ADVENTIST MEDICAL CENTER COLLEGE Page |1

BACTERIOLOGY PRELIM NOTES

Definition of Terms:
Anaerobic no oxygen Culture-plant
Aerobic- with oxygen Fastidious not easily grow
Aerosol- air transmitted Gram stain test for the presence of
Agar- extract from seaweed(liquid bacteria
when heated, solid when cold) Gram positive blue/purple
Bacilli- rod-shaped Gram negative - red
Cocci- spherical Hemolytic
Colony- group from one kind Inoculation - plant
Mordant chemical that increases the
affinity of the stain
HISTORY
Robert Hooke father of cytology
Anton Van Leeuwenhoek father of microbiology
Linnaeus - nomenclature
Jenner-vaccine
Robert Koch- Germ theory
Louis Pasteur- disproved abiogenesis
Elie Mechnikoff- phagocyotosis
Watson and Crick-DNA

Prokaryotes vs. Eukaryotes

CELLULAR NON-CELLULAR
Prokaryotic Eukaryotic Virus
True Nucleus +
Cell wall +
Chromosome Haploid Diploid
DNA/RNA Either
Cell Division Binary Fission Mitosis Assemble/ Disassemble
Ribosome 70s 80s
Organelles
Size 1-5 nm 3-25nm 0.25nm

CLASSIFFICATION OF BACTERIA BASED ON MORPHOLOGY

Cocci - spherical
1. Staphylococci clusters
2. Streptococci chains
3. Pneumococci diplococci/ two
Bacilli Rods
1. Mycobacterium Tuberculosis
2. Pseudomonas
3. Salmonella typhi
4. Clostridium tetani
Spirochetes Spirals
1. Treponema
2. Leptospira

METABOLIC CHARACTERISTICS
1. Oxygen H202, O2-
ENZYMES : Catalase 2H2O2 2H2O+ O2
Peroxidase 2H2O2 2H2O+ O2
Superoxide dismutase (SOD) 2H+ + 40- 2H2O2

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ORGANISM OXYGEN REQUIREMENT PATHWAY ENZYMES

Obligate Aerobe Glycolysis Catalase
Peroxidase
SOD
Facultative anaerobe Primarily Aerobic Fermentation SOD
Catalase
Microaerophilic Low Oxygen Fermentation SOD
Aerotolerant Primarily Aerobes Fermentation SOD
Obligate anaerobe Cant live without oxygen Fermentation None

2. Carbon Source
Phototrophs light
Chemotroph organic
Autotroph inorganic source
Heterotroph organic, glucose
Chemoheterotroph requires glucose, lipid, protein
Capnophilic - 10% CO2 required
Halophilic requires salt, salt-loving
NaCl (0.85%) human
1-3% - vibrio
6.5% - enterococcus
7.5% - staphylococci

3. Temperature
Psychrolic - cold, 2-10 oC, bacteria: Listeria, Yersinia
Mesophilic moderate, 20-40 oC (RT= 20-30 oC), most clinically important
Thermophilic - heat
Hyperthermophilic extreme temperature
e.g. bacteria: Bacillus stearothermophilus, Bacillus Subtilis
Other:
42oC Campylobacter
5oC Listeria
35-37oC

4. Hemolytic Pattern
Alpha partial hemolytic, green
Beta clearing of colony
Gamma no hemolysis
Alpha-prime Alpha+Beta

5. pH requirement
acidophile 3.0-6.5 pH, bacteria: Lactobacillus Lactonica lactis
alkalophile 8-10 pH, bacteria: Vibrio
neutral/slightly alkaline (7.35-7.45), optimum

GRAM STAIN
1. Gram positive blue/purple
2. Gram negative red
Exception:
Mycobacteria
Spirochetes
Mycoplasma

