix, Nocardia, Actinomyces
3. All spiral organism are reported as gram (-)
4. Yeasts are gram (+)
Gram positive cocci (aerobes) Micrococcus, Staphylococcus, Streptococcus

Gram positive cocci (anaerobes) Peptococcus, Peptostreptoccocus, Sarcina
Gram negative cocci (aerobes) Branhamella, Neisseria
Gram negative cocci (anaerobes) Veilonella
Gram positive bacilli (aerobes) Bacillus, Corynebacterium, Erysipelothrix, Listeria, Mycobacterium,
Nocardia
Gram positive bacilli (anaerobes) Actinomyces, Clostridium, Propionobacterium
Gram negative bacilli (aerobes) Acinetobacter, Aeromonas, Alcaligenes, Bordetella, Brucella
Enterics, Francisella, Legionella, Pasteurella, Pseudomonas, Vibrio
Gram negative bacilli (anaerobes) Fusobacterium, Bacteroides
NOT Gram stain
1. Chlamydia/Ricketssia intracellular
2. Mycoplasma/Ureaplasma no cell wall
3. Spirochete cant resolve by bright field
Note: Acridine orange (nucleic acid)

BURKES MODIFICATION OF GS for metachromatic granules
ACID FAST STANING METHODS
Mycolic acid responsible for acid fast resistance

Acid fast organisms
Mycobacteria, Nocardia spp
Cryptosporidium modified AFS (No heating, 1% H2SO4)
Others: Rhodococcus, Tsukamurella, Legionella micdadei, Isospora, Sarcocystis, Cyclospora, Gordonia
Purpose Ziehl-Neelsen (Hot) CAM Kinyoun (Cold) (C-A-M) Auramine-Rhodamine

Best AFS Tissue AFS (Fluorochrome)
SENSITIVE AFS
Primary Carbol-Fuchsin Carbol-Fuchsin Auramine-Rhodamine
Mordant Heat Pheno, Tergitol
Decolorizer 3% acid alcohol 3% acid alcohol 0.5% acid alcohol
Counterstain Methylene blue Malachite green 0.5% KMNO4 quenching agent
Result AFO-Red AFO red AFO yellow fluorescence
NAFO-Blue NAFO green NAFO NO fluorescence
*Screening at 400 magnification and confirm all suspicious (red) organisms at 1000 magnification
Other acid fast stain
Pappenheims (urine) = M.smegmatis (blue), M.tuberculosis (red)

Baugmartens (tissue) = M.leprae (red), M.tuberculosis (blue)
Fite Faraco stain = M. leprae (hematoxylin as counterstain)
Special Stain
Metachromatic granules culture on Loefflers, Pai medium and stain (Neissers, Albert, Ljubinsky, LAMB)
Spore culture on Bap and stain (Dorners, Acetic acid, Wirtz-Conklina
Capsule specimen (Hiss/India Ink)
Flagella culture (Gray Leifson, Fischer-Conn)
Nuclei Acid Fuelgen method
Ricketssia Gimenez, Macchiavelo, Giemsa
Bacillus anthracis MFadyean
Spirochetes Levaditis, Fontana-Tribondeux
Calcouflour whitet binds to chitin cell wall (fungi yellow green fluorescence)
Acridine orange - stains nuclei acid (fungi green fluorescence; bacteria-orange/red fluorescence)
LPCB Aman stain for fungal structure
Non-staining methods
1. LANA (L-alanine-4-nitronilide) 2. Strings test

(+) Yellow color 3% KOH
For gram negative (+) string formation
For gram negative
Culture the bacteria first
Types of Microscopy
Resolution extent to which detail of the object is maintained (cellular details)
Resolving power closest distance the two objects can be distinguished from each other
1. Bright-Field
GS, AFS, KOH, most common microscope
Used for stained and unstained samples
2. Dark-Field
Motility of Spirochetes
Also used for Fluorescent stains
Higher resolving power than bright field
3. Phase Contrast
Inclusion bodies seen on virus and Chlaymydia, living cells/natural state, Microlymphocytotoxicity
test for HLA
Good for KOH mount
4. Fluorescent
Requires fluorescent stain (Calcofluor white, acridine orange)
UVL provided by mercury arc lamp
Substitute: Dark Field microscopy
a. Electron Microscopy viral morphology
b. Transmission Electron Microscopy internal structure
c. Scanning Electron Microscopy external structure
Sterilization
Sterilization sporocidal, all organisms are killed
Prions no nuclei acid, most resistant to sterilization, CJD, agent,
Envelope virus most sensitive to sterilization, most easily destroyed
Killing of MTB in sputum
o Boiling - 10 minutes
o Direct sunlight 20-30 hours
o Dried sputum 0 6-8 months
o 5% phenol 24 hours
Sterilization Method by MOIST HEAT
1. Autoclave/Steam Under pressure

Autoclave tape Fractional Sterilization
1st day = VEGETATIVE CELLS
o Heat sensitive indication
2nd day = SPORES
o Control: B.stearothermophilus 3rd day = REMAINING CELLS
o Blackline if it reaches 121C
Denatures protein
121C 15lbs psi for 15 minutes Best sterilization and waste disposal
Sterilize bacteriologic media (gauze)
121C at 15psi for 60 minutes Sterilize most contaminated microbiological material
132C for 30 to 60 minutes Infectious medical waste
2. Inspissation
Sterilize CHON containing medium (L-J, Loefflers)
75-80C for 2 hours on 3 days
3. Tyndallization 100C for 30 minutes on 3 days
4. Boiling
100C for 30 minutes
Non-sporocidal (disinfection)
5. Pasteurization
for milk
Phosphatase - test for success of pasteurization, phosphatase should be negative
Grade A milk 75,000 before pasteurization 15,000 after pasteurization
Bacteria seen on unpasteurized milk: Listeria, Brucella, Y.enterocolitica, M.bovis
a. Low Temperature Holding (LTH) 62C for 30 minutes
b. High Temperature Short Time (HTST) 72C for 15 seconds
Sterilization Method by DRY HEAT
1. Hot Air Oven

QC: Bacillus subtilis
Dry hot air at 170-180C for 2 hours
Glass wares, cotton swabs, oils, powders
2. Incineration not used
3. Cremation - to control disease
4. Flaming needles, use 70% alcohol prior to flaming
5. Gas
Used for machines
For heat labile materials: ethylene oxide
For heat labile solution: cellulose membrane filtration
Others
1. Cold temperature/freezing for preservation, bacteriostatic

2. Lyophilization/freeze drying best preservation for bacteria for many years
3. Osmotic pressure food preservation, use of high concentration of salt and sugar
4. Dessication food preservation, aka dehydration
5. Ultraviolet Light
Acts on DNA (air and water)
Reduce airborne infection
For hospital room, laboratory, BSC
Produces pyrimidine dimer DNA that can lead to cancer
6. Ionizing radiation - disposables
7. Filtration
Air and water
HEPA filter: BSC 0.3um
Cellulose membrane liquid filter, 0.22um
8. Gamma radiation - sterilization of plastic wares/plates
Disinfection/Antiseptic
Disinfection non-sporocidal, some organisms are killed, kills pathogen
Disinfection for living things, Antiseptic for non-living things
1. 10% Sodium hypochlorite (Chorox)

Spillage disinfectant
10-30 minutes contact
2. Iodine/iodophor sporocidal, best antiseptic for blood culture
3. 70% ethyl alcohol non-sporocidal, cannot destroy viruses
4. 3-6% Hydrogen peroxide cleansing of wound
5. 1% silver nitrate
Credes prophylaxis
Prevent gonococcal opthalmia neonatorium
6. Dyes inhibits gram positive, anti-fungal
7. Formaldehyde - reject for bacterial culture because aldehyde is sporocidal
8. Glutaraldehyde (Cidex)
cold sterilant
for surgical equipment sterilization
9. Phenol (Carbolic Acid) - standard disinfectant
10. Lysol (Cresol)
11. Zephiran benzalkonium chloride
12. Quats (Quaternary ammonium)
Inactivated by organic materials
Note:
Standard precautions - Blood and body fluids precautions must be observed for all patients blood and
body fluid specimen
Universal precautions all human blood and all other body fluids that contain visible blood
precautions must be observed
Antimicrobial Agents
Antagonistic = 1>2 (single drug)
Synergistic = 2>1 (combined drugs)
Minimum Bactericidal Concentration or Minumum Lethal Concentration
Antibiotics derived from bacteria or fungi, not used for viruses
o Eg. Penicillin, Cephalosporin, Streptomycin
o Streptomyces fungus like bacteria, common source of antiobiotics (Ex. Streptomyces, Nystatin)
Chemotherapeutics chemically synthesized (SXT)
Broad spectrum wide range of bacteria (Tetracyline- for gram pos and gram neg)
Narrow spectrum - limited number of bacteria (Van for gram pos only, not sensitive for gram neg)
Bactericidal Penicillin, Vancomycin, Aminoglycoside, Quinilnes, Metronidazole, TB drugs (RIZES)
Rifampin, Isoniazid, Pza, Ethambutol, Streptomycin
Bacteriostatic Chloramphenicol, Tetracyclin, Erythromycin, Clindamycin, SXT
Cations effect
o Increased Ca and Mg ions False resistant to aminoglycoside in P.aeruginosa
o Increase thymidine or thymin False resustatn to SXT in Enterococci
A. Cell Wall inhibitors sensitive for gram positive organisms

1. Penicillin Penicillum notatum (gram pos)
2. Cephalosphorin Cephalosporium (Acremonium)
3. Vancomycin Streptomyces, treatment for MRSA (Penicillin R, Vancomycin S)
4. Aztreonam Chromobacterium violaceum
5. Imipenem - carbapenems
6. Penicillinase Resistant Pen = Methicllin, Cloxacillin, Nafcillin, Oxacillin
B. Cell Membrance Inhibitors
1. Bacitracin
2. Colistin, Polymixin (Bacillus)
3. Amphotericin B, Nystatin (Streptomces), Imidazole, Clotrimazole (Anti-fungal)
Structure destroyed by antibiotics: cell membrane (cell wall is not destroyed)
C. Ribosomes (CHON) Inhibitors broad spectrum, for gram pos and gram neg
1. Aminoglycosides gentamicin, kanamycin, tobramycin, netilmicin, amikacin, streptomycin
2. Tetracycline, chloramphenicol bone marrow suppression
3. Erythromycin (macrolide)
substitute for patients with allergy to penicillin
eg.Clindamycin, Lincomycin
D. Nucleic Acid (DNA) Inhibitor
1. Mitomycin, Quinolones (levofloxacin, norfloxacin)
2. Metronidazole
anaerobes, anti-protozoa, for amoebiasis
G.vaginalis
Ex. Flagyl
3. Sulfonamide-Trimethoprim (SXT) inhibits folic acid (Ex. Rifampin)
Antibiotic Susceptibility Testing

1. Micro/Macrobroth Dilution
for anaerobes
MIC and MBC
Reference method
2. Agar dilution many organisms vs single drug
3. Disk diffusion
one organism vs multiple drugs
for aerobes or facultative aerobes
4. Epsilometer test (E-test)
antibiotic strip diffusion MIC test
filter paper strip with antibiotic
MIC ellipse zone at intersection
5. Automated System Vitek
6. Disk Elution Test for Mycobacteria
7. Schlichter Test
Serum bactericidal test
Measure activity of the antibiotic in the patients own serum against an infecting organism
Kirby Bauer Disk Diffusion disk agar diffusion, aerobes/facultative, use of filter paper disk
Standard inoculum 1.5 x 108 organisms/mL
Medium Mueller-Hinton Agar
pH 7.2-7.4
Depth 4mm
Condition Aerobic, no CO2
Temperature 35-37C (MRSA 35C)
Incubation time 16-18 hours
Standard McFarland Std (1% H2SO4 & 1.175% BaCl2)
0.5 concentration for bacteria
1.0 concentration for fungi
Antiobiotic Disk 6mm
Bacterial count method Petroff-Hausser
AST Media
General AST media Mueller-Hinton Agar
MRSA MHA + 2% NaCl
S.pneumoniae and N.meningitides MHA + 5% Sheeps Blood
Haemophilus Haemophilus Test Medium
Neisseria GC Agar
Mycobacteria Middlebrook 7H10
Anaerobes Wilkins-Chalgren broth and agar
Notes to remember:
Disk distance -15mm

