Microbiology Boards Lecture 1

MICROBIOLOGY REVIEWER
INSTRUMENTS
1. INCUBATOR set at 35-37C
Quality Control: Monitoring of temperature at 35C for MRSA, not at 37C. 2. Robert Koch
Also for viral culture (37C) 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: 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)
Father of Microscopy (+) Capsular Swelling (due to Ag-Ab reaction)
Prepared By: Mrs. Alicia Aldave

Side Notes Edited by: Kerwin Faustino I1
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 doesnt 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 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 its 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)
Cannot be seen in gram stain, Spirochetes cannot be seen in brightfield
microscope. Only see in Dark-Field Microscope.
BACTERIAL PHYSIOLOGY 3. Temperature:
a. Psychrophilic = 0-20C (refrigerator)
Ex. LISTERIA M. and YERSINIA E.
1. Oxygen Requirement:
a. Obligate Aerobe = With oxygen Listeria agent of food poisoning
Ex. MYCOBACTERIA Food contaminated with Listeria (mostly found in ref)
b. Obligate Anaerobe = Without oxygen 1. Coleslaw
Ex. BACTEROIDES FRAGILIS (normal flora 2. Milk (Mam: Ano ang gatas? Milk yan!) and
c. Facultative Anaerobe = With or without oxygen Cheese
Ex. PATHOGENS Yersinia e. blood bank contaminant (presence of bubbles in the blood
(most pathogenic bacteria are incubated AEROBICALLY) bag)
d. Microaerophiles = Low Oxygen (5% O2 + 10% CO2 + 85% N2) b. Mesophilic = 20-40C (Pathogenic) Optimal pH (37C)
Ex. CAMPYLOBACTER c. Thermophilic = 40-60C
- Materials providing CO2: Gas Pack, Candle Jar (3% CO2), 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:
Ex. LACTOBACILLUS a. Acidophilic
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
FACULTATIVE/AEROTOLERANT grows EITHER/ANYWHERE. Gardnerella vaginalis (vaginosis) since it is alkaline
b. Neutrophilic Pathogenic Bacteria
Case Analysis: Ex. E. COLI
In the case of bronchial washing, an organism grows on slide (Gram Stain (+)) but c. Basophilic VIBRIO
Culture () (No growth).
Remember that if Gram Stain (+), should be Culture (+)! 5. Salt Concentration - Halophilic
Ex. S. AUREUS = 7.5% NaCl
Rationale: Bronchial washing is an aerobic sample. The bacteria is possibly ENTEROCOCCUS = 6.5% NaCl
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. Krebs 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
Anaerobic process.
BACTERIAL GROWTH CURVE Huckers Stain (Crystal Violet + Ammonium Oxalate) Gram stain for FUNGI
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 to the organism.
b. Depletion of nutrients
c. Adverse environmental conditions Decolorizer is the most critical step in gram staining (commonly mistaken)
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 the 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 Crystal Violet) Size: Yeast > Cocci
3. Spirochete since it is very small, it cant 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 at Mabagal
Any organism that cannot be gram stained, once gram stained, they are
considered GRAM NEGATIVE. Acid Fast Organisms: Mycobacteria, Nocardia, Cryptosporidium
Hiss Stain Capsular Stain
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
Baumgartens M. leprae vs. M. tuberculosis b. Spore Dorner, Wirtz Conklin, Schaeffer Fulton
Fite Faracos M. leprae (hematoxylin) (Dx: Skin Biopsy) c. Metachromatic Granules Alberts, LAMB, Neisseria
d. Flagella Leifson, Grays
M. smegmatis for uncircumcised patients e. Nucleic Acid Fuelgen
M. leprae causative agent of leprosy (Hansens 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 -20C/Freezer
Ziehl-Neelsen: Best Method 4. Working Culture 4C, 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 of 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
Air drying is done first before HEAT FIXATION to prevent AEROSOL formation.
70% Ethyl Alcohol with SAND for sputum to prevent aerosol formation.
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
Sheeps 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
a. TCBS Vibrio (Stool) Needle aspiration for both AEROBIC and ANAEROBIC
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. 35C 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
3. GC Agar Gram (-) cocci
4. GENTAMICIN BAP Strep. Pneumoniae
Transport Medium: CLINICAL SPECIMEN
1. Cary Blair stool pathogens (for enteric pathogens, VIBRIO) 1. Blood (BHIB)
2. Stuarts 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 (4C 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 37C)
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 121c at 15lbs/psi for 15 min
Culture media, bandages, gauze
7. Throat Swab Sore throat 2. INSPISSATION
- BAP (Strep for hemolysis) 75-80C for 2 hours on 3 days
- Modified Thayer Martin (Neisseria gonorrhea, but still best in genital) Destroys HIGH PROTEIN containing medium (LJ, Loefflers)

