MICROBIOLOGY REVIEWER

INSTRUMENTS
1. INCUBATOR – set at 35-37’C
35- 37’C
 Quality Control: Monitoring of temperature at 35’C for MRSA, not at 37’C. 2. Robert Koch
 Also for viral culture (37’C)  Germ Theory
 18-24 hrs (aerobic culture)  “It is the organism that causes human disease”
 24-48 hrs (anaerobic culture)  First to isolate bacteria (Pure Culture)
2. DURHAM TUBE NOTE: Culture is a definitive test/gold standard
 For water bacteriology 3. Louis Pasteur – Father of Modern Micro
 Gas detector 4. Ehrlich – First to use dyes for stain
3. INOCULATING NEEDLES (<5cm)
 Bent Wireloop:
Wireloop: Used for Fungal Culture CHARACTERISTIC OF BACTERIA
 Calibrated Wireloop: 1. Prokaryotic
a. For quantitative technique: important in colony count in urine  No nuclear membrane, no mitochondria, small than Eukaryotic
samples  Fungi is Eukaryotic (same as human)
b. Diameter Size: 2mm (0.001 ml urine = 1,000 loop factors)  Virus is NEITHER prokaryotic nor eukaryotic
c. 70% Ethyl Alcohol – used as a disinfectant, better than using fire 2. Has both DNA and RNA
for disinfection 3. Multiply by Binary Fission
d. 70% Ethyl Alcohol with SAND – used for SPUTUM specimen, used 4. Measured in Micrometer (um)
to dislodge the stickiness of the sputum since flame can cause 5. Cell wall (except Mycoplasma)
aerosol formation when heated.  Main composition: Peptidoglycan
 Nichrome Inoculating Needles– contains iron, can cause false positive in  Mycoplasma
oxidase test. Use an applicator stick instead of inoculating needle for o is the smallest bacteria
oxidase test. o first bacteria to be cloned
4. COTTON SWAB o no cell wall, reason why it is resistant to antibiotic like Penicillin,
 Only small amount of organism is obtained (carrier state) and not gram stained
 Toxic to Neisseria o Incubated aerobically (CO2, CAP)
 Charcoal is added in culture media to remove the toxicity of cotton 6. Classification
 2 Swabs needed for:  Phenotype – Observable
st
1. Culture (1 )  Genotype – DNA type (PCR)
nd
2. Gram Stain (2 ) 7. Size: 0.4 – 2um
5. TYPING SERA
 For Salmonella-Shigella PARTS OF BACTERIA
 Detects Antigen 1. CAPSULE – aka. Slimy Layer
a. O – Somatic  Mucoid colony in culture
b. H – Flagellar  Virulence Factor and Anti-phagocytic
6. TUBERCULIN SYRINGE – used for Mantoux Test or Purified Protein Derivative o OPSONIN (IgG) – antibody that facilitates phagocytosis of
(PPD): a method for the skin test for Tuberculosis encapsulated bacteria
7. PASTEUR PIPETTE – transfer liquid  Capsular Antigen (K Ag or Vi Ag)
o Vi Ag = Salmonella typhi, causative agent for Typhoid Fever ( Mary,
HISTORY the first person to spread typhoid fever)
1. Anton Van Leeuwenhoek o Neufeld Quellung Test – serologic test for capsular antigen
 First to describe the bacteria Reagent: Anti-Sera (Antibody against capsular antigen)
 o Bacteria commonly used: Gene Transfer of Bacteria: (For Gram Neg Bacilli)
a. Streptococcus pneumoniae 1. Conjugation
b. Neisseria meningitides  Plasmid mediated
c. H. influenzae  Sex Pili required
o (Serotyping) – used for identification and vaccine making 2. Transduction – bacteriophage mediated (virus infecting bacteria)
2. CELL WALL Ex. Corynebacterium diptheriae – gene is carried upon by a
 Responsible for bacterial morphology bacteriophage
 Gives shape to bacteria
 Basis of gram stain 3. Transformation – naked DNA (virulent  avirulent
o Gram (+): PURPLE Ex. Streptococcus pneumoniae
 Thick Peptidoglycan (reason why it cannot be decolorized
by alcohol/insoluble) 5. METACHROMATIC GRANULES
 Teichoic Acid  Cannot be seen in gram stain, special stain is needed (such as Methylene
o Gram (-): RED Blue)
 LPS  Food reserves (Corynebacterium) – Babes Ernst/Volutin Granules
 Thin Peptidoglycan (soluble, easier to be decolorized)
 Periplasm 6. RIBOSOMES
Note:  For protein synthesis; 70S for Bacteria, 80S for Fungi
 Since MYCOPLASMA doesn’t have cell wall, its shape is PLEOMORPHIC, can
be bacilli, coccobacilli, etc. 7. PILI OR FIMBRIAE (For Gram Negative)
 ENDOTOXIN – found at LPS a. Common Pili – bacterial adherence
 EXOTOXIN found at gram (+), is more dangerous than Endotoxin since - attaches on the epithelium attracting phagocytes (PMNs for Neisseria
Exotoxin is focused one site, while ENDO is systemic. gonorrhea  PUS *Tulo)
 Botulinum Toxin – most potent toxin to human b. Sex Pili – gene transfer

3. PLASMA MEMBRANE 8. ENDOSPORES

 Site for energy synthesis  Calcium dipicolinate or dipicolinic acid
 Osmotic or permeability barrier  For RESISTANCE; Endospores of Fungi is for Reproduction
o Hypertonic/Hypotonic solution cannot be destroyed by bacteria  Bacillus, Clostrdium
 Regulate transport of nutrients in and out of the cell  Autoclave is the best sterilization method because it is sporicidal killing the
spores (Bacillus
(Bacillus is used as a control for sterilization)
4. NUCLEOID  Iodine = Sporicidal; Alcohol = Non-Sporicidal; Soap = Germicidal
2 Types of DNA:
1. CHROMOSOME: DS-DNA 9. FLAGELLA
2. PLASMID: Extrachromosomal DNA a. Monotrichous – 1 polar flagella ( Vibrio, Pseudomonas aeruginosa)
b. Amphitrichous – 2 polar flagella ( Spirillum minor)
Drug Resistance: c. Lophotrichous – group or tufts flagella ( Burkholderia,
1. Chromosome Mediated Drug Resistance – MRSA Stenotrophomonas)
2. Plasmid Mediated Drug Resistance – ESBL (+) Organisms: (Gram d. Peritrichous – flagella around, MOST COMMON ( E. coli,
Negative Rods) enterobacteriaciae)
- more dangerous since it’s a transfer DNA  H Antigen (Flagellar Ag) – used for motility test
Transfer DNA: “PLASMID”
Transfer Gene: “TRANSPOSON” (Transfer ng Puson hahaha) 10. AXIAL FILAMENTS (For Spirochetes)
 o Bacteria commonly used: Gene Transfer of Bacteria: (For Gram Neg Bacilli)
a. Streptococcus pneumoniae 1. Conjugation
b. Neisseria meningitides  Plasmid mediated
c. H. influenzae  Sex Pili required
o (Serotyping) – used for identification and vaccine making 2. Transduction – bacteriophage mediated (virus infecting bacteria)
2. CELL WALL Ex. Corynebacterium diptheriae – gene is carried upon by a
 Responsible for bacterial morphology bacteriophage
 Gives shape to bacteria
 Basis of gram stain 3. Transformation – naked DNA (virulent  avirulent
o Gram (+): PURPLE Ex. Streptococcus pneumoniae
 Thick Peptidoglycan (reason why it cannot be decolorized
by alcohol/insoluble) 5. METACHROMATIC GRANULES
 Teichoic Acid  Cannot be seen in gram stain, special stain is needed (such as Methylene
o Gram (-): RED Blue)
 LPS  Food reserves (Corynebacterium) – Babes Ernst/Volutin Granules
 Thin Peptidoglycan (soluble, easier to be decolorized)
 Periplasm 6. RIBOSOMES
Note:  For protein synthesis; 70S for Bacteria, 80S for Fungi
 Since MYCOPLASMA doesn’t have cell wall, its shape is PLEOMORPHIC, can
be bacilli, coccobacilli, etc. 7. PILI OR FIMBRIAE (For Gram Negative)
 ENDOTOXIN – found at LPS a. Common Pili – bacterial adherence
 EXOTOXIN found at gram (+), is more dangerous than Endotoxin since - attaches on the epithelium attracting phagocytes (PMNs for Neisseria
Exotoxin is focused one site, while ENDO is systemic. gonorrhea  PUS *Tulo)
 Botulinum Toxin – most potent toxin to human b. Sex Pili – gene transfer

3. PLASMA MEMBRANE 8. ENDOSPORES

 Site for energy synthesis  Calcium dipicolinate or dipicolinic acid
 Osmotic or permeability barrier  For RESISTANCE; Endospores of Fungi is for Reproduction
o Hypertonic/Hypotonic solution cannot be destroyed by bacteria  Bacillus, Clostrdium
 Regulate transport of nutrients in and out of the cell  Autoclave is the best sterilization method because it is sporicidal killing the
spores (Bacillus
(Bacillus is used as a control for sterilization)
4. NUCLEOID  Iodine = Sporicidal; Alcohol = Non-Sporicidal; Soap = Germicidal
2 Types of DNA:
1. CHROMOSOME: DS-DNA 9. FLAGELLA
2. PLASMID: Extrachromosomal DNA a. Monotrichous – 1 polar flagella ( Vibrio, Pseudomonas aeruginosa)
b. Amphitrichous – 2 polar flagella ( Spirillum minor)
Drug Resistance: c. Lophotrichous – group or tufts flagella ( Burkholderia,
1. Chromosome Mediated Drug Resistance – MRSA Stenotrophomonas)
2. Plasmid Mediated Drug Resistance – ESBL (+) Organisms: (Gram d. Peritrichous – flagella around, MOST COMMON ( E. coli,
Negative Rods) enterobacteriaciae)
- more dangerous since it’s a transfer DNA  H Antigen (Flagellar Ag) – used for motility test
Transfer DNA: “PLASMID”
Transfer Gene: “TRANSPOSON” (Transfer ng Puson hahaha) 10. AXIAL FILAMENTS (For Spirochetes)
BACTERIAL PHYSIOLOGY 3. Temperature:
a. Psychrophilic = 0- 20’C (refrigerator)
Ex. LISTERIA M. and YERSINIA E.
1. Oxygen Requirement:
 Listeria – agent of food poisoning
a. Obligate Aerobe = With oxygen
 Food contaminated with Listeria (mostly found in ref)
Ex. MYCOBACTERIA
1. Coleslaw
b. Obligate Anaerobe = Without oxygen
2. Milk (Mam: “Ano ang gatas? Milk yan!”) and
Ex. BACTEROIDES FRAGILIS (normal flora
Cheese
c. Facultative Anaerobe = With or without oxygen
 Yersinia e.
e. – blood bank contaminant (presence of bubbles in the blood
Ex. PATHOGENS
(most pathogenic bacteria are incubated AEROBICALLY) bag)
d. Microaerophiles = Low Oxygen (5% O 2 + 10% CO 2 + 85% N 2) b. Mesophilic = 20-40’C
20-40’C (Pathogenic) Optimal pH (37’C)
Ex. CAMPYLOBACTER c. Thermophilic = 40- 60’C
- Materials providing CO 2: Gas Pack, Candle Jar (3% CO 2), Campy Gas Ex. THERMUS AQUATICUS – source of DNA in PCR Polymerase
e. Aerotolerant Anaerobes = Not destroyed by O2, anaerobic but can tolerate
oxygen 4. pH Requirement:
a. Acidophilic
Ex. LACTOBACILLUS
Ex. LACTOBACILLUS
NOTE: In growth culture:  Produces lactic acid
 AEROBIC BACTERIA grows at the SURFACE;  Normal flora of GIT and Vagina
 ANAEROBIC BACTERIA grows at the BOTTOM;  Increased in: Pregnancy (protection against UTI)
 MICROAEROPHILES grows at the MIDDLE;  Promotes Candidiasis (fungi are also acidophilic) but inhibits
Gardnerella vaginalis (vaginosis) since it is alkaline
 FACULTATIVE/AEROTOLERANT grows EITHER/ANYWHERE.
b. Neutrophilic – Pathogenic Bacteria
Ex. E. COLI
Case Analysis:
Analysis :
c. Basophilic – VIBRIO
In the case of bronchial washing, an organism grows on slide (Gram Stain (+)) but
Culture (–) (No growth).
Remember that if Gram Stain (+), should be Culture (+)! 5. Salt Concentration - Halophilic
Ex. S. AUREUS = 7.5% NaCl
ENTEROCOCCUS = 6.5% NaCl
Rationale: Bronchial washing is an aerobic sample. The bacteria is possibly
anaerobic, and the bronchial washing might be exposed to oxygen already, the
reason why there is no growth in the agar. Bronchial aspirate should be the BACTERIAL METABOLISM
specimen. 1. Respiration (Aerobic Process)
a. Kreb’s cycle – aerobic process
2. Nutritional Requirement: b. Electron Transport Chain – aerobic process
a. Autotrophs/Lithotrophs = inorganic compound as carbon source. Ex. CO2 c. Glucose  CO2 and H2O
b. Heterotrophs/Organotrophs = organic compound as carbon source 2. Oxidation (Aerobic Process)
= mostly pathogenic bacteria a. Glucose  Acid
Ex. Glucose Ex. NFO (Non Fermentative Organism) such as PSEUDOMONAS oxidizes sugar to
produce acid.
3. Fermentation (Anaerobic Process)
a. Glycolysis (Embden Meyerhoff Pathway)
b. Glucose  Acid/Alcohol

NOTE: Oxidation and Fermentation BOTH produces ACID, but differ in Aerobic and
BACTERIAL GROWTH CURVE  Hucker’s Stain (Crystal Violet + Ammonium Oxalate) – Gram stain for FUNGI
Hucker’s
1. LAG Phase / Adjustment Phase / Adaptation Phase (Gram +)
 Increase in cell size NOT in number
 Increase of enzyme and metabolic activity of bacteria I. GRAM STAIN (PRESUMPTIVE NOT CONFIRMATORY)
2. LOG Phase / Exponential Phase Purpose Reagents Gram Positive Gram Negative
 Increase in growth rate (cell division/binary fission) Primary V (Crystal Violet) Purple Purple
 Susceptible to antimicrobial agents (Best time to do AST) Mordant I (Iodine) Purple Purple
3. STATIONARY Phase / Plateau Phase Decolorizer A (95% Acetone-Alcohol) Purple Colorless
 No net growth rate (death = live cells)
Counterstain S (Safranin) Purple Red
 Increase death rate
 Cell death starts due to
NOTES:
a. Toxin and waste products
 Mordant is alkaline in pH, increases affinity of the dye t o the organism.
b. Depletion of nutrients
 Decolorizer is the most critical step in gram staining (commonly mistaken)
c. Adverse environmental conditions
4. DEATH Phase / Period of Decline  No MORDANT (Iodine): Gram (+) bacteria can be mistaken as Gram (-)
 No net growth rate (death = live cells)  No DECOLORIZER (Alcohol): Gram (-) bacteria can be mistaken as Gram (+)

Gram (+) becomes Gram (-)

STAINING PROCEDURE
1. Over decolorization, Old dying
2. Use of acidic iodine as mordant
A. Direct – stains the bacteria
3. Penicillin, Omit iodine
1. Simple = 1 dye, Basic dye is used to stain bacteria
 Crystal Violet
Gram (+) becomes Gram (-)
 Methyl Red 1. Under decolorization
2. Differential = 2 dyes (used to differentiate G + and - ) 2. Thick smear
 Gram Stain (both direct and Indirect – you can gram stain not only the
specimen but also the colony in t he agar media) Gram Stain – General Rule
 AFB 1. All cocci are gram +ve, EXCEPT: / Gram Negative Cocci (NVM)
3. Special = Bacterial structure, Metachromatic granules Neisseria, Veilonella, Moraxella
B. Indirect/Relief/Negative – background is stained, for capsules 2. All bacilli are gram -ve, EXCEPT: / Gram Positive Bacilli
a. India Ink / Borris Method Mycobacteria, Corynebacteria
b. Nigrossin Methods Clostridia, Nocardia, Actinomyces
Bacillus, Lactobacillus, Listeria, Erysiphilothrix
Do NOT Gram Stain: 3. All spiral organisms are reported as Gram (-)
1. Chlamydia/Rickettsia – intracellular (stain cannot reach inside the cell) 4. Yeasts and Fungi are Gram (+). Yeasts are differentiated to cocci based on SIZE.
2. Mycoplasma/Ureaplasma – cell wall less (walang kakapitan ang Cry stal Violet) Size: Yeast > Cocci
3. Spirochete – since
since it is very small, it can’t be gram stained.
II. ACID FAST STAINING METHODS – SCREENING not DIAGNOSTIC
NOTE: “Acid Fast” = Acid alcohol resistant
 Acridine Orange – a fluorescent dye that binds to bacterial DNA, which can be - not decolorized by acid alcohol retaining the RED color of CARBOLFUCHSIN
used to stain Mycoplasma (no cell wall but with nucleic acid), since it has DNA. due to the presence of MYCOLIC ACID (a long chain of fatty acid that makes
For Nucleic Acid. More sensitive than Gram stain. Mycobacteria the bacteria with the highest amount of lipid)
 Fluorescent stain is more sensitive than gram stain and AFB. Mycobacteria = “Mataba
“ Mataba at Mabagal”
M abagal”
 Any organism that cannot be gram stained, once gram stained, they are
A. MYCOBACTERIA - Acid Fast Methods used for differentiation of Mycobacteria: III. SPECIAL STAIN (such as Rickettsia, Chlamydia, Mycoplasma, Ureaplasma)
 Pappenheims – M. smegmatis vs. M. tuberculosis a. Capsule – Negative stain
 Baumgarten’s – M. leprae vs. M. tuberculosis b. Spore – Dorner, Wirtz Conklin, Schaeffer Fulton
 Fite Faraco’s – M. leprae (hematoxylin) (Dx: Skin Biopsy) c. Metachromatic Granules – Albert’s, LAMB, Neisseria
d. Flagella – Leifson, Gray’s
M. smegmatis – for uncircumcised patients e. Nucleic Acid – Fuelgen
M. leprae – causative agent of leprosy (Hansen’s disease) f. Polar bodies – Wayson
g. Rickettsia – Gimenez, Macchiavelo
Specimen: Sputum, Urine, Stool (due to Intestinal MTB) h. Spirochetes – Levaditi, Fontana, Tribondeau