150mm 12 discs; 100mm 5-6discs
Ignore swarming
Indirect relationship between organism and zone of inhibition
o Susceptible: decrease organism, increase zone of inhibition
o Resistant: increase organism, decrease zone of inhibition
False Sensitive False Resistance

Delay of 15 minutes before incubation Delay of 15 minutes before disc application
Increase drying Incrase moisture
Thin medium Thick medium
AST For Mycobacteria
MDR-TB to INH, Rifampin

XDR-TB to INH, Rifampin, and Quinolone
1. Disc elution
2. Bactec (RIA) and MGIT (IFA) AST and Rapid culture system
3. Gene Expert ID and AST
Drug Resistance Mechanism

1. Inactivates the antibiotic (beta-lactamase) destroys penicillin
2. Decrease permeability
3. Acquired resistance SUPERBUGS: chromosome (MRSA) and plasmid mediated (ESBL)
4. Intrinsic resistance
Novobiocin : S.saprophyticus
SXT and Tetracycline : P.aeruginosa
5. Modifies target site
6. Multi-drug resistance (MDR) pump
Drug Resistant Test Resistant (+) Result

GRAM NEGATIVE BACTERIA
Extended-Spectrum Beta Lactamase Penicillin Keyhole effect
(ESBL) Monobactams Elleptical clearing on Cefotazime-Clavulanic
Cephalosporin acid-Aztreonam disk
AmpC Beta Lactamase g(-) and g(+) Penicillin Cefoxitin screen
Cephalosporin Flattened edge zone on Cefotaxime disk
Modified Hodge Test Carbapenems (imipenem) Carbapenemase screening
(+) Clover leaf like
Swab MHA with E.coli
Klebsiella pneumonia gram neg bacilli
Metallo-Beta Lactamase (MBL) Test Penicillin Keyhole between imipenem and EDTA
Cephalosporin Pseudomonas aeruginosa and
Cephamycin S.maltophilia gram negative bacilli
Carbapenems
GRAM POSITIVE BACTERIA
mecA mediated Oxacillin Resistance Oxacillin and penicillin Oxacillin and Penicillin resistant
MHA with NaCl
Inducible Clindamycin Resistance Macrolide (Erythromycin) Staph and Strep
Clindamycin Flattening (D-zone) of clindamycin zone
Automation in Diagnostic Microbiology
Advantages: High Sensitivity, Reduce TAT, Replaces manual procedure
1. Vitek 1 and 2
2. Vitek-MS
3. MALDI-TOF
4. MicroScan WalkAway system
5. BD Phoenix nephelometry
BacT/Alert culture system only
Vitek 2 Compact ID and AST

Using pure colonies, prepare an inoculum with the appropriate McFarland standard to the ID tube
Has ID/AST cards
Data program: Advanced Expert System or Innovative Data Processing
Gram positive 0.50-0.63 McF
Gram negative 0.50-0.63 McF
Yeast 1.80-2.22 McF
Anaerobes 2.70-3.30 McF
Neisseria/Haemophilus (NH) 2.70-3.30 McF
*Fungi are larger than bacteria
*Anaerobes and NH requires hign concentration because they are fastidious organisms
Troubleshooting
Misidentification confirmation test using different method

Unfamiliar/Uncommon isolates referral to reference center, seek review results from supervisor
No automated microbial ID result do conventional method
Quality Control
Quality Control Quality Assurance
o routine (internal QC) o External QC, annually
o use ATCC, CLSO o Check performance of MT done by
o check validity of test DOH-RITM
o must have +/- controls
Quality Control Frequency
Daily Oxidase, catalase, gram stain, refrigerator, incubator
Weekly Antibiotic disks, autoclave, reagents
Semi-annual Centrifuge rpm
Annual BSC airflow, analytical balances (Accuracy)
Monthly Rheostat control, BSC
Each use Gas Pak jar, ONPG
Stock culture storage
Working culture storage
*Note: New drugs r reagents must first undergo 30 days QC before reducing it to weekly
Family Micrococcaceae
Genera: Staphylococcus, Micrococcus, Planococcus, Stomatococcus
Laboratory Procedures
Catalase Test Staphylococcus Streptococcus

3% H2O2 + -
(+) Effervescence, bubbles
BAP gives false positive in catalase test
Coagulase Test Staphylococcus CONS
Medium: rabbit/human plasma with EDTA + -
(+) clot after 4 hours S.epidermidis
1. Slide screen for bound coagulase/clumping factor S.saprophyticus
2. Test tube confirm for free coagulase
Mannitol Fermentation Test (MSA)
Inhibitory agent: 7.5% NaCl
Indicator: Phenol red
(+) Yellow and (-) Red
DNAse Test
Detects deoxyribonuclease
1. Dye Method
A. Methyl green (+) clear zone (-) no zone
B. Toluidine blue (+) pink zone (-) no zone
2. HCl Precipitation no precipitation after addition of 1N HCl
Presence of DNAse (+) clear zone (-) no clearing
Positive controls
o S.aureus gram positive
o S.marcescens gram negative
Novobiocin (5ug) Test S.epidermidis S.saphrophyticus
Differentiate CONS (S) >16mm (R) < 16mm
16mm measurement
Modified Oxidase Test Micrococcus Staphylococcus
Rgt: tetramethyl p-phenylene diamine dihydrochloride + -
in DMSO
(+) blue/purple (-) no color change
(+) Micrococcus luteus
Staphylococcus A Coagglutination Test
Antigen detection
S.aureus (cowan strain) with protein A as inert particles to which antibody (Fc fragment) binds
Detects specific bacterial antigens
o S.pneumoniae, N.meningitidis, N.gonorrheae, H. influenzae
3% H2O2 Staphylococci Citrate gives false positive because it uses citrate

30% H2O2 - Superoxol catalase for N.gonorrhoea and and releases calcium
Mycobacteria More than 4 hours delay on coagulase test gives false
15% H2O2 - anaerobes negative due to staphylokinase which dissolves clot
No growth on coagulase test after 4 hours re-
incubate at RT overnight before reporting as
negative
Micrococcus Staphylococcus
O/F test Oxidative Fermentative *Note: Stomatococcus Modified oxidase (-), Lysostaphin (R),
Modified oxidase Furazolidone (R)
Bacitracin
Furazolidone
Lysostaphin
Staphylococcus aureus
Staphylococcus species
Virulence Factors/Enzyme SBAP, MAC, CAP
Gram stain: gram positive in clusters
Protein A cell wall, anti-phagocytic, virulence BAP: pin-head, opaque, cream, yellow, white
Enterotoxin (Exotoxin) food poisoning Screen: Catalase
Beta hemolysin synergistic to CAMP factor of Confirmatory: Coagulase
group B streptococci
Leukocidin-Panton Valentine Yellow-orange colony due to lipochrome
Exfoliatin (epidermolysin) Skin scalded syndrome Catalase (+)
(Ritters disease) Coagulase (+)
Beta-lactamase MRSA drug resistance Nitrate and VP (+)
DNAse Gelatin (+)
Staphylokinase dissolve clot PYR (-)
Hyaluronidase spreading factor #1 diseases of SAU

Gelatinase Skin infections
o Gelatin Amino acid Ocular pathogen (sty)
o End product: amino acid Wound
o @ refrigerator temperature (+) gel will liquefy (-) gel will solidify Osteomyelitis
Note: Catalase is not a pathogenic/virulence factor Food poisoning
Nosocomial infection
Diseases
TSST-1 (Toxic Shock Syndrome)

Skin scalded syndrome (Ritters disease)
Carbuncles, furuncles folliculitis, cellulitis, bacteremia, endocarditis on IV drug users, septic arthritis
Toxigenic diseases: SSS, food poisoning, TSS (use of tampons)
Staphylococcus aureus like
1. S.intermedius slide coagulase (+), VP/aceotoin (-)

2. S.lugdunensis slide coagulase (+), PYR (+)
3. S.haemolyticus beta hemolytic, coagulase (-)
Laboratory Diagnosis
1. Gram stain gram positive in clusters

2. Culture
BAP, Chapman, Tellurite Glycine, P agar, PEA, Columbia CAN
Vogel-Johnson (with tellurite) = (+) black colonies
3. Catalase positive
4. Coagulase best confirmatory
5. Mannitol Fermentation Test yellow colonies
6. DNA hydrolysis
7. Latex Agglutination Test for Protein A confirmatory
Coagulase Negative Staphylococcus
1. Staphylococcus epidermis
Skin flora, blood culture contaminant, bacteremia
Prostethic valve endocarditis
Novobiocin senstitive
2. Staphylococcus saprophyticus
UTI
Novobiocin resistant
Staphylococcus aureus Coagulase Negative Staphylococcus

S.epidermidis S.saprophyticus
Colony Yellow White White
Catalase + + +
Coagulase + - -
Mannitol + - +/-
Novobiocin S S R
DNAse + - -
Phosphatase + + -
Gelatinase + + +
Gram Positive Streptococci

Gram positive cocci in chain or pairs
Catalase negative Staph Strep
Pinpoint colonies
Facultative anaerobes Catalase + -
Capnophilic (5-10% CO2)
Medium of choice: Sheeps Blood Agar Hemolysis +/- +
Selective medium: PEA
CO2 - +
Classification Colony Pinhead Pinpoint
A. Smith and Browns Classification

1. Alpha Streptococcus
Incomplete (greenish zone) hemolysis
S.pneumoniae, Viridans strep
Note: alpha prime: zone of alpha hemolysis surrounded by zone of beta hemolysis after
refrigeration
Streptococcus pneumoniae Optochin (S), Bile solubility (+)

Streptococcus viridans Optochin (R), Bile solubilty (-), Vanco (S)
2. Beta Streptococcus
Complete (coloress zone) hemolysis
S.pyogenes, S.agalactiae, Groups C,F,G
Group A Streptococci (S.pyogenes) Bacitracin (S), PYR (+), SXT (R)

Group B Streptococci (S.agalactiae) CAMP (+), Hippurate HOH (+), SXT (R)
Group C, F, G SXT (S)
3. Gamma Streptococcus
No hemolysis Group D Streptococci Bile Esculin HOH (+)
E.faecalis, E.faecium, S.bovis (Group D)
B. Lancefield Classification
1. Group A = S.pyogenes
2. Group B = S.agalactiae
3. Group C = S.equisimilis, S.zooepidemicus, S.equi, S.dysgalactiae, S.equisimilis
4. Group D
Enterococci = E.faecalis, E.faecium, E.durans, E.avium
Non-Enterococci = S.bovis, S.equinus
Note: Confimatory tests for Strep
PYR (+) = Group A strep and Group D enterococci 1. Lancefield test

Hippurate (+) = Group B strep and Group D enterococci 2. Flourescent Ab test
To differentiate group D enterococci from the rest, do bile 3. Phadebact agglutionation test
esculin. 4. Neufeld-Quellung Strep. Pneumonia,
detection of capsular antigen
Laboratory Tests
Bacitracin Susceptibility ID of S.pyogenes

Taxo A (+) zone of inhibition
0.04 units
PYR (L-pyrrolidonyl B-napthylamide) ID of S.pyogenes, Enterococcus
Rgt: p-dimethlyaminocinnamaldehyde
(+)red
CAMP Test CAMP factor of S.agalactiae synergistic reaction Beta lysine of S.aureus
(+) arrow head zone beta hemolysis
Hippurate HOH Test S.agalactiae S.pyogenes
ID of S.agalactiae + -
Rgt: Na hippurate and Ninhydrin
(+) purple (-) colorless Na hippurate + ninhydrin rgt --hippuricase benzoic acid + glycine
Bacitracin/SXT Organism Bacitracin/Taxo A SXT
Differentiate beta hemolytic Group A S R
strep Group B R R
Group C, F, G S S
Bile Esculin HOH Test
Hydrolysis of esculin
40% bile Group D streptococci is able to grow in the presence of 40% bile and
Indicator: Ferric ammonium hydrolyze esculin turning the indicator Ferric ammonium citrate a
citrate black color
(+) blackening of medium
(-) no blackening
Optochin Test/Taxo P (5ug) S.pneumoniae S.mitis
ID of S.pneumoniae (S) >14mm (R) <13mm
Ethylhydrocupreine HCl (Taxo P)
(+) >14mm
(-) <13mm
Bile Solubility Test S.pneumoniae Viridans Strep/E.faecalis
Colonies are hydrolyzed by bile Positive Negative
salt or sodium desoxycholate BAP: 10% sodium desoxycholate (+) lyzed colony (-) intact colony
resulting to lysis of colonies Tube: 2% sodium desoxycholate (+) clear (-) turbid
Lancefield Test
Slide agglutination = (+) Strep group
Presence of carbohydrate on cell wall
Vancomycin Resistance Pediococcus S.viridans
ID of Pedicoccus/Leuconostoc SBE Van (R) SBE Van (S)
(causes SBE)
Leucine Aminopeptidase (LAP) E.faecalis and Pediococcus Leuconostoc
Incubate at RT for 5minutes + -
(+) red
(-) yellow/no color change
MRS Broth Test Leuconostoc Pediococcus
(+) gas (-) no gas + -
Gram Negative cocci
Genera included
Neisseria (Streptococcus) - aerobic

