New - Micro-2023-Part-2

REVIEW NOTES in BACTERIOLOGY # 2 – REVISED MAY 2023
STAPHYLOCOCCUS, MICROCOCCUS & RELATED ORGANISMS
GRAM (+) COCCI, CATALASE POSITIVE

Catalase Test – to differentiate Staphylococci & Micrococci from Streptococci Reagent: ___________________________
(+) result ___________________________________; use of colonies from BAP __________________________________
Cocci in GRAPE LIKE CLUSTERS Cocci in TETRADS / CUBOIDAL PACKETS

“Staphylococci” “Micrococci”
IDENTIFICATION TESTS for REAGENT / MEDIA (+) RESULT

Staphylococcus aureus
COAGULASE TEST Rabbit’s Plasma collected
using EDTA
Slide Method:
Detects BOUND COAGULASE or CLUMPING FACTOR Use of Citrated plasma will
cause
Tube Method:
Detects FREE COAGULASE ________________________
Requires incubation at 37 degC
Uses MSA
MANNITOL FERMENTATION TEST 7.5% salt Yellow halo around the colonies
Phenol red
HCl precipitation method Clearing of agar around the colonies

DNase TEST or THERMONUCLEASE TEST
Dye Method With Methyl green- clear zone around the

Use of Toluidine Blue colonies
Methyl Green
With Toluidine Blue __________________
S. aureus is also
_____________________on BAP; _________________to 0.2-0.4 units of Bacitracin (TAXO A); VP ______ and PYR ______
COAGULASE NEGATIVE STAPHYLOCOCCI

Staphylococcus epidermidis and Staphylococcus saprophyticus
S. aureus Staphylococcus epidermidis Staphylococcus
saprophyticus
PYR (-); Glucosidase (+)
Colony
Catalase Test
Coagulase Test
Mannitol Fermentation
Hemolysis on BAP
NOVOBIOCIN SUSCEPTIBILITY S – more than 16 mm R – less 16 mm
(5 ug)
DNAse Test
Phosphatase + -
Gelatinase + + +
pathogenesis Normal skin flora Most common cause of UTI
Causes UTI, stitch abscess Causes pyelonephritis and
Prosthetic Heart Valve infection cystitis in those with indwelling
catheters
VIRULENCE FACTOR Slime production / Biofilm Formation Uncertain
pg. 1 PREPARED BY : MA. CRISTINA SJ LIWANAG (MAM LIGHT)

IDENTIFICATION TEST for Modified Oxidase Test _____________________________________________________

Micrococci Uses tetramethyl p – phenylenediamine dihydrochloride in DMSO dimethylsulfoxide,
(+) result _______________________________________________________________
Staphylococcus Micrococcus
“Strict Aerobe”
Aerobic growth
Anaerobic growth
Lysostaphin Susceptibility
Modified Oxidase Test
MICRODASE DISK
Bacitracin Susceptibility
Furazolidone /Furoxone Susceptibility
Catalase Test
Benzidine Test negative Positive
Glucose Utilization/ OF medium
PATHOGENESIS & SPECTRUM OF DISEASE – Staphylococcus aureus