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BACTERIAL STRUCTURE
1. Cell Wall (Peptidoglycan)
Techoic Acid - the affinity of the stain, G(+), crystal violet
2. Outer membrane Gram (-) rich in lipid
3. Lipopolysachharide (LPS) Gram Negative ( virulence)
Endotoxin dead
4. Periplasmic Space Gram negative, resistance,
bacteria: Lactobacilllus (Lactamase) resistance
5. Capsule protects from phagocytosis, covers antigen of bacteria,
bacteria: Neisseria meningitidis cause meningitis
6. Cell membrane energy production, cytochrome electron transport, polymoxin
7. Ribosome repair, site of protein synthesis, erythromycin
8. Plasmid nonchromosomal DNA, carries resistant gene
9. Pili/Fimbriae 2 functions:
Attachment e.g. E.Coli
Conjugation
Flagella
a. Atrichous w/out flagella
b. Monotrichous vibrio
c. Apmhitrichous both ends
d. Lopotrichous tuft of flagella
e. Petrichous surrounded by flagella
10. Endospore dormant/nonvegetation, resilient
11. Toxin
Endotoxin Gram(-), DEAD
Exotoxin gram(-)/gram(+), ALIVE, exaletanus plasmin, e.g. Botulinum flaccid
Enterotoxin holds NaCl

GROWTH CURVE the growth in bacteria is in number, NOT SIZE

1. Lag Phase adjustment
2. Log/Logarithmic/Exponential Phase ideal collection, most important, most sensitive in
antibiotic
3. Stationary/Plateau Phase Dead or Alive
4. Death Phase number of deaths exceeds number of new cells formed

BACTERIAL METABOLISM
Respiration w/ O2
Krebs Cycle
ETC (Elecrone
Glucose-CO2 and H2O
Oxidation w/ O2 Gluc-acid
Fermentation w/o O2
EMP(Embden Meyerhof Pathway)
Gluc-acid/alcohol
BACTERIAL GENETICS
Genotype genetic makeup/complete genome
Phenotype expressed(G+/G-)/observable
Plasmid extrachromosomal DNA
Transposon jumping genes w/n plasmid
Mutations change in nucleotide sequence
a. Point mutation one
b. Frameshift mutation more than one
DNA TRANSFER same objective but different mechanism
Transformation naked DNA
Transduction phages, mediated by bacteria infected by virus (bacteriophages)
Conjugation direct transfer through pili

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BACTERIAL CONTROL
Sterilization all include spores, kills all forms of life
Disinfection pathogenic/flora
a. Disinfectant living, e.g. NaOCL (Hypo) 1:10
b. Antiseptics non-living, e.g. Alcohol
KILLING DEPENDS ON:
Type
Number
Chemical concentration e.g. NaOCl (1:10)
Duration of contact
Alcohol(1 min)
HIV (2 min)
HBV (10 min)
Organic matter/oil present
KILLING DEPENDS ON:
Mycolic acid acid and lipid in Mycobacterium Tuberculosis, refers to mycobacterium
Spores/dormant
Vegetative bacterial feeding stage, easy to kill
Enveloped virus unstable , note used to be exposed
Naked virus more resistant t
Chitin Fungi
LEVEL OF DISINFECTION/STERILIZATION
High sporicidal(kills the spores), tubercolocidal
Intermediate Tuberculocidal, NOT spores. e.g. Boiling
Low NOT sporicidal nor tuberculocidal. e.g. Alcohol
METHODS OF DISINFECTION AND STERILIZATION
1. Physical
Incineration dry heat, highest temp
waste disposal (870-980C)
Dry Heat Oven 160-180C / 1.5-3hrs. For glasswares
Cremation Disease control
Flaming needle/culture
Autoclave moist heat/steam under pressure. For media
15psi/121C/15-20mins
Boiling 100C/15-30mins (MTB)
Pasteurization 63C/30mins or 72C/15secs for milk
2. Filtration 100% sterility, thin membrane filters(0.22-0.45u cellulose nitrate) or cellulose
diacetate(.015-12u) for vaccines and antiobiotic
3. Chemical methods- mostly disinfectant
Formaldehyde/glutaraldehyde cold sterilant
Alcohol disinfectant (70%)
Phenol standard disinfectant/cell membrane
Zephiran (Bezalkonium Cl) use for Alcohol measurement of blood
Iodine halogen
NaOCl Hypo/Chlorox

OTHER DISINFECTANTS
Freezing
Lyophilization best for culture preservation/years
UVL pyrimidine/DNA, use in biosafety cabinet, targets DNA
H2O2 agua oxinada
Detergents cell membrane