Moraxella (Branhamella) aerobic Pelvic Inflammatory Disease (PID)
Veilonella anaerobic
1. C.trachomatis
Characteristics 2. N.gonorrhea
3. M.hominis
Gram negative intra(extra) diplococcic
Oxidase and catalase positive
5-10% CO2
Grow well on CAP
N.gonorrhoeae = (+) CAP (-) BAP
N.meningitidis = (+) CAP (+) BAP
Pigmented Neisseria: N.subflava, flavescens
o Flavin yellow
o Non-pathogenic/normal flora
Neisseria gonorrhoeae
Characteristics Diseases
Kidney/coffee bean shaped in PMN Gonorrhea (Clap) Epididymis
Virulence pili Opthalmia neonatorium Arthritis, PID
Salphingitis Fitz-Hugh Curtis
1. Ferments glucose (dextrose)
2. PPNG (Penicillinase producing N.gonorrhoeae)
3. Gram stain and culture on BAP and CAP
4. Selective media
TMA CAP-VCN Martin Lewis CAP-VCAnT
M.TMA CAP-VCNT NYCA yeast extract with VCAmT
5. Oxidase/Taxo N
Screening (+) purple
Rgt: 1% tetramethyl-p-phenylenediaminedihydrochloride
Taxo N ------cytochrome oxidase---- Indophenol blue (+) purple
(+) Neisseria, Moraxella, Aeromonas, Pseudomonas
6. Superoxol Catalase Test
30% H2O2
(+) Neisseria gonorrhoeae
7. CHO Utilization/Fermentation Test
Confirmatory Non-CO2 incubator
Ferments glucose Media: Cysteine Trypticase Agar + phenol red
Definitive test and speciates Neisseria Ferments glucose only yellow color
8. Beta lactamase test
Held on primary culture because plasmid is lost on subculture
Done on bacteria resistant to penicillin
Best substrate: Nitrocefin
(+) color change
a. Chromogenic cephalosphorin test (+) pink/red (-) yellow
b. Iodometric test iodine + Pen (+) colorless (-) purple
c. Acidimetric test phenol red + Pen (+) yellow (-) red
Acceptable specimen Neisseria Antibiotics inhibits

Pus and secretion from: urethra, cervix, prostate, Vancomycin = gram positive
rectal mucosa, throat Colistin = gram negative bacilli
Gastric washing and joint fluid Nystatin = yeast
Trimethoprim lactate = swarming proteus
Anisomycin = yeast
Amphoterecin = yeast
Neisseria menigitidis
Characteristics Diseases Laboratory Diagnosis

Virulence: capsule, endotoxin, pili, Meningitis Throat swab, NPS, blood, CSF,
IgA protease Meningococcemia Swabs skin
Serotypes: A,B,C,Y,W135 capsule DIC Transport: Amied medium at RT
Ag Waterhouse-Freiderichsen Direct plating on BAP, CAP
syndrome hemorrhage in Gram negative, kidney shaped
adrenal gland diplococcic
CAP: smooth, gray-brown,
mucoid
Screen: Oxidase positive
Confirm: ferments glucose and
maltose
Moraxella catarrhalis

Gram negative diplococcic 3rd cause of otitis media Oxidase positive
URT commensal LRT: bronchopulmonary Reduces NO3 NO2
Honey puck colony URT: Sinusitis DNAse positive best to diff
from other Moraxella spp
Assacharolytic
Beta-lactamase (+)
Disease: Otitis Media Butyrate Disk (Tributyrin HOH) Test

1. S.pneumoniae (+) Blue color M.catarrhalis
2. H.influenzae (-) no color change N.gonorrhea
3. M.cattarhalis
Oxidase Carbohydrate DNAse TMA

N.gonorrheae + Glucose - +
N.meningitidis + Glucose, Maltose - +
M.cattarhalis + None + +
Glucose Maltose Lactose Sucrose

Neisseria meningitidis + + - -
Neisseria gonorrheae + - - -
Neisseria secca (wrinkled colony) + + - +
Neisseria lactamica (ONPG +) + + + -
Moraxella catarrhalis (hockey puck colony) - - - -
Mycobacteria
Acid fast bacilli due to mycolic acid acid alcohol resistant
Slow growers expect M.fortuitum and M.chelonei
Much granules
Aerobic, non-sporeformer, non-motile
Three groups
A. Mycobacterium tuberculosis complex causes TB

1. M.tuberculosis pulmonary TB
2. M.bovis intestinal tuberculosis, BCG vaccine source
3. M.africanum pulmonary TB in Africa
B. Mycobacteria other than tuberculosis/Non-Tb mycobacteria
C. Mycobacterium leprae agar negative, grows on cell free media
M.tuberculosis M.bovis
Niacin + -
Nitrate + -
TCH R S
Catalase - V
AFB Grading National Standard (DSSM) DOTS Directly Observed Treatment Strategy
0 = No AFB/300 fields DSSM Direct Sputum Smear Microscopy
+n = 1-9 AFB/100 fields SPOT-AM-SPOT (DOH 2013 = 2 sputum) 1 morning, 1 random Sx
1+ = 10-99 AFB/100 fields 2x3cm ideal size of the smear
2+ = 1-10 AFB/in at least 50 fields Dry prior to heat fix to prevent aerosol
3+ = >10 AFB/in at least 20 fields 70% alcohol with sand used loop
Detection rate of 70% and cure rate of 85%
300 fields examined before negative result
Accdg to DSSM, salivary sample is acceptable
Mycobacterium tuberculosis
Obligate aerobe
Require 5% CO2 for growth
Virulence:
o cord factor responsible for clumping of cells seen in smear/culture
o sulfatides
1. Gram Stain qualify specimen

Decontamination-Digestion
o NALC-NaOH best
o Zephiran Trisodium PO4
o 6% oxalic acid if contaminated by Pseudomonas
o Dithiothreitol (sputulysin)
2. Acid fast
3. Culture
Glycerol carbon source of Mycobacteria
1. Agar Base Media
a. Dubols Oleic Acid Albumin Medium
b. Mitchisons Medium
c. Middlebrook 7H10-7H11 AST clear media
2. Egg-Base Media malachite green which inhibits normal flora
a. Petragnani Medium
b. Lowenstein-Jensen medium BEST
o MTB buff colored, cauliflower non-pigmented colonies
c. American Thoracic Society Medium (ATS)
d. Dorset Egg Medium
3. Liquid media: Rapid culture
a. Bactec 12B
b. Septi-Chek AFB
c. Middlebrook 7H9 (broth) rapid culture for MGIT
d. Middlebrook 7H12 (agar)
Biochemical Test for Mycobacteria
Niacin Test M.tuberculosis M.bovis

Principle: Niacin + Niacin Ribonucleotide + Aniline + -
dye + Cyanogen Bromide = Yellow (+); no color (-)
Best medium: L-J medium
Catalase Test M.kansasii M.tuberculosis
Heat stable at 68C + -
Medium: Tween 80
Reagent: 30% H2O2
Tween 80 + Mycobacteria + 30% H 2O2 + heat at
68C = (+) >45mm height of gas bubbles
Nitrate Reduction Test M.tuberculosis M.marinum
a. HCl M. kansasii M.simiae
b. Sulfanilamide M.fortuitum M.chelonei
c. N-napthtylethylene diamine + -
(+) pink/red
Tween 80 HOH Test M.kansasii M.avium
Px: Tween 80 lipase HOH of Tween 80 M.scrofolaceum
(+) red, (-) no red/amber + -
Tellurite Reduction Test M.avium M.kansasii
Px: Tellurite Black metallic tellurium + -
Used to ID M.avium
Arylsulfatase Test
For rapid growers (2-3 days)
Tripotassium phenolphthalein disulfide/sulfate acted upon by arylsulfatase to produce free phenolphthalein
(+) pink/red
(+) M.fortuitum, M.chelonei
TCH Susceptibility Test M.bovis M.tuberculosis
Susceptible Resistant
Mycobacterium Other Than Tuberculosis or Non-TB Mycobacteria
Based on: Photoreactivity, Pigment production, Rate of Growth and biochemical test
A. Photochromogents Rounyons I B. Scotochromogens Rounyuns II

M.kansasii = NO3 (+), yellow, pneumonia M.scrofulaceum (scrofula) = Tween 80 (-),
M.marinum = NO3 (-), 30C, swimming pool Urease (+)
granuloma, seawater M.gordonae (Tap water bacillus) Tween 80
M.asiaticum (+), Urease (-)
M.simiae = Niacin (+),NO3 (-), M.szulgai also photochromogen at 25C
M.xenopi also non-photochromogen
M.flavescens
C. Nonphotochromogens Rounyons III D. Rapid Growers Rounyuns IV
M.avium AIDS, tellurite (+) M.fortuitum = NO3 (+)
M.intracellulare battey bacillus M.chelonei = NO3 (-)
M.ulcerans Buruli M.phlei = CO2 (+)
M.xenopi (Hot Cold Water tap) 30C and 42C M.smegmatis = urine AFB (+), Pappenheim (+)
M.triviale, M.haemophilum beta hemolysis Hgb vs MTB (-)
M.malmoense, M.terrae, M.gastri
Rapid Culture for Mycobacterium

M.fortuitum M.chelonei 1. Bactec 460 Middlebrook 7H12
Arylsulfase + + Radioimmunoassay
MAC w/o crystal violet + + Px: 14C Palmitic acid + orgs = 14C CO2
Nitrate + - Result: more than 10 growth index
5% NaCl and Iron uptake + -
2. Mycobacteria Growth Indicator Tube
Fluorometric based
Middlebrook 7H9
3. Bactec 12B + NAP
Inhibition test (NAP inhibits MTB)

P-nitro acetylamino
betahydroxyporpiophenone (NAP) = no growth
on MTB
Mycobacterium leprae

Hansens bacillus Tropism to peripheral nerves Lepromine skin test that uses killed
Lepra macrophage Leprosy (Hansens disease) M.leprae
containing AF bacilli a. Lepromatous/Mutibacillary Sample: tissue
Cigarette-packet/picket- Lepromine (-) Culture foot pads of armadillo (cold
fence Many AFB condition)
NOT Culturable in agar CMI (-) Fite Faraco Stain uses hematoxylin
(in vitro) Leonine face Phenolase test separates M.leprae from
Hydrolyze 3,4-dihydroxy- b. Tuberculoid/Paucibacillary other mycobacteria with the use of DOPA
phenylalanine (DOPA) Lepromine (+) Wade Fite Technique used to detect
Few AFB AFB in paraffinied tissues
CMI (+) Treatment: Dapsone
Nodules
Other Mycobacteria
1. M.genavensi disseminated infection in AIDS, Bactec (+)

2. M.paratuberculosis
Crohns disease
Jones bacillus
Nocardia spp
Partially acid fast Pneumonia Sx: Tissue, sputum
1. Nocardia asteroides Modified acid fast 1%H2SO4, no heat
2. Nocardia brasiliensis Urease (+)
Gram positive branching rod fungus-like bacteria
Casein hydrolysis
Nocardia Actinomyces o N.asteroides (+)
Oxygen Aerobic Anaerobic o N.brasiliensis (-)
Acid fast AFO Non-AFO Sensitive to antibiotics should be placed on media
Catalse + - without antibiotics
Urease + -
Sulfur granules +/- +
Corynebacteria
Characteristics Laboratory Diagnosis
NON-acid fast BAP raised, translucent, gray colonies
Diphteroids normal flora Catalase positive
Club shape, chinese letters, palisade, Listeria Corynebacteria
X and V letters shape Motility + -
Babes-Ernst Metachromatic granules Esculin HOH + -
NON-motile, NO spore, NO capsule Salicin + -
Pleomorphic gram (+) rods CAMP +
Corynebacterium diptheriae
Kleb Loefflers Bacillus

