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 4°C 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 - 20-40°C, (pathogenic)
 Thermophilic - 40-60°C, (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)
 Kreb’s 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 35-37’C
 18-24 hours: aerobic culture
 24-48 hours: anaerobic culture
 Bacteria: 35’C, Virus: 37’C, Fungi: 25-30’C
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:
 Stuart’s: 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 (35-37’C)

 Urine preservative: boric acid
 Nutrients to maintain the growth of organism  Urine, stool, swab, viral specimens, sputum,
 Buffer to maintain pH foreign devices: 4’C
 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
 Bartlett’s 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 – can’t resolve by bright field

 Note: Acridine orange (nucleic acid)

 BURKE’S 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)

 Baugmarten’s (tissue) = M.leprae (red), M.tuberculosis (blue)
 Fite Faraco stain = M. leprae (hematoxylin as counterstain)

Special Stain

 Metachromatic granules – culture on Loeffler’s, Pai medium and stain (Neisser’s, Albert, Ljubinsky, LAMB)
 Spore – culture on Bap and stain (Dorner’s, 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 – M’Fadyean
 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. String’s 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 121’C
 Denatures protein

121’C 15lbs psi for 15 minutes Best sterilization and waste disposal
Sterilize bacteriologic media (gauze)
121’C at 15psi for 60 minutes Sterilize most contaminated microbiological material
132’C for 30 to 60 minutes Infectious medical waste
2. Inspissation
 Sterilize CHON containing medium (L-J, Loeffler’s)
 75-80’C for 2 hours on 3 days
3. Tyndallization – 100’C for 30 minutes on 3 days
4. Boiling
 100’C 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) – 62’C for 30 minutes
b. High Temperature Short Time (HTST) – 72’C for 15 seconds

Sterilization Method by DRY HEAT

1. Hot Air Oven

 QC: Bacillus subtilis
 Dry hot air at 170-180’C 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
 Crede’s 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 patient’s 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 patient’s 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 35-37’C (MRSA – 35’C)
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% Sheep’s 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 (+)
(Ritter’s 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 (Ritter’s 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: Sheep’s Blood Agar Hemolysis +/- +
 Selective medium: PEA
CO2 - +

Classification Colony Pinhead Pinpoint

A. Smith and Brown’s 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

Laboratory Diagnosis
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

Characteristics Diseases Laboratory Diagnosis

 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

Laboratory Diagnosis

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. Dubol’s Oleic Acid Albumin Medium
b. Mitchison’s 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 68’C + -
 Medium: Tween 80
 Reagent: 30% H2O2
 Tween 80 + Mycobacteria + 30% H 2O2 + heat at
68’C = (+) >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 – Rounyon’s I B. Scotochromogens – Rounyun’s II

 M.kansasii = NO3 (+), yellow, pneumonia  M.scrofulaceum (scrofula) = Tween 80 (-),
 M.marinum = NO3 (-), 30’C, swimming pool Urease (+)
granuloma, seawater  M.gordonae (Tap water bacillus) – Tween 80
 M.asiaticum (+), Urease (-)
 M.simiae = Niacin (+),NO3 (-),  M.szulgai – also photochromogen at 25’C
 M.xenopi – also non-photochromogen
 M.flavescens
C. Nonphotochromogens – Rounyon’s III D. Rapid Growers – Rounyun’s 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) – 30’C and 42’C  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

Characteristics Diseases Laboratory Diagnosis

 Hansen’s bacillus  Tropism to peripheral nerves  Lepromine – skin test that uses killed
 Lepra – macrophage  Leprosy (Hansen’s 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
 Crohn’s disease
 Jones bacillus

Nocardia spp
Characteristics Diseases Laboratory Diagnosis
 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 Loeffler’s 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
 Loefller’s serum agar  Potassium tellurite - gray to black colonies
 Pao coagulated egg  Cystine tellurite BAP - gun metal gray colony)
 Clauberg  Potassium tellurite inhibits normal flora
 MacLeod’s 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 Hoffman’s 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