SPECIMEN COLLECTION
Collected during acute phase/before treatment
Written order/site Sterile/non-sterile, aerobic or anaerobic
Culture/usual flora
Compared with diagnosis

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SPECIMEN COLLECTION METHODS

1. Aspirate
Tissues synovial fluid, abscess, pericardial, peritoneal
Blood 1:10 / 0.025% SPS / 2-3 cultures during 24hrs (at least 10ml)
70% Alcohol + 2% Iodine (allowed to dry, not palpated)
Hemolysis (sign of bacterial growth), turbidity/BAP and CAP
CSF by doctors, never refrigerated (N and H)
BAP,CAP, Mac, India ink
2. Swab least desirable especially anaerobic bacteria
URT throat / sputum
LRT sputum less than 25 EC/greater than 10 Neutrophils
NALC (digestant) / NaOH (decontaminant) Gold standard for MTB
Wound clean, explore, obtain fresh material/quantity
Avoid drying
Requires H2O to survive
Vaginal swab (G-)
3. Urine clean perianal/catheterized
Midstream discard first few mL
Suprapubic direct to the bladder
4. Stool not Gram stained 6. Genital tracts dont expose to cold
Sterile container NOT temp
necessary Men 3-4cm swab into the
Wide-mouthed container urethra
Without urine Women no lubricants, physician
1 hour collection uses speculum
5. Throat depressed before swabbing

PRESERVATION, STORAGE AND TRANSPORT

Notes:

Neisseria Delays: Refrigerate except

Room temp (20 - 30C) CSF
Sensitive Blood
Most fastidious Swab for Neisseria
Special collection and Boric acid for urine
environment
Never use cotton for culture

Major Concerns why preserve:

Inaccurate quantitation
Death due to temp
Overgrowth
Oxygen protection for anaerobes
Loss thru dyingSafety for transport

USE OF ANTICOAGULANTS AND PRESERVATIVES

Citrate/EDTA never to be used for culture (possible contamination)
Heparin for virus
Antibiotic Removal Device (ARD) neutralizes the antibiotic
SPS (sodium polyanetholsulfonate) most common
SAS (Sodium amylsulfate)
STORAGE
Freezing (-70C) virus/ if delayed within 4 days
Ref (4C) urine ,viral blood, swabs
RT (25C) fungi
Incubator (37C) CSF, bacterial blood, agar plates

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SPECIMEN LABELING AND REJECTION

Request
Patients ID/names
Source
Initial diagnosis/history,
Test requested
Suboptimal Specimens - rejected
Syringe with needles attached
Leaking
Stools with urine/barium
Non-matching request and specimen:
Non-invasive recollect
Invasive process if responsible person signs a waiver
Anaerobes from inappropriate source
Older than 2 hrs. /unpreserved / 24hrs. urine
Ref blood/CSF
Dried-up
Specimen in formalin
Non-sterile containers
QNS (Quantity Not Sufficient)
Greater than 25 EC/LPF/less than 10 neutrophils(saliva)
STAINS BACTERIAL CRAYONS
Simple One dye
Different Quellung test
Indirect/negative India ink
Gram Stain
a. 1 stain Crystal violet (stains Gram+ and Gram- blue/purple)
b. Mordant Iodine ( the affinity of the stain)
c. Decolorizer Acetone alcohol (removes CV in G- )
d. 2 stain/counter stain Safranin (stains G- red) and (G+ purple)
NOTE:
WBC and RBC pink (internal control)
Factors affecting false positive and false negative result:
a. G(+) becomes G(-) false negative
Over decolorization, old, dying
Acidic Iodine
Penicillin
b. G(-) becomes G(+) false positive
Under decolorization
Thick Smear

GRAM STAIN GENERAL RULE

All cocci are G(+) except:
Neisseria
Veillonella
Moraxella (Branhamella)
All bacilli are G(-) except:
My Mycobacteria
Corny Corynebacterium
But Bacilli
Colorful Clostridium
Lab Lactobacillus
Li Listeria
Fe Erysiphilothrix
Nocardia and actimyces
All spiral organism are reported as G(-)
Yeasts are G(+)