Virulence: exotoxin, heatl labile A and B
Disease: Diptheria grayish, pseudomembrane on tonsils, pharynx , larynx
Bul-neck appearance
Laboratory Diagnosis (Sx: oropharyngeal swab, nasopharyngeal swab, skin swab, throat swab)
1. Culture
BAP
Loefllers serum agar Potassium tellurite - gray to black colonies
Pao coagulated egg Cystine tellurite BAP - gun metal gray colony)
Clauberg Potassium tellurite inhibits normal flora
MacLeods Tinsdale black colony
with brown halo
2. Gram Stain gram positive bacilli in V, X, or chinese letter
3. LAMB stain metachromatic granules
4. POSITIVE: catalase and DNAse
5. NEGATIVE: urease
6. CHO fermentation test
7. ELEK test
8. Schick test
9. Culture similar to C.pseudotuberculosis and C.ulcerans
C.ulcerans - closely resembles diphteriae which produces diphtheria like illness
C.pseudotuberculosis - horses, goat, sheep
Note: Arthrobacter culture similar to Brevibacterium
Corynecbacterium spp
Urease Nitrate Starch Reduce tellurite Pathogenic to

C.diptheriae - + +/- + Humans
C.ulcerans (mastitis in cattle) + - + +
C.pseudoutuberculosis + +/- - + animals
*Reduce tellurite produce gray to black colony
Diptheroids
Normal flora of skin, oral, conjunctiva and GUT

Non-toxigenic
Does not produce gray back colony on tellurite media
Endocarditis due to bacteremia
Diptheroids Feature Disease

C.xerosis Ferment dextrose, saccharose Conjunctivitis
(sucrose), and maltose
C.pseudodipheriticum Hoffmans bacillus Endocarditis through dental
Oral flora procedure
C.jeikeium JK bacillus Prostethic valve endocarditis
Drug resistant
Skin flora PVE
Gram (+) bacilli: C.jeikeium
Gram (+) cocci: S.epidermidis
Diptheroids Feature Disease

C.amycolatum Skin, conjunctiva, oral flora Endocarditis
C.auris Human flora Otitis media
Leifsonia aquatic Grash water Bacteremia
Kurthia Environment Bacteremia
C.minutissimum Coral red fluorescence Erythrasma
Arcanobacterium haemolyticum Reverse CAMP (+) with S.aureus - Bacteremia
inverted triangle
C.urealyticum Skin flora, urease (+) UTI
Normal flora of GUT
Rhodococcus (Corynebacterium) euqi
Pleomorphic (rod-cocci or cocci-rod)

24 hours incubation: cocci to rod or rod to cocci
Salmon pink colonies
CAMP test (+) with S.aureus = arrow head zone
Spore-forming (Bacillus and Clostridia)
Bacillus anthracis

Largest pathogenic Malignant pustule Needs bicarbonate medium for capsule
bacteria cutaneous anthrax, black formation
Anthrax bacillus eschar McFadyeans capsular stain
Gram positive rods in chain Woolsorters pulmonary PLET (Polymixin Lyozyme EDTA
forming bamboo and anthrax, Ragpickers disease, Thallous Acetate) selective media
square end most dangerous Medusa head colony, inverted pine tree
Non-motile, spore-forming, Gastroenteritis intestinal Catalase positive
zoonotic anthrax BAP: string of pearl test (0.05 units PEN)
Virulence factor: exotoxin Ascoli test serologic precipitation test
and capsule (D glutamate) Presumptive test: Penicillin
susceptibility test (10 units PEN)
Definitive test: PCR
Bacillus Clostridium BAP

O2 Aerobic Anaerobic
Catalase + - B.anthracis = non, hemolytic, irregular, serrated, swirling
Gas - + P.aeruginosa = beta-hemolytic, serrated, moist colony
B.subtilis = beta-hemolytic, ground glass, dry colony
*Note: gram positive dry colony; gram negative moist colony
Bacillus cereus
Fried rice bacillus (spores on rice grain) Food poisoning /gastroenteritis
Virulence: exotoxin (cholera like toxin) Blood bank contamination at RT
Bacillus subtilis
Quality control for sterilization Eye infection in heroin addicts
Gram positive rod in chain
Central spore
Common laboratory contaminant
Bacillus stearothermophilus
New name: Geobacillus Flat sour spoilage in canned goods
No gas but with acid
Bacillus anthracis Bacillus cereus

Motility - +
Capsule + -
Hemolysis - + beta-hemolytic
Growth at 45C - +
Salicin ferment - +
Penicillin G S R
Gelatin (HOH) and PEA - +
3 types of Clostridium
Clostridium
Obligate anaerobe, gram positive, With endospore Neurotoxic = C.tetani and C.botulinum (most severe)
Habitat: human and animal Histotoxic = C. perfringens and C.septicum
Saccharolytic except C. tetani and C. septicum Enteric = C.difficile
Clostridium perfringens/Clostridium welchii

Old name: Bacillus welchii Gas gangrene myonecrosis Chopped meat agar = growth + gas
Encapsulated, non-motile, Food poisoning BAP: target or double zone of hemolysis
double hemolysis enterotoxins o Inside: B-hemolysis (theta toxin)
Box car shape bacillus Necrotic enteritis Pig-bel o Outside: alpha hemolysis (alpha toxin)
Source: wound contact Nagler test/Lecithinase
with soil o due to alpha toxin, lecithinase,
phospholipase C
o Media: McClung/Neomycin Egg Yolk
o (+) opalescence on agar w/o anti-toxin
o (-) no opalescence on agar w/ anti-toxin
Reverse CAMP test
o S.agalactiae and C.perfringens
o (+) arrow head zone of beta hemolysis
Stormy fermentation of milk
o (+) coagulate casein/clotting of protein
+ gas
o Litmus Milk test (pH)
Acid pink
Alkaline - blue
Clostridium botulinum
Canned good bacillus (home made) Flaccid paralysis
Virulence: botulinum toxin which block the Wound botulism spore on wound
release of acetylcholine flaccid paralysis Infant botulism honey bee, floppy baby syndrome
Botulinim, a neutoxin and the most potent SIDS sudden infant death syndrome, crib death
exotoxin
Clostridium tetani

Terminal oval spore Spastic paralysis Clinical findings basis of diseases
Tennis racket, drumstick Lockjaw look for gram Morphology test gram stain (useful for
Tack head bacillus positive bacilli anaerobes)
Assacharolytic Risus sardonicus o Terminal oval spore
Virulence: exotoxin Opisthotonus arching of o Tennis racket, drumstick
(tetanolysin and the back o Tack head bacillus
tetanospasmin) binds to
ganglioside receptors and Note: Clostridium ramosum round terminal
inhibit neurons in CNS spore, ferments glucose
spastic paralysis
Clostriudium difficile

Colon flora Antiobiotic (Clindamycin) Direct detection of toxin from the stool by
Major cause of diarrhea associated enzyme immunoassay
in hospital pseudomembranous Tissue/Cell culture/Cytotoxin assay gold
enterocolitis standard for toxin identification of
C.difficile
Medium: Cycloserine Cefoxitin Fructose
Agar (CCFA)
o yellow color (fructose fermentation)
o horse manure odor
o indicator: Phenol red
Motility Capsule Lecithinase Lipase Lactose Glucose
C.perfringens - + + - + +
C.botulinum + - - + - +
C.tetani + - - - - -
C.difficile + - - - - -
*Lipase (+) = C.botulinum, C.novyii, C.sporogenes
Anaerobic Bacteriology
Collection: needle aspiration (never swab)
Media: (note: reduced media O2 is reduced)
o Shaedler
o Lombard Dowell Agar (LD)
o Wilkins-Chalgren broth and agar AST media for anaerobes
o Anaerobic PEA gram positive anaerobes
o THIO enriched broth with hemin and Vit K to enhance growth of anaerobes
o Bacteroides Bile Esculin (BBB) B.fragilis, (+) blackening due to bile and esculin
o Anaerobic Kanamycin Vancomycin Blood Agar gram negative anaerobes
Methods to promote anearobiosis
o Gas Pak Jar or Mcintosh Fildes Jar, Brewer, Torbal Jar
o Cooked meat medium/Chopped cooked meat medium
o Anaerobic glove boc and chamber
o Pre-reduced Anaerobically Sterilized (PRAS)
o Thioglyollate = for aerobic, anaerobic and microaerophilic
Resazurin (pink)
Boil for 10 minutes to drive off oxygen
Storage: RT
Gas Liquid Chromatography definitive test for anaerobes used for acid analysis
Marker of Anerobiosis Gas Pak Jar

Methylene blue Rasazurin Placed in CO2 incubator
Aerobic Blue Pink Gas Pak envelope = H+ and CO2 gas O2 + H = H2O
Anaerobic Colorless Colorless Anerobiosis in Gas Pak can be confirmed by:
1. Moisture in jar
2. Indicator turns colorless
Kanamycin Vancomycin Characteristics of Anaerobes

Colistin Pattern Brick Red Fluorescence Prevotella, Porphyromonas
RRR B.fragilis Red Fluorescence Veillonella
RSR Porphyromonas Pitting of Agar B.ureolyticus
RRS Prevotella Double Zone Hemolysis C.perfringens
SRS B.urealyticus Swarming C.tetani, C. septicum gram positive
Fusobacterium (if gram negative Proteus)
SSR Clostridium Molar tooth colony, Sulfur granules Actinomyces israelli
Breadcrumb colony F.nucleatum
Gram positive anaerobic bacilli non-spore former
1. Actinomyces 5. Lactobacillus
Fungus like bacteria Doderlein bacillus (other name of
Ray fungus Lactobacillus acidophilus)
Actinomyces bovis lumpy jaw High during pregnancy
Actinomyces Israeli draining sinus tract with sulfur Inhibits G.vaginalis
granules, molar tooth colony, catalase negative Promote C.albicans
2. Bifidobacterium dentium GIT, oral flora Catalase negative
3. Eubacterium lentum GIT, oral flora 6. Mobiluncus
4. Propionebacterium (Coryne) Vaginitis (G.vaginalis motile)
Acne, skin flora
Blood culture contamination
RBC contamination
Anaerobic diptheroid
Catalase and indole positive
Gram negative anaerobic bacilli GIT flora
1. Bacteroides fragilis
Predominant bacteria of GIT (colon flora) - has greater concentration in GIT than E.coli
Needs 20% bile, black on BBE, capsulated, catalase positive
2. Porphyromonas asaccharolytica
Black pigment, red fluorescence on UVL
Vancomycin Sensitive, assacharolytic
3. Prevotella melaninogenica
Black pigment, red fluorescence on UVL
Vancomycin Resistant, saccharolytic
4. Fusobacterium nucleatum
Breadcrumb colonies
Fusiform rod, spindle shape, bacilli with pointed ends
5. Fusobacterium necrophorum
Vincents angina and Lemierres disease
Colony: opalescent with speckles on stereoscope
Positive for Chartreuse fluorescence
6. Bacteroides ureolyticus pitting of agar
Note: Borrelia vincentii trench mouth and Vincents angina (has synergistic infection with F.necrophorum)
Gram positive anaerobic cocci
1. Peptostreptococcus anaerobius SPS sensitive, indole negative, catalase negative
2. Peptostreptococcus assacharolyticus catalase negative
3. Peptostreptococcus niger Staphylococcus-like, catalase positive
Gram negative anaerobic cocci
1. Veillonella parvula fluoresce red on UVL, jaw surgery, mouth flora
2. Megasphera
3. Acidaaminococcus
Gram Negative bacilli