B. NOCARDIA and CRYPTOSPORIDIUM – Modified Acid Fast Note: (Non Staining Method)
= uses 1% H2SO4 as Decolorizer instead of Acid Alcohol  STRING TEST = uses 3% KOH. Presence of STRING LINE = Gram (-) Bacteria.
= Cold method (no heat required)
TYPES OF MICROSCOPY
Nocardia Specimen: Sputum (since Nocardia is an agent of Pneumonia) 1. Brightfield
Cryptosporidium Specimen: Stool (mostly for patients with HIV) 2. Darkfield – motility of Spirochetes; confirm Primary Syphilis
3. Phase Contrast – used for living cells and inclusion body ( virus and Chlamydia can
ACID FAST: produce inclusion body); also used for HLA TYPING
Purpose Ziehl-Neelsen Kinyoun Rhodamine- 4. Fluorescent
(Hot) (Cold) Auramine  For Bacteria: Acridine Orange: Red; Aura-Rauda: Yellow (PEPTIDOGLYCAN)
(C-A-M) (C-A-M) (Fluorochrome)  For Fungi: Calcofluor White binding in the CHITIN CELL WALL
Primary Carbolfuchsin Carbolfuchsin Auramine-  For Serology: Immunofluorescent Test
(10 min) Rhodamine 5. Electron – with the highest magnification
Start timing when a. TEM – Transmission (internal structure)
Steam appears  requires a stain: Phosphotungstic Acid (Negative Stain)
Mordant Heat Phenol, Tergitol b. SEM – Scanning (external/surface structure)
(3 min)
Decolorizer 3% Acid Alcohol 3% Acid Alcohol 0.5% Acid Alcohol TYPES OF CULTURE
Counterstain Methylene Blue Malachite Green 0.5% KMNO4 1. Pure Culture – most important! Where Identification and AST is done.
(30 sec) Quenching Agent a. Streak Plate (best method)
Result AFO – Red AFO – Red AFO (+) - Yellow b. Pour Plate
NAFO – Blue NAFO – Green Fluorescence c. Selective Medium
NAFO – No fluor, d. Animal Inoculation
2. Mixed Culture – 2 or more bacterial species
NOTE: 3. Stock Culture – for Quality control; stored at - 20’C/Freezer
 Ziehl-Neelsen: Best Method 4. Working Culture – 4’C, from Stock Culture
 Kinyoun : Used in tissue samples
 Rhodamine Auramine: Most sensitive method According to Consistency:
 Heating removes the fat allowing the penetration of the stain to the cell wall a. Liquid (broth) – used to increase number o f bacteria, mostly for swab specimens
 Acid Alcohol Composition: (HCl + 95% Ethyl Alcohol); For Nocardia: 1% H2SO4 since swab sp. have only small amount of bacteria
 KMNO4 (Quenching Agent) – absorbs fluorescence b. Semi-solid = 0.5 – 1% agar (motility), for SIM. (Motile = Hazy; NonMotile = Clear)
 LED Fluorescent Microscopy – new fluorescent stain, more sensitive than c. Solid = 2-3% agar (plated media)
Auramine Rhodamine d. Biphasic = Both liquid and solid ( Castaneda) = Blood Culture media for Brucella
Types of Culture Media: 5. BASITRACIN CAP H. influenzae
1. General Purpose Media – NON FASTIDIOUS 6. CYSTINE BLOOD GLUCOSE AGAR Francisella
a. BAP (Blood Agar Plate) 7. CYSTINE TELLURITE BLOOD AGAR C. diptheriae
- good for hemolysis study 8. CYSTINE TRYPTICASE AGAR Neisseria (Confirm)
- Contains X Factor (HEMIN – Heat stable) 9. CHARCOAL CEPHALEXIN BLOOD AGAR B. pertusis
- for both: Gram (+) White Dry Colony 10. BCYE Legionella pneumophila
Gram (-) Gray Moist Colony 11. McCOY C. trachomatis
 Sheep’s Blood – Streptococcus 12. TSB Brucella spp (Aerobes)
 Horse Blood – Haemophilus hemolyticus/parahemolyticus 13. THIOGLYCOLLATE Aerobes/Anaerobes
 Human Blood – beta hemolysis of Gardnerella vaginalis 14. Potato Blood Glycerol Agar B. pertusis
b. NA (Nutrient Agar)
2. Enriched Media – FASTIDIOUS NOTE:
a. CAP (Chocolate Agar Plate)  TSB is mostly for Aerobes. Brucella spp. are Obligate Aerobe. Brucella
- for culture only not for hemolysis study causes Brucellosis, Endocarditis; Specimen: Blood; Media: Castaneda)
- Contains X and V Factor (NAD – Heat labile)  Thioglycollate is for both Aerobes and Anaerobes
- Does not contain Chocolate but LYSED RBC  Glycerol in PBGA is made up of egg = LJ Medium
- Horse Blood – best source of blood for CAP
- good for Neisseria
b. BCYE SPECIMEN HANDLING AND COLLECTION
3. Enrichment (Broth)– enhance the growth of bacteria
- Increase the LAG phase of NORMAL FLORA Types of Specimen
- Decrease the LOG phase of PATHOGEN
 Sterile
a. Selenite F, APW, THIO
 None Sterile
4. Differential
 Aerobic (24 hours); Anaearobic (48 hours)
a. BAP – differentiates alpha, beta, gamma hemolysis
b. Mac – differentiates lactose from non-lactose fermenters (Important
Collection
for differentiation of pathogenicity of Enterobacteriaciae, NLF are
 Swab
pathogenic than LF)
 Cotton – toxic for NEISSERIA, good for VIRUS (Countertoxicity: Charcoal)
c. EMB, XLD, HEA
 Calcium alginate - toxic for VIRUS, good for NEISSERIA
5. Selective (inhibitory agents)
 Bronchial washing – for AEROBIC culture
- pathogenic organisms are needed in a non-sterile specimen
 Needle aspiration – for both AEROBIC and ANAEROBIC
a. TCBS – Vibrio (Stool)
b. TMA (Thayer Martin Agar) – Neisseria  Catheterization – for sterile urine
c. CBAP - Campylobacter  Intubation – for gastric samples ( H. pylori = Urea Breath Test)
Inhibitory Agents – ANTIBIOTICS
DYES, BILE SALT = Inhibits Gram + (For Gram Neg only) Delays Refrigerator except:
a. Mac – contains crystal violet and bile salt (selective to gram -) 1. CSF – immediately processed
b. EMB – contains dyes (Eosin and Methylene Blue) a. Room Temp – transport temperature
ALCOHOL (PEA) = Inhibits Gram – (For Gram Pos only) b. 35’C – storage temperature (incubator)
a. CNA (Collistin Nalidixic Acid) 2. Blood
3. Urogenital Swab of N. gonorrhea – sensitive to cold temperature (do not ref)
Common Culture Media: 4. Boric acid – preservative for URINE culture
1. PEA Gram (+) bacteria 5. Cary Blair – rectal swab
2. COLUMBIA CNA Gram (+) bacteria
Transport Medium: CLINICAL SPECIMEN
1. Cary Blair – stool pathogens (for enteric pathogens, VIBRIO) 1. Blood (BHIB)
2. Stuart’s –Viral Transport Medium - requires TWO to THREE blood culture to rule out bacteremia
3. Amies – respiratory - 1:10 (1ml of Blood to 10ml of Broth media)
4. Transgrow – Neisseria - Antibiotic Removal Device (ARD) – this will remove the antibiotic the patient
5. JEMBEC – Neisseria is taking
6. Todd Hewitt – GROUP B Strep S. agalactiae (vaginal swab) - Collection Time:
 Before antibiotic treatment
Biologic Safety Cabinet  During acute stage of infection
 HEPA Filter – air sterilization, holds bacteria in the air SPS – anticoagulant (0.25% SPS); needed since the clotting of blood will trap the
 Negative Pressure – takes infectious air outside the BSC bacteria
 Note: Not required in AFS, but for culture and sensitivity  Anti-complimentary and anti-phagocytic: preventing hemolysis
1. Class I  Neutralizes: aminoglycosides (antibiotics) and bactericidal effect of
- air velocity 75 linear feet/min serum
- with product (culture) contamination  Inhibits: G. vaginalis, Neisseria, S. monoliformis, P. anaerobius
- exhaust air through ONE HEPA filter  NOTE: 1% GELATIN counteracts SPS
2. Class II (Vertical Laminar Flow) Bacterial Growth in Blood: (+) Hemolysis, turbidity, pellicle, bubble formation
- air velocity 75-100 linear feet/min If (+), Subculture in: BAP, CAP, Mac
- no product (culture) contamination
- exhaust and recirculated air through TWO HEPA filters (+)BAP (+)BAP (-)BAP
- MUST for MICRO lab/hospitals (tertiary) (+)CAP (+)CAP (+)CAP
a. IIa = exhausts air inside the room
(+)MAC (-)MAC (-)MAC
b. IIb = exhausts air outside the building Gram (-) Gram (+) Neisseria gonorrhea
3. Class III Haemophilus influenzae
- Maximum protection
Neisseria g. = Genital specimen
- Supply and exhaust air through TWO HEPA filters
Haemophilus i. = respiratory and CSF specimen
- For BSL Level IV (viruses)
After 7 days: Negative
Classification of Biologic Agents (Risk Level of Organisms): th
 Blood Culture Contaminant: Staph. epidermidis (5 day (+));
 7 Days before reporting negative: Bacteremia (typhoid)
Biosafety Level I No risk M. gordonae, BSC Class I
 21 Days before reporting negative: Brucellosis, Endocarditis, SBE
B. subtilis
(HACEK)
Biosafety Level II Moderate risk Y. pestis
B. anthracis
Note: Brucella is a FASTIDIOUS organism therefore hard to culture, requiring 21
Biosafety Level III High risk Mycobacteria BSC Class II
days.
(With Treatment) Brucella
Francisella, Molds 2. Urine
Biosafety Level IV High Risk Viruses BSC Class III - Catheterized (bedridden), midstream (female), suprapubic (anaerobic
(No Treatment) culture)
- Quantitative Technique / Colony Count (BAP for (G+), Mac for (G-)): only
Note: applicable for MIDSTREAM Collection
 B. anthracis and Y. pestis are agent of bioterrorism yet easily destroyed by penicillin. >100,000 CFU – significant for UTI
 Francisella and Brucella are laboratory acquired infections <10, 000 CFU – not significant
 Agents causing UTI: 8. Vaginal, Urethral Swab
(+)BAP (+)Mac = Gram NEG= (#1) E. coli, (#2) Proteus - CAP
(+)BAP (-)Mac = Gram POS= S. saprophyticus, Enterococcoci, C. urealyticum - Modified Thayer Martin
- Gram Stain
3. CSF – not refrigerated; immediately processed
- Neisseria meningitidis and Haemophilus influenzae 9. TB culture
- Culture Media: - Requires BSC Level II
 CAP (most important culture media for CSF) - NALC-NaOH (gold standard)
 BHI(to enhance the growth of the organism since they are FASTIDIOUS)  NALC – digestant for sputum (N-acetyl L-cysteine)
 BAP, Mac  NaOH (2-4% NaOH) – digestant and decontaminant
- India Ink method: CAPSULE of Cryptococcus meningitis - Oxalic Acid – used when specimen is contaminated by Pseudomonas, such
- Latex: CAPSULAR ANTIGEN of Cryptococcus meningitis as URINE and STOOL
- Clorox (Anti-formin) – cannot clear Mycobacteria, but can destroy virus.
NOTE: Centrifuge Urine and CSF for 2000rpm for 10 minutes. Sediments are - CENTRIFUGE: for 15 mins at 3000rpm ( 4’C Temp. of centri for TB culture =
used for culture. “Ref Centrifuge” to prevent aerosol)
Culture Media:
4. Wound  Lowenstein-Jensen (Green) – best culture medium (test tube) for TB
 Gram (+): S. aureus (#1 cause)  Middlebrook 7H11, 7H10 - can be used as AST media
 Gram (-): Pseudomonas, Vibrio, Enterobacteriaciae TAT before reporting as NEGATIVE:
 Culture Media: - LJ Medium = 8wks (through incubation at 37’C)
 Thioglycollate (Anaerobes causing wound infection, FOUL odor) - BACTEC: 2-3 days
 Gram stain - DNA Test: 2-3 hours
 BAP, Mac - (+) 2-3 weeks, growth is seen
NOTE: BACTEC = uses radiometric method
5. Stool – NOT Gram Stain
- Selective Media is needed for Pathogenic Organisms: STERILIZATION AND DISINFECTION
 Aeromonas, Campylobacter, Salmonella, Shigella, Vibrio  Sterilization – standard for microbiology; both the pathogenic and non-
 MacConkey, BAP + ampicillin pathogenic organisms are destroyes
 CBAP, SSA, Selenite F  Disinfection – only the pathogenic is destroyed
 TCBS, Alkaline Peptone, HEA  Moist Heat – destroy bacteria through coagulation of protein
Tests:  Quality Control:
 Oxidase Test o Moist Heat: Bacillus stearothermophilus
 Biochemical Test (Screening) o Dry Heat: Bacillus subtilis
 Serologic Typing(Confirmatory): E.coli, Salmonella, Shigella, Vibrio
STERILIZATION METHOD – MOIST HEAT
6. Respiratory – (sputum, NPS) 1. AUTOCLAVE – steam under pressure, BEST sterilization procedure
- BAP (S. pneumoniae = Rusty sputum) - Autoclave tape – indicator
- BCA (Basitracin Chocolate Agar): H. influenzae - Bacillus stearothermophilus – used for quality control of autoclave
- Mac, GBA (Gentamicin BA) (WEEKLY BASIS)
- Amies, Do gram stain and AFS  121’c at 15lbs/psi for 15 min
 Culture media, bandages, gauze
7. Throat Swab – Sore throat 2. INSPISSATION
- BAP (Strep for hemolysis)  75-80’C for 2 hours on 3 days
Modified Thayer Martin (Neisseria gonorrhea, but still best in genital)
 3. TYNDALLIZATION 6. Ionizing Radiation – disposables (gloves, microwave oven, catheter)
- 100’C for 30min on 3 days 7. Filtration (air and water)
4. BOILING – non sporicidal, only kills vegetative. a. HEPA filter – filter for Air (0.3 um)
- 100’C for 30min b. Cellulose Membrane – Liquid (0.22um)
5. PASTEURIZATION – only the pathogens are destroyed in t he milk sample
- 63'C for 30min / 72'C for 15secs DISINFECTANT – ANTISEPTIC
- 75,000–15,000 – count of bacteria before and after milk pasteurization 1. 10% Sodium Hypochlorite (Clorox) – best disinfectant, destroys HIV and HBV
- 10,000 - count of bacteria for certified milk 2. Iodophor – combination Iodine and Detergent, best skin antiseptic (sporicidal)
 Pseudomonas syncyanea – causes BLUE MILK 3. 70% Ethyl Alcohol – non sporicidal
 Flavobacterium synxanthium – causes YELLOW MILK 4. 1% Silver Nitrate – (eyedrop) prevents Prophylaxis; prevents gonococcal
Tests: opthalmia neonatorum
 Phosphatase Test – Test for pasteurization 5. 3% Hydrogen Peroxide – used in catalase test; antiseptic
Result: (-): Success of Pasteurization 6. Dyes – inhibits gram positive
 Methylene Blue Reduction Test – detects the presence of bacteria in 7. Formaldehyde – sporicidal, preservative for tissue samples
milk sample 8. Glutaraldehye – sporicidal, sterilize surgical instruments
Result: (+) Colorless; (-) Blue 9. 5% Phenol (Carbolic Acid) – standard (“benchmark”) disinfectant
 Litmus Milk Test – detects the pH of milk 10. Lysol (Cresol) – disinfectant
11. Zephiran (Benzalkonium chloride) – Merthiolate; antiseptic
Note: 12. Quats – (Quarternary ammonium compound) inactivated by organic materials
1. Autoclave, Inspissation and Tyndallization are SPORICIDAL
2. Inspissation and Tyndallization are forms of Fractional Crystallization requiring 3 ANTIMICROBIAL AGENTS
days. 1. Antagonistic = 1>2 (The effect of 1 drug is better than the combined effect of 2
Day 1 – Destroys Vegetative Cells drugs)
Day 2 – Destroys Spores 2. Synergistic = 2>1 (The combined effect of 2 drugs is better than the effect of 1
Day 3 – All cells are destroyed drug)
3. MIC = Minimum Inhibitory Concentration – Lowest concentration of drug to kill
STERILIZATION METHOD – DRY HEAT bacteria
1. Hot Air Oven = Drying 4. MBC/MLC = Minimum Bactericial/Lethal
 Dry heat method 5. Beta Lactamase – if the bacteria is found to be resistant to Penicillin, perform
 Bacillus subtilis – used for quality control of Oven Beta Lactamase test)
 Dry hot air at 170-180’C for 2 hours
 Glasswares, cotton swabs, metallic instruments, oils, powders A. Cell Wall Inhibitors
2. Incineration = Waste Disposal Broad Spectrum (Inhibits both Gram + and -)
3. Cremation = Control communicable disease 1. Penicillin (Penicillum notatum) – inhibits Peptidoglycan synthesis
4. Flaming = Needles, burning organism into ashes 2. Cephalosporin (Cephalosporium)
5. Gas-Ethylene Oxide = Heat Labile 3. Cycloserine
4. Imepinem, Carbapenems
Others: 5. Penicillinase Resistant Antibiotics = Methicillin, Cloxacillin, Nafcillin
1. Cold Temperature/Freezing – preserve reagents, bacteriostatic – inhibits the
growth of bacteria Narrow Spectrum
2. Lyophilization/Freeze Drying – BEST to preserve microbial cultures 1. Vancomycin (Streptomyces) = inhibits Gram (+) ONLY;
3. Osmotic Pressure – foods (bacteriostatic)  MRSA = Penicillin (R); Vancomycin (S)
4. Dessication – foods 2. Basitracin (Bacillus subtilis); inhibits Gram (+) ONLY
5. Ultraviolet Light – acts on the DNA of the organism (air and water) and leads to
B. Cell Membrane Inhibitors 7. Disc Elution Test = Susceptibilty test for Mycobacteria
1. Colistin – Inhibits GRAM (-) ONLY - Antibiotic is FIRST applied in the agar, B acteria is the LAST to be a pplied
2. Polymyxin (Bacillus subtilis) - Inhibits GRAM (-) ONLY (unlike in Kirby Bauer vice versa).
3. Amphoteracin B (Streptomyces) – Anti-FUNGAL - Requires 1 drop of inoculum in t he four quadrants
4. Nystatin – Anti-FUNGAL - (S) = No Colony (R)= With Colony
NOTE: Antifungal Agents – targets the CELL MEMBRANE o MDR-TB (Multi-Drug Resistant TB) = Mycobacteria that is resistant to
primary drugs ISONIAZID and RIFAMPICIN
C. Ribosomes (Protein) Inihibitors (All Broad Spectrum) o XDR-TB (Extensively Drug Resistant TB) = Mycobacteria that is resistant
1. Aminoglycosides (-cin, Gentamicin) – antibiotic that has the HIGHEST DRUG to ALL DRUGS + QUINOLONES
RESISTANCE, affected with the addition of Calcium and Magnesium in Mueller
Hinton Agar II. Antibiotic Susceptibility Testing (AST) Media
2. Tetracycline - All are CLEAR Media, making zone o f inhibition and colonies easily seen
3. Cloramphenicol - BAP cannot be used since it’s a dark medium
4. Erythromycin (Macrolide) – “wonder drug” a. MHA – general AST media
5. Clindamycin – Antibiotic associated enterocolitis affecting Clostridum difficile b. MHA + 2% NACL – MRSA
NOTE: Pseudomonas aeruginosa – bacteria that has the highest drug resistance, also c. MHA + 5% Sheep’s Blood – Strep
#1 seen in ICU (nosocomial) d. Heamophilus Test Medium (MHA + Yeast Extract)
e. GC Agar (Gonococci Agar)– Neisseria
D. Nucleic Acid (DNA) Inhibitor f. Middlebrook 7H10 – Mycobacteria
1. Mitomycin, Quinolones (-floxacins) – acts on the DNA
2. Metronidazole – Anti-PROTOZOA, Anti ANAEROBES III. Disk Diffusion – Kirby Bauer (Semi Quantitative)
3. Sulfonamide-Trimetophrim (SXT) - inhibits FOLIC ACID (needed for DNA  STANDARD INOCULUM 1.5 x 108
Synthesis)  MEDIUM Mueller Hinton Agar (MHA)
4. Rifampin – anti TB Drug  pH 7.2 – 7.4
 DEPTH 4mm (standard thickness of agar)
I. Methods of Antibiotic Susceptibility Testing  CONDITION Aerobic, No CO 2 (to prevent increase in pH)
1. Micro/Macrobroth Dilution  TEMPERATURE 35-37’C (MRSA-35’C)
- recommended for ANAEROBIC BACTERIA  INC. TIME 16-18 hours
- reference method for MIC and MBC (Antibiotic is being diluted)  STANDARD 0.5 McFarland (1% H 2SO4 and 1.175& BaCl 2)
- as dilution increases, the concentration of the antibiotic decreases
 ANTIBIOTIC DISK 6mm
2. Agar dilution = many organisms vs single drug
NOTE:
3. Disk Diffusion = one organism vs multiple drugs (MOST COMMON)
 Petroff-Hauser = Bacterial Counting Chamber
4. E Test (Epsilometer Test)
 McFarland for Fungi: 2.0
- antibiotic strip diffusion MIC test
- uses filter paper/strip
IV. Zone of Inhibition
- incorporated with DECREASING concentration of antibiotic
 6mm = Resistant
5. VITEK/Automated System
 Standard distance between 2 antibiotic disk = >15mm (to avoid overlapping of
- both identification and susceptibility test
zone of inhibition)
- gives you the exact amount of antibiotic to inhibit the growth of organism
 <15 min = antibiotic disks should be applied on the agar after streaking. Delay
Errors:
causes SMALLER ZONE
- No Identification result : Do the manual/conventional method
 Antibiotic Disk = responsible for the zone of inhibition NOT the organism
- Doubtful/Unfamiliar of the ID result: Endorse/refer to the supervisor
 Presence of Swarming = Ignore. Continue measuring the zone of inhibition.
- Misidentification of result: Do confirmatory test using other method
6. Microstat Walk-Away System = combination of Identification, Susceptibility  Presence of Double Zone = OUTER ZONE is measured not the inner zone.
Presence of Colony Inside the Zone do gram stain
False Resistant GRAM POSITIVE COCCI
1. Heavy inoculum (The higher the bacteria, the smaller the zone of inhibition)
2. Thick Medium
I. STAPHYLOCOCCI
3. Delay in Disc Application (should be applied <15min)
4. Ca/Mg – aminoglycoside (P.aeruginosa)
CATALASE
5. Thymine-Thymidine-SXT (Enterococci)
(+) (-)
Staphylococci Streptococci
False Sensitive
I
1. Light inoculum (The lesser the bacteria, the larger the zone o f inhibition)
COAGULASE
2. Thin medium
3. Delay in Incubation (growth of organisms are affected
(+) (-)
4. Presence of CO2 (Increased pH) – False sensitive in Tetracycline
S. aureus MOD. OXIDASE/BACITRACIN