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ACID-FAST STAIN
Differentiate AFO(Acid Fast Organism) from NAFO (Non-acid Fast Organism)
Mycolic Acid resists acid decolorization
Brightfield Fluorescence
Method Ziehl-Neelsen Kinyoun Auramine-Rhodamine
(C-A-M) (C-A-M)
1 Stain Carbol-Fuchsin (red) Carbol-Fuchsin Auramine-Rhodamine
Mordant Heat Phenol (increased
detergent)
Decolorizer 3% acid alcohol 3% acid alcohol 0.5% acid alcohol
2 Methylene Blue Malachite Green 0.5% KMnO4 quenching
Result AFO-red AFO-red AFO yellow fluorescence
NAFO - Blue NAFO - Blue NAFO No fluorescence

MICROSCOPY (OIL IMMERSION)

Magnification times an image magnified
Resolution power that two objects are really separate
Types of Microscope
Bright-field light background(0.2 um) Electron (0.001 um)
Dark-field dark background(Spiro) a. TEM (transmission) internal
Phase contrast HLA typing/living b. SEM (scanning) external
Fluorescent

UNIVERSAL PRECAUTIONS
Barrier protection gloves, masks, lab gown, puncture resistant biohazard container(color)
Color coding for waste category:
Black non-infectious dry waste
Green non-infectious wet waste
Yellow infectious and pathological waste
Yellow with black band chemical waste
Red sharp and pressurized containers
Engineering controls protects while working(BSC) from aerosols
Heat
UV
HEPA (High efficiency particulate air) removes 0.3 um particle

BIOSAFETY CABINET CLASSES

a. Class I - Open(unsterilized air circulates), exhaust air sterilized by HEPA filters, negative
b. Class II - Both air entering, circulating and exhaust is fertilized (routine)
c. Class III Entirely enclosed, highest level of safety, infectious materials are handled with gloves.
1st HEPA sterilizes supply air, 2nd HEPA for exhaust air

CULTURE MEDIA
Media contains nutrients for growth, isolation and ID, can be classified based on:
Physical State/Consistency
Solid 1.5% agar added
Semi-Solid 0.5-1% agar
Liquid no agar
Agar polysaccharide extract from seaweed/algae
Composition
Synthetic/commercial exact composition is known
Non-synthetic not exact/serum or blood/meat extract broth
Tissue culture living tissues/ immortal cells(cancer cells)
Chick embryo/eggs
A549 from lung cancer
Hela cell cervical cancer
Hep 2 cells laryngeal cancer

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Dispensing/Distribution
Plated dispensed on sterile petri dish(EMB, Mac, BAP)
Preparation: Weigh Dissolve Autoclave Dispense
Tube on sterile tubes
Preparation: Weigh Dissolve Dispense Autoclave
Broth
Agar slant
Slant and butt
Butt
Use
Non-selective (CAP/BAP)
Selective inhibitory agents that does not affect the desired org. (stool)
Differential characteristics on medium(BAP)
Enriched for fastidious organism / CAP
Selective enrichment/broth media supports small numbers
Transport prevents replication
ROUTINE CULTURE MEDIA
BAP(Blood Agar Plate) fastidious/hemolysis/defibrinated blood(V factor)
CAP(Chocolate Agar Plate) Haemophilus/Neisseria/Isovitalex(X and V factors)
Thayer-Martin Agar(TMA) Modified chocolate/antibiotics added
Bacteria:
N. gonorrhoeae
N. meningitidis
Antibiotics:
Vancomycin G(+)
Colistin G(-)
Nystatin fungi (yeast)
Modified TMA added trimethoprim(proteus inhibitor)
Martin Lewis agar Neisseria / anisomycin in place of colistin
Eosin Methylene blue lactose fermentation
E and M indicators/ M inhibits G+
Mac Conkey same use w/ EMB
Bile salts and CV inhibits G+/ Neutral red (indicator)
Mannitol Salt agar(MSA) G+ isolation/ CPS not CNS
7.5% NaCl/Phenol red indicator
Phenyl Alcohol agar G+ isolation/PE inhibits G-
Selenite Broth - S and S/ Na hydrogen selenite inhibits most enterics
Xylose lysine desoxycholate - S and S/high NaCl inhibits enterics
Thioglycolate aerobes and anaerobes
ANAEROBIC CULTURE SYSTEM
Resazurin white(reduced) to pink(+)/used in PRAS
Methylene blue blue to white/pink(+)
Catalyst Palladium
Methods
Anaerobic jar 5-10% CO2
Prereduced anaerobically sterilized system (PRAS)
Anaerobic glove box
METHOD ENVIRONMENT
Aerobic Culture O2 21%
CO2 0.03%
Candle Jar O2 15%
CO2 5-10%
Anaerobic Jar O2 0%
CO2 5-10%
H2 5-10%
N2 80-90%