S. aureus is a NORMAL FLORA of anterior nares, nasopharynx
VIRULENCE FACTORS DISEASES
LIPASE Initiates skin infection NON-TOXIN MEDIATED
HYALURONIDASE Enhances ability of organism to invade tissues Skin Infections:
a.k.a Spreading factor Boils, carbuncles, furuncles, cellulitis
DURAN RAYNAL FACTOR Wound infections
BETA LACTAMASE Responsible for S. aureus resistance to penicillin
PENICILLINASE Bullous Impetigo
COAGULASE Causes bacterial cell to agglutinate in plasma Sty
Converts fibrinogen into fibrin
Considered as MARKER of VIRULENCE
STAPHYLOKINASE Promotes fibrinolysis, causes dissolution of clot
DNase decreases viscosity of exudates allowing more
THERMONUCLEASE mobility
BETA HEMOLYSIN Causes beta hemolysis TOXIN MEDIATED
Sphingomyelinase C
HOT -COLD LYSIN
PROTEIN A Food Poisoning
Toxic Shock Syndrome
ENTEROTOXINS A &B
Scalded Skin Syndrome/Ritter’s disease
TSST / TOXIC SHOCK SYNDROME Causes Toxic shock syndrome
TOXIN
or
PYROGENIC EXOTOXIN C
EXFOLIATIN Causes skin desquamation/exfoliation in
SCALDED SKIN SYNDROME / RITTER’s Disease
Pemphigus neonatorum (localized)
PVL – PANTON VALENTINE Destruction of WBCs
LEUKOCIDIN
CATALASE Catalyzes decomposition of hydrogen peroxide
(Not a virulence factor) into water and oxygen
Test for Beta Lactamase Production: Cephalosporinase test
uses __________________________, substrate ______________________Positive result __________________

To treat S. MRSA / ORSA TESTS TO DETECT MRSA
aureus Methicillin Resistant S. aureus
infections, we Oxacillin Resistant S. aureus
use penicillinase
resistant drugs:
Oxacillin Strain of S. aureus resistant to Methicillin, (1) Use of CHROM AGAR – selective and differential for MRSA
cloxacillin Nafcillin and Oxacillin. MRSA = ROSE TO MAUVE COLONY COLOR
methicillin
Non-MRSA ________________________________________
Resistance of Staphylococcus to penicillinase
resistant penicillin is due to PBP2a (Penicillin (2) CEFOXITIN DISK DIFFUSION TEST
binding protein 2 (in cell wall) encoded by Induces expression of PBP2A
________________________________ 3) Oxacillin Screen Agar – use of MHA with 6 ug Oxacillin with

4% NaCl
Characteristics of MRSA: Growth indicates RESISTANCE to Oxacillin/Methicillin
Penicillin RESISTANT (3) GOLD STANDARD for MRSA detection

Oxacillin RESISTANT
Cefoxitin Test POSITIVE ______________________________________
Treatment for MRSA ___________________________________________
PYR VP TEST SLIDE COAGULASE TUBE COAGULASE

CLUMPING FACTOR
S. aureus + + +
S. lugdunensis + + V/-
Causes bacteremia & endocarditis
Mannitol Fermentation + and Narrow Beta Hemolytic
S. intermedius - V V
primarily isolated in animals
MISCELLANEOUS
CATALASE TEST (+) Control – S. aureus (-) Control – S. Pyogenes

COAGULASE TEST (+) Control – S. aureus (-) control – S. epidermidis
✓ Important marker of S. aureus ____________________

✓ Colonies with an odor similar to an OLD SOCK on MSA ______________________
✓ Bacitracin is also known as ____________________________
✓ Able to tolerate 7.5% NaCl ____________________________p
✓ S. aureus produces MEDIUM SIZED, BUTYROUS colonies
✓ CNA – Columbia Colistin Nalidixic acid – can be used to isolate gram + cocci
✓ S. intermedius and S. hyicus causes DOG BITE INFECTION / canine pyoderma
STREPTOCOCCUS, ENTEROCOCCUS & SIMILAR ORGANISMS
GRAM (+) COCCI, CATALASE NEGATIVE

LAP TEST (Leucine Amino Peptidase) ___________________________________________________________________
Substrate: Leucine-beta-naphthylamide Reagent: cinnamaldehyde (+) result: ___________ (-) no color or slight yellow
Streptococci are non-motile & capnophilic

Starting Point of Streptococcus identification ____________________________________
Medium of Choice _____________________________ Selective Medium ________________________________
ALPHA HEMOLYTIC BETA HEMOLYTIC GAMMA HEMOLYTIC