Perform AST for the following Organisms:

1. Pathogenic Organism
+/S -/R
2. Drug Resistant Organism
Micrococcus Coag. Negative Staph
3. Opportunistic Organism
I
NOVOBIOCIN
QUALITY CONTROL
 Quality Control – routine (internal QC)
- checking media and reagents with specific organisms
S R
 Quality Assurance – external QC, annually S. epiderdimis S. saprophyticus
 QC Frequency:
a. Daily QC
- Oxidase, Catalase, Gram Stain
- Incubator, Ref/Freezer, Water Bath A. Diagnostic Tests:
b. Each use 1. CATALASE TEST (Presumptive Test)
- Gas Pak jar, ONPG  Catalase enzyme on 3% H 2O2
c. Weekly QC
 Performed on slide. Not performed on BAP since blood contains catalase =
- Antibiotic, Autoclave, Biochem
false positive
d. Monthly (30-Day) QC
 (+) Gas bubbles
- New drugs and reagents
 (+) Staphylococcus; (-) Streptococcus
e. ATCC
- Reference strains for QC
2. COAGULASE TEST (Confirmatory Test for S. aureus)
f. -20’C or -70’C
 Bacterial colonies  Clot ( 4 Hours)
- Stock culture storage
a. Slide (Screening) – BOUND coagulase
- -70’C (Virus and PCR)
b. Test Tube (Confirmatory) – FREE coagulase
g. 2-8’C
 Medium: Rabbit’s Plasma with EDTA (not citrate because there are some
- Working cultures storage
bacteria that can utilize citrate, e.g. Enterococci, Campylobacter)
 (+) Staphylococcus aureus
3. MANNITOL FERMENTATION TEST B. Staphylococcus aureus Properties:
 Mannitol Salt Agar (7.5% NaCl)  Protein A – cell wall, antiphagocytic, virulence
 Indicator: Phenol Red  Enterotoxin – food poisoning
 (+)Yellow (S. aureus) - Acid Production  Beta Hemolysin
 (-) Red  Leukocidin – Panton Valentine
 Exfoliatin (epidermolysis) – SSS
4. DNASE TEST  TSST-1 – Toxic Shock Syndrome
Two Methods:  Beta Lactamase – drug resistance
1. Toluidine Blue – pink zone (Presence of DNAse)  DNAse – dissolves clot
Methyl Green – clear zone (Presence of DNAse)  Hyaluronidase – spreading factor
2. HCl Precipitation – no precipitation after 1N HCl (Presence of DNAse)  Gelatinase
DNAse (+) = Clear Zone  Lipase – fat splitting enzyme
DNAse (-) = No Clearing
Bacteria used as a POSITIVE CONTROL for DNAse Test: C. Staphylococcus aureus Identification:
1. Staphylococcus aureus (Gram + cocci)  Yellow orange colony – lipochrome
2. Serratia marcescens (Gram – bacilli)
 Catalase (+), Coagulase (+), Oxidase (-), Nitrate and VP (+), Gelatin (+), PYR (-)

5. NOVOBIOCIN TEST (5 units)

Staph like Organisms SIMILARITY TO S. aureus DIFFERENCE
 CNS differential test
S. intermidius Slightly Coagulase (+) VP (-) (Acetoin)
 ID. of S. saprophyticus
S. lugdunensis Slightly Coagulase (+) PYR (+)
 R = <16mm
S. haemolyticus Beta Hemolytic Coagulase (-)

6. MODIFIED OXIDASE TEST NOTE: It’s is better to perform TEST TUBE METHOD than slide method because S.
 Reagent = Tetramethyl p-phenylene diamine dihydrochloride in DMSO intermidius and S. lugdunensis are Coagulase (-) in t est tube method for coagulase test.
(Dimethyl Sulfoxide)
 (+) Blue/Purple ( Micrococcus luteus) Diseases :
 (-) No color change  #1 SKIN INFECTIONS: Carbuncles, furuncles, folliculitis, cellulitis, impetigo, skin
scalded syndrome (SSS), TSS (Tampons)
7. STAPH A COAGGLUTINATION TEST  #1 WOUND, #1 OSTEOMYELITIS, #1 NOSOCOMIAL
 A means Protein A that is found at the cell wall of S. aureus.  Bacteremia, endocarditis, Food posining
 Staph aureus (cowan strain) with protein A as inert particles to which Lab Diagnosis:
antibody (Fc fragment) binds 1. Gram Stain – can be applied directly in the wound swab
 Detects specific bacterial Ag (Strep. Pneumoniae, N. meningitides, N. 2. Culture
 BAP (Slightly beta haemolytic, pinhead colony)
gonorrhea, H. influenzae)
 Vogel-Johnson (brown to black colony because s. aureus can reduce
Micrococcaceae Tellurite present in VJ agar; Corynebacterium can also reduce Tellurite.
Difference: S. aureus is cocci while Corynebacterium is bacilli)
Micrococcus Staphylococcus
 Chapman, Tellurite Glycine, P Agar, PEA, Columbia CNA
O/F Test Oxidative Fermentative
3. Catalase (+) Coagulase (+)
Modified Oxidase + -
4. Mannitol Fermentation Test (+): Yellow
Basitracin S R
5. DNA Hydrolysis Test
Furazolidone R S
6. Latex Agglutination test for protein A (Co nfirmatory Test) : (+)Agglutination
Lysostaphin R S
Note: Stomatococcus: Mod. Oxidase (-), Lysostaphin (R) and Furazolidone (R)
D. Coagulase Negative Staph: II. STREPTOCOCCI
1. STAPH. EPIDERMIDIS
 Skin flora, Blood culture contaminant
 Causes PROSTHETIC HEART VALVE, ENDOCARDITIS ALPHA HEMOLYSIS
 Novobiocin Sensitive, Non Hemolytic, O xidase (-) I
OPTOCHIN
2. STAPH. SAPROPHYTICUS (S) (R)
S. pneumoniae Group D Strep
 Causes UTI, sexually transmitted
S. viridans
 Novobiocin Resistant I
BILE ESCULIN
Note: S. aureus and S. epidermidis are both Novobiocin sensitive. S. saprophyticus is the
only Novobiocin resistant (+) (-)
Group D Strep S. viridans
Coagulase Negative
S. aureus
S. epidermidis S. saprophyticus
Colony Yellow White White
Catalase + + +
Coagulase + - -
Mannitol + - +/- BETA HEMOLYSIS
I
Novobiocin S S R
BACITRACIN
DNAse + - - (S) (R)
Phosphatase + + - Group A Strep Group B, C, D, F, G Strep
Gelatinase + + + I
BILE ESCULIN

(+) (-)
Group D Strep Group B (CAMP +)
Group C, F, G (SXT +)

GAMMA HEMOLYSIS
I
BILE ESCULIN
(+) Group D Strep
I
6.5% NaCl
PYR
(+) (-)
Enterococci Non-Enterococci
A. Diagnostic Tests: (Presumptive Test Only) 8. VANCOMYCIN RESISTANT
1. BACITRACIN SUSCEPTIBILITY / TAXO A (0.04 Units)  ID of Pediococcus/Leuconostoc (Streptococcus-like organisms that are
 ID of S. pyogenes resistant to Vancomycin)
 (+) Zone of Inhibition
Tests for Differentiation Pediococcus Leuconostoc
2. PYR (L-pyrrolidonyl B-napthlamide) LEUCINE AMINOPEPTIDASE (LAP) (+) Red (-) Yellow/No Color change
 ID of S. pyogenes, Enterococcus MRS BROTH (-) No Gas Production (+) Gas in Durham Tube
 Rgt: p-dimethylaminocinnamaldehyde
 (+) Red 9. PYRUVATE BROTH
 Incubate: 35’C for 48 hours
3. CAMP TEST  (+) Yellow (E. faecalis)
 CAMP factor of S. agalactiae synergistic reaction to beta lysine of S. aureus  (-) Green (E. faecium)
 (+) Arrow head zone beta hemolysis
NOTE:
4. HIPPURATE HYDROLYSIS TEST  All of the above are only PRESUMPTIVE test.
 ID of S. agalactiae  The CONFIRMATORY test for Strep is LANCEFIELD TEST (Serologic Typing) except for S.
 Rgt: Sodium Hippurate and Ninhydrin pneumoniae since it doesn’t have lancefield classification.
 (+) Purple  The CONFIRMATORY test for S. pneumoniae is NEUFELD QUELLUNG TEST.
 S. agalactiae
 Listeria B. Streptococcus Characteristics:
 Campylobacter  Gram (+) cocci in chain, pairs
 (-) Colorless, Pink  Catalase (-), “Pinpoint” colonies
 Oxidase (-) (Remember that Oxidase (+) are usually for Gram Negative)
5. OPTOCHIN TEST / TAXO P (5 units)  Facultative Anaerobes
 ID of S. pneumoniae  Capnophilic (5-10% CO2)
 5ug ethylhycrocupreine HCl (Taxo P)  Medium of Choice: S heep’s Blood Agar
 (+) >14 mm zone of inhibition (S. pneumo)  Selective Medium: PEA
 (-) <13mm zone or no zone (S. mitis)
C. Streptococcus Classifications:
6. BILE SOLUBILITY TEST 1. Smith and Brown’s Classification
 Incubate: 35’C for 30 mins a. Alpha Streptococcus
 Rgt: Sodium Desoxycholate (Bile salt) – able to destroy gram positive  Incomplete Hemolysis
bacteria  (+) Greenish Zone
 BAP (10% Bile Salt)  S. pneumoniae, S. viridans
 (+) Lyzed Colony (S. pneumoniae) Note: ALPHA PRIME – a small zone of alpha hemolysis surrounded by zone
 (-) Intact Colony (S.viridans / E. faecalis) of beta hemolysis after refrigeration
 Tube Method (2% Bile Salt)
 (+) Clear b. Beta Streptococcus (Common)
 (-) Turbid  Complete Hemolysis
 (+) Clear/Colorless Zone
7. BILE ESCULIN HOH TEST  S. pyogenes, S. agalactiae, Grps. C F G
 40% Bile
 Ferric NH4 Citrate reacts with esculetin c. Gamma Streptococcus
 2. Lancefield Classification (CONFIRMATORY) b. GROUP B Strep (Streptococcus agalactiae)
- Carbohydrate on cell wall (Grp. A, B, C …)  Beta Hemolytic
a. GROUP A Strep (Streptococcus pyogenes)  Vaginal flora, URT
- pus forming, flesh eating bacteria  #1 Neonatal Meningitis (acquired through vaginal delivery), Septicemia
- requires anaerobic incubation to detect hemolysis Lab Diagnosis:
1. CAMP Test: (+) Arrow Head zone of BH
Characteristics: 2. Hippurate Hydrolysis Test: (+) Purple
 M protein – found at the cell wall, antiphagocytic, virulence factor
 Streptolysin O – O2 labile, Ag, Sub surface (needs Anaerobic incubation c. GROUP C, F, G Strep
to detect hemolysis)  Animal pathogens that cause endocarditis ( Specimen: Blood)
 Streptolysin S – O2 stable, Non Ag  Beta Hemolytic,
 Erythrogenic toxin – causing scarlet fever  Basitracin Resistant
 Streptokinase – dissolves clot (treatment to prevent AMI)  SXT Sensitive
 Hyaluronisade – spreading factor  Group C: S. equimilis, S. equi, S. dysagalactiae
 Bacitracin Sensitive
Basitracin SXT Organism
Diseases: S R Group A Strep
1. Pharyngitis – “Sore throat” (Specimen: Throat swab) R R Group B Strep
2. AGN, RF (Rheumatic Heart Disease) - since it cross reacts with R S Group C, F, G Strep
myocardial antigens
3. Scarlet Fever d. GROUP D Strep
a. Dick’s Test (red) – skin test where toxin is given 1. Enterococcus
b. Schultz-Charlton (rash fade) – immunity test  E. faecalis, E. faecium, E. durans
4. Erysipelas Drug resistant (VRE), UTI


5. Impetigo
2. Non-Enterococcus
6. Wound, Burn
 S. bovis – colon cancers;
7. Toxic Shock Syndrome, Pyoderma, Necrotizing Fascitis
 S. equinus
 Easier to treat than Enterococcus (Penicillin Sensitive)
Lab Diagnosis:
Lab Diagnosis:
1. Gram Stain – Gram (+) cocci in chain
 Bile Esculin Hydrolysis Test
2. Culture – BAP (BH)
 (+) Blackening; Bile Esculin Medium
3. Catalase – Catalase (-) (no gas bubbles)
 Differentiates Group D from other Strep
4. Basitracin (Taxo A) – sensitive (any zone)
 Presumptive test for Group D
5. SXT Test – resistant
6. PYR Test – PYR (+) Red
7. Lancefield Typing – Group A

NOTE:
 A Protein = S. aureus
 M Protein = S. pyogenes
 Dick’s Test = S. pyogenes
 Schick’s Test = Corynebacterium diptheriae
 Erysipelas/Scarlet Fever= S. pyogenes
Erysiperloid = Erysipelothrix
D. Streptococcus pneumoniae: F. Pediococcus/Leuconostoc (Strep-like Organisms)
 Gram (+) diplococci, “ Lancet Shaped”  Vancomycin resistant test – differentiate Pediococcus from Strep viridans
 No Lancefield classification Note: Leuconostoc is S. viridans-like/ Aerococcus is Enterococcus-like
 Alpha haemolytic, Virulence: Capsule TESTS Pediococcus Leuconostoc Aerococcus
 Nasopharynx and Oropharynx Vancomycin R R S
 Inhibited by OPTOCHIN; Bile Soluble Bile Esculin + + +/-
PYR - - +/-
Diseases: 6.5% NaCl + + +
1. #1 Adult Bacterial Meningitis ( Specimen: CSF) MRS Broth - (+) Gas N/A
2. Pneumococcal pneumonia = (“Rusty Sputum”) LAP (+) Red - N/A
3. #1 Otitis Media
Lab Diagnosis: Beta Hemolytic Strep
1. Gram Stain – Gram (+) diplococci TESTS Group A Group B Group C, F, G
2. Culture – Gentamicin Blood Agar (alpha, mucoid colony) Basitracin S R R
3. Optochin (Taxo P) - >14mm zone SXT R R S
4. Bile Solubility CAMP - + -
a. 10% Sodium Desoxycholate (BAP) (+) Lysis Colony PYR + - -
b. 2% Sodium Desoxycholate (tube) (+) Clear Bile Solubility - - -
5. Mouse Virulence Test - death
6. Francis Test – skin G. Abiotrophia spp
7. Neufeld Quellung Test – capsular swelling (confirmatory)  Nutritionally variant streptococci (NVS)
 Strep that will not grow in BAP or CAP
NOTE:  Requires Vit. B6 (pyridoxine); Staph streat test (+)
 #1 Neonatal Meningitis = S. agalactiae
 #1 Adult Meningitis = S. pneumoniae
GRAM NEGATIVE COCCI
Genera included:
E. Streptococcus viridans:
 Aerobic: Neisseria and Moraxella
 Not classified under LANCEFIELD (same as S. pneumoniae)
 Anaerobic: Veilonella
 OPTOCHIN Resistant, Bile Insoluble
 Gram (-) Intra (extra) diplococci
 Normal flora of URT, GIT, GUT
 Oxidase (+) (Presumptive test for gram negative organism) , Catalase (+)
 Best Media: CAP (Requiring 5-10% CO2)
Species:
1. S. mitis (mitior)  Charcoal is added in culture media to remove the toxicity of cotton
2. S. sanguis - subacute endocarditis (SBE) acquired through dental procedure  Pathogenic: N. gonorrhoeae, N. meningitides
3. S. mutans - dental plaques/caries  Pigmented Neisseria: N. subflava, flavescenes (Yellow color due to Flavin)