Diagnostic Tests
Oxidase Test P.aeruginosa E.coli

Cytochrome oxidase (indophenol blue) + -
Filter paper method: Tetramethyl p-phenylene
diamine dihydrochloride
(+) bluish purple
Nitrate Reduction Test
Px: NO3 NO2
Rgt: Sulfanilic acid and Alpha-napthylamine
(+) red; (-) colorless
If negative, do detection of unreduced nitrate.
o Add zinc dust powder
o Px: NO2 N2
o Rgt: sulfanilic acid + alpha-napthylamine + zinc dust
o (+) for NO2: colorless = A.faecalis
(-) for NO2: red
O-Nitrophenyl-Beta-D-Galactophyranoside (ONPG) E.coli Sal.typhimurium
ONPG beta galactosidase orthonitrophenol + -
(+) yellow (-) change in color
ONPG Test: Lactose beta galactosidase (BG) Galactose
A Lactose Fermentation test, a pathogenicity test for ENTERICS
Rapid lactose fementer ONPG (+) Permease, BG 24 hours
Late lactose fermenter ONPG (+) BG 48 hours
Non-lactose fermenter ONPG (-) Pathogenic Sal, Shigella, Yersinia, Plesio
Colorless colony
*Permease as a carrier enzyme, for rapid reaction
Lysine Ornithine Arginine (LOA) Test
Uses 4 tubes (3 for LOA, 1 for control) Lysine Decarboxylase Cadaverine
Indicator: Moellers decarboxylase medium with
Ornithine Decarboxylase Putrescine
bromcresol purple
Uses mineral oil for anaerobic organisms Arginine Dihydrolase Citrulline
(decarboxylase and dihydrolase)
(+) purple (-) yellow
(+) LDC K.pneumoniae (-) LDC E.cloacae
Differentiating Enterics, Vibrio, Aeromonas and Plesiomonas
Triple Sugar Iron
Glucose + Lactose + Sucrose + Iron
o Glucose:Lactose:Sucrose = 1:10:10
o Lactose to diff enterics
o Sucrose to diff vibrio
Indicators
o pH: Phenol red (red to yellow)
o H2S: Ferrous sulfate (black)
o Gas = splits medium (aerogenic)
TSI reactions
o A/A = 2-3 sugars fermented (LF)
o K/A = glucose fermented (NLF) Ex. Pseudomonas
o K/K = no sugar fermented (NFO)
Slant: aerobic; Butt; anaerobic
*No A/K because acid is first seen on butt not on slant
*K/K not seen on Enterics because all enterics ferments sugar
Lysine Iron Agar
1. Lysine Decarboxylation = butt, anaerobic
(+) purple (-) yellow
2. Lysine Deamination = slant, aerobic
(+) red (-) purple
3. Indicator
pH: bromcresol purple
H2S: Ferric NH4 citrate
LIA reaction: K/K = (+) LDC K/A = (-) LDC
R/A = (+)LD
Indole Test
Tryptophan tryptophanase Indole
Kovacs/Ehrlichs reagent
(p-dimethylaminobenzaldehyde)
(+) red ring using SIM medium
Rapid Spot Indole Test
Filter paper strips impregnated with p-
diaminocinnamaldehyde
Screening for indole production
(+) blue
Methyl Red Test E.coli E.cloacae
Mixed acid of glucose fermentation + -
Indicator: Methyl red; medium: MRVP
pH below 4.4
(+) red (-) yellow
Vogues Proskauer (+) KESH (Klebsiella, Enterobacter, Serratia, Hafnia)
Butylene glycol of glucose fermentation KESH = (+)VP, (-)MR
Acetoin or acetylmethylcarbinol
Barrits method: alpha napthol and KOH
Coblentz method: alpha napthol and 40% KOH
in creatine
(+) red (-) yellow
Utilization Tests
Source: Carbon
End product: NH3 (ammonia)
Indicator: bromthymol blue
(+) blue (-) green
1. Citrate Test (+) K.pneumoniae (-) E.coli
2. Acetate Test (+) E.coli (-) Shigella
3. Acetamide Test (+) P.aeruginosa (-) S.maltophilia
4. Malonate Test (+) Citrobacter (-) E.coli
(+) KECH (Klebsiella, Enterobacter, Citrobacter, Hafnia)
Urea Hydrolysis Test/Urease
Urea + H2O CO2 + H2O +2NH3 = NH4CO3
Indicator: phenol red
Christensens agar-Stuart Urea Broth
(+) pink (-) yellow
End product: NH3 and CO2
Phenylalanine Deaminase (PAD) P.vulgaris E.coli
Phenylalanine PAD PPA + 10% FeCl3 + -
(+) green
(-)
Seen on slant because deaminase production is
aerobic
KCN Broth Test
(+) turbid = KEPSC ( Klebsiella, Enterobacte, Proteus, Providencia, Citrobacter)
(-) clear
String Test
ID of Vibrio cholera
0.5% sodium deoxycholate
(+) string like
Esculin HOH Test K.pneumoniae S.flexneri
(+) black (-) yellow + -
MUG Test (UVL) E.coli P.aeruginosa
4 methylumbelliferyl-beta-D-glucoronide + -
Fluoremetric method more preferred
(+) electric blue fluorescence
(-) no fluorescence
Colorimetric method: (+) yellow
Gelatin Hydrolysis Test P.vulgaris E.aerogenes
(+) gel liquifies + -
(-) gel liquefies
End product: amino acid
Enteric Media
Medium Inhibitory CHO Indicator LF NLF

EMB Eosin Y Lactose Eosin Y Red/Pink GMS Colorless
Methylene Blue Methylene Blue
MAC Crystal violet Lactose Neutral red Red/Pink Colorless
Bile Salt
XLD Bile Salt Xylose, Lactose, Sucrose Phenol red Yellow Red/colorless
HEA Bile Salt Salicin, Lactose, Sucrose Bromthymol blue Yellow Green/Colorless
DCA Bile Salt Lactose Neutral red Red/Pink Colorless
SSA Bile Salt Lactose Neutral red Red Colorless
Brillant green
BSA Brillant green Glucose Bismuth sulfite Black colony Salmonella
TCBS Bile Salt Sucrose Bromthymol blue Yellow Green
Brillant Green Agar H2S indicator

o Other Salmonella spp. except S.typhi Ferrous sulfate/ferrous NH4 sulfate = TSI, BSA
o Brillant green inhibitory agent Ferric citrate = SSA
o Phenol red pH indicator Ferric ammonium citrate = XLD, HEA, LIA
Bismuth Sulfite Agar (BSA)/Wilson Blair = Sal.typhi Lead Acetate = SIM, paper strip test
Tetrathinate broth = Salmonella spp Media without H2S indicator = EMB and MAC
Selenite F broth = Salmonella and Shigella
Enterobacteriaceae
Laboratory ID of Enterobacteriaceae Media Colonies Organisms

Stool, blood, CSF, swab, sputum, urine, wound SSA Colorless Shigella
BAP-MAC (Selective, Differential) Colorless with black Salmonella
Enrichment broth to selective media MAC Red/Pink E.coli
o Selenite to SSA EMB GMS E.coli
o APW to TCBS Red LF (K.pneumoniae)
Glucose fermenter Colorless NLF (Shigella, Sal)
Screen: (-) oxidase (+) catalase and nitrate TCBS Yellow Vibrio cholera
Confirmatory: serotyping SMAC Bluish-green V.parahaemolyticus
Generalities
Gram negative enteric coccobacilli, short, plump All are motile (peritrichous) at 37C except SKY
bacilli o Klebsiella, Shigella, Yersinia pestis
Non-sporeformers o Yersinia motile at 25C
Facultattive anaerobes All are AEROGENIC except Salmonella typi, Yersinia,
Antigenic Shigella and Providencia (ProSSY)
o cell wall (O) somatic, heat stable All are CATALASE POSITIVE except S.dysenteriae
o flagella (H) flagellar, heat labile All are CYTOCHROME OXIDASE NEGATIVE except
o capsule (K) capsular, heat labile Plesiomonas
K1 E.cloi; Vi S.typhi All are NITRATE REDUCERS except Photorabdus
BAP/CAP: large moist gray colonies and Xenorabdus
All are gamma hemolytic except E.coli Most are commensal flora of the intestinal tract
All are non-encapsulated except Klebsiella and except Salmonella, Shigella and Yersinia
Enterobacter
All are GLUCOSE FERMENTERS
Rapid Lactose (18-24 hours) Late Lactose (>48 hours) Non-Lactose
EKE CHYSSS SPEMPSY
Escherichia Citrobacter Salmonella except Sal. enteritica
Klebsiella Hafnia subsp. Arizonae
Enterobacter Yersinia Providencia
Salmonella enteritica subsp. arizonae Edwardsiella
Shigella sonnei Morganella
Serratia Proteus
Shigella except S.sonnei
Yersinia except Y.enterocolitica
Deaminase positive H2S positive Vogues Proskauer positive

PPM black SPaCEd SHEK
Proteus Salmonella Serratia
Providencia Proteus Hafnia
Morganella Citrobacter Enterobacter
Edwardsiella Klebsiella
Urease producers
Rapid Urease Late Urease
PPM CKEYS
Proteus Citrobacter
Providencia rettgeri Klebsiella
Morganella morganii Enterobacter
Yersinia
Serratia
Rapid Lactose Fermenters (Coliforms)
Escherichia coli

Colon bacillus #1 cause of UTI, gram TSI: A/A
Pathogenic when it negative sepsis IMVIC: + + - -
produces toxin #2 neonatal meningitis (K1 LOA: + + -
E.coli that is non-lactose, antigen) EMB: greenish metallic sheen
non-motil and anerogenic: Nosocomial, wound, MUG (+) except E.coli 0157:H7
Alkalescens dispar bacteremia, pneumonia Limulus test
o Detect bacterial endotoxin
o Derived from limulus horse crab
Escherichia coli infections biotype
Enterotoxigenic E.coli (ETEC) Enteropathogenic E.coli (EPEC) Enteroinvasive E.coli (EIEC)

Travellers (turista) diarrhea Infantile diarrhea (pathogenicity Dysentery
Cholera like toxin or heat labile island) Shigella-like diarrhea
enterotoxin EPEC O111, O114 Invasive
Watery diarrhea Bloody stool with mucus
ETEC O6, O8, O25 EIEC O124, 143, 164
Sereny Test
o Virulence test
o Organism is injected on
mouses conjunctive
o (+) kerativa conjunctivitis
Enterohemorrhagic E. coli (EHEC) Enteroaggregative E.coli
Verotoxin E.coli (VTEC) Acute and chronic diarrhea
Hemolytic Uremic Syndrome (HUS) bloody urine Aggregative adhesion fimbriae
Hemorrhagic colitis bloody stool Stacked brick pattern of cells
Shigella-like toxin
VEROTOXIN
E.coli O0157:H7 = SMAC (-), MUG (-)
E.coli Shigella
MAC LF NLF
Acetate + -
Motility + -
Lysine Ornithine Arginine

Enterobacter E.aerogenes + + -
E.gergoviae = urease positive + + -
TSI: A/A
IMVIC: - - + + Hafnia alvei + + -
Urease negative except E.gergoviae E.cloacae (-) LDC control - + +
UTI, wound, septicemia E.sakazakii = yellow - + +
Pantoea agglomerans = yellow - - -
Klebsiella pneumonia

Friedlanders bacillus Pneumonia, wound, TSI: A/A
Capsulated meningitis, UTI LIA: K/K
Non-motile IMVIC: - - + +
Urease and malonate (+)
MAC: mucoid, lactose fermenter
(+) String test due to mucoid colony
LDC VP and Urease Indole
K.pneumoniae + + -
K.oxytoca + + +
K.ozaenae + - -
K.rhinoscleromatis - - -
*Biochemically inert (all negative) : K.rhinoscleromatis
Biochemical Test Result of Rapid Lactose Fermenters
TSI LIA I M V C U
Escherichia coli A/A + gas K/K + + - - -
Klebsiella pneumonia A/A + gas K/K - - + + +
Enterobacter aerogenes A/A + gas K/K - - + + -
Enterobacter cloacae A/A + gas K/A - - + + -
Late Lactose Fermenters

Arizona spp Citrobacter
Old name of salmonella Cross react with Salmonella but LDC (+)
New name: Salmonella arizona LDC (-)
Only lactose fermenting salmonella TSI: A/A + H2S
TSI: A/A + H2S LIA: K/A
LIA: K/K ONPG (+)
ONPG (+) C. diversus neonatal meningitis
C.freundii UTI, pneumonia, endocarditis
TSI Indole Malonate C.freundii S.typhi