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INOCULATION TECHNIQUES
Streaking accurate colony count. 2 purposes:
Quantitative
Isolated Colony
Semi Quantitative dilution streaks: yields isolates from pure colony
Key Characteristics presumptive Dx, reduces cost, QC on automation
Colony size (staph,strep,fungi)
Pigments
Staph (gold) Serratia (red)
Pseudomonas (green-yellow) Salmonella (Dark/Black)
E. coli ( ) TSI (yellow)
Shape/surface appearance (proteus,Kleibsiella)
Hemolysis
Odor pseudo (grapelike)
ANTIMICROBIAL THERAPY
Antibiotic chemical
Antimicrobial therapy use to treat Disease
(-cidal) kill
(- static) inhibits growth
Synergistic (1+1=3)
Antagonism ( 2 + 1 = 1)
Additive (1+1=2)
Indifference (A + B =A)
Broad spectrum Both G(+)/G(-) ; e.g. Tetracyclin
Narrow Spectrum either G (-) / G (+)
S Sensitive I Intermediate R Resistant

BACTERIAL ACTIVIES ON:

1. CELL WALL
B lactam antibiotics binds with transpeptidase (peptidoglycan interrupted)
Penicillin
Cephalosporin
Monobactams
B lactamase combined w/ B lactam drugs
Clavulanic acid
Sulbactam
Tazobactam
Other antibiotics
Vancomycin MRSA (methicillin-resistant Staphylococcus aureus)
Bacitracin S. pyogenes
Cycloserine Clostridia
2. CELL MEMBRANE
Polymyxin active against G(-) bacteria (phospholipids)
Bacitracin active against G(+) bacteria
3. PROTEIN SYNTHESIS interference in bacterial protein synthesis at 30s
Aminoglycoside G(-) and Staph, 30s(pseudomonas)
Tetracycline 30s (Broad spectrum)
Macrolides 50s
Chloramphenicol 50s (aplastic anemia)
4. METABOLITES prevents the synthesis of folic acid
Sulfonamides primarily treatment of UTI
Trimethoprim treatment of Chronic UTI
5. NUCLEIC ACID inhibition of bacterial DNA and RNA
Rifampin
Quinolones
Metronidazole
Naldixic acid

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GRAM POSITIVE COCCI (MICROCOCCACEAE)

TEST STAPHYLOCOCCUS MICROCOCCUS
Coagulase V Neg
Oxidation-Fermentation F O

Bacitracin Resistant Sensitive

Lyostaphin Sensitive Resistant
Modified Oxidation - +

CLINICALLY SIGNIFICANT SPECIES:

STAPHYLOCOCCI Gram (+) cocci Catalase (+)
S. Aureus - golden yellow colony, G+ in clusters
Gram(+) cocci Catalase(+) Coagulase(+)
Virulence Factors:
Enterotoxin A-E food poisoning
Enterotoxin F/Exotoxin C/TSST-1 Toxic shock syndrome
Exfoliatin SSS(skin scalded syndrome)
Protein A antiphagocytic/binds Fc portion
Leukocidin Panton-Valentine factor
Enzymes catalase, coagulase, DNAse, hyaluronidase, lipase, B-lactamase
S. Epidermidis nosocomial UTI(most common)/slime adheres to prosthetic heart valves
Gram(+) cocci Coagulase(-) Novobiocin (S)
S. Saprophyticus UTI among sexually active women/adheres to urogenital tracts low numbers
are significant
Gram(+) cocci Coagulase(-) Novobiocin (R)
Micrococcus
Gram (+) cocci Catalase (+) Modified Oxidation (+)
PRESUMPTIVE AND DIAGNOSTIC TESTS Staphylococci
Microscopic exam Gram(+) cocci in clusters
Culture Golden yellow/white(MSA 7.5% NaCl)
Catalase 3% H2O2(reagent) /+ gas(staph)/bubbles
Modified oxidase (microdase) (+ in 30sec)
Coagulase clot formation
Slide screening/bound coagulase (Clumping Factor)
NSS + org
Tube confirm/free or unbound (Coagulation Factor)
rabbits plasma/human plasma (pt and ptt normal) + 37C for 4hrs(clot)/
BAP avoid touching the agar
Latex coagulation CF and protein A
5 ug Novobiocin Resistance/Zone of Inhibition = 16mm
Antibiotic Penicillin(w/o B-lactamase)
Methicillin PRSA
Vancomycin MRSA

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STREPTOCOCCI

BETA-HEMOLYTIC BACTERIA (LANCEFIELD GROUPS) carbohydrate composition of bacterial antigens

found in their cell wall
Group A
S. Pyogenes
Disease: Tonsilitis Rheumatic Fever Acute Glomerulonephritis (AGN)
Test:
CAMP (-) absence of arrowhead; reagent Cyclic adenosine monophosphate
PYR (+) red color result; reagent P-dimethylamino-cinnamaldehyde
Taxo A (Bacitracin Test) Sensitive (no zone of inhibition)
Hippurate Hydrolysis no color change; reagent Sodium Hippurate
Group B
S. Agalactiae
Disease:
Neonatal Meningitis
Otitis Media
Test:
CAMP (+) arrowhead result; reagent - Cyclic adenosine monophosphate
Hippurate Hydrolysis (+) purple/violet result; reagent Sodium Hippurate

ALPHA-HEMOLYTIC BACTERIA (NON-LANCEFIELD GROUPS)

S. Pneumoniae / Pneumococci (diplococci)
Lancet-shaped
Disease:
Pneumonia
Acute Bacterial Meningitis
Test:
Bile Solubility (+) clearing result; reagent 10% Desoxycholate(BAP) and
2% Desoxycholate(Tube)
Taxo P (Optochin Test) sensitive (Zone of Inhibition = 14mm)
Quellung (+) swelling result; reagent antibody
India Ink
Viridans
Test:
Bile Solubility (-) no clearing of turbidity 10% Desoxycholate(BAP) and
2% Desoxycholate(Tube)

Quellung (-) no swelling

Taxo P (Optochin Test) resistant (Zone of Inhibition = 14mm)
S. Sanguis
o Disease: Subacute endocarditis
S. Mutans
o Disease: Dental Cavities

GAMMA-HEMOLYTIC BACTERIA
Group D
Enterococcus: E. Faecalis
Test:
Bile Esculin (+) Black color result; reagent Oxgall (Bile Salts)
6.5% NaCl (+) turbidity result / yellow color
PYR (+) Red color result; reagent P-dimethylamino-cinnamaldehyde
Non-enterococcus: S. Bovis
Test:
Bile Esculin (+) Black color result; reagent Oxgall (Bile Salts)
6.5% NaCl (-) clear result/ no growth / no color change
PYR (-) yellow/orange color result ; reagent P-dimethylamino-cinnamaldehyde

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Note:
Nutrionally Variant Stretococci (NVS)
Abiotrophia without life, needed vitamin B6 to grow