3. TYNDALLIZATION 6. Ionizing Radiation disposables (gloves, microwave oven, catheter)
- 100C 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)
- 100C for 30min b. Cellulose Membrane Liquid (0.22um)
5. PASTEURIZATION only the pathogens are destroyed in the milk sample
- 63'C for 30min / 72'C for 15secs DISINFECTANT ANTISEPTIC
- 75,00015,000 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 170-180C 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
mutation, reduction of airborne infection, also used in ICU.

B. Cell Membrane Inhibitors 7. Disc Elution Test = Susceptibilty test for Mycobacteria
1. Colistin Inhibits GRAM (-) ONLY - Antibiotic is FIRST applied in the agar, Bacteria is the LAST to be applied
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 the 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 of inhibition and colonies easily seen
3. Cloramphenicol - BAP cannot be used since its 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% Sheeps 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 CO2 (to prevent increase in pH)
1. Micro/Macrobroth Dilution TEMPERATURE 35-37C (MRSA-35C)
- recommended for ANAEROBIC BACTERIA INC. TIME 16-18 hours
- reference method for MIC and MBC (Antibiotic is being diluted) STANDARD 0.5 McFarland (1% H2SO4 and 1.175& BaCl2)
- 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
- Misidentification of result: Do confirmatory test using other method Presence of Swarming = Ignore. Continue measuring the zone of inhibition.
6. Microstat Walk-Away System = combination of Identification, Susceptibility Presence of Double Zone = OUTER ZONE is measured not the inner zone.
Testing and Automated System Presence of Colony Inside the Zone = do gram stain
In a 100mm agar = 5-6 antibiotics ; In a 150mm agar = 12 antibiotics
Side Notes Edited by: Kerwin Faustino I 10
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 of 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% H2O2
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. -20C or -70C
Bacterial colonies Clot (4 Hours)
- Stock culture storage
a. Slide (Screening) BOUND coagulase
- -70C (Virus and PCR)
b. Test Tube (Confirmatory) FREE coagulase
g. 2-8C
Medium: Rabbits 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: Its 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 test 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
gonorrhea, H. influenzae) BAP (Slightly beta haemolytic, pinhead colony)
Vogel-Johnson (brown to black colony because s. aureus can reduce
Micrococcaceae Tellurite present in VJ agar; Corynebacterium can also reduce Tellurite.
Micrococcus Staphylococcus Difference: S. aureus is cocci while Corynebacterium is bacilli)
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 (Confirmatory 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, Oxidase (-) 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: 35C 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 doesnt 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: Sheeps 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 Browns Classification
Incubate: 35C 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
Incubate: 35C for 48 hours No Hemolysis
(+) Blackening of Agar (E. faecalis); (-) No Blackening of Agar (S. mitis) (+) No zone (E. faecalis, E. faecium, S. bovis (Grp. D)
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. Dicks 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
Dicks Test = S. pyogenes
Schicks 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
E. Streptococcus viridans: Genera included:
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 (+)
Species: Best Media: CAP (Requiring 5-10% CO2)
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 45C + + Diseases: gonorrhea, opthalmia neonatorum, Fitz-Hugh Curtis, PPNG
Growth 10C + - (Penicillinase Producing Neisseria gonorrhea); salphingitis, epididymitis
Hippurate HOH +/- -