Characteristics Diseases Laboratory Diagnosis

 Largest pathogenic  Malignant pustule –  Needs bicarbonate medium for capsule
bacteria cutaneous anthrax, black formation
 Anthrax bacillus eschar  McFadyean’s – capsular stain
 Gram positive rods in chain  Woolsorter’s – pulmonary  PLET (Polymixin Lyozyme EDTA
forming bamboo and anthrax, Ragpicker’s 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

Characteristics Diseases
 Fried rice bacillus (spores on rice grain)  Food poisoning /gastroenteritis
 Virulence: exotoxin (cholera like toxin)  Blood bank contamination at RT

Bacillus subtilis

Characteristics Diseases
 Quality control for sterilization  Eye infection in heroin addicts
 Gram positive rod in chain
 Central spore
 Common laboratory contaminant

Bacillus stearothermophilus

Characteristics Diseases
 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 45’C - +
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

Characteristics Diseases Laboratory Diagnosis

 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

Characteristics Diseases
 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

Characteristics Diseases Laboratory Diagnosis

 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

Characteristics Diseases Laboratory Diagnosis

 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
 Vincent’s angina and Lemierre’s disease
 Colony: opalescent with speckles on stereoscope
 Positive for Chartreuse fluorescence
6. Bacteroides ureolyticus – pitting of agar
Note: Borrelia vincentii – trench mouth and Vincent’s 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: Moeller’s 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
 Kovac’s/Ehrlich’s 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 37’C except SKY
bacilli o Klebsiella, Shigella, Yersinia pestis
 Non-sporeformers o Yersinia – motile at 25’C
 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

Characteristics Diseases Laboratory Diagnosis

 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)

 Traveller’s (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
mouse’s 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

Characteristics Diseases Laboratory Diagnosis

 Friedlander’s 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

Characteristics Diseases Laboratory Diagnosis

 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

Characteristics Diseases Laboratory Diagnosis

 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 (boyd’s) 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)

Characteristics Diseases
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 22’C but  Enterocolitis
not at 35’C  Arthritis
 Cold enrichment at 4’C (Listeria)  Erythema nodosum
 Yersinia g(-), Listeria g(+)  Appendicitis and blood bag
 CIN agar: Bull’s 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

Characteristics Diseases Laboratory Diagnosis

 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   Pfeipper’s 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

Characteristics Diseases Laboratory Diagnosis

 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

Characteristics Diseases Laboratory Diagnosis

 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

Characteristics Diseases Laboratory Diagnosis

 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

Characteristics Diseases Laboratory Diagnosis

 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 37-42’C o Found in poultry CAMPY-BAP, CCDA
 Zoonotic  Campylobacter fetus
o Animal abortion
 Campylobacter coli
o Gastroenteritis (diarrhea)

37’C 42’C 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

Characteristics Diseases Laboratory Diagnosis

 Formerly Campylobacter  Peptic ulcer  Oxidase, catalase, urease (+)
pylori  Gastritis
 Microaerophilic  Cancer

C.jejuni H.pylori
Oxidase + +
Catalase + +
Microaerophilic + +
Urease - +
Growth at 42’C + -

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 42’C P.aeruginosa P.fluorescens
 (+) growth at 35’C and 42’C + -
Cetrimide Test P.aeruginosa E.coli
 35’C 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

Characteristics Diseases Laboratory Diagnosis

 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 42’C
 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 42’C
 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

37’C 42’C 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
 Whitmore’s bacillus Glander’s like medium
 Vietnamese time bomb pneumonia  OF test = +/-
(bioterrorism)  lactose oxidizer
 Arginine (+)
 Growth at 42’C
B.mallei  Only non-motile  Glander’s 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 - +
Characteristics Diseases Laboratory Diagnosis
 #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 42’C + -
OF Glucose + -
Old name Herella vaginocola Mima polymorpha
Alkaligenes faecalis