C.freundii A/A + H2S - + MAC LF NLF
C.diversus/koseri A/A + + LDC - +
C.amalonaticus A/A + -
Biochemical Test Result of Late Lactose Fermenters
TSI LIA I M V C U
Arizona spp A/A + gas + H2S K/K + H2S - + - + -
Citrobacter freundii A/A + gas + H2S K/A - + - + -
Citrobacter diversus A/A + gas K/A + + - + -
*To differentiate: use LIA
Non-Lactose Fermenters
Proteus, Providencia, Morganella (Group Proteeae) Proteus
PAD (+), Lysine deamination (+) Swarm on BAP and CAP but not on MAC
Urease (+) exept Providencia alcalifacien #2 cause of UTI
LOA : - - - except Morganella and P. mirabilis (ornithine Renal stone due to urease virulence factor
positive) Cross react with rickettsia
Indole (+) expcept P. mirabilis Species:
o P.vulgaris indole (+), Ox2, Ox19, OxK
o P.mirabilis indole (-), OxK
Laboratory tests:
o TSI: K/A + H2S
o PAD (+)
POSITIVE FOR DIENESS PHENOMENON
TSI Urease Ornithine

M.morgani K/A + + Morganella Providencia
Providencia stuartii K/A + - Ornithine + -
Providencia rettgeri K/A + - Citrate - +
Providencia alcalifaciens K/A - -
Proteus mirabilis K/A + H2S + +
Biochemical test result of PMP Group Proteeae
TSI LIA I M V C U
Proteus vulgaris K/A + gas + H2S R/A + + - + +
Proteus mirabilis K/A + gas + H2S R/A - + - + +
Providencia rettgeri K/A + gas R/A + + - + +
Morganella morganii K/A + gas R/A + + - - +
*Urease (+) = PAD (+)
------------------------------------------------------------------------------------------------------------------------------------------------------
Salmonella

Aerogenic except Sal.typhi Salmonellosis pea soup stool SSA and BSA: black colony
and Sal. gallinarum Salmonella typhi (+) Selenite F and tetrathinate broth
Motile except S.gallinarum o Thyphoid fever, meningitis, TSI: K/A + H2S
and S.pullorum osteomyelitis LDC (+)
Serotype: Kauffman o 1st wk: blood Sal.paratyphi
white scheme 2nd wk: stool (carrier state) o Only Salmonella negative for
Antigens for S.typhi o To confirm typhoid fever, do H2S and LDC
o Vi, O and H antigen bone marrow culture If salmonella is negative for somatic
o Vi heat labile Salmonella paratyphi A and B Ag (O-Ag), do heating. It will destroy
Related to Citrobacter o Paratyphoid fever Vi Ag which covers the somatic Ag. Vi
(LDC negative) Salmonella paratyphi C Ag is heat labile.
o S.cholera suis Confirmatory test for typhoid fever:
o Septicemia most severe Culture not Typhi-dot
Salmonella enteritidis
o Gastroenteritis on poultry
Salmonella typhimurium
o Food poisoning
Shigella

Almost negative to all Dysentery - bloody stool with Colorless on SSA
biochemical tests = mucus Acetate (-)
biochemically inert Shigella dysenteriae common TSI: K/A
Non-motile cause of communicable diarrhea LIA: K/A
Related to E.coli (acetate LDC (-)
positive) LOA: - - - except S.sonnei (LOA - + -)
Shigella sonnei cross Culture
react with Plesiomonas o fresh stool with mucus flecks
shigelloides o rectal swab of ulcer - best
O Ag Mannitol Ornithine Salmonella Shigella

and ONPG Motility + -
S.dysenteriae (shiga) A - - H2S + -
S.flexneri (strong) B + - LOA + + + - - -
S.boydii (boyds) C + - Indole - +/-
S.sonnei (duval) D + + Invasive - +
Blood Culture + -
Related to Citrobacter E.coli
Biochemimcal test result of Salmonella, Shigella, and Serratia
TSI LIA I M V C U
Salmonella typhi K/A + small H2S K/K - + - - -
Salmonella enteritidis K/A + gas K/K - + - + -
+ large H2S
Shigella dysenteriae K/A K/A - + - - -
Shigella sonnei K/A K/A - + - - -
Shigella flexnerii K/A K/A + + - - -
Shigella boydii K/A K/A + + - - -
Serratia marcescens K/A or A/A K/K + - + - +
Edwardsiella tarda
Diseases Laboratory Diagnosis

diarrhea Lysine decarboxylase (+)
wound TSI: K/A + gas + H2S (Salmonella)
bacteremia To diff: Edwardsiella indole (+), Salmonella indole (-)
IMVC: + + - - (E.coli)
To diff: Edwardsiella (K/A , NLF); E.coli (A/A, LF)
Yersinia spp (Pasteurella)
Yersinia pestis Plague bacillus Rat flea bite
Stain: Safety pin Bubonic, pneumonic, septicemic
Growth pattern: Stalactite PLAGUE
Bipolar bodies (Wayson) Black death (bioterrorism in Europe)
V and W antigens
Non-motile
Urease and ornithine (-)
Yersinia enterocolitica Oxidase (-) Seen on unpasteurized milk
Psychrophilic: Motile at 22C but Enterocolitis
not at 35C Arthritis
Cold enrichment at 4C (Listeria) Erythema nodosum
Yersinia g(-), Listeria g(+) Appendicitis and blood bag
CIN agar: Bulls eye colony contaminant
CIN (+): Yersinia, Aeromonas
Y.enterocolitica Oxidase neg
A.hydrophilia Oxidase pos
Zoonotic
Yersinia pseudotuberculosis LOA: - - - Acute mesenteric Lymphadenitis
Urease (+) Septicemia
Pseudotubercles : animal pathogen
Yersinia pestis Yersinia enterocolitica Yersinia pseudotuberculosis

Motility - + +
Urease - + +
Ornithine - + -
Sucrose - + -
TSI K/A A/A K/A
Vibrionaceae
Classification of Vibrio cholera O1 (VCO1)
Vibrio, Aeromonas, Plesiomonas *VCO1 El tor more common, pandemic cholera
All are oxidase (+), catalase (+), indole (+) agent
All ferment glucose Biotype Classical El Tor
Polar flagella Polymixin Susceptibility S R
All are pathogenic Lysis by bacteriophage + -
To differentiate from enterics Chicken RBC agglutination - +
Vibrio oxidase (+) Hemolysis of sheep RBC - +
Enterics oxidase (-) Vogues proskauer test - +
Vibrio spp
Comma shape, Oxidase (+) except V.mitschnikovii

Motile (monotrichous) LOA: + + -
O129 susceptible Nitrate reduction (+)
Halophilic except V.cholerae and V.mimicus MAC: colorless colony except V.vulnificus
Alkaliphilic
Vibrio cholerae

Non-halophilic Rice watery stool (cholera) TSI: A/A , Indole (+)
1-3% NaCl Seen in flood and drinking water String test (+): 0.5% Na
Choleragen cholera desoxycholate
toxin which increases Sucrose fermenter yellow on TCBS
CAMP loss of water Pfeippers phenomenon lysis of
dehydration V.cholerae
Transport medium: Cary Blair
Serotype Ogawa Inaba Hikojima
Anti-Ogawa + - +
Anti-Inaba - + +
Laboratory ID of V.cholerae 01
1. Darkfield microscopy 3. Oxidase test
2. Culture 4. O129 Sensitivity test = SENSITIVE
TCBS Thiosulfate Citrate Bile Salt Sucrose 5. Polymixin B susceptibility test
TTGA Tellurite Taurocholate Gelatin Agar 6. Cholera Red Test: Nitroso-Indole Test
APW Alkaline Peptone Water (6-8hours) NO3 (+) Indole (+)
Vibrio parahaemolyticus

Halophilic (8%NaCl) Gastroenteritis seafoods in TSI: K/A , Indole (+)
Japan LOA: + + -
Non-sucrose fermenter green on
TCBS
Kanagawa positive beta
hemolysis on Wagatsuma agar
Biolumiscent bacteria = Vibrio harveyi, Vibrio fischeri, Vibrio leognhati
Disease 8% NaCl TCBS CHO Oxidase

V.cholerae Cholera - (1-3%) Yellow sucrose +
V.alginolyticus Gastroenteritis + Yellow Non-sucrose +
V.parahemolyticus Gastroenteritis + Green Arabinose +
V.vulnificus Septicimia - Green Lactose +
Wound
Aeromonas

Non-halophilic Red leg disease O129 Sensitivity test = RESISTANT
Opportunistic Diarrhea TSI: A/A + gas
(freshwater) Wound (+) for CODE: Catalase, Oxidase,
Septicemia DNAse, Esculin HOH
BAP with ampicillin: beta hemolysis
LOA: + - +
Plesiomonas

Non-halophilic Diarrhea O129 Sensitivity test =
Opportunistic Wound RESISTANT/SENSITIVE
(freshwater) Septicemia TSI: K/A or A/A (glucose and
inositol)
LOA: + + +
(+) for ICO
o Inositol, Catalase, Oxidase
Vibrio Aeromonas Plesiomonas
NaCl + - -
Motility + + +
Oxidase + + +
O129 Sensitivity S R S/R
LOA + + - + - + + + +
DNAse, Hemolysis, Esculin HOH - + -
Campylobacter

Spiral or curved rods Campylobacter jejuni Oxidase and catalase (+)
Sea gull wing o Guillain-Bare syndrome - paralysis Indole, acetate (+) except C.lari
Darting motility o #1 cause of gastroenteritis o (+) result: purple
Microaerophilic WORLDWIDE Selective: Skirrows, Butzler,
Growth at 37-42C o Found in poultry CAMPY-BAP, CCDA
Zoonotic Campylobacter fetus
o Animal abortion
Campylobacter coli
o Gastroenteritis (diarrhea)
37C 42C Nalidixic Cephalotin Hippurate Catalase Oxidase Hippurate Indoxyl

C.jejuni + + S R + Acetate
C.coli + + S R - C.jejuni + + + +
C.fetus + - R S - C.coli + + - +
C.lari + + - -
Helicobater pylori

Formerly Campylobacter Peptic ulcer Oxidase, catalase, urease (+)
pylori Gastritis
Microaerophilic Cancer
C.jejuni H.pylori
Oxidase + +
Catalase + +
Microaerophilic + +
Urease - +
Growth at 42C + -
Non-fermentative Organisms
Diagnostic Tests
O-F Test Open tube (w/o Close tube (with

Determine the action of bacteria to CHO mineral oil) mineral oil)
Fermentative: close; Oxidative: open Oxidizer Yellow Green
Semi-solid medium: Hugh & Leifson medium (Pseudomonas)
o 1% glucose, 1% agar, peptone
o High CHO (acid), low peptone (alk) Fermenter Yellow Yellow
Indicator: bromthymol blue (E.coli)
(+) yellow acid Non-utilizer Green Green
(-) green no acid can only become (Alkaligenes)
Uses mineral oil for fermentative tube to prevent oxidizer
entry of oxygen
Growth at 42C P.aeruginosa P.fluorescens
(+) growth at 35C and 42C + -
Cetrimide Test P.aeruginosa E.coli
35C for 7 days + -
Pseudomonas spp
Oxidase positive except S.maltophilia OF: Yellow (O), Green (F) = oxidizer
Motile except B.mallei (non-motile) Opportunistic infection
MAC: colorless colony Pyocyanin blue green
TSI: K/K or neutral reaction Fluorescein yellow green
Pseudomonas aeruginosa

Bacillus pyocyanus Blue pus agent Sx: respiratory specimens
Motile (monotrichous) #1 cystic fibrosis, ventilator Oxidase and catalase (+)
Fruity grape like odor associated pneumonia and meningitis LDC (-)
Corn tortillas odor #1 ICU isolate, #1 NFO, #1 Growth at 42C
Pyocyanin, fluorescein or opportunistic Acetamide carbon source
pyoverdin #2 burn OF test = +/-,
Fluorescein P.putida and Wound (ecthyma gangrenosum) Oxidize glucose
P.fluorescens Swimmer ear (otitis externa) (+) iodine preparation
Dermatitis Jacuzzi hot tub syndrome, resistant to disinfection
can thrive up to 42C
Endocarditis, UTI
Laboratory of P.aeruginosa
Cetrimide (+) on selective media
BAP: gray, spreading, serrated, metallic sheen, mucoid, beta-hemolytic
o Similar growth with B.anthracis and B.subtilis
o Mucoid feature differentiates Pseudomonas from B.subtilis
o PAE mucoid colony, B.subtilis dry colony
MAC: colorless with green pigment
MHA: bluish-green colonies due to pyocyanin
(+) NO3, Urease, Gelatin HOH
LOA: - - +
Glucose oxidizer
37C 42C Pyocyanin Fluorescein Gelatin HOH and Proteolysis