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 NO3 to NO2
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 Color
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:
c. Acidimetric Test Phenol Red and Pen: (+) Yellow N. secca produces wrinkled colony
N. lactamica ONPG (+)
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 O2 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 68C (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 68C
Dubois Oleic Acid Medium Result: >45mm height of gas bubbles
b. Mitchisons 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 - ROUNYOUNS I = YELLOW
1. M. kansasii
5. Biochemical Tests for Mycobacteria 2. M. marinum
3. M. asiaticum, M. simiae
a. NIACIN TEST B. Scotochromogens - ROUNYOUNS 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 - ROUNYOUNS 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 Taps)
a. HCl 5. M. triviale, M. haemophilum
b. Sulfanilamide 6. M. malmoense, M. terrae, M. gastri
c. N-napthtylethylene diamine D. Rapid Growers- ROUNYOUNS 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)
(+) M. bovis (susceptible); (-) M. tuberculosis (resistant) (Pappenheims Method: M. tb = RED; M. smeg = BLUE)
(-) M. tuberculosis (resistant)
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
ROUNYOUNS 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 30C Fluorometric Based (less harmful)
ROUNYOUNS II SCOTOCHROMOGENS ORANGE / YELLOW POSITIVE Result: Fluorescence
Scrofula agent PPL: Increased bacteria = Increased O2 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)
ROUNYOUNS III NON POSITIVE Result: No growth
CREAM / BUFF COLORED
PHOTOCHROMOGENS
M. avium Chronic Pulmonary C. MYCOBACTERIUM LEPRAE (Hansens 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 4C and
(Hot and Cold Water Disease: Leprosy (Hansens Disease)
45C
Taps)
ROUNYOUNS 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- Crohns Disease
3. Rhodococcus equi - pleomorphic (rodcocci / vice versa) every 24 hours, and
pink colonies (+); CAMP Test (+) with S. aureus beta hemolytic
Note: CAMP TEST = S. agalactiae (Arrow head); Rhodococcus equi (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),
Cause: PNEUMONIA (Sputum) d. Loefflers serum agar
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 Loefflers 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 Loefflers 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 (-)
4. CHO ferment: dextrose & maltose (+) C. pseudodiphthericum + None +
5. ELEK test: definitive test (confirmatory test) (Hoffmans bacillus)
6. Schicks 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 woods 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 + McFadyeans 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. Woolsorters / Rag Pickers Disease - pulmonary anthrax
Catalase + + 3. Gastroenteritis bloody diarrhea (intestinal anthrax)
Oxidase
Motility + (22C) Lab Diagnosis:
Growth at 4C + 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
6. Penicillin Susceptibility Test (10 Unit) Presumptive Test (Susceptible)
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 motile)
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 45C + 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
Difference: P. aeruginosa (moist colony); B. subtilis (dry colony) Beta Hemolysis(Complete)
(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
Note: lecithinase
(+) 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
C. tetani Oval (Assachorylytic) + 6. Lombard Dowell agar (LD Agar)
C. ramosum Round + (Saccharolytic)
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 = CO2 + H (Enclosed) Actinomyces israeli draining sinus tract with sulfur granule; Molar Tooth
When opened: O2 (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 (O2 has been removed) - Black Colony on Bacteroides Bile Esculin Medium
b. Resazurin: Pink Colorless (O2 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 Vincents 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
Horse odor (CCFA) C. difficile

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 for 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. typhimurium)
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; Moellers Decarboxylase Medium
Cytochrome oxidase (indophenol blue) o Indicator: Bromceresol Purple
(+) P. aeruginosa (Bluish Purple) MINERAL OIL: Prevents the entry of O2
(-) 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 H2S) 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
YELLOW Hafnia
SLANT
Lactose Arizona NEG (-) LYSINE DEAMINASE PURPLE
A/A Sucrose Citrobacter K/A
ACID Glucose E. coli
NEG (-) LYSINE DECARBOXYLASE YELLOW
BUTT YELLOW Enterobacter
Klebsiella
POS (+) LYSINE DEAMINASE RED
R/A
Non Lactose Fermenter NEG (-) LYSINE DECARBOXYLASE YELLOW
ALKALINE H2S (+)Blackening due to Ferric Ammonium Citrate
SLANT RED P-M-P
Salmonella
K/A Glucose Shigella Note: Summary of Some Indicators on Agars and Tests:
ACID Serratia 1. Phenol Red
BUTT YELLOW Yersinia a. TSI
b. XLD
ALKALINE Non Fermentative Organism c. MSA
SLANT RED Pseudomonas d. Christensens 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 Kliglers Iron Agar (G-L-S Iron) 3. Neutral Red