Characteristics Diseases Laboratory Diagnosis

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

Alkaligenes faecalis Acinetobacter

Oxidase + -
Motility + -

Moraxella lacunata

Characteristics Diseases Laboratory Diagnosis

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

Characteristics Diseases Laboratory Diagnosis

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

Eikenella corodens

Characteristics Diseases Laboratory Diagnosis

 Bleach like odor  Human bite woud – “Clenched fist”  MAC (-)
 SBE agent

Kingella spp.

Characteristics Diseases Laboratory Diagnosis

 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

Characteristics Diseases
 Pfeiffer’s bacillus  Doesn’t cause influenza
 Virulence:  3rd cause of bacterial meningitis
o Capsule type B–Hib 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

Characteristics Diseases Laboratory

 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
Characteristics Diseases
 Koch week’s 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.

Characteristics Diseases Laboratory

 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  Bang’s disease
o Found in animal placenta  Animal abortion
 Endocarditis
 Laboratory acquired infection
Dye inhibition Test
Urease CO2 Thionine Fuchsin Abortion
B.abortus (Bang’s) + + - + Cattle
B.melitensis + - + + Goat and sheep
B.suis + - + - Swine
B.canis + - + - Dog
*Note: B.abortus and B.suis = H2S (+)

Francisella tularensis

Characteristics Diseases Laboratory

 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 men’s disease  (-): oxidase, urease, MAC
 Used in bioterrorism  Deerfly, lemming, rabbit fever,  Forshay Skin Test – tularemia
water-trapper’s  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

Characteristics Diseases Laboratory

 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

Characteristics Diseases
 Obligate intracellular (rabbit’s 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 Weil’s 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

Characteristics Diseases Laboratory

 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 Fletcher’s – rabbit serum + fatty acid; 30’C
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: Bull’s eye rash
 Secondary stage – meningitis, cardiac
 Tertiary stage – arthritis
 Culture on Barber Stoenner Kelly (BSK) at 33’C
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: 4’C
inflammatory disease  Swab:Dacron/Rayon on sucrose phosphate buffer
 Lyphogranuloma venerium (LGV) – buboes
 Reiter’s 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

Characteristics Diseases Laboratory

 Non-glycogen inclusion body  MOT: inhalation of bird’s 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

Characteristics Diseases Laboratory

 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

Characteristics Diseases Laboratory

 Pleuropneumonia-like orgs  Primary atypical pneumonia (PAP)  Mulberry (fried egg) colony
 Aerobic with CO2  Walking pneumonia  Selective: PPLO agar, Edward
 Hayflick’s
 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

Characteristics Diseases Laboratory

 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

Characteristics Diseases
 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

Characteristics Diseases Laboratory

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

Streptobacillus monoiformis

Characteristics Diseases Laboratory

 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

Characteristics Diseases Laboratory

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

Characteristics Diseases Laboratory

 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 Carrion’s 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

Characteristics Diseases
 Destroy RBC  Carrion’s disease
 Vector: sandfly  Verruga peruana – skin eruption
 Oroya fever – anemia

Legionella pneumophila

Characteristics Diseases Laboratory

 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: 4’C
and water cooling system  Storage: 70’C
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

Characteristics Diseases Laboratory

 Gram positive rod  MOT: in-utero (pregnancy)  SBAP: beta hemolysis
 Motile at RT  Neonatal meningitis  Cold enrichment at 4’C
 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

Characteristics Diseases Laboratory

 Gram positive rod  Erysipiloid (butcher’s 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 25’C + -
Hemolysis Beta Alpha
Vogues proskauer + -
H2S production - +
Bile esculin and hippurate + -
Gluconate + -
Media McBride, Cold enrichment BAP

Tropheryma whipplei

Characteristics Diseases Laboratory

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