P.aeruginosa + + + + To diff P.fluorescens from P.putida
P.fluorescens + - - + +
P.putida + - - + -
Burkholderia spp
Characteristics Diseases Laboratory

B.cepacia Motile (lopotrichous) #2 cystic fibrosis Oxidase and LDC(+)
Earthy or dirt like odor Pneumonia OFPBL
Sepsis o OF test: Yellow
Onion bulb rot in plants o Polymixin B
Foot rot in humans o Basitracin
o Lactose oxidizer
PC Agar
Pink colony on MAC
Yellow B.cepacia and
P.stutzeri (wrinkled col)
B.pseudomallei Motile (lopotrichous) Melioidosis or Wrinkled colony on ashdown
Whitmores bacillus Glanders like medium
Vietnamese time bomb pneumonia OF test = +/-
(bioterrorism) lactose oxidizer
Arginine (+)
Growth at 42C
B.mallei Only non-motile Glanders disease OF test = +/-
pseudomonad (horses) Glucose, maltose, lactose
oxidizer
Pseudomonas stutzeri
Brown/buff colored wrinkled colony Non-lactose fermenter

(+) 6.5% NaCl Arginine (-)
(+) for unreduced nitrite test (NO2 N2 gas)
Stenotrophomonas maltophilia
Old name: Xanthomonas

Motile (lopotrichous) Culture:
Ammonia-like odor o SBAP: lavender green colony
Biochemical tests o TSA: yellow pigment
o Oxidase (-), DNAse and LDC (+) o Heart infusion agar with tyrosine:
o OF test: +/- brown pigment
o Glucose and maltose fermenter
Shewanella putrefaciens
Only pseudomonads that can produce H2S

TSI: K/K + H2S
Oxidase (+)
Other non-fermentative organisms: TSI: K/K no sugar, NF Acinetobacter Pseudomonas

Motility - +
Acinetobacter Oxidase - +
#2 NFO Drug resistant Oxidase (-)
Gram negative UTI Catalase (+)
coccobacilli Wound BAP: round, opaque, mucoid col
Non-motile Diarrhea MAC: purple colony
NOT reduce nitrate Nosocomial pathogen
Mistaken as Neisseria
(oxidase positive)
A.baumanii/A.anitratus A.lwoffi
Growth at 42C + -
OF Glucose + -
Old name Herella vaginocola Mima polymorpha
Alkaligenes faecalis

Apple like fruity odor UTI, wound diarrhea Assacharolytic = OF test (-/-)
Motile (peritrichous) Oxidase and catalase (+)
Alkaligenes faecalis Acinetobacter

Oxidase + -
Motility + -
Moraxella lacunata

Morax axenfield Blepharoconjuctivitis Oxidase and catalase (+)
Mistaken as Neisseria Assacharolytic
(CHO +) MAC (-)
Chryseobacterium meningosepticum

Yellow flavin Neonatal meningitis (+) oxidase, DNAse, gelatin HOH,
Non-motile Sepsis indole
Old: Flavobacterium Nebulizers MAC (-)
New: Elizabethkingiae
Eikenella corodens

Bleach like odor Human bite woud Clenched fist MAC (-)
SBE agent
Kingella spp.

Pits the agar Causes SBE (HACEK group) Ferment glucose
1. K. denitrificans non-hemolytic
o denitrificans = Nitrate (+)
2. K.kingae beta-hemolytic
Oxidase Catalase MAC

Acinetobacter - + +
Alkaligenes + + +
Flavobacterium + + -/+
Moraxella + + -
Kingella + - -
Eikenella + - -
Parvobacteria
Gram negative bacilli or coccobacilli CO2 requiring
Fastidious MAC negative
Aerobic
Haemophilus spp
Gram negative coccobacilli Satellitism H.influenzae

Oxidase (+/-) Medium: CAP horse blood + 5% CO2
Require X (hemin) and V (NAD) No growth on MAC
Haemophilus influenzae
Pfeiffers bacillus Doesnt cause influenza
Virulence: 3rd cause of bacterial meningitis
o Capsule type BHib vaccine Major cause of acute epiglottitis
o IgA, Protease, LPS. Pili Cystic fibrosis
Satellite around SAU on BAP Otitis media, conjunctivitis, pneumonia, sepsis, cellulitis
Affects both URT and LRT
Laboratory ID of Haemophilus influenzae Growth Factor tests for Haemophilus
Horse blood bacitracin agar selective for 1. Porphyrin test = X factor (ALA) test
H.influenzae, prevents growth of P.aeruginosa 2. X and V strip test using MHA
Medium of choice for hemolysis: horse BAP a. XV growth = HAI
Sx: blood, CSF, NPS, throat swab, body fluids (RT) H.influenzae, H.aegypticus, H. haemolyticus
Swab on Amies transport medium b. V and XV growth = H.parainfluenzae
BAP, CAP, MAC, Bacitracin Chocolate Agar (for c. X and XV growth = H.ducreyi
unsterile specimens) 3. Satellistim = BAP with S.aureus
GS: gram negative coccobacilli (Substitute: C.albicans, S.pneumoniae) as V factor source
CAP: grayish, dew drop, mousy odor 4. Beta hemolysis on horse BAP =
Porphyrin test: negative H.haemolyticus/H.parahaemolyticus
Satellitism test: growth near S.aureus
Porphyrin Test (X factor)
Beta lactamase test: positive - control
Delta aminolevulenic acid (ALA) Protoporphyrin
Screen: oxidase positive
(Porphyrin-Heme/X factor)
Confirmatory: growth on X and V disk
(+) red fluorescens
Haemophilus ducreyi

School of red fish - clusters of Chancroid soft chancre, painful Sx: genital specimen
gram negative rod X factor requiring
Growth on CAP + vancomycin
Haemophilus aegypticus
Koch weeks bacillus Pink eye conjunctivitis
Brazilian purpuric fever
Differential test for Haemophilus
X V Porphyrin
H. influenzae + + -
H. aegypticus + + -
H. haemolyticus + + -
H. parainfluenzae - + +
H. parahaemolyticus - + +
H. paraphrophilus - + +
H. ducreyi + - -
H. aphrophilus - - +
Bordetella pertussis
Characteristics Laboratory
Capsulated Sx: nasopharyngeal swab
Obligate aerobe Dacron swab
Whopping cough bacillus Best time: Catarrhal, paroxysmal, convalescence
Requires cysteine and methionine for growth
MAC (+) except B.pertusis
Culture Media: (Cough Plate Method)
1. Potato blood glycerol agar or Bordet Gengou = mercury drop/pearl-like
2. Best medium: Regan Lowe/Charcoal Horse Blood with Cephalexin and Amphotericin) add charcoal to
detoxify
3. Jones Kendrich charcoal and yeast extract
4. Charcoal Cephalexin Blood Agar (CCBA)
5. Stainer and Scholte
6. Casamino Broth
Motile Urease Oxidase MAC, BAP
B. pertusis - - + - Human patho
B. parapertusis - + - + Animal
B. bronchiseptica + + + + pathogen
(kennel cough)
Brucella spp.

No capsule Brucellosis Culture: TSB, Wisconsin,
Obligate aerobe Undulant fever, Malta fever, Castaneda broth
Non-motile Mediterranean fever, Gibraltar Sample: blood, urine, stool,
Zoonotic Eryhtritol fever, Cyprus fever lymph node, sputum
o Enhances growth Bangs disease
o Found in animal placenta Animal abortion
Endocarditis
Laboratory acquired infection
Dye inhibition Test
Urease CO2 Thionine Fuchsin Abortion
B.abortus (Bangs) + + - + Cattle
B.melitensis + - + + Goat and sheep
B.suis + - + - Swine
B.canis + - + - Dog
*Note: B.abortus and B.suis = H2S (+)
Francisella tularensis

Old name: Bacterium tulareri MOT: inhalation, insect bite Sx: blood, lymph node
Non-motile ingestion Require cysteine and cysteine
Capsulate Tularemia Ohara (+): catalase, beta-lactamase
Aerobe Market mens disease (-): oxidase, urease, MAC
Used in bioterrorism Deerfly, lemming, rabbit fever, Forshay Skin Test tularemia
water-trappers Rapid test: PCR
Most common laboratory acquired Culture media:
infection o Glucose Cysteine Blood
Agar (GCBA)
o Peptone Cystein Agar
(PCA)
o Cysteine Heart Agar (CHA)
Pasteurella multocida

Gram negative coccobacilli Multiple killing (+): oxidase, catalase, indole,
Capsulated, non-motile Animal bite wound nitrate, glucose
Bipolar staining, safety pin Cat bite infection Growth on BAP but not on MAC
appearance Shipping fever in cattle
Musty or mushroom-like Pneumonia, endocarditis,
odor meningitis, arthritis
Diagnostic Test for HACEK

Gram negative coccobacilli, fastidious, requires CO2, MAC (-), SBE (Sx: blood)
Oxidase Catalase Features
Haemophilus aprophilus +/- - No X and V
Actinobacillus/Aggregatibacter actinomycetemcomitan - + Dots and dashes of Moore
Star like colony
Cardiobacterium hominis + - Indole (+)
Forms rosette
Eikenella corodens + - Assacharolytic
Kingella kingae + - Twitching motility

Miscellaneous bacteria
Treponema pallidum
Obligate intracellular (rabbits testicles)s Syphilis
Acquired by sexual contact 1. Primary
Jarisch-Herxheimer Rxn o Hard chancre
o Phenomenon where large quantities of toxin are o painLESS
released as bacteria dies during treatment 2. Secondary
o Post (arsenic) treatment reaction o Condylomata lata
o Tx: Penicillin o Skin rash
Other treponemes caused by skin disease, not STD: o High Ab titer (highly infectious)
o T.pertenue = yaws/framboise 3. Latent asymptomatic, do serology
o T.carateum = pinta 4. Tertiary
o T.endemicum = bejel o Gummas, granuloma formation
5. Congenital syphilis
o Stillbirth, abortion
Spirochetes Diagnosis Disease
Treponema blood RPR (+) Serology Syphilis, Yaws, Pinta, Begel
Leptospira DAP cell wall Culture Weils disease, Infectious jaundice
Borrelia blood/BM Giemsa, Lyme, Relapsing fever
serology
Laboratory
Non-cultivatable on agar medium
1. Dark Field Microscopy corkscrew motility
2. Levaditi Silver Impregnation
3. Serology
a. Screen: VDRL, RPR, TRUST (regain test)
b. Confirmatory: FTA-ABS, TPHA, MHA-TP, HATTS (Treponemal Ab test)
c. (+) RPR, (-) TPHA = Biologic false positive, not true syphilis
Leptospira interrogans icterohemorrhagica

Hook ends spiral Weil Specimen:
Obligate aerobes, zoonotic disease/Leptospirosi o 1st week: blood/CSF (Acute)
Note: L.biflexa non- s (animal urine) o 2nd week: urine (Chronic)
pathogenic Culture media
o Fletchers rabbit serum + fatty acid; 30C
for 6 weeks
o Noguchi
o EMJH Ellinghaussen McCullough Johnson
Harris
Laboratory Diagnosis of Leptospirosis
1. Dark field microscopy corkscrew motility
2. Culture confirmatory/definitive test
3. Serology
a. MAT Macroscopic Agglutination Test - screen
o Serum + Ag (killed leptospira)
o (+) agglutination
b. MIT Microscopic Agglutionation Test confirm
o Serum + Ag (live leptospira)
o (+) agglutination using darkfield microscope
Borrelia blood spirochete
Relapsing Fever due to Lyme disease by B. burgdorferi