Enterococcus Non Enterococcus A. Neisseria gonorrhoeae

Bile Esculin + +  Aka: “CLAP” (Since clapping requires 2 hands = diplococci)
6.5% NaCl + -  Kidney (coffee) bean shaped in PMN
PYR + -  Oxidase positive (PRESUMPTIVE) and ferment glucose (dextrose)
Penicillin R S (CONFIRMATORY)
Basitracin R R  Virulence – “pili” (N. meningitides – capsule and endotoxin)
Growth 45’C + +  Diseases: gonorrhea, opthalmia neonatorum, Fitz-Hugh Curtis, PPNG
Growth 10’C + - (Penicillinase Producing Neisseria gonorrhea); salphingitis, epididymitis
Laboratory Diagnosis: B. Neisseria meningitides
1. GRAM STAIN – the presence of any diplococci inside or outside a PMN is an - Gram negative diplococci that produces mucoid colony in culture media
indicative of Neisseria gonorrhea  Carrier: nasopharynx
 Virulence: Capsule and enotoxin (N. gonorrhoeae – pili)
2. CULTURE: CAP (+); BAP (-) (NON STERILE)  Diseases:
Selective Media  Meningitis, Meningococcemia
 Chococolate Agar Containing Media:  Waterhouse Freiderichsen – hemorrhage of adrenal gland
1. Thayer Martin Agar – VCN  Specimen: Blood and CSF
2. Modified TMA – VNCT – Trimethoprim lactate  Serotypes: A, B, C, Y, W135 (Capsular Ags)
3. Martin Lewis – VCAT (Anisomycin)
Laboratory Diagnosis:
 Yeast Extract Agar (Clear Media): 1. Gram Stain
o New York City Agar – VCAT (Amphoteracin B) 2. Culture: CAP (+); BAP (+)
3. Oxidase Test (+) Purple
Antibiotics: 4. Carbohydrate Fermentation – Glucose and MALTOSE
 Vancomycin = inihibits Gram (+)
 Colistin = inihibits Gram (+) bacilli C. Moraxella catarrhalis/Branhamella
 Nystatin = inhibits Yeasts  Oxidase Test (+)
 Trimetophrim = inhibits swarming of Proteus  Reduce NO 3 to NO 2
 DNAse (+) (Best); M. lacunata and M. denitrificans = DNAse (-)
3. OXIDASE TEST / TAXO N – Presumptive Test  “Hucky Puck Colony ”
 Procedure: Rub the colony into a filter paper. (+) PURPLE 
rd
3 cause of otitis media
 The reagent is already incorporated into the filter paper or the reagent is Lab Diagnosis:
placed on the colony itself.  Butyrate Esterase Disc Test (Tributyrin Hydrolysis) = (+) Blue Co lor
 Reagent = 1% tetramethyl-p-phenylenediaminedihydrochloride  Assacharolytic (not degrading any sugar) ;
 Positive Control: Pseudomonas (easily grows)  Beta Lactamase Producer : Penicillin resistant
 (+): Neisseria, Moraxella, Aeromonas, Pseudomonas  Also grows in Nutrient Agar.

4. CARBOHYDRATE FERMENTATION TEST – Confirmatory Test Oxidase Sugar/CHO DNAse TMA

 Media: CTA (Cysteine Trypticase Agar + Phenol Red) N. gonorrheae + Glucose - +
 Phenol Red – indicator detecting acid in this test N. meningitidis + Glucose - +
 No need for CO2 requirement! CO2 is only for culture. Maltose
 Fermentation of GLUCOSE only: (+) YELLOW M. catarrhalis + None + +

5. SUPEROXOL CATALASE TEST Carbohydrate Fermentation Test

 Reagent = 30% H2O2 GLU MAL LAC SUC
 (+) Neisseria gonorrhea N. meningitides + + - -
N. gonorrheae + - - -
6. BETA LACTAMASE TEST: (+) Color Change N. secca + + - +
 Performed when Penicillin resistant. Held on Primary Culture (since Plasmid N. lactamica + + + -
is lost during subculture) M. catarrhalis - - - -
a. Chromogenic Cephalosporin Test: (+) Pink/Red
b. Iodometric Test – Iodine and Pen: (+) Colorless
NOTE:
Acidimetric Test Phenol Red and Pen: (+) Yellow
ACID FAST ORGANISMS  Decontamination-Digestion
- performed only on sputum (since it is not sterile), therefore
CSF does not need this process anymore since it is a sterile
“Acid Fast” = Acid alcohol resistant
specimen.
- not decolorized by acid alcohol retaining the RED color of CARBOLFUCHSIN
a. NALC-NaOH (2-4%) + Na citrate (GOLD STANDARD)
due to the presence of MYCOLIC ACID (a long chain of fatty acid that makes
 Na Citrate – removes the metallic compound
Mycobacteria the bacteria with the highest amount of lipid)
b. Zephiram Trisodium PO4
c. 6% Oxalic Acid – used when the specimen is contaminated by
I. MYCOBACTERIA (Mycobacteria = “Mataba at Mabagal”) Pseudomonas such as urine and stool sample
d. Dithiothreitol (sputulysin) – lyses the sputum and the Acid Fast
 ACID FAST BACILLI (mycolic acid) Stain
 Growth: 2-3 weeks
 8 weeks before reporting as NEGATIVE 2. Acid Fast Stain
 Slow growers (except M. fortuitum, M. chelonei) = 1 week only  AFB GRADING NATIONAL STANDARD
 “Much granules”; aerobic non sporeforemer; non motile 0 - No AFB / 300 fields
+n - 1-9 AFB / 100 fields
3 GROUPS: 1+ - 10-99 AFB / 100 fields
A. MYCOBACTERIUM TUBERCOLISIS COMPLEX - causes TB 2+ - 1-10 AFB / field in at least 50 fields
1. M. tuberculosis- pulmonary TB 3+ - >10 AFB / field in at least 20 fields
2. M. bovis- intestinal tuberculosis (BCG) (Acquired through drinking Note:
unpasteurized milk)  Spot-Morning-Spot – requires 1 morning sputum specimen used to view
3. M. africanum- pulmonary TB (Africa) Acid Fas Organisms by DSSM (Direct Sputum Smear Microscopy)
 Ideal Size of Sputum Smear = 2x3cm
B. MOTT (Mycobacteria Other Than Tuberculosis / Non Tuberculosis  300 Fields = 2 Lines (should be read before declaring 0 or No AFB)
Mycobacteria)
- can cause pneumonia but not tuberculosis
150 Fields
C. MYCOBACTERIUM LEPRAE 150 Fields
- cannot be grown on agar
- obligate intracellular
3. Skin test
A. MYCOBACTERIUM TUBERCULOSIS (Koch Bacillus) - Sensitive and simplest test for cell-mediated immunity (Type IV)
 Obligate aerobe requiring slight opening of LJ Media to allow O 2 to enter; require - Ex. Tuberculin Test
5-10% CO2 for growth
 Cauliflower, Buff-colored (non-photochromogen) colonies in LJ 4. Culture – still needed even if (+) in Gram Stain. Remember that when you
 Virulence: do culture, you should also do susceptibility test since there are drug-
 Cord factor (causes sticking of mycobacteria, producing cording effect resistant mycobacteria.
on Acid Fast Stain)
 Grading System (3+) Note:
 Sulfatides  Type of media for susceptibility test for mycobacteria:
 Laboratory Diagnosis: Agar Based (Middlebrook 7H11)
1. Gram Stain - qualify specimen (accept or reject specimen)  Type of media of the best culture media for mycobacteria:
a. Sputum: <10 Epithelial Cells; > 25 PMN Egg Based (LJ)
b. Saliva: >10 Epithelial Cells; <25 PMN
 A. AGAR BASED MEDIA d. CATALASE TEST at 68’C (Heat Stable Catalase)
a. Middlebrook 7H11 - (for AST)  Medium: Tween 80
- clear media used for susceptibility testing  Reagent: 30% H2O2
- Wrinkled colony of MTB  PPL:Tween 80 + Mycobacteria + 30% H2O2 + Heat at 68’C
Duboi’s Oleic Acid Medium  Result: >45mm height of gas bubbles
b. Mitchison’s Medium (+) M. kansasii, M. avium
(-) M. tuberculosis
B. EGG BASED MEDIA: Malachite Green
- Type of sterilization: INSPISSATION e. TWEEN 80 HYDROLYSIS TEST
a. Lowenstein Jensen Medium (BEST)  Patient: Tween 80  HOH of Tween 80
- Malachite Green serves as an INHIBITORY AGENT since (+) M. kansasii (red)
sputum is non-sterile (-) M. avium
- Glycerol: serves as the CARBON SOURCE for LJ
- Take note that Nocardia can also grow in LJ other than f. TELLURITE REDUCTION TEST
Mycobacteria spp.  Patient: Telurite  Black Metallic Tellurium
- Cauliflower, Buff-colored (yellow brown) colonies in LJ (+) M. avium (blackening of media)
b. Petragnani Medium (-) M. kansasii
c. American Thoracic Society Medium
d. Dorset Egg Medium g. ARYSULFATE TEST
 Tripotassium phenolphthalein disulfide / Sulfate acted upon by
C. LIQUID MEDIA Arylsulfatase to produce Free Phenolphthalein
- For RAPID CULTURE SYSTEM (+) M. fortuitum-chelonei (pink/red)
a. Bactec 12B
b. Septi-Chek B. MOTT (ATYPICAL MYCOBACTERIA)- NTM
c. Middlebrook 7H9 A. Photochromogens - ROUNYOUN’S I = YELLOW
1. M. kansasii
5. Biochemical Tests for Mycobacteria 2. M. marinum
3. M. asiaticum, M. simiae
a. NIACIN TEST B. Scotochromogens - ROUNYOUN’S II = ORANGE/YELLOW
 Requires colony coming from the LJ media 1. M. scrofulaceum (scrofula)
 PPL: Niacin + Niacin Ribonucleotide + Aniline Dye + Cyanogen 2. M. szulgai
Bromide = YELLOW (+) 3. M. gordonae (Tap Water Bacillus)
(+) M. tuberculosis (yellow) C. Non photochromogens - ROUNYOUN’S III = CREAM/BUFF COLORED
(-) M. bovis 1. M. avium (#1 NTM) or
2. M. intracellulare (Battery Bacillus)
b. NITRATE REDUCTION TEST 3. M. ulcerans (Buruli)
 Reagents: 4. M. xenopi (Hot and Cold Water T aps)
a. HCl 5. M. triviale, M. haemophilum
b. Sulfanilamide 6. M. malmoense, M. terrae, M. gastri
c. N-napthtylethylene diamine D. Rapid Growers- ROUNYOUN’S IV (<7days)
(+) M. tuberculosis (pink/red) 1. M. fortuitum
(-) M. bovis 2. M. chelonei
3. M. phlei- provide CO2
c. TCH SUSCEPTIBILITY TEST 4. M. smegmatis- confused with MTB in urine (formerly known as M. lacticola)
PHOTOCHROMOGENS

TWEEN 80 HYDROLYSIS
NON PHOTOCHROMOGENS (Mycobacterium terrae Complex - MTC)
(+) (-)
M. kansasii M. simiae
M. marinum
CATALASE
M. asiaticum
(>45mm)
I
(+) (-)
NITRATE REDUCTION
M. terrae Complex M. avium Complex
M. triviale M. xenopi
I
(+) (-)
5% NaCl
M. kansasii M. marinum (+UREA)
M. asiaticum

(+) (-)
M. triviale M. terrae Complex

SCOTOCHROMOGENS

NITRATE REDUCTION

(+) (-)
M. szugai M. scrofulaceum
M. gordonae RAPID GROWERS
I
UREASE
ARYLSULFATASE
(+) (-)
M. scrofulaceum M. gordonae (+) (-)
M. fortuitum – chelona M. smegmatis
I
NITRATE REDUCTION
NON PHOTOCHROMOGENS (Mycobacterium avium Complex - MAC) 5%NaCl
IRON UPTAKE

UREASE
(+) (-)
(+) (-) M. fortuitum M. chelonae
M. avium M. xenopi
M. intracellulare
(MAC)
 MOTT SUMMARY OF DIFFERENTIATION AUTOMATED TEST FOR MYCOBACTERIUM
ROUNYOUN’S I PHOTOCHROMOGENS YELLOW 1. Bactec 460 Middlebrook 7H12 (RIA)
14 14
M. kansasii Chronic Pulmonary  PPL: C Palmitic Acid + Organisms = CO2 (measured)
“Yellow Bacillus” Disease Nitrate (+)  POSITIVE Result: More than 10 growth index
(Pneumonia)
Swimming Pool Nitrate(-) 2. Mycobacteria Growth Indicator Tube (MGIT)
M. marinum
Granuloma Grows well at 30’C  Fluorometric Based (less harmful)
ROUNYOUN’S II SCOTOCHROMOGENS ORANGE / YELLOW  POSITIVE Result: Fluorescence
Scrofula agent  PPL: Increased bacteria = Increased O 2 Consumption = Fluorescence
Urease (+)
M. scrofulaceum (Cervical
Tween 80 (-)
Lymphadenitis) 3. Bactec 12B + NAP (Growth Inhibition Test)
M. gordonae (Non Pathogenic) Urease (-)  p-nitro acteylamino beta
“Tap Water Bacillus” Tween 80(+)  NAP - inhibits M. tuberculosis (Sensitive)
ROUNYOUN’S III NON  POSITIVE Result: No growth
CREAM / BUFF COLORED
PHOTOCHROMOGENS
M. avium Chronic Pulmonary C. MYCOBACTERIUM LEPRAE (Hansen’s Bacillus)
M. avium Catalase (+)
complex Disease (among  AFB, “cigarette-packet / picket fence ”
Tellurite (+)
- differentiated AIDS patients)  Obligate Intracellular
by Nucleic Acid M. intracellulare  Not cultivated in agar (in vitro)
Amplification (Battery Bacillus)  Hydrolize DOPA
Test/PCR  Tropism to peripheral nerves
M. xenopi Wound Infection
Grows at 4’C and
(Hot and Cold Water Disease: Leprosy (Hansen’s Disease)
45’C
Taps)
ROUNYOUN’S IV RAPID GROWERS GROWS <7 DAYS Lepromine Test – skin test for leprosy
Both: M. fortuitum All Positive a. Lepromatous:
Arylsulfatase (+) Differentiated by: Lepromine (-), many AFB; characterized by LEONINE FACE
 Nitrate 2 Types of Reaction:
Grows on M. chelonei Reduction All Negative 1. Fernandez = Early
MacConkey w/o  5% NaCl 2. Mitsuda = Late
Crystal Violet  Iron Uptake b. Tubercoloid
Lepromine (+), few AFB; good prognosis than Lepromatous
Photoreactivity/Photosensitivity
 Uses 2 LJ media (light and dark tube) Laboratory Diagnosis
a. Light Tube – without alumunim foil 1. Clinical Findings- basis of diagnosis
b. Dark Tube – with aluminum foil 2. Culture - foot pads of armadillo (since their feet are cold, t. leprae like cold
 Remove the aluminum foil in the dark tube when there is growth on the light tube, environment)
and simultaneously check for the growth of the 2 tubes. 3. Fite Faraco Stain Treat: Dapsone

OTHER MYCOBACTERIA:
1. M. genavensi- disseminated infections in AIDS; BACTEC (+)
2. M. paratuberculosis- Crohn’s Disease
3. Rhodococcus equi - pleomorphic (rod cocci / vice versa) every 24 hours, and
pink colonies (+); CAMP Test (+) with S. aureus beta hemolytic
II. NOCARDIA 8. Culture on :
 Partially acid fast (1% H2SO4) (Modified Acid Fast) a. Blood Agar (White and dry colony)
 Urease (+) Gram (+)branching rod b. Tinsdale (black colony w/ brown halo)
 FUNGUS LIKE BACTERIA (Just like Actinomyces) c. Potassium tellurite (gray to black colony),
d. Loeffler’s serum agar
 Cause: PNEUMONIA (Sputum)
e. Pai coagulated egg
 Best Media: Casein Medium
f. Clauberg Mclead
 Also grows in LJ Media. To differentiate with Mycobacteria = GRAM STAIN. Nocardia is
g. Cystine Tellurite BAP (CT-BAP) ( gun metal gray colony) – media
fungus like.
used to differentiate the biotypes of C. diptheriae based on the
 Differentiate Species by: CASEIN HYDROLYSIS
size of their colonies
o N. asteroids (-)
*Potassium tellurite : inhibits normal flora
o N. brasiliensis (+)

III. CORYNEBACTERIA BIOTYPE OF C. diphtheria (CT-BAP)

 Pleomorphic Gram (+ )rods 1. C. gravis: gray, large, Beta hemolytic, starch/glycogen fermentation (+)
 Has similarity with Listeria. 2. C. intermidius: black, small, non hemolytic
 Arrangement: 3. C. mitis : medium-size, black, beta hemolytic, starch/glycogen (-)
o Clubshape, X, Y, V L, chinese letters
o Palisade appearance (side by side) “PATHOGENS” Urease Nitrate red. Starch
 Babes-Ernst (metachromatic granules) - All ferment DEXTROSE and MALTOSE HOH
 Normal Flora: Oral Cavity, Skin, Genital (just like Candida albicans)
C. diphtheriae (–) + –
 Non motile, No spore, capsule
C. ulcerans + - +
 BAP: raised, transluscent gray colonies
C. pseudotuberculosis + +/- –
 Catalase (+)

1. Corynebacterium diphtheriae (Kleb Loeffler’s Bacillus)

Note:
 Virulence factor: Exotoxin Labile A,B
 C. diphtheria is the only human pathogen
 Disease: Diphtheria (grayish pseudomembrane on tonsils and pharynx
 C. ulcerans and C. pseudotuberculosis are animal pathogens
(Upper respiratory infection)
o C. ulcerans = mastitis in cattle
Specimen:
o C. psedutuberculosis = TB like infection in animals
 Throat Swab (Pharyngitis or Diptheria)
 Starch Hydrolysis
 if Diptheria, culture in Loeffler’s Medium to see the
 used to differentiate C. ulcerans and C. pseudotuberculosis
Metachromatic Granules
 (+) Disappearance of Blue color (C. ulcerans)
 Reagent: Iodine
Lab Diagnosis:
1. LAMB – Metachromatic granules (Pai Loeffler)
2. Tellurite Media: Gray to black colony (since it can reduce Tellurite)
“DIPTHEROIDS” (Normal Flora) Urease CHO ferment Nitrate red
3. Urease (-): (Presumptive test) The only pathogenic corynebacteria which is
C. xerosis – Glucose, Maltose, Sucrose +
Urease (-)
C. pseudodiphthericum + None +
4. CHO ferment: dextrose & maltose (+)
(Hoffman’s bacillus)
5. ELEK test: definitive test (confirmatory test)
6. Schick’s test – skin test for diphtheria C. jeikeium + Glucose –
7. Culture similar to C. pseudotuberculosis, C. ulcerans . (Arthrobacter culture
similar to Brevibacterium)
Note:
 Glucose = Dextrose
 Sucrose = Saccharose
 All of them are normal flora but can cause ENDOCARDITIS
o Specimen: Blood
 C. xerosis – causes conjunctivitis
 C. pseudodiptheriticum – oral flora
 C. jeikeium – common for patients with HIV ; cause of Prosthetic Valve
Endocarditis (like S. epidermidis); drug resistant (beta lactamase producer)
 C. urealyticum – Urease (+), causes UTI

2. Corynebacterium minutissimum
 agent of erythrasma
 Diagnosis: “coral red fluorescence” on wood’s lamp (skin infection) Red
Fluorescence due to PORPHYRIN.