BACTERIAL TESTS
1. Gram Staining differentiate bacteria into two large groups
Reagent:
1o stain Crystal Violet
Mordant Iodine
Decolorizer acetone alcohol
Counter stain Safranin
Result:
Positive purple
Negative red
2. Catalase Test differentiate Staphylococcus from Streptococcus
Reagent
H202 Hydrogen Peroxide
Result
Positive bubbling reaction; bacteria Staphyloccus / Micrococcus
Negative no bubbling reaction; bacteria Streptococcus
3. Modified Oxidation differentiate Staphylococcus from Micrococcus
Result:
Positive Micrococcus
Negative Stapylococcus
4. Coagulase Test differentiate S. Aureus from Catalase Negative Staphlylococcus
Reagent:
Rabbit plasma/Human plasma
Slide Coagulase Test clumping factor
Result:
Positive clumping cocci; bacteria S. Aureus
Negative no clumping; bacteria S. Epidermidis / S. Saprophyticus
Tube Coagulase Test coagulation factor
Result:
Positive coagulate; bacteria S. Aureus
Negative no coagulation; bacteria S. Epidermidis / S. Saprophyticus
5. Novobiocin differentiate S. Epidermidis from S. Saprophyticus
Reagent:
Novobiocin (Zone of Inhibition = 16mm)
Result:
Sensitive S. Epidermidis
Resistant S. Saprophyticus
6. Hemolysis used to classify streptococci species
Reagent:
Blood Agar Plate
Result:
Alpha partial hemolysis / green; bacteria S.Pneumoniae and S. Viridans(2)
Beta complete hemolysis / yellow ; bacteria S. Pyogenes and S.Agalactiae and
other Lancefield Groups
Gamma no hemolysis/unchanged; bacteria Group D Streptococcus: E. Faecalis
and S. Bovis
7. Bacitracin(TAXO A) differentiate S. Pyogenes and Group B Streptoccus: S. Agalactiae
Reagent:
Bacitracin (No Zone of Inhibition)
Result
Sensitive S. Pyogenes
Resistant S. Agalactiae

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8. CAMP Test differentiate Group B Streptococcus: S. Agalactiae from other Lancefield group
Named after 3 researchers who discovered ( Christie, Atkins, Munch-Petersen )
Reagent:
Cyclic adenosine monophosphate
Result:
Positive arrowhead; bacteria Group B: S. Agalactiae
Negative absence of arrow head; bacteria other Lancefield groups
9. Bile Solubility Test identify S. Pneumoniae from bile-insoluble Streptococci
Reagent:
10% Desoxycholate (BAP)
2% Desoxycholate (Tube)
Result:
Positive clearing of turbidity; bacteria S. Pneumoniae
Negative no clearing of turbidity; bacteria bile-insoluble Streptococi
10. Optochin Test (TAXO P) - identify S. Pneumoniae from Optochin-resistant Streptococci
Reagent:
Optochin (Zone of Inhibition 14mm)
Result:
Sensitive S. Pneumoniae
Resistant Optochin-resistant Streptococci
11. Bile Esculin Test identify Group D Streptococcus from other Streptococci
Reagent:
Oxgall (bile salts)
Result:
Positive black color; bacteria Enterococcus: E. Faecalis/Nonenterococcus: S. Bovis
Negative no black color form; bacteria other Streptococci
12. 6.5% NaCl Test - differentiate Enterococcus from Non-Enter.
Reagent:
Sodium Chloride
Result:
Positive turbidity / yellow color; bacteria Enterococcus: E. Faecalis
Negative no turbidity / no color change; bacteria Non-enterococcus: S. Bovis
13. PYR (Pyrrolidonyl Arylamidase) Test presumptive identification of group A Beta-Hemolytic
Streptococci and differentiate Enterococcus: E. Faecalis and Non-Enterococcus: S. Bovis
Reagent:
P-dimethylamino-cinnamaldehyde
Result:
Positive color red; bacteria S. Pyogenes and Enterococcus: E. Faecalis
Negative yellow/orange color; bacteria Non-Enterococcus: S. Bovis
14. Hippurate Hydrolysis Test presumptive identification Group B streptococci: S. Agalactiae
Reagent:
Sodium Hippurate
Ninhydrin Reagent
Result:
Positive purple/violet color; bacteria S. Agalactiae
Negative no color change; bacteria S. Pyogenes

END

Prelim Exam Type of Test

Multiple Choice 50%
Identification 30%
Matching type/ True or False 10 %
Book 10%

Martin Clyde G. Paglinawan | BACTERIOLOGY PRELIM NOTES

ADVENTIST MEDICAL CENTER COLLEGE P a g e | 14

Martin Clyde G. Paglinawan | BACTERIOLOGY PRELIM NOTES