RDA Russells 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:
Kovacs/Erlichs 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
acetylmethylcarbinol
E. cloacae) Serratia
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 Simmons 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 (-) GREEN Enterobacter
Blue
Citrobacter
Hafnia
Product: (NH4)2CO3
16. UREA
HYDROLYSIS TEST
(Alkaline Ammonium Christensens 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 medium 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
somatic/cell wall (O) heat stable; most common in serologic typing serological tests
flagella (H) heat labile (destroyed)
Vi Ag removed by Boiling or Heating (for S. typhi) Enterotoxigenic E. coli Travellers 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 (EHEC) syndrome (HUS), - 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 stool = 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
Greenish metallic sheen on EMB rd
(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 = + + (+)
Proteus Morganella- Providencia P. alcalifaciens UREASE NEGATIVE!! (All PMP group are UREASE (+)!!! Huhu.
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 stone 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 + +
Salmonella

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 25C)
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.
mirabilis) LOA: ++- LIA: K/K (LDC +)

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
K/A, GAS( +), H2S (+) K/A Pasteurella)
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 + Bulle 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) V. cholerae Cholera Sucrose Yellow
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
Plesiomonas is the only INOSITOL (+)
c. TTGA
BAPA (Blood Agar Plate w/ Ampicillin) selective for Aeromonas (Beta Hemo.)
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 42C; 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 37C 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
Diarrhea, Media: Cetrimide Agar/Pseudosel Agar(containing detergent)
C. jejuni + - Skirrow
abortion (+) growth (P. aeruginosa)
CBAP (-) no growth (E. coli)
Peptic ulcer
H. pylori + + Urea agar
Gastritis
PSEUDOMONAS SPP Wound Ecthyma gangrenosum (Black)
All are OXIDASE (+) and DNASE (-) except S. maltophilia Swimmers 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)
O-F = Oxidizers (Yellow (O) Green (F)) 2. Burkholderia cepacia
Opportunistic infection (Found in environment) Contaminants in Alcohol!
Associated with IV Drug Users (Important cause of Iatrogenic 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:
Grape like / Corn Tortillas Odor (Apple Like = Alkaligenes faecalis) Pink colony on Mac (LACTOSE OXIDER)
Resistant to Disinfectants! Yellow Colony on (OFPBL) or PC agar
NF Organism that has the ability to produce mucoid colony due to its Slime YELLOW PIGMENT producer (similar to P. stutzeri)
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 42C 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) Glanders disease (horses)
#2 burn (#1 S. aureus) O-F = +/- (Glucose, Maltose, Lactose)
Nosocomial commonly seen in Cadaver

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 +
Non Fermentetative Organism that causes UTI (together with Alkaligenes)
Resistant to AMA (BETA LACTAMASE Producer)
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 (Pfeiffers 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 (Its 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 shouldnt 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 33C
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 ) Kochs 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. aegypticus, and H.
haemolyticus, they grow on X & V but does not grow in X Factor or V Factor alone!
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 (Sheeps Blood) or Bordet Gengou agar - Commonly reported laboratory acquired infection
Mercury Drop or Pearl Like Colony
not good since Sheeps 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 Bangs 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 (Bangs) + + +
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 trappers 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 Weils
(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 (+) Weils 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 (rabbits testicle)
Mercury Drop (Pearl-Like) Colony = Bordetella Killed at 4C 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,
patay ang ulo mo pati puso Aldave)

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 30C for 6 weeks
Fletchers Medium (contains rabbits 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: 4C
Antigen: KILLED Leptospira + Specimen: Serum
Storage Temperature: -70C
(+) Agglutination
Lab Diagnosis:
2. MIT (Microscopic Agglutination Test) Glycogen inclusion body (Halberstadter Prowazeik)
Gold Standard o If Glycogen is present = Iodine
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 Hayflicks
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
Cold agglutinin

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 Carrions 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
Transport = 4 C Storage = -70 C
O
Dots and dashes of Morse code Legionella micdadae Acid Fast organism
4. Streptobacillus monoliformis
Bacilli in chain, monoliformis = can produce L forms (defective cell wall, artificial
loss of cell wall, reversible) = can have the ability to produce fried egg colony)
Water Bacteriology
Lab Diagnosis: 1. Presumptive Test
DFA Legionella Ag Lactose Broth / Lauryl Tryptose Broth + Water Incubate at 35C 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 35C for 24-48 hours = Acid
+ Gas (+)
12. Listeria monocytogenes
Gram (+) rod, Multiple Tube Fermentation
Motile at RT not at 35C, 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 4C (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 44C = (+) 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 (Butchers 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 Coxiella proteolytic action on coagulated
Catalase + -
O FMDV milk
Motile (25 C) + - Cowpox virus Alcaligenes viscosus slimy or
Hemolysis Beta Alpha S. pyogenes ropy milk
VP + -
H2S prod. - +
Bile esculin & hippurate + -
Gluconate Utilization + (blue) - (green)
Media McBride, Cold enrichment BAP