3. Arcanobacterium haemolyticum
 Beta hemolysis on SBAP, lipase and lecithinase(+)
 (+) reverse CAMP with S. aureus
 (+) Inverted Triangle / Triangle Type of Hemolysis

Note:
 CAMP (S. aureus) = S. agalactiae, R. equi
 Reverse CAMP (S. aureus) = Arcano
 Reverse CAMP (S. agalactiae) = C. perfringens
GRAM POSITIVE BACILLI
LACTOBACILLUS ACTINOMYCES
Gram Stain Gram (+) Rod Gram (+) Branching
SPORES Rod
(-) (+) O2 Anaerobic Anaerobic
Corynebacterium Bacillus (Aerotolerant)
Listeria Clostridium
Sulfur Granules – +
Lactobacillus
Actinomyces
Acid + –
Kythria Catalase – –
Erysiphelothrix
I
CATALASE TEST BACILLUS
1. Bacillus anthracis (Anthrax Bacillus)
 Gram (+) Rods, chain – Bamboo shape appearance
(+) (-)  Largest bacteria (Smallest is Mycoplasma)
Corynebacterium Lactobacillus
 Non motile, spore forming, zoonotic (acquired from animal source)
Listeria Actinomyces
Kuthria  Virulence factor:
Erysiphelothrix o Exotoxin (edema and lethal)
o Capsule
 made up of Glutamic Acid (D Glutamate)
BACILLUS CLOSTRIDIUM  BICARBONATE MEDIUM (Enhances the capsule formation of B.
O2 Aerobic Anaerobic anthracis)
Catalase + –  McFadyean’s Reaction – presumptive test for the presence of Capsule
Gas – + (Foul Odor) (+) Pink = Capsule; Blue = Bacilli (due to Methylene Blue)

Disease:
1. Malignant pustule – black eschar
CORYNEBACTERIUM LISTERIA 2. Woolsorter’s / Rag Picker’s Disease - pulmonary anthrax
Catalase + + 3. Gastroenteritis – bloody diarrhea (intestinal anthrax)
Oxidase – –
Motility – + (22’C) Lab Diagnosis:
Growth at 4’C – + 1. Selective Medium: PLET (Contains antibiotic: POLYMYXIN from its name
Esculin – + Polymyxin Lysozyme EDTA Thalous Acetate)
VP (Acetoin) – + 2. COLONY
Methyl Red + - a. Medusa Head Colony
b. Lion Head Colony
c. Serrated Irregular Colony
d. Inverted Pine tree (Grows on GELATIN TUBE MEDIA)
NOCARDIA ACTINOMYCES 3. STRING OF PEARL TEST (0.05 units PEN) – BAP (Microscopically seen)
Catalase + – 4. ASCOLI TEST = serologic precipitation test: (+) Precipitation Ring
O2 Aerobic Anaerobic 5. Serologic Tests:
AF AFO NAFO PCR – best method for diagnosis of anthrax
Urease + – Fluororescence Ab test
ELISA
 Note: Summary of Tests for some organisms: CLOSTRIDIUM
 Ascoli = B. anthracis  Obligate anaerobe gram (+), endospore
 Anton = Listeria  Habitat: human and animal
 Casein = Nocardia  Saccharolytic (fermentation since anaerobic process) except: C. tetani, C. septicum
 Nagler = C. perfringens
3 Types pf Clostridium
Note: 1. Neurotoxic: C. tetani and C. botulinum (more dangerous since they act on CNS)
 Inverted Pine Tree = B. anthracis 2. Histotoxic: C. perfringens and C. septicum
 Umbrella = L. monocytogenes 3. Eneteric: C. difficile
 Pipe Cleaner/Test Tube Brush = Erysiphelothrix
1. Clostridium perfringens (C. welchii)
2. Bacillus cereus (Fried Rice Bacillus/Spore Rice Grain)  The only ENCAPSULATED clostridium (The only Non mot ile)
 Virulence: Exotoxin (Cholera like T)  Double Hemolysis
 “Box car shape bacillus”
B. anthracis B. cereus  SUBTERMINAL SPORE
Motility – +  Source: wound contamination with soil
Capsule + –
Hemolysis Non Hemolytic Beta Hemolytic Diseases:
Growth 45’C – +  Gas gangrene, myonecrosis (skin infection)
Salicin Ferment – +  Food poisoning, enterotoxins
Penicillin G S R  Necrotic enteritis
Gelatin Hydrolysis & PEA – +
Lab Diagnosis:
Note: 1. CHOPPED MEAT – growth + gas (anaerobic growth)
 Once a bacteria has CAPSULE = NON MOTILE! - anaerobic broth. Promotes spore formation
- Growth: Turbidity
3. Bacillus subtilis - 48 hours growth of anaerobes
 Gram (+) Rod in chain, central spore
 Common laboratory contaminant 2. BAP – Target or Double Zone of Hemolysis
 Source of antibiotics (Exclusive for C. perfringens)
 Eye infection in heroin addict
 Used as QC in OVEN Alpha Hemolysis (Partial/Incomplete)
 BAP – large, flat dull, beta hemolytic, ground glass (ALPHA TOXIN)
 Has similarity with P. aeruginosa
Beta Hemolysis(Complete)
Difference: P. aeruginosa (moist colony); B. subtilis (dry colony)
(THETA TOXIN)
4. Bacillus stearothermophilus
3. NAGLER TEST – Lecithinase Test (Alpha Toxin)
 Flat sour spoilage; acid without gas;
 Due to alpha, lecithinase C, phospholipase C
 QC for AUTOCLAVE
 Media: Mc Clung or Neomycin Egg Yolk – medium to demonstrate
lecithinase
Note:
(+) OPALESCENCE on agar without anti toxin
 Flat sour spoilage = B. stearothermophilus
 Bloated sour spoilage = C. botulinum
 4. REVERSE CAMP TEST – uses S. agalactiae (Group B Strep) Note:
(+) Arrow Head Zone of Beta Hemolysis  Flaccid Paralysis – C. botulinum (Nanlalambot)
 Spastic Paralysis – C. tetani (Naninigas)
5. STORMY MILK FERMENTATION
(+) COAGULATE CASEIN/ Clotting of protein Note: Swarmers!
 Proteus (Gram – )
Note:  C. tetani (Gram + )
 CAMP (S. aureus) = S. agalactiae, R. equi  C. septicum (Gram +)
 Reverse CAMP (S. aureus) = Arcano
 Reverse CAMP (S. agalactiae) = C. perfringens 4. Clostridium difficile
 Colon flora
2. Clostridium botulinum (Canned Good Bacillus)  Antibiotic (clindamycin) associated pseudomembranous enterocolitis
 Virulence: Toxin heat labile – block release of acetylcholine ( flaccid (violent diarrhea)
paralysis; Virus = Poliovirus)
 Botulinum Toxin (neurotoxin) Lab Diagnosis
- most potent toxin in human, acts on CNS  Direct detection of toxin (stool)
- used as BOTOX  Toxin detection by EIA
 Cytotoxin assay
Diseases: (Not Cultured)  Culture: CCFA – yellow (horse manure) Indicator: Phenol Red
 Wound Botulism – spore on wound
 Infant botulism – honey bee (floppy baby)
 Botulism = most sever food poisoning

Note: Food Poisoning (Wag mag aral sa CSB, maraming nafofood poison!)
 C. perfringens/ C.tetani
 S. aureus
 B. cereus

3. Clostridium tetani (Tackhead Bacillus)

 TERMINAL SPORE(tennis racket, drumstick)
ANAEROBIC BACTERIOLOGY
- 5% CO2 + 10H2 + 85% N2
 Asaccharolytic
- FOUL ODOR
 Swarming!
Growth: 48 hours
 Has special affinity to Iron.
Collection:
 Virulence: Exotoxin (tetanolysin and tetanospasmin) binds to ganglioside
a. needle aspiration
receptors and inhibit neurons in CNS – spastic paralysis
b. suprapubic aspiration
 Lock Jaw, Risus sardonicus (Sardonic Smile), Opisthotonus (Arching of back),
Media:
Neonatal Tetanus
1. Chopped meat
2. Thioglycollate - aerobic and anearobic
Lab Diagnosis:
3. Reduced media
 Clinical findings – basis of diagnosis - reduced oxygen
 Terminal spore = OVAL TERMINAL SPORE - anaerobic BAP, Schaedlar, Bacteroides bile esculin
4. Laked kanamcin vancomycin BAP (LKVBA)
Terminal Spore Fermentation of Glucose Gelatin 5. Anaerobic PEA, egg yolk agar
Methods to Promote Anaerobiosis: A. GRAM (+) ANAEROBIC BACILLI
 Anaerobic Jars 1. Actinomyces – fungus-like bacteria
o Gas Pak Jar (Anaerobic Jar) - Catalase (-)
 Incubation Time: 48 Hours  Actinomyces bovis – lumpy jaw
 Gas Pak Envelope = CO 2 + H (Enclosed)  Actinomyces israeli – draining sinus tract with sulfur granule; Molar Tooth
 When opened: O 2 (that enters) react with H  H2O Colony
(Moisture): Indicator that the O2 has been removed 2. Bifidobacterium dentium – Oral and GIT (Causing Periodontitis)
o Mcintosh Fildes Jar 3. Eubacterium lentum - Oral and GIT (Causing Periodontitis)
o Torbal 4. Propionebacterium acne (Formerly known as Corynebacterium acne)
o Brewer - anaerobic diptheroid
- skin flora; blood culture contaminant
Note: Candle Jar is not anaerobic jar! It cannot be used for anaerobic organisms. - Catalase (+) and Indole (+)
 Chopped Meat 5. Lactobacillus
- anaerobic broth. Promotes spore formation - GIT and Vaginal Flora
- Growth: Turbidity - Increased during Child bearing years
 Thioglycollate medium - Inhibits Gardnerella vaginalis; Promotes Candidiasis
- Indicator: Resazurin (Pink). Seen at the surface, once it goes down, indicates - Catalase (-)
presence of O2. 6. Mobiluncus – motile organism causing vaginitis (G. vaginitis)
- Note: When exposed to O2, there will be no growth. In case of exposure,
BOIL or AUTOCLAVE to remove O2
- Stored at: Dark Room at Room Temp
B. GRAM (–) ANAEROBIC BACILLI (GIT FLORA)
 PRAS – Pre-Reduced Anaerobically Sterilized (Method: Roll Tube of Hungate) 1. Bacteroides fragilis (Colon Bacillus)
- needs 20% bile (Bile resistant)
Indicators of Anaerobiosis: - Specimen: peritoneal fluid, instestinal abscess
a. Methlyene Blue: Blue  Colorless (O 2 has been removed) - Black Colony on Bacteroides Bile Esculin Medium
b. Resazurin: Pink  Colorless (O 2 has been removed) - Catalase (+), Indole (-)
 All Bacteroides are Indole (-) except: Bacteroides
Identification: Kanamycin Vancomycin Colistin (KVC) Thetaiotamicrons
- presumptive test to Identify anaerobic bacteria 2. Porphyromonas and Prevotella:
- B. fragilis is the most important anaerobic bacteria since it a beta lactamase a. Porphyromonas asaccharolytica – black pigment, red fluorescence on UVL,
producer (penicillin resistant), which is seen in the GIT (normal flora). Also Van (S)
requires 20% Bile for growth (Bile Resistant) b. Prevotella melaninogenica – black pigment, red fluorescence on UVL, Van
- Porphyromonas is Vancomycin (S); (Bile Sensitive) (R)
- Prevotella is Vancomycin (R); (Bile Sensitive) 3. Fusobacterium
- Catalase (-), Indole (+)
Characteristics of Anaerobes a. F. nucleatum – breadcrumb colonies; fusiform rod (spindle shaped)
Brick red fluorescence Prevotella, Porphyromonas b. F. necrophorum – Synergistic effect with Borrelia vincentii (a spirochete
Red fluorescence Veillonella which is a trench mount agent ) causing Vincent’s angina (giginvitis/gum
Pitting of agar B. ureolyticus disease)
Double zone hemolysis C. perfringes 4. Bacteroides ureolyticus – pitting of agar
Swarming C. tetani, C. septicum (Gram +)
Molar tooth colony, sulphur granules Actinomyces israelii
Breadcrumb colony F. nucleatum
C. GRAM (+) ANAEROBIC COCCI 2. NITRATE REDUCTION TEST(Nitrate Broth)
1. Peptostreptococcus anaerobius – SPS sensitive , Catalase (-) indole (-)  PPL: NO3 → NO2
2. Peptostreptococcus asaccharolyticus - Catalase (-), indole (-)  Reagent: sulfanilic acid and a-napthylamine = (+) RED COLOR
3. Peptococcus niger (Staph-like) - Catalase (+)  If (-) COLORLESS: add ZINC DUST/POWDER to detect the presence of unreduced
nitrate
CATALASE INDOLE o Turns colorless: positive
Propionebacterium + + o Turns red: negative (unreduced nitrate)
Bacteroides spp. + -
Bacteroides thetaiotamicrons + -
3. NITRITE REDUCTION TEST
Fusobacterium spp. - +
 PPL: NO2 → N2 gas (DURHAM TUBE – Inverted Test tube f or Gas Production)
Peptostreptococcus anaerobius - -
 Reagent: sulfanilic acid, a-napthylamine + zinc dust
Peptostreptococcus asaccharolyticus - -
(+) COLORLESS (A. faecalis)
Peptococcus niger + -
(-) RED (A. piechaudii)
NOTE:
 H2O2 for the Catalase Test for Anaerobic Bacteria = 15% H2O2 (not 3%)
4. ONPG TEST
 ONPG via B-galactosidase to Orthonitrophenol
D. GRAM (-) ANAEROBIC COCCI  B-galactosidase degrades Lactose to Galactose
1. Veillonella parvula – fluoresce red UVL; oral flora; nitrate (+) o Rapid Lactose Fermenter – ONPG (+); B-galactosidase + Permease
2. Megasphera o Late Lactose Fermenter – ONPG (+); B-galactosidase only
3. Acidaaminococcus o Non Lactose Fermenter – ONPG (-)
Note: Lactose Fermentation Test: Pathogenicity test for Enterobacteriaceae
(Where NON LACTOSE are mostly pathogenic)
DEFINITIVE Test for Anaerobic Bacteria: (+) yellow (E.coli)
 GLC/HPLC (detects acid) (-) change in color (S. typh imurium)

Diagnostic test for GRAM (-) BACILLI: 5. LOA TEST / AMINO ACID ENZYME TEST
Note: Growth in MacConkey Agar indicates growth of Gram (-) Bacilli. BUT some  (+) purple (-) yellow
Gram (-) Bacilli does not grow in MacConkey. The following are few examples:  3 Enzymes:
a. Haemophilus – requires X and V Factor (not in Mac) 1. Decarboxylase (anaerobic): Lysine to Cadaverine
b. Brucella/Legionella – needs special growth factor (most bacteria 2. Dehydrolase (anaerobic): Ornithine to Putrescine
ending with –ella does not grow in Mac, except for Salmonella) 3. Deaminase (aerobic – Slant): Arginine to Citrulline
“De” – removal of amino group
1. OXIDASE TEST (Presumptive Test for Gram Neg Bacilli)  MEDIUM; Moeller’s Decarboxylase Medium
 Cytochrome oxidase (indophenol blue) o Indicator: Bromceresol Purple
 (+) P. aeruginosa (Bluish Purple)  MINERAL OIL: Prevents the entry of O 2
 (-) E. coli  Total of 4 Test Tubes (with 1 being the Control)
 (+) Lysine Decarboxylase: K.pneumoniae
Note:
 (-) Lysince Decarboxylase: E.cloacae
Oxidase test: Presumptive Test for Gram – Bacilli
Oxidative-Fermentative Test: Presumptive Test for Non Fermentative Organism
6. TRIPLE SUGAR IRON (TSI) 7. LYSINE IRON AGAR (LIA)
 Lysine deamination – slant (aerobic)
 Glucose + Lactose + Sucrose + Iron (Reacted by H 2S)  Lysine decarboxylation – butt (anaerobic)
o G:L:S Ratio (1: 10: 10)
 pH indicators:  Indicators:
a. Phenol Red = red to yellow (Acid Production) a. Bromcresol Purple
b. Ferrous Sulfate (H2S Production) = black b. Ferric NH4 Citrae (H2S Production) = black
 TSI Reaction:
NEG (-) LYSINE DEAMINASE PURPLE
K/K
ACID
Lactose Fermenter POS (+) LYSINE DECARBOXYLASE PURPLE
SLANT YELLOW  Hafnia
Lactose  Arizona NEG (-) LYSINE DEAMINASE PURPLE
A/A Sucrose Citrobacter K/A
Glucose