Borrelia recurrentis epidemic, louse bite #1 in US
Borrelia anserine, toricatae, parkeri endemic, tick bite Tick bite of Ixodes dammini
Diagnosis: Wright/Giemsa blood/bone marrow Primary stage erythema chronicum migrans
o ECM: Bulls eye rash
Secondary stage meningitis, cardiac
Tertiary stage arthritis
Culture on Barber Stoenner Kelly (BSK) at 33C
for 6 weeks
Best method: Giemsa stained blood smear
Chlamydia spp.
Old name: Bedsonia, New name: Chlamydophilia

Grows on cell culture, not on agar media (also applies for rickettsia)
Obligate intracellular parasite
Energy ATP parasite
Diagnosis: presence of inclusion body using Giemsa stain
o Elementary body infectious Non gonococcal urethritis (NGU)

o Reticulate body reproductive 1. Chlamydia trachomatis
2. Ureaplasma urealyticum
Chlamydia trachomatis 3. Mycoplasma genitallum
Diseases Laboratory
TRIC agent: Trachoma and Inclusion conjunctivitis Frie test skin test for LGV
= #1 cause of bacterial conjunctivitis Sensitive to sulfonamide
#1 nongonococcal urethritis and pelvic Transport: 4C
inflammatory disease Swab:Dacron/Rayon on sucrose phosphate buffer
Lyphogranuloma venerium (LGV) buboes
Reiters syndrome 1. Iodine or Giemsa stain of glycogen containing
inclusion bodies Halberstaedter prowazeik
o Iodine: brown; Giemsa: purple
2. McCoy - best medium, gold standard
3. Direct Fluorescence Antigen chlamydia Ag
4. PCR/NAAT definitive test
Chlamydia psittaci

Non-glycogen inclusion body MOT: inhalation of birds droppings Resistant to sulfonamide
Parrot fever or psittacosis Giemsa stain inclusion boday
(ornithosis) cannot be stained by iodine due
Man: pneumonia to absence of glycogen
Chlamydia pneumoniae

Human to human TWAR: Taiwan Acute Respiratory (+) Human lines and Hep-2 cell
transmission Pneumonia Immunofluorescence Test
Guillain Barre Syndrome
Rickettsiae
Rickettsia, Orientia, Ehrlichia
Rickettsia
Obligate intracellular except Coxiella

Arthropod borne (MOT: insect bite) Laboratory Diagnosis
Cross react with Proteus (Weil-felix) 1. Special stain = Gimenez, Macchiavelo, Castaneda, Giemsa
2. Culture = embryonated egg (best), cell culture
Rocky Mountain Spotted Fever
3. Weil Felix test = Rickettsial Ab
o Rickettsial disease
o Causes bleeding, indicated by the presence of rashes
Excluded from Ricketssiae
1. Coxiella Extracellular
2. Bartonella (Rochalimea) agar (+)
Vector: ticks
Site of multiplication: endothelial cells
Rickettsia spp Vector Disease

R. rickettsi Tick Rocky Mountain Spotted Fever
R. akari Mite Rickettsial pox
R. typhi Rat flea Endemic murine typhus fever
R. prowazekii Louse Epidemic typhus fever
Brill Zinsser disease recerudescence typhus
Orientia tsutsugamushi Chigger Scrub typhus
Bartonella quintana Louse Trench fever
Ehrlichia chaffensis Tick Monocytic ehrlichoisis
Ehrlichia equi (morulae) Tick Granulocytic ehrlichoisis
Coxiella burnetti Tick/Inhalation Q fever
Ehrlichia chaffensis and Ehrlichia equi bacteria that destroy WBCs
Ehrlichia
Transmitted by tick
Diagnostic: morulae
Destroys leukocytes seen inside WBC
Sennetsu fever
Mycoplasma and Ureaplasma

Mycoplasma
Wall-less, pleomorphic, non-motile, smallest

Dienes stain (methylene blue) : Fried egg or Mulberry colony
Require sterol for growth except acholeplasma
Penicillin resistant
Equal to size of Pox virus (300nm)
Mycoplasma pneumonia

Pleuropneumonia-like orgs Primary atypical pneumonia (PAP) Mulberry (fried egg) colony
Aerobic with CO2 Walking pneumonia Selective: PPLO agar, Edward
Hayflicks
Cold agglutination test/DFA
o Anti-I confirmed by
hemagglutinin test, cold
agglutinin
Grows on CAP
Confirm: Hemadsorption Test
Best: inhibitin of growth by
specific antisera
*Mycoplasma grows on CAP; Ricketssia and chlamydia only grow on cell culture
Mycoplasma hominis
Diseases Laboratory
Post abortal fever Large fried egg colony
Post partum fever Media: A7/A8, NYCA, SP4 (arginine)
Pelvic inflammatory disease
Ureaplasma urealyticum

T-strain (tiny fried egg) Non-gonococcal urethritis Urease (+) - brown
Media: A7/A8, NYCA, SP4 (urea)
No haze/turbidity in broth
Gardnerella (Coryne/Haemophilus) vaginalis
Gram variable bacilli (gram negative or gram positive) Bacterial vaginosis grayish, fouls smelling
Tx: Metronidazole
Nugen scoring system used for diagnosis of vaginosis
Laboratory
Oxidase an catalase (-)
SPS sensitive
Hippurate and starch HOH (+)
Clue cells on cytology exam - are vaginal epithelial cells with gram negative bacilli or coccobacilli
Best for clue cell demonstration: GS, Pap, Wet mount
Whiff/Sniff test
o 10% KOH
o (+) fishy amin like odor
Selective media
o Human blood tween 80 agar (HBTA) best for demonstration of G.vaginalis hemoysis
o V agar (vaginalis)
o Columbia CNA
Calymmatobacterium (Klebsiella) granulomatis

Safety pin, capsule Donovanosis: granuloma inguinale Donovan bodies giemsa stain,
Non-motile MOT: sexual contact macrophage with gram negative
rod
Streptobacillus monoiformis

Bacilli in chains Rat bite fever animal bite Heart infusion medium: fried
String of beads, fluff balls Haverhill disease - ingestion egg colony
(broth) L-forms - defective cell wall
that produces fried egg colonies
SPS sensitive
Medium: whole blood, serum,
ascetic fluid presence of cotton
ball or bread crumbs
*Spirillum minus/major rat bite fever, spiral, sodoku fever
Chromobacterium violaceum

Violet colored (violacein) abscess, cellulitis NH4 cyanide
Opportunistic MAC - NLF
Quorum sensing control
Capnocytophaga spp. (gingivalis)

Gliding motility Periodontal disease (oral flora) SBAP: yellow, fusiform rod
Spreading colony
Bartonella henselae
Diseases Laboratory
Cat scratch disease Warthin Starry silver
Bacillary angiomatosis impregnation
Peliosis hepatitis
MOT Disease
B. bacilliformis Sandfly Carrions disease
B. Quintana Body louse Trench fever, endocarditis
B. henselae Cat scratch Major Cat scratch disease
Endocarditis
B. clarridgeiae Cat scratch Minor Cat scratch disease
NO endocarditis
B. elizabethae Fleas Endocarditis
Bartonella bacilliformis
Destroy RBC Carrions disease
Vector: sandfly Verruga peruana skin eruption
Oroya fever anemia
Legionella pneumophila

Philadelphia strain Broadstreet pneumonia Require L-cysteine and iron for
Short gram negative rods Pontiac fever growth with yeast extract
Aerobic, motile MOT: inhalation of contaminated Oxidase and catalase (+)
Found in air conditioning water Transport: 4C
and water cooling system Storage: 70C
Other laboratory methods
1. DFA Legionella Antigen 3. Stain: Dieterle Silver stain
2. Culture: black
BCYE best, blue-green col/cut glass colony, 4 days incubation 4. Rapid test for legionella: Urine
Feeley-Gorman brown colony Ag test
Note:
Legionella micdadei AFO, Pittsburgh pneumonia
Legionella bozemanii WIGA agent, pneumonia
Specimen: BAL (best), urine, sputum, blood, stool
*Cysteine positive = Francisella, Bordetella, Legionella
Listeria monocytogenes

Gram positive rod MOT: in-utero (pregnancy) SBAP: beta hemolysis
Motile at RT Neonatal meningitis Cold enrichment at 4C
Tumbling motility on broth Sepsis McBride (+)
Umbrella like motility on Infanseptica granulomatous Anton test ocular virulence test
semi-solid Food poisoning - coleslaw CAMP test with S.aureus = (+)
Inverted Christmas tree Fetal abortion block/rectangular hemolysis
motility Still birth Oxidase (-)
Listerolysin O Ferments glucose, salicin, and
o O2 labile hemolysin trehalose
*Organisms that can grow on cold enrichment: Listeria (gram positive) and Yersinia (gram negative)
Erysiphilothrix rhusiophatiae

Gram positive rod Erysipiloid (butchers cut, diamond Catalase (-)
Non-motil cut, red disease)
H2S production naturally
producing H2S on BAP even
without indicator
Test tube brush, pipe cleaner,
bottle brush
Listeria monocytogenes Erysiphilothrix rhusiophatiae

Catalase + -
Motile at 25C + -
Hemolysis Beta Alpha
Vogues proskauer + -
H2S production - +
Bile esculin and hippurate + -
Gluconate + -
Media McBride, Cold enrichment BAP
Tropheryma whipplei

Gram positive Whipple disease PAS stained vacuoles or
actinomycete/bacilli Brain biopsy disease macrophages
PCR amplification of bowel
biopsy

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BACTERIOLOGY

Pioneer notes from Ms. A.Aldave and some added notes

Classification of Biologic agents (Biosafety Level) Bioterrorism categories

Chain of infection: Source, Transmission, Host

1. Adherence factors pili/fimbriae

Bacterial Growth Factors

Psychrophilic/cryophilic = 0-2 C, (refrigerator)

Bacterial Growth Curve

Bacterial Gene Transfer

I. According to physical state/consistency:

Lowenstein Jensen Media MTB

Equipment, Supplies, and Instrumentation

Collection, transport, processing and staining of specimens

1. Sterile without normal flora

1. Collect before antibiotic theraphy

1-2 hours delay

Room temperature (not ref): CSF, blood, body fluids,

Note: Transport Medium CSF transport (RT), storage (35-37C)

Purpose Reagents Gram (+) Gram (-)

Gram (+) becomes gram (-)

Gram (-) becomes gram (+)

Gram stain general rule

1. All cocci are gram (+) except Neisseria, Veilonella, Moraxella

Gram positive cocci (aerobes) Micrococcus, Staphylococcus, Streptococcus

NOT Gram stain

Note: Acridine orange (nucleic acid)

Mycolic acid responsible for acid fast resistance

Purpose Ziehl-Neelsen (Hot) CAM Kinyoun (Cold) (C-A-M) Auramine-Rhodamine

Other acid fast stain

Pappenheims (urine) = M.smegmatis (blue), M.tuberculosis (red)

1. LANA (L-alanine-4-nitronilide) 2. Strings test

Sterilization Method by MOIST HEAT

1. Autoclave/Steam Under pressure

Sterilization Method by DRY HEAT

1. Hot Air Oven

1. Cold temperature/freezing for preservation, bacteriostatic

1. 10% Sodium hypochlorite (Chorox)

A. Cell Wall inhibitors sensitive for gram positive organisms

Antibiotic Susceptibility Testing

Disk distance -15mm

False Sensitive False Resistance

AST For Mycobacteria

MDR-TB to INH, Rifampin

Drug Resistance Mechanism

Drug Resistant Test Resistant (+) Result

Vitek 2 Compact ID and AST

Misidentification confirmation test using different method

Catalase Test Staphylococcus Streptococcus

3% H2O2 Staphylococci Citrate gives false positive because it uses citrate

Hyaluronidase spreading factor #1 diseases of SAU

TSST-1 (Toxic Shock Syndrome)

Staphylococcus aureus like

1. S.intermedius slide coagulase (+), VP/aceotoin (-)

1. Gram stain gram positive in clusters

Coagulase Negative Staphylococcus

Staphylococcus aureus Coagulase Negative Staphylococcus

Gram Positive Streptococci

Classification Colony Pinhead Pinpoint

A. Smith and Browns Classification

Streptococcus pneumoniae Optochin (S), Bile solubility (+)

Group A Streptococci (S.pyogenes) Bacitracin (S), PYR (+), SXT (R)

Note: Confimatory tests for Strep

PYR (+) = Group A strep and Group D enterococci 1. Lancefield test