PRION-CAUSED DISEASES
Scrapie (Sheep or goat)
Kuru
Creutzfeldt-Jakob Disease (CJD)
Gerstmann-Straussler Scheinker Syndrome (GSS)
Bovine Spongiform Encephalopathy (Madcow)
35C 18-24 Hours 35C up to 48 Hours 35c up to 7 days

*BOCLiTXDP *NIMViBiMPyS *ACE MONULL
Bacitracin Nitrite Reduction Acetamide

Optochin Indole Acetate
CAMP MR /V-P Citrate
LIA Bile Cetrimide
TSI Esculin Esculin Hydrolysis
X and V Factor MRS Broth Motility Test
DNA Hydrolysis Pyruvate Broth O/F Test
PAD Salt Tolerance Nitrate Reduction
Urease Test
LOA Decarboxylase
Litmus Milk
Incubation Period of Certain Tests

TESTS INCUBATION PERIOD
ONPG, Coagulase 35C 4 hours
Bile Solubility 35C 30 mins
Butyrate disk and LAP Test RT 5 Mins
Catalase RT 10 sec/asap
Oxidase RT 10 sec
Spot Indole RT 30 sec
Microdase and PYR RT 2mins
MUG Test 35C 2 hours
Hippurate HOH 35C 2 hours and 30 min
Growth at 42C 24 hours


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MICROBIOLOGY REVIEWER

Prepared By: Mrs. Alicia Aldave

3. PLASMA MEMBRANE 8. ENDOSPORES

Gram (+) becomes Gram (-)

Prepared By: Mrs. Alicia Aldave

Prepared By: Mrs. Alicia Aldave

Prepared By: Mrs. Alicia Aldave

Prepared By: Mrs. Alicia Aldave

Prepared By: Mrs. Alicia Aldave

Prepared By: Mrs. Alicia Aldave

Prepared By: Mrs. Alicia Aldave

Enterococcus Non Enterococcus A. Neisseria gonorrhoeae

Prepared By: Mrs. Alicia Aldave

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

5. SUPEROXOL CATALASE TEST Carbohydrate Fermentation Test

Prepared By: Mrs. Alicia Aldave

Prepared By: Mrs. Alicia Aldave

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

1. Corynebacterium diphtheriae (Kleb Loefflers Bacillus)

Prepared By: Mrs. Alicia Aldave

Prepared By: Mrs. Alicia Aldave

Prepared By: Mrs. Alicia Aldave

3. Clostridium tetani (Tackhead Bacillus)

Prepared By: Mrs. Alicia Aldave

Prepared By: Mrs. Alicia Aldave

KIA Kliglers Iron Agar (G-L-S Iron) 3. Neutral Red

4. Bromcresol Purple = LIA

Prepared By: Mrs. Alicia Aldave

Prepared By: Mrs. Alicia Aldave

Note: Summary of H2S Indicators:

Prepared By: Mrs. Alicia Aldave

Prepared By: Mrs. Alicia Aldave

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

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

Disease: Citrobacter Spp. Differentiation:

Prepared By: Mrs. Alicia Aldave

Prepared By: Mrs. Alicia Aldave

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

Y. pestis Y. enterocolitica Y. pseudotuberculosis

Prepared By: Mrs. Alicia Aldave

PYOCYANIN TEST GROWTH AT 42C 3. Burkholderia pseudomallei

Prepared By: Mrs. Alicia Aldave

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

Prepared By: Mrs. Alicia Aldave

Prepared By: Mrs. Alicia Aldave

Other Treponemes A. Relapsing fever due to

Prepared By: Mrs. Alicia Aldave

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

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

2. Calymmatobacterium (Klebsiella) granulomatis 11. Legionella pneumophila

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35C 18-24 Hours 35C up to 48 Hours 35c up to 7 days

Bacitracin Nitrite Reduction Acetamide

Incubation Period of Certain Tests

Prepared By: Mrs. Alicia Aldave

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