E. coli
NEG (-) LYSINE DECARBOXYLASE YELLOW
ACID 

BUTT YELLOW Enterobacter


POS (+) LYSINE DEAMINASE RED
 Klebsiella R/A
Non Lactose Fermenter NEG (-) LYSINE DECARBOXYLASE YELLOW
ALKALINE
SLANT RED  P-M-P H2S (+)Blackening due to Ferric Ammonium Citrate
 Salmonella
K/A Glucose
 Shigella Note: Summary of Some Indicators on Agars and Tests:
ACID Serratia 1. Phenol Red
BUTT YELLOW 

a. TSI
 Yersinia
b. XLD
ALKALINE Non Fermentative Organism c. MSA
SLANT RED  Pseudomonas d. Christensen’s Urea
K/K ALKALINE
None
e. BGA
BUTT RED
H2S Producer 2. Bromthymol Blue
H2S (+)Blackening due to Ferrous Sulfate  Salmonella a. HEA
 Proteus b. SCA
Gas (+)Splits Medium (Space) Aerogenic Organisms c. TCBS
d. Utilization Tests (Citrate, Acetate, Acetamide, Malonate)

 KIA – Kligler’s Iron Agar (G -L-S Iron) 3. Neutral Red

 RDA – Russell’s Double Agar (G -L) a. MacConkey
b. SSA
c. DCA

4. Bromcresol Purple = LIA

 DESCRIPTION REAGENT MEDIA INDICATOR POSITIVE NEGATIVE POSITVE NEGATIVE

Indole from
SIM
a. Amino Acid:
Kovac’s/Erlich’s Reagent H2S Indicator: Lead
8.INDOLE TEST Tryptophan
= PDAB Acetate
(+)RED (-) YELLOW
b. Enzyme:
Tryptophanase
Filter paper strips (Anaerobic
impregnated with Bacteria)
9. RAPID SPOT PDAB
INDOLE TEST
(+)BLUE
Screening for indole
production.
MRVP (broth) (Aka: (pH below 4.4); E. cloacae
10. METHYL RED Mixed acid of glucose
TEST (Opposite VP) fermentation
“Clark And Lubs Methyl Red (+)RED (-) YELLOW (E.coli)
Dextrose”)
BARRITS METHOD: (KESH)
A-napthol and 40% KOH  Klebsiella,
(GRAM NEG BACILLI –  Enterobacter
PRODUCT: Acetoin or
E. cloacae)  Serratia
acetylmethylcarbinol
11. VOGUES Hafnia
PROSKAUER TEST COBLENTZ METHOD: (+)RED (-) YELLOW 

A-napthol and 40% KOH

in creatine
(GRAM POS COCCI –
Strep. mutans)
Utilize Citrate as (P. aeruginosa) (E. coli)
12. CITRATE Simmon’s Citrate Bromthymol
UTILIZATION TEST
source of carbon and
Agar Blue
(+)BLUE (-) GREEN
energy
Acetate → alkaline pH (E. coli) (Shig.
13. ACETATE
(blue) Bromthymol flexneri)
UTILIZATION TEST
Acetate → acid pH Blue
(+)BLUE (-) GREEN
(green)
14. ACETAMIDE Acetamide → NH3 (P. aeruginosa) (S.
Bromthymol
UTILIZATION TEST (BLUE)
Blue
(+)BLUE (-) GREEN maltophilia)

(KECH)
15. MALONATE  Klebsiella,
Bromthymol
UTILIZATION TEST
Blue
(+)BLUE (-) GREEN  Enterobacter
 Citrobacter
 Hafnia
Product: (NH4)2CO3
16. UREA
HYDROLYSIS TEST
(Alkaline Ammonium Christensen’s Urea Phenol Red (+)RED/PINK (-) YELLOW
Carbonate)
17. PHENYLALANINE DEAMINASE (PAD) ENTERIC MEDIA (Selective and Differential)
 Organism is found at the slant since it is an aerobic enzyme Medium Inhibitory CHO Indicator LF NLF
 Presumptive test for Proteus, Morganella, Providencia EMB Eosin Y Lactose Eosin Y Red/purple Colorless
Methylene Methylene
blue blue
18. KCN BROTH TEST
Mac CV, bile salt Lactose Neutral red Red/pink Colorless
 (+) turbid (growth of organism) (-) clear (Salmonella )
XLD Bile salt Xylose Phenol red Yellow Red
o Klebsiella Lactose (E. coli) (Shigella or
o Enterobacter Sucrose NLF)
o Proteus/Providencia Black
o Serratia (Salmonella)
o Citrobacter HEA Bile salt Salicin Bromthymol Yellow Green
Lactose blue (Shigella or
Sucrose NLF)
19. STRING TEST
Black
 ID of V. cholerae (Salmonella)
 Reagent: 0.5% Na deoxycholate DCA Bile salt Lactose Neutral red Red/pink Colorless
 (+) String Like (V. cholerae) SSA Bile salt Lactose Neutral red Red Colorless
(E. coli) (Shigella or
20. ESCULIN HYDROLYSIS TEST NLF)
Black
 (+) Black (K. pneumoniae)
(Salmonella)
 (-) Yellow (S. flexneri) BSA Brilliant Glucose Bismuth Black colony – Salmonella
green sulphite typhi
21. MUG TEST (UVL) TCBS Bile salt Sucrose Bromthymol Yellow Green
 (+) Blue Fluorescence = E.coli (exceot. E. coli 0157 H7) blue (Vibrio)
 (-) No Fluorescence = P. aeruginosa Brilliant Brilliant Phenol Red Black colony - For other
Green A. Green Salmonella spp not S.
typhi
22. GELATIN HYDROLISIS TEST
 (+) Gel Liquifies = P. vulgaris Note:
 (-) Gel Solidifies ; E. aerogenes  All of the above inhibits the growth of GRAM (+), promoting the growth of GRAM (-
)
 BSA and TCBS are the only m edium above that do not have Lactose.
 BSA cannot differentiate LF from NLF, only selective to Salmonella typhi

Note: Summary of H2S Indicators:

1. TSI, and BSA = Ferrous Sulfate
2. LIA, XLD and HEA = Ferric NH4 Citrate
3. SIM = Lead Acetate
4. SSA = Ferric Citrate
ENTEROBACTERIACEAE
 All are glucose fermenters (A/A or K/A only). K/K (no sugar fermented) is not possible. E. coli vs. E.coli O157:H7
 All are motile (peritrichous flagella) except Klebsiella (capsulated) and Shigella  a. MUG
(noncapusulated) o E. coli = MUG (+)
 Presumptive Tests: o E. coli O157:H7 = MUG (-)
a. All are catalase (+) and reduce NO3 → NO2  b. MacConkey with Sorbitol
b. All are oxidase (-) except PLESIOMONAS o E. coli = (+)
Plesiomonas is related to Proteus and differentiatied by Oxidase Test o E. coli O157:H7 = (-) COLORLESS (Does not ferment sorbitol)
(Plesiomonas: Oxidase (+) while Proteus: Oxidase ( -))
 Selective: Mac, EMB, DCA, SSA, BSA E. coli Infections – Biotypes
 Ag:
 capsule (K) – heat labile (destroyed) Note: Biotype – based on serological tests; Serotype – based on
serological tests
 somatic/cell wall (O) – heat stable; most common in serologic typing
 flagella (H) – heat labile (destroyed)
 Vi Ag – removed by Boiling or Heating (for S. typhi) Enterotoxigenic E. coli Traveller’s diarrhea; E. coli O6;O8;O25
(ETEC) Watery diarrhea; Cholera-like toxin or heat
Lactose Fermenters Non Lactose Fermenters labile enterotoxin
Increases CAMP
 E. coli  P-M-P
activity = loss of Na, K
 Klebsiella  Serratia
and H2O causing
 Arizona  Salmonella*
dehydration, causing the
 Citrobacter  Shigella*
watery diarrhea
 Enterobacter  Yersinia*
Enteropathogenic E. coli Infantile diarrhea E. coli O111, O114
 Hafnia  Plesiomonas* – oxidase (+)
(EPEC) among children
*Pathogenic in Stool (pathogenecity
island)
Growth on MacConkey:
Enteroinvasive E. coli Dysentery (shigella) E. coli O124, 143, 164
Rapid Lactose Fermenters = Pink Colonies within 24 hours
(EIEC) like diarrhea
Late Lactose Fermenters = Pink Colonies within 48 hours
(invasin); Sereny test - virulence
Bloody with Mucus test for EIEC.
1. Escherichia coli (Colon Bacillus) (LF)
stool (+) Keratoconjuctivitis in
mice
Diseases:
Enterohemorrhagic E. coli Hemolytic uremic E. coli O157:H7
 #1 cause of UTI, #1 Bacteriuria(Cystitis) #1 Gram (-) sepsis syndrome (HUS),
(EHEC) - Burger is the source of
 #2 neonatal meningitis (K1 antigen) Or hemorrhagic colitis; this strain
 Nosocomial, wound, bacteremia, pneumonia Verotoxin E. coli (VTEC) Bloody urine and = MUG and Mac w/
 Specimen: Blood diarrhea. Sorbitol (-)
 Toxin: Endotoxin (systemic) Shigella like toxin –
Note: Normally seen in stool! Pathogenic in sto ol = diarrhea. verotoxin
Enteroaggregative E. coli Acute & chronic diarrhea (aggregative adhesion
Biochemical Reaction: (EAEC) fimbriae)
 TSI: A/A GAS(+) IMVIC: + + – – (Indole +) st
Note: For identification of E. coli O157:H7, once found colorless on Mac w/ Sorbitol (1 ),
 LOA: + + – nd
perform biochemical test (2 ), then serological test
rd
 Greenish metallic sheen on EMB (Slide Agglutination: Bacteria + Anti-sera = (+) Agglutination) (3 ).
2. Enterobacter (LF)
 TSI: A/A GAS(+) IMVIC: – – + + 4. Arizona spp. (LLF) – related to Salmonella (Salmonella Arizona)
 Urease (-) except E. gergoviae  Salmonella that is a Lactose Fermenter (Since Salmonella is a NLF)
 UTI, wound, septicaemia  TSI: A/A GAS(+) H2S (+), ONPG (+), LIA: K/K (LDC +)

LOA Reaction of Enterobacter 5. Citrobacter (Citrate) (LF)

Lysine Ornithine Arginine  TSI: A/A GAS(+) H2S ( +/- ), LIA: K/A (LDC – ), ONPG: (+)
E. aerogenes (+) + (–)  Related to Salmonella
E. gergoviae + + –
Hafnia alvei + + – UREASE LDC
E. cloacae (–) + (+) Citrobacter + –
E. sakazakii – + + Salmonella – +
E./ Pantoea agglomerans – – –

Notes: Species:
 E. aerogenes and E. gergoviae is differentiated by Urease: i. C. freundii
o E. aerogenes = Urease (-) - UTI, pneumonia, endocarditis
o E. gergoviae = Urease (+) - can cross-react(Ag sharing) with Salmonella typhi
 E. aerogenes commonly used as (+) Control for Lysine Decarboxylase
 E. cloacae commonly used as (-) Control for Lysine Decarboxylase and (+) UREASE Mac
Control for Arginine Dehydrolase C. freundii + LF
 E. aerogenes and E. cloacae can also be differentiated by Arginine test. S. typhi – NLF
 E. sakazakii and agglomerans are Yellow Pigment Producers

3. Klebsiella pneumoniae (LF) ii. C. diversus (C. koseri)

 TSI: A/A (+)GAS; - INDOLE (+) (Like E. coli)
 LIA: K/K (LDC +) - nursery outbreak neonatal meningitis
 LOA: + – – IMVIC: – – + + - mistaken as E. coli
 Differentiated with Enterobacter by its:
o Mucoid Colony; Entero - Dry INDOLE LDC
o Non Motility; Entero - Motile C. freundii + –
 String Test (+) Urease (+) Malonate (+); = SUM (+) E. coli + +
 All Klebsiella are Indole (-) except K. oxytoca – Indole (+) (Mistaken as E.
coli; can be differentiated by its Mucoid Colony.

Disease: Citrobacter Spp. Differentiation:

 Pneumonia, wound, meningitis, UTI TSI Indole Malonate
C. freundii A/A, GAS (+) H2S – +
Lysine decarboxylase VP & Urease Indole C. diversus/koseri A/A, GAS (+) + +
K. pneumoniae + + - C. amalonaticus A/A, GAS (+) + –
K. oxytoca + + +
K. ozaenae malonate (-) + - -
K. rhinoscleromatis malonate (+) - - -
 6. Proteus-Morganella-Providencia Group – NLF Species:
 ALL are PAD (+)  P. vulgaris – Indole Positive 
 ALL are Urease (+) except Prov. alcalifaciens - (Source of OX2 and OX19 – used in Weill Felix Test for Rickettsial
 ALL are Indole (+) (like E. coli and C. diversus) except P. mirabilis Antibody Detection)
 ALL are LOA (– – –) except: Morganella and P. mirabilis (ornithine +)  P. mirabilis – INDOLE NEGATIVE!! (All PMP group are INDOLE (+)!!! 
 Lysine deamination (+) = LIA: R/A (First mentioned to be R/A) Huhu.
 ALL can cause WOUND INFECTIION - (Source of OXK)
- Common Isolate than P. vulgaris
Providencia
 Citrate (+), PAD(+)IMVIC = + + – (+)

 P. alcalifaciens – UREASE NEGATIVE!! (All PMP group are UREASE (+)!!!  Huhu.
Proteus – Morganella- Providencia

TSI: K/A, GAS( +), H2S (+) TSI: K/A, GAS( +) Morganella
(Swarming) (NO Swarming)  PAD (+), IMVIC: + + – (–), (Like E. coli)

PROTEUS PROVIDENCIA  Has similiarity with the swarmer Proteus (P. vulgaris) = Ornithine (+)
MORGANELLA (Morganella and P. vulgaris = the only Ornithine (+) in PMP Group)
I
CITRATE
Morganella vs. Citrobacter! FIGHT!!
(+) (-) CITRATE Mac
PROVIDENCIA MORGANELLA Morganella – NLF
Citrobacter + LF

Proteus
 SWARM on BAP , (Burnt Chocolate Agar) TSI Urease Ornithine
 TSI: K/A GAS(+) H2S (+); LIA: R/A; PAD (+) M. morgani K/A Gas(+) + +
 Related to PLESIOMONAS! Differentiated by Oxidase Test! Plesiomonas is the Prov. Stuartii K/A Gas(+) +/ – –
only Oxidase (+) of all the Enterobacteriaceae spp.) Prov. Rettgeri K/A Gas(+) + –
Prov. Alcalifaciens K/A Gas(+) – –
OXIDASE P. vulgaris K/A Gas(+) H2S + –
Proteus – P. mirabilis K/A Gas(+) H2S + +
Plesiomonas +
7. Salmonella (NLF)
 #2 UTI, renal stone (urinary calculi) = due to UREASE = virulent factor that can  MacConkey = Colorless
lead to alkaline product Ammonium Carbonate leading to renal st one  Producing Black Colony in the ff:
 Considered as CONTIMINANT (If present in urine, perform colony count. If no  SSA
growth = CONTAMINANT, if there is growth = UTI.  XLD
 HEA
Proteus vs. Salmonella! FIGHT!!  BSA
UREASE KCN  TSI: K/A GAS(+) H2S (+)(Like Proteus) LIA: K/K
Proteus + +
Salmonella – – REMATCH: Proteus vs. Salmonella! FIGHT!!
UREASE KCN
Proteus + +
  All are GAS PRODUCER (Aerogenic) except S. typhi, S. gallinarum  Related to E. coli but acetate (+)
(TSI: K/A H2S(+)  Dysentery
 All are MOTILE except S. gallinarum, S. pullorum  Dx: culture – fresh stool with mucus flecks and rectal swab of ulcer
 All are H2S(+) and LDC (+) except S. parathyphi A
 All are LOA (+ + +) SHIGELLA SPP. O Ag Mannitol Ornithine ONPG
S. dysenteriae A - -
Salmonella spp K/A GAS(+) H2S (+) LDC (+) S. flexneri B + -
S. paratyphi A K/A GAS(+) H2S (-) LDC (-) S. boydii C + -
S. typhi and S. gallinarium K/A GAS(-) H2S (+) LDC (+) S. sonnei D + +
Salmonella Shigella
Motility + –
S. typhi Serology H2S + –
 Antigens of S. typhi: Vi, O, H Ag (Requires serologic typing) LOA +++ –––
 Without serologic typing, reported as “ Salmonella spp.” LIA K/K LDC(+) K/A LDC (-)
 Heating removes Vi Ag! Indole – +
 Somatic (O) Group where S. typhi is (+) = Group D (causing typhoid fever)
 S. paratyphi A = (H2S and LDC -) Invasive – +
 Related to Citrobacter. Take note that Salmonella is generally LDC + Blood culture + –
Related Citrobacter E. coli (due to Indole +)
UREASE LDC
Citrobacter + – Note:
Salmonella – +  S. dysenteriae is also known as Shiga Bacillus

 S. sonnei is the only Ornithine (+) among all Shigella spp.

Species and Diseases:  S. sonnei cross reacts with Plesiomonas shigelloides.

 S. typhi (MARY = spread the typhoid fever!!!!) OXIDASE

o Typhoid Fever S. sonnei –
st
Specimen: 1 week = Blood P. shigelloides +
nd
2 week = Stool (Carrier Sample)
o Meningitis, Osteomyelitis
 S. paratyphi A and B – paratyphoid fever 9. Serratia marcescens (NLF)
 S. paratyphi C (S. cholera suis) – septicimia
 S. enteritidis – #1 gastroenteritis in Philippines (poultry)  Normal in the stool but still need to do Susceptibility Test (since it is an
 Other spp – food poisoning Opportunistic Infection)
 Ability to produce RED PIGMENT (prodigiosin) at ROOM TEMP (22 or 25’C)
8. Shigella spp (NLF) at MUELLER HINTON AGAR or NUTRIENT AGAR.
 Biochemically inert/Negative (Almost no reaction – Walang kakwenta  ENZYMES:
kwentang bacteria! o Dnase: (2 (+) Controls for DNAse Test – S. aureaus & S. marcescens)

o Non motile, o Lipase

o Colorless on SSA (Since it cannot produce H2S) o Gelatinase

o Acetate (-)  Opportunistic and Drug resistant (beta lactamase producer)

 TSI: K/A only. LIA: K/A (LDC –)  Nosocomial (UTI, bacteremia, pneumo)
 Note: Citrobacter, S. paratyphi A, Shigella are ALL LDC (–)  TSI: A/A or K/A GAS (+/-)(Can be LACTOSE or NON-LACTOSE) NaSIRAan na
 ALL are LOA ( ) except S. sonnei (ornithine +) (Like Morganella and P. SIYA, hindi na niya alam kung ano siya. Serratia! Haha weh.
 11. Yersinia (NLF)
Differential Test for Salmonella, Shigella, and Serratia
A. Y. pestis (plague bacillus) = Coccobacilli!
TSI  “Safety pin ”, Bipolar Bodies - Wayson Stain is used (Similar to
Pasteurella)
K/A, GAS( +), H2S (+) K/A
SALMONELLA SHIGELLA  Antigens: V and W Ag
SERRATIA  STALACTITE (+): When Yersinia grown in a broth, it grows like a
I Stalactite
 UREASE (–) (Like Entrobacter, P. alcalifaciens, Salmonella)
(Non Motille) (Motile)  LOA (– – –) (Like P. vulgaris, Providencia, Shigella)
SHIGELLA SERRATIA
 The only Entero spp. that is acquired through insect bite ( Rat flea bite);
(Red Pigment)
 Bioterrorism Agent
Disease: Bubonic/Pneumonic/Septicemic plague; Black death

B. Y. enterocolitica
O O
10. Edwardsiella tarda (NLF)  Motile at 22 C but not at 35 C
 Pathogenic in stool, can also be contaminant (like Proteus)  Blood Bank Contaminant
 TSI: K/A GAS(+) H2S (+) (Similar to Salmonella). Differ in INDOLE.  TSI: A/A, Ornithine (+), Urease (+), ONPG (+)
O
Note: Indole (+) (Like: E.coli, C. diversus, PMP, Shigella)  Cold enrichment at 4 C (Similar to Listeria that grows at ref temp)
 Acquired by drinking unpasteurized milk
INDOLE Disease : Appendicitis, Enterocolitis
Edwardsiella +  “Bull’e eye colony” on CIN (a selective media for Y. enterocolitica and
Salmonella – Aeromonas). To differentiate, OXIDASE Test.

 IMVIC: + + – – (Similar to E.coli) OXIDASE

Y. enterocolitica –
Mac Aeromonas +
Edwardsiella NLF
E. coli LF
C. Y. pseudotuberculosis
 UREASE (+)
 Lysine decarboxylase (+)
 LOA (– – –)
 Diarrhea, wound, bacteremia
Disease: Mesenteric lymphadenitis , sepcticemia

Y. pestis Y. enterocolitica Y. pseudotuberculosis

Motility – + (22’ C) +
Urease – + +
Ornithine – + –
Sucrose – + –
VIBRIONACEAE – Vibrio, Aeromonas, Plesiomonas 3. O129 SENSITIVITY TEST (S) = (+) Zone of Inhibition
 All are OXIDASE (+) CATALASE (+), INDOLE (+)
4. POLYMIXIN B SUSCEPTIBILITY TEST
 All ferment glucose; polar flagella
- to detect the Biotype
 Classical (S) = (+) Zone of Inhibition
VIBRIO
 El Tor (R)
 “Comma Shaped”, motile (monotrichous)
 “Darting Motility”
5. CHOLERA RED TEST – Nitrate (+) and Indole (+) = (+)RED
 OXIDASE (+) except V. mitschnikovii
 HALOPHILIC except V. cholerae, V. mimicus (seen
2. V. parahemolyticus
 O129 Susceptible
 HALOPHILIC (8% NaCl), BETA HEMOLYTIC, Indole (+), LOA: ++-
 Vinegar – used to counteract Vibrio
 NON SUCROSE fermenter (green TCBS)
 Alkaliphilic, LOA: ++-, Nitrate reduction (+)
 KANAGAWA (+): Beta Hemolysis on Wagatsuma Agar
 #1 Gastroenteritis in Japan (seafood)
1. V. cholerae
 NON HALOPHILIC (1-3% NaCl), Indole (+)
 Rice watery stool (cholera)
Disease 8% NaCl Sugar TCBS
 Transport Medium: CARY-BLAIR
Fermented
 STRING TEST (+) (0.5% Na desoxycholate) Cholera Sucrose Yellow
V. cholerae –
 SUCROSE Fermenter (Yellow on TCBS ) (Rice watery (Non
 Vibrio cholera O-1: Pandemic Cause of cholera (Global) stool) Halophilic)
Biochemical Tests V. alginolyticus Gastroenteritis + Sucrose Yellow
BIOTYPE CLASSICAL EL TOR V. Gastroenteritis + Arabinose Green
Polymixin susceptibility S R parahemolyticus
Lysis by bacteriophage + – V. vulnificus Septicimia, + Lactose Green
Chicken RBC agglutination – + wound
Hemolysis of sheep RBC – +
VP test – + VIBRIO AEROMONAS PLESIOMONAS
NaCl reqt + – –
Note: El Tor – common cause of cholera
Motility + + +
Serologic Tests Oxidase + + +
Serotype Ogawa Inaba Hikojima O129 S R S
Anti-Ogawa + – + LOA ++– +–+ +++
Anti-Inaba – + +
Dnase, – + –
Lab Diagnosis of V. cholera O-1 Beta-BEM
1. DARKFIELD Microscopy TSI A/A or K/A A/A (+)Gas (+)H2S A/A or K/A
- “Darting Motility”
Disease All cause diarrhea, wound, septicimia

2. CULTURE
Note:
a. APW (Alkaline peptone water): (6-8 hrs) An enrichment broth
 Aeromonas and Plesiomonas is seen in Fresh water
needed to be subcultured in TCBS
 Vibrio and Plesiomonas can be LF or NLF
b. TCBS Selective media for Vibrio
CAMPYLOBACTER curved, seagull wing Diagnosis:
 Curved, S Shape, Seagull Wing Urea Breath Test
 Oxidase, catalase (+) - Rapid test for H. pylori
13 13
 Urease (-), Indoxyl Acetate (+) - C  Urea  If Urease (+)  CO2 (Exhaled) (Uses radiation)
 “Darting Motility” (Similar to Vibrio) Differ in: Scintillation – counter radiation
o Morphology (S-Shape vs Comma Shape)
o Microaerophilic (5% O2 10% CO2 85% N2)
 Growth at 42’C; Zoonotic NON FERMENTATIVE ORGANISMS
 Suturella wadworthiensis – resembles Campylobacter  Gram (-) Rod
Species  TSI = K/K, Oxidase (+/-), Mac(+/-)
 C. jejuni  Obligate Aerobes (48-72 hours incubated)
- #1 cause of Gastroenteritis in US  Grow best at 37’C except: P. fluorescens and P. putida at RT;
- Guillain Barre syndrome (paralysis)  Requires AST (drug resistant)
- Hippurate differentiates C. jejuni from other spp.
 C. fetus Lab Diagnosis for NFO
- associated with animal abortion 1. O-F TEST (2 Test Tubes)
 Important to detect the type of action of carbohydrates (Increase CHO,
Note: Summary of #1 Gastroenteritis Decreased Peptone)
 Salmonella enteritidis - #1 in Philippines  Media: HUGH AND LEIFSON MEDIUM (Semi-solid)
 Vibrio parahemotlyticus = #1 in Japan - inoculate first the organism before the mineral oil, since it has an
 Campylobacter jejuni = #1 in US inhibitory effect to the bacteria
 1% glucose, 1% agar, low peptone
O O
37 C 42 C Nalidixic Cephalothin Hippurate  BROMTHYMOL BLUE – indicator
C. jejuni + + S R +  (+) YELLOW (acid) (-) GREEN (no acid)
C. coli + + S R -
C. fetus + - R S - OPEN MEDIUM CLOSED MEDIUM ORGANISMS
(Oxidative) (Fermentative)
Helicobacter pylori OXIDIZER Yellow Green NFO
 Formerly “Campylobacter pylori, but became “Helicobacter” since its is Helical + –
not curved FERMENTER Yellow Yellow Enterobacteriaceae,
 Pylori = “Pylorus” (seen in stomach + + Vibrio
 Causes Peptic Ulcer and Gastritis NON UTILIZER Green Green NFO
 Urease (+) - differentiate H. pylori from C. jejuni (Assacharolytic) – –
 It can also grow in Butzler/Skirrow Media since it is still a member or
O O
Campylobacter 2. GROWTH at 42 C = (+) growth at 35 & 42 C
 Used to differentiate CONTAMINANTS
Differentiation of Campylobacter & Helicobacter (P. aeruginosa and P. fluorescens)
Oxidase,  (+) P. aeruginosa (-) P. fluorescens
catalase, Urease Media Disease
O
microaerophile 3. CETRIMIDE TEST = 35 C for 7 days
Butzler  Media: Cetrimide Agar/Pseudosel Agar (containing detergent)
Diarrhea,
C. jejuni + - Skirrow  (+) growth (P. aeruginosa)
abortion
CBAP  (-) no growth (E. coli)
PSEUDOMONAS SPP  Wound – Ecthyma gangrenosum (Black)
 All are OXIDASE (+) and DNASE (-) except S. maltophilia  Swimmer’s Ear – “Otitis Externa”
 All are MOTILE except B. mallei  Contact lens infection; Sore Eyes
 MacConkey (+)  Dermatitis – “Jacuzzi”
 TSI: K/K (No Sugar Fermented since NFO)
2. Burkholderia cepacia
 O-F = Oxidizers (Yellow (O) – Green (F))
 Contaminants in Alcohol!
 Opportunistic infection (Found in environment)
 Associated with IV Drug Users (Important cause of I atrogenic infections)
1. Pseudomonas aeruginosa – B. pyocyaneus  Motile = LOPHOTRICHOUS
 Motile = MONOTRICHOUS  Lysine decarboxylase (+) (P.aeruginosa is LDC - )
 O-F test = +/- (Oxidise GLUCOSE)
“Blue pus Agent” Colonies:


 Pink colony on Mac (LACTOSE OXIDER)

 Grape like / Corn Tortillas Odor (Apple Like = Alkaligenes faecalis)
 Yellow Colony on (OFPBL) or PC agar
 Resistant to Disinfectants!
 YELLOW PIGMENT producer (similar to P. stutzeri)
 NF Organism that has the ability to produce mucoid colony due to its Slime
Layer (differ in B. subtilis by its dry colony)
PIGMENT COLONY OXIDIZER
Biochemical Tests: B. cepacia Yellow Pink/Yellow Lactose Oxidizer
 CETRIMIDE (+), ACETAMIDE (+) Mac (+) NITRATE REDUCTION(+) P. stutzeri Yellow Wrinkled Non-Lactose Oxidizer
 TSI: K/K, LIA: K/A (Lysine Decarboxylase – )
 Pseudosel Agar (Cetrimide Agar) is used for the selective isolation of Diseases:
Pseudomonas aeruginosa.  #2 Cystic fibrosis

O
PYOCYANIN Test and GROWTH at 42 C (used to differ from P. fluorescens –  septicaemia, pneumonia
also a blood bag contaminant)  Earthy or Dirt Like Odor (drug R)

PYOCYANIN TEST GROWTH AT 42’C 3. Burkholderia pseudomallei

P. aeruginosa + +  Motile = LOPHOTRICHOUS
P. fluorescens – –  Melioidosis or Glanders like
 “Vietnamese Time Bomb”
 Lactose Oxidizer
Pigments:  Wrinkled colony (ashdown medium) (Similar to P. stutzeri)
a. PYOCYANIN (Blue-Green Color) – best characteristic to identify P.
aeruginosa and differentiate it from other Pseudomonas spp. COLONY OXIDIZER
b. FLUORESCEIN (Yellow-Green Color) (also pigments of P. putida, P. B. pseudomallei Wrinkled Lactose Oxidizer
fluorescens; P. stutzeri Wrinkled Non-Lactose Oxidizer
c. Pyorubin, Pyomelanin, Pyoverdin
O
 Growth at 42 C (like P. aeruiginosa)
Diseases:  O-F = +/- (Lactose Oxidizer)
 #1 Opportunistic Infection in Immunocompromised Patients
 #1 Nonfermentative Organism 4. Burkholderia mallei
 #1 Cystic fibrosis (Punong puno ng plema. “Siya ay plema na naging  Only NON MOTILE Pseudomonas
tao!” – Aldave)  Glander’s disease (horses)
 #2 burn (#1 S. aureus)  O-F = +/- (Glucose, Maltose, Lactose)
 5. Pseudomonas stutzeri 2. Alkaligenes faecalis
 Brown (buff colored) Wrinkled Colony  Oxidase & catalase (+)
 6.5% NaCl (+), NO2-N2 gas, Non-Lactose Oxidizer  Motile (PERITRICHOUS FLAGELLA)
 Asaccharolytic = O/F test -/-
6. Stenotrophomonas maltophilia  APPLE like “fruity” odor
 The only OXIDASE (-) and DNAse (+)  UTI, wound, diarrhea
 Lysine Decarboxylase (+) (like B. cepacia)
 Associated with Catheter Infection (Iatrogenic Infection) 3. Moraxella lacunata – Morax Axenfield
 Nosocomial Infection  Blepharoconjunctivitis (Conjuctivitis Agent)
 O-F = +/ (Glucose, Maltose)  Oxidase (+) & catalase (+)
 Ammoniacal Odor  Asaccharolytic ;
 Lavender Green colony  MacConkey (-)(All -ella are negative in Mac, except Salmonella)
 Mistaken as Neisseria (gram – CB)
7. Shewanella putrefaciens
 TSI: K/K H2S; Oxidase (+) CHO FERMENTATION
 The only H2S Producer Pseudomonas M. lacunata –
Neisseria +
Summary of Sugar Oxidized
Pseudomonas Spp. SUGAR OXIDIZED 4. Chryseobacterium (Flavobacterium) meningosepticum
Pseudomonas aeruginosa Glucose  (+) Oxidase, (+) Dnase, (+) Gelatin Hydrolysos, (+) indole (Generally positive
Burkholderia cepacia Lactose in biochemical tests)
Burkholderia pseudomallei Lactose
 Yellow Pigment Producer (FLAVIN) (Chrys = “ Kris Aquino – Yellow”
Burkholderia mallei Glucose, Lactose, Maltose
 (like B. cepacia and P. stutzeri)
Pseudomonas stutzeri Non-Lactose
Stenotrophomonas maltophilia Glucose, Maltose
 Non motile; MacConkey (-)
 Neonatal meningitis, bacteremia

Other Non-Fermentative Organisms 5. Eikenella corrodens

 MacConkey (-)
1. Acinetobacter
 Human bite wound “clenched fist”
 #2 Isolate of NFO next to Pseudomonas
 Corrodes agar, bleach like odor
 Mistaken as Neisseria (gram – CB)
OXIDASE 6. Kingella spp
Acinetobacter –  Cause SBE (HACEK); pits the agar
Neisseria +  MacConkey (-)

 OXIDASE (-), CATALASE (+), Non Motile

OXIDASE CATALASE MOTILITY Oxidase Catalase Mac
Acinetobacter – + Non Motile Acinetobacter – + +
Enterobacteriaceae – + Motile Alkaligenes + + +
Flavobacterium + + –/+
 Growth on MacConkey Moraxella + + –
a. A. anitratus (oxidizer) aka: Herella vaginocola Kingella + – –
b. A. iwoffi (non oxidizer) aka: Mima polymorpha Eikenella + – –
PARVOBACTERIA  H. aprophilus is the only Haemophilus that can grow anywhere (X Factor, V
 Gram (-) coccobacillus, fastidious (hard to culture) Factor, and X&V Factor)
 NO GROWTH IN MacCONKEY!
1. H. influenzae (Pfeiffer’s bacillus) – GNCB
A. Haemophilus  HiB – Haemophilus influenza TYPE B (Serologic Type)
 Oxidase (+/-)  Formation of SATELITE around S. aureus in BAP media
 Medium: CAP (Horse Blood) + 5% CO2  Used as a POSITIVE CONTROL for BETA LACTAMASE Test
 Requires X (hemin) and V (NAD)
Disease:
 Satellitism (H. influenzae)
 #1 Acute Epiglottis
 Note: NOT cause of influenza (It’s the flu virus)
 #3 Meningitis (CSF), (#1 Strep. Pneumo, #2 N. meningitides)
 Gram Stain: Gram Negative Cococcobacilli
 Pneumonia (among children) (Sputum)
Differential test for Haemophilus  Otitis media, cystic fibrosis, conjunctivitis, pneumonia, URT infection
Porphyrin X Factor V Factor  RARE isolate of wound infection
H. influenzae – + +
Lab Diagnosis:
H. aegypticus – + +
1. CULTURE: CAP, Levinthal Agar, Fildes – “Dew Drop Colony”, Mousy
H. haemolyticus – + +
Odor
H. parainfluenzae + – +
2. X AND V TEST in MHA
H. parahemolyticus + – +
3. SATELLITISM (+) in BAP with S. aureus
H. paraphrophilus + – +
- BAP (contains X Factor)
H. ducreyi – + – - S. aureus (supplies the V Factor)
H. aphrophilus + – – Sources of V factors:
Note: 1. S. aureus
 The hemolysis of H. haemolyticus and parahemolyticus is seen in Horse Blood 2. Strep pneumo
Agar. 3. Neisseria
 Growth Factor Tests: X and V Test, Porphyrin Test, Sattelitism 4. Candida albicans
o X AND V TEST – presumptive test for Haemophilus 4. Porphyrin test (-) (Since it requires X factor)
- the media shouldn’t have X and V Factor (Ex. MHA)
- The X and V factor should be external source (not yet
incorporated on the agar) 2. H. ducreyi (chancroid) – X factor
o PORPHYRIN TEST (X Factor) - Delta Aminolevulinic Acid (ALA) is  Soft chancre; (hard Chancer = Syphilis)
converted to Protoporphyrin (Porphyrin) = (+) Red Fluorescence. Those  “School of red fish” = cluster of gram negative rods
who are (+) in Porphyrin does not require X Factor.  Specimen: Genital sample (non sterile)
 Growth on CAP + Vancomycin (since the specimen is non sterile)
Example: (CSF should prioritize CAP as its Medium for most probably causative  Grows at 33’C
agents are Neisseria or Hemophilus)  Treatment: Erythromycin
1. If CAP (+) BAP (-) Mac (-) = (H. influnzae)
2. Perform X and V Factor Test in MHA
st
1. Put the bacteria (1 ) 3. H. aegypticus (X and V)
nd
2. Apply the X, V, and X & V Factors (2 )  Koch’s week bacillus
If the bacteria grows near X, means that bacteria requires X factor for  Pink eye conjunctivitis ; Brazilian purpuric fever
growth, but could also grow in X & V. If the bacteria grow in V, bacteria requires V
factor, but could also grow in X & V. For the case of H. influenza, H. ae gypticus, and H.
B. Bordetella pertussis C. Brucella spp
 Capsule, obligate aerobe  No capsule, obligate aerobe, non motile
 Requires CYSTEINE and METHIONINE  Zoonotic – Erythritol (found on animal placenta) (Important cause of ANIMAL
 “Whooping Cough bacillus” ( Lower Respiratory Infection) ABORTION)
 Vaccine: DPT (Diptheria Pertussis Tetanus Toxoid) o B. abortus – cattle
 3 Stages: o B. melitensis – goat and sheep
o Catarrhal o B. suis – swine
o Paroxysmal – best time to do collection of specimen o B. canis – dog
o Convalescence  Used as Ag for Febrile Agglutination Test
 Nasopharyngeal swab (carrier). Since B. pertussis is toxic to cotton, it requires Disease:
Charcoal for the media to remove toxin. - Brucellosis, Endocarditis
Culture media: (NEVER GROW ON BAP, CAP, MAC) - Undulant Fever, Malta Fever
 Potato Blood Glycerol Agar (Sheep’s Blood) or Bordet Gengou agar - Commonly reported laboratory acquired infection
– “Mercury Drop or Pearl Like C olony”
– not good since Sheep’s blood is the source Specimen: Blood
 Regan Lowe (charcoal horse blood) – best since it has charcoal and
horse blood is the source. Culture Media:
 Charcoal cephalexin blood agar (CCBA) – better than PBGA  Castaneda broth (biphasic media)
 Jones Kendrich (charcoal, yeast extract)  TSB (aerobic since Brucella is aerobic)
 Stainer and Scholte, Casamino broth  W medium (Wiskottsin Medium)
 CAP
 Rose Bengal (+), 2-mercaptoethanol aggt
Spp Motile Urease Oxidase Mac, BAP  B. abortus and B. suis are H2S producers.
B. pertussis – – + –  B. abortus causes Bang’s Disease.
B. parapertussis – + – +  B. abortus is the only Brucella that requires CO2
B. bronchiseptica + + + +
Uses Fuchsin and Thionine Dye Inhibition Test for differentiation
 (-) No growth = Inhibited
Note:  (+) With growth = Not Inhibited
 B. pertussis is the only human pathogen. The other two are animal pathogen.  B. abortus is inhibited by Thionine
 B. bronchiseptica is KENNEL COUGH BACILLUS  B. suis and B. canis is inhibited bu Fuchsin
 Oxidase and Urease are important biochemical tests to differentiate bordetella  B. melitensis is not inhibited by Thionine or Fuchsin
spp.
 Clue: B. pertussis is OPPOSITE of B. parapertussis in Urease, Oxidase and Differential test for Brucella
Mac,BAP while B. bronchiseptica is (+) on all tests. Urease CO2 Thionine Fuchsin
B. abortus (Bang’s) + + – +
B. melitensis + – + +
B. suis + – + –
B. canis + – + –
D. Francisella tularensis HACEK: Agent of SUB BACTERIAL ENDOCARDITIS (SBE),
 Requires CYSTINE (not Cysteine) BAP (+) CAP (+), Requires CO2, (+) Mac ( -)
 Related to Pasteurella (Zoonotic) (Non motile, ENCAPSULATED) All can be seen in Blood Culture = Endocarditis
 Obligate aerobe
 CATALASE (+) BETA LACTAMASE (+) Description OXIDASE CATALASE
 OXIDASE (-) UREASE (-)
H Haemophilus aprophilus Does not require X and V +/ – –
Disease: Actinobacillus Star-like Colony – +
 Tularemia (deerfly, lemming, rabbit, water rat trapper’s disease) – the ONLY A Actinomyctemcomitans
PARVOBACTERIA that can be acquired through insect bite (Vector-borne) (Aggregatibacter)
 Lymphadenopathy (Specimen = Lymph Node) C Cardiobacterium hominis Indole (+) + –
 Lab acquired infections Eikenella corrodens Assacharolytic + –
E
K Kingella kingae Twitching Motility + –
Culture media:
 GCBA – GLUCOSE CYSTEIN BLOOD
 PCA – PEPTONE CYSTEIN AGAR
 CHA – CYSTEINE HEART AGAR SPIROCHETES
CATALASE DIAGNOSIS DISEASE
E. Pasteurella multocida – GNCB Treponema – Serology Syphilis, yaws, bejel, pinta
 “Multocida” = Multiple Killing Leptospira + Culture Weil’s
 (Non Motile, ENCAPSULATED like Francisella) Borrelia – Giemsa serology Lyme, relapsing fever
 Bipolar stain “safety pin” (Similar to Yersinia)
 MOTL Note:
o Yersinia - Insect bite  All of the above do not grow in culture
o Pasteurella – Animal Bite (oral flora of certain animals)  Leptospira is easily cultured
 Oxidase (+) Catalase (+) Glucose (+)  Weil’s Disease (aka Infectious Jaundice) is leptospirosis.
 Ornithine (+) Indole (+) Urease (+)  Borrelia is Vector BorneLyme Disease is Borreliosis.
 Grow on BAP but not in MacConkey (-ella)  Giemsa is used as a stain for Borrelia. Giemsa is color Purple.
 Animal bite wound infection, pneumonia, endocarditis, meningitis, arthritis
1. Treponema pallidum
Note:  Non cultivable on agar medium
 Dew Drop Colony = H. influenzae  Obligate intracellular (rabbit’s testicle)
 Mercury Drop (Pearl-Like) Colony = Bordetella  Killed at 4’C after refrigeration
 Cysteine and Methionine = Bordetella
 Fuchsin and Thionine = Brucella
Syphilis (Syn – Together) (Pillis – Love) = So this is a product of LOVE ♥♥♥
 Cystine = Francisella
 Primary – hard chancre (can be seen orally)
 L-Cysteine = Legionella pneumoniae
 Secondary – condylomata lata (genital lesion), skin rash – BEST time to do
 Sterol = M. pneumoniae
serologic test (many Ab present)
 Latent – asymptomatic (serology)
 Tertiary – gummas
 Congenital syphilis – stillbirth, abortion
End Result: Cardiovascular Disease and Neurosyphilis (“Kaya pag nagmahal ka,
 Torch Test - serologic test for congenital infections 3. Borrelia (Blood spirochete)
Treatment: Penicillin - can be seen on blood smear
st
1 treatment for Syphilis : Salvarsan (associated Hexheimer Reaction – a post - the spirochete that is arthropod-borne/ acquired through insect bite
treatment reaction) - Spirochete that can be seen in haematology (due to the use of Giemsa Stain)

Other Treponemes A. Relapsing fever due to

 T. pertenue – yaws  Borrelia recurrentis – (EPIDEMIC Relasping Fever) Louse Bite
 T. carateum – pinta  Borrelia anserina, toricatae, parkeri – (ENDEMIC Fever) Tick bite
 T. endemicum – bejel Diagnosis: Presence in Blood or Bone Marrow
Stain: Giemsa or Wright Stain
Lab Diagnosis
 Darkfield microscopy – Corkscrew Motility (CONFIRMATORY) B. Lyme disease (B. burgdorferi)
 Levaditi Silver impregnation – brown to black  #1 Tick Bite Borne Disease in America
 Serology  Vector: Tick bite (Ixodes dammini)
O
a. VDRL, RPR, TRUST (both presumptive and confirmatory) (reagin test) -  1 stage – Erythema Chronicum Migrans (ECM)
O
FLOCCULATION  2 stage – meningitis
O
b. FTA-ABS (best), TPHA, MHA-TP, HATTS (Trep. Ab test)  3 stage – arthritis
O
 Culture on Barber Stoenner Kelly (BSK) at 33 C for 6 weeks
2. Leptospira interrogans icterohemorrhagica
 Leptospira that is Spiral with hook ends (Like Question Mark – “interrogative”) CHLAMYDIA (Former Name: Bedsonia; Now: Chlamydophila)
 Cause Weil Disease (coming from animal urine) Obligate INTRACELLULAR (Like Virus), Have both DNA and RNA

st
o 1 week – blood, CSF (acute infection)
nd  “ENERGY PARASITE” (Can only live inside the cell)
o 2 week – urine (chronic infection)
 Inclusion body – Diagnostic only (Giemsa Stain)
 L. biflexa – non pathogenic leptospira
o Reticulate body – reproductive that will undergo binary fission
o Elementary body - infectious
Culture media: Incubate at 30’C for 6 weeks
 Fletcher’s Medium (contains rabbit’s serum and Fatty acid) 1. Chlamydia trachomatis
 Noguchi
 TRIC agent (Trachoma, Inclusion Conjunctivitis) – leads to blindness
 EMJH (Ellinghaussen McCullough Johnson Harris)
 LGV (Lympho Granuloma Venerium (#1 STD in US)
o Frei Test (Skin Test) = Intracellular – Cell Mediated Immunity
Serologic Tests:
 NGU (Non Gonococcal Urethritis)
1. MAT (Macroscopic Agglutination Test)
 Sensitive to sulfonamid
– screening test
 Transfer Temperature: 4’C
– Antigen: KILLED Leptospira + Specimen: Serum
 Storage Temperature: -70’C
– (+) Agglutination
Lab Diagnosis:
 Glycogen inclusion body ( Halberstadter Prowazeik )
2. MIT (Microscopic Agglutination Test)
o If Glycogen is present = Iodine
– Gold Standard
o If no Glycogen = Giemsa
– Antigen: LIVE Leptospira + Specimen: Serum
– Darkfield Microscope  McCoy (best medium) – a cell media (Gold Standard)
– (+) Agglutination  DFA (Direct Fluorescent Antibody) – Chlamydia Ag
 PCR/NAAT – best method
 Shell Vial Culture System
- a rapid culture system not only to Chlamydia but also to virus Rickettsia spp Vector Disease
- vial that contains coverslip that has monolayers of McCoy and then add the R. rickettsi Tick RMSF
organism. After centrifugation and incubation, transfer the coverslip to the slide R. akari Mite Rickettsialpox
and add Iodine stain. R. typhi Rat flea Murine typhus
a. Brown inclusion Body (Iodine Stain) = Chlamydia (Endemic Typhus Fever)
b. Fluorescent Stain = Green R. prowazekii Louse Epidemic typhus,
Brill-Zinsser
2. Chlamydia psittaci Orientia tsutsugamushi Chigger Scrub typhus
 MOT: Inhalation (Bird Droppings) Rochalemia quintana Louse Trench fever
 Parrot fever or psittacosis (ornithosis) Ehrlichia chaffensis Tick Monocytic E.
 Man: pneumonia Ehrlichia equi (morulae) Tick Granulocytic E.
 Non glycogen “inclusion body” (Giemsa) – Levinthal Cole Lillie Coxiella burnetti Tick and Inhalation Q fever
 Resistant to sulfonamide
Note:
Note:  Brill-Zinsser disease is a secondary infection
 C. trachomatis – Glycogen inclusion body = Halberstadter Prowazeik  Rochalemia quintana ( now Bortanella quinta) – agar (+); trench fever
 C. psittaci – Non-Glycogen inclusion body = Levinthal Cole Lillie  Borrelia vincentii – trench mouth

3. Chlamydia “Chlamydophyla” pneumoniae (TWAR –Taiwan Acute Respiratory) MYCOPLASMA

 Human to human (pneumonia); growth on human lines & Hep-2 cell  Smallest organism
 Immunofluorescence test (DFA), PCR (Best Method)  Wall-less (pleomorphic); cannot be gram stained. But if gram stained, they are
gram negative coccobacilli or bacilli.
 Fried egg/Mulberry – if the organism has no cell wall, it has the ability to
produce fried egg appearance of colony.
Rickettsia – Ehrlichia, Orientia, Anaplasma o M. pneumoniae - Respiratory
 Obligate intracellular (like Chlamydia) except Coxiella o M. hominis - Genital
 Arthropod borne – commonly TICKS (Chlamydia is sexually, bird and human o U. urealyticum Genital
transmitted)  Requires STEROL (for the rigidity of cell membrane) for growth except
 Cross react with Proteus (weil-felix) acholeplasma. Penicillin resistant because they lack cell wall (not destroyed by
 Site of Multiplication: Endothelial Cell penicillin)
 Can also cause DIC  Size: 10-100 microns (Colonies cannot be seen macroscopically)
Lab Diagnosis
 Special stain – Gimenez, Macchiavelo 1. M. pneumoniae (Eaton agent)
 Culture – EMBRYONATED EGG is the best culture media  Pleuropneumonia like organism (PPLO)
 Weil-Felix test – Rickettsial Ab  Primary Atypical Agent or WALKING PNEUMONIA (can easily be transferred
 Immunofluorescence – more specific test from one person to another)
 Fried egg colony (Incubate aerobically w/ CO2)
 Selective Media: PPLO agar, Edward Hayflick’s
Ehrlichia  Can grow to Chocolate Agar (better) and Blood Agar
 MORULA (Diagnostic of Ehrlichia) Tick transmitted  Confirm: HEMADSORPTION test (attachment of organism to RBC)
 Can destroy leukocytes! Serologic test:
 Sennetsu fever  DFA
  BEST: Inhibition of growth by specific antisera  String of beads, fluff balls (broth)
2. M. hominis  Fried egg in HEART INFUSION (HI) , SPS sensitive
 Large fried egg colony  Rat bite fever, Haverhill disease
 Genital mycoplasma
 Media: A7/A8, NYCA (like Neisseria) SP4-arginine 5. Spirillum minor/minus
 Rat bite fever
Disease:  Soduko Fever
 Post abortal
 Past partum fever 6. Chromobacterium violaceum
 Pelvic Inflammatory Disease (PID)  Violet colored organism, VIOLACEIN;
- Sexually transmitted organisms like Neisseria gonorrhea, Chlamydia  Contaminant
trachomatic and M. hominis is asscociated with PID  NH4 cyanide, Mac – NLF

3. Ureaplasma urealyticum 7. Cardiobacterium hominis (HACEK organism)

 SMALLEST MYCOPLASMA (Smallest bacteria)  Cause SBE, tear drop shape
 T strain (tiny fried egg), Urease (+): (brown); NGU
 Media: A7/A8, NYCA, SP4-urea 8. Capnocytophaga spp
 Broth: NO HAZINESS since very small despite the growth  Gliding motility (Spreading Colony), Fusiform rod (Like Fusobacterium)
 Periodontal disease (oral flora)
 Nitrate (+), Esculin Hydrolysis (+)
 Catalase (-) and Odixase (-)
Miscellaneous Bacteria
1. Gardnerella /Haemophilus/Corynebacterium vaginalis 9. Bartonella henselae
 Oxidase & catalase (-)  Cat scratch (Cat bite = Pasteurella)
 Not a vaginal flora! (Pathogenic)  Bacillary angiomatosis
 Cause bacterial vaginosis  Peliosis hepatitis
 CLUE CELLS are squamous epithelial cells which can be gram positive or negative
(for cytologic exam) 10. Bartonella bacilliformis
 Sniff Test: Fishy amine like odor (alk pH – whiff/sniff)  Destroy RBC, vector: sandfly
o (Vaginal Discharge + 10% KOH) then smell. Hmmm AMOY FISHY! LOL  Carrion’s disease
 Media: human blood tween 80 agar, V agar, Columbia CAN  Verruga peruana – skin eruption
 Treatment: Metronidazole  Oroya fever – anemia

2. Calymmatobacterium (Klebsiella) granulomatis 11. Legionella pneumophila

 Safety pin, ENCAPSULATED, NON MOTILE, Non cultivable on agar.  Short gram (-) rods, aerobic
 Diagnosis: Donovan bodies (Giemsa ) = macrophage with a gram negative rod  Catalase (+) Oxidase (+) Motile
 Disease: Granuloma inguinale (donovanosis)  Require L-cystine and iron for growth
 Isolated from Air conditioning, water cooling
3. Actinobacillus actinomycetemcomitans  Broadstreet pneumonia & pontiac fever
Star like colony (like aggregatibacter) cause SBE O O

 Transport = 4 C Storage = -70 C
 Dots and dashes of Morse code  Legionella micdadae – Acid Fast organism

4. Streptobacillus monoliformis
 Water Bacteriology
Lab Diagnosis: 1. Presumptive Test
 DFA – Legionella Ag  Lactose Broth / Lau ryl Tryptose Broth + Water Incubate at 35’C for 24
 Culture: Hours = Gas (+); 48 hours (-)
o BCYE (Buffered Charcoal Yeast Extract) = BEST MEDIA - blue green 2. Confirmatory Test
colony/cut glass colony  EMB / Endo Agar + Inoculum (24 Hours Inc) = Colony
o Feeley-Gorman Agar – brown Colony 3. Compeleted Test
 Stain: Deiterle silver stain = black  Lactos Broth Fermenn tube + Org Incubate at 35’C for 24 -48 hours = Acid
+ Gas (+)
12. Listeria monocytogenes
 Gram (+) rod, Multiple Tube Fermentation
 Motile at RT not at 35’C, tumbling motility (broth), umbrella like motility (semi-  Gold Standard Test (5 Test Tubes)
solid medium)  Reported in MPN (Most Probable Number)
 B-hemolysis on SBAP  Positive: >1.1 MPN/100mL; Negative: <1.1 MPN/100MI
 Cold enrichment at 4’C (like Yersinia) (+)
 Media: McBride Stages of MTFT
 Virulence: Listerolysin O – O2 labile hemolysin 1. Presumptive Test – Triple Strength Lactose Tube Broth + H2O = (+) Gas (Durham
 Anton test – ocular virulence test (+) Keratoconjunctivitis Tube); (-) No Gas after 48 hours
 CAMP test with S. aureus (+) = Block Hemolysis 2. Confirmatory Test for Total Coliforms – Brilliant Green Lactose Broth = (+) Gas
Disease: 3. Confirmatory Test for Fecal Coliforms = EC Broth at 44’C = (+) Gas
 Meningitis, sepsis, infantiseptica granulomatous,
 Food poison (coleslaw, cheese, chicken, unpasteurized milk) Milk Bacteriology
 Fetal abortion
PATHOGENS NORMAL FLORA
13. Erysiphilothrix rhusiophatiae  Salmonella  P. syncyanea – Blue Milk
 Gram (+) rod, non motile, H2S producer  V. cholerae  F. synxanthum – Yellow Milk
 Catalase (-), Test tube brush (Pipeline Brush) (Semi-Solid)  B. abortus  P. aeruginosa – Blue Green Milk
 Erysipiloid (Butcher’s cut or Diamond Cut)  C. diptheriae  S. marcescens – Red Milk
 M. bovis/tb  S. lactis – Souring of Milk
Miscellaneous Gram (+) Bacilli
 B.anthracis  B. subtilis – hay bacteria;
Listeria monocytogenes Erysiphilothrix rhusiophatiae proteolytic action on coagulated
 Coxiella
Catalase + - milk
O  FMDV
Motile (25 C) + - Alcaligenes viscosus – slimy or
 Cowpox virus 

Hemolysis Beta Alpha ropy milk

 S. pyogenes
VP + -
H2S prod. - +
Bile esculin & hippurate + -
Gluconate Utilization + (blue) - (green)
Media McBride, Cold enrichment BAP