S. pneumoniae & S. pyogenes & S. agalactiae Group D Streptococci
VIRIDANS streptococci Enterococci

✓ _______________________Classifies Streptococci as to hemolysis on BAP
✓ _______________________ partial / incomplete hemolysis on BAP, greening of agar
✓ _______________________ complete hemolysis on BAP; clear zone around the colonies
✓ _______________________ no hemolysis on BAP
✓ __________________________________ colony surrounded by inner alpha outer beta hemolysis
✓ LAP TEST (+) – Enterococcus, Streptococcus, Pediococcus
✓ LAP TEST (-) – Aerococcus & Leuconostoc
DIFFERENTIAL TESTS for ALPHA HEMOLYTIC STREPTOCOCCI – S. pneumoniae & VIRIDANS

CAPSULAR SWELLING BILE SOLUBILITY TEST OPTOCHIN DISK TEST
TEST
NEUFELD QUELLUNG Media: ______________________ Optochin is also known as ___________
To be added: sodium desoxycholate
(+) result __________________________________ Chemical name of Optochin is –
Ethylhydrocuprein hydrochloride
(-) result __________________________________
Considered SUSCEPTIBLE using
If on broth (2% sodium desoxycholate)
(+) result if on broth ____________________________ 6mm disk ______________
(-) result _____________________________________ 10 mm disk _____________
S. pneumoniae S. pneumoniae S. pneumoniae
VIRIDANS VIRIDANS VIRIDANS
S. pneumoniae VIRIDANS Streptococci

✓ Normal flora of nasopharynx and oropharynx (URT)
✓ LANCET or bullet shaped diplococci ✓ Not classified under Lancefield
✓ Requires 5-10% CO2 ✓ Produces glucans & dextrans
✓ Young colonies are __________________________ ✓ Regarded as the ___________________________
Noted after 24 hrs of incubation
✓ Aged colonies (colonies after 48 hrs incubation) assume ✓ # 1 cause of _______________________________
a _______________________________, ✓ Mouse Virulence test (-)
_______________________, colonies with Checker ✓ Classified as Viridians:
appearance. Doughnut (umbilicated)

1) S. mitis group – found in the oral cavity
✓ # 1 cause of ________________________________ 2) S. mutans group – most commonly isolated Viridans
3) S. salivarius group
✓ Agent of __________________________________
✓ Can cause Lobar pneumonia (rusty colored sputum)
✓ Mouse Virulence Test (+) ✓ # 1 contributor to dental carries & most common
member of S. mutans group __________________
✓ S. mitis – frequent cause of SBE
BETA HEMOLYTIC S. pyogenes, S. agalactiae
LANCEFIELD CLASSIFICATION – most significant in identifying and classifying Beta Hemolytic Streptococci; based on the extraction of C
carbohydrate from the Streptococcal cell wall
IDENTIFICATION TEST for REAGENT RESULT

GROUP A - Beta Hemolytic Streptococci
Streptococcus pyogenes
PYR TEST (+) Result _________________________
Pyrrolidonyl – alpha-naphthylamide (-) result: orange or no color change
Bacitracin Disk Test TAXO A
Purpose of Bacitracin Disk Test

1) __________________________________________________________
2) __________________________________________________________

IDENTIFICATION TEST for REAGENT / MEDIA RESULT
GROUP B - Beta Hemolytic Streptococci
Streptococcus agalactiae
HIPPURATE HYDROLYSIS TEST To detect BENZOIC ACID use
Detects hydrolysis of sodium hippurate to _______________________________
benzoic acid and glycine
Alternatively, (+) Result ________________________
The positive result is due to enzyme: To detect Glycine use
Hippuricase/hippurate hydrolase ________________________________
(+) Result:
ENHANCED hemolysis as shown by an
CAMP TEST Media ___________________________
arrow head zone of beta hemolysis
(Christie, Atkins, Munch, Peterson) Known organism ___________________
(-) Result:
Group A Group B Group C, F, G

S. pyogenes S. agalactiae Group C – S. dysgalactiae subsp equisimilis
Group F – S. anginosus group
Bacitracin S R R
SXT R R S
Trimetophrim sulfamethoxazole
CAMP
Hippurate
PYR
✓ ___________________________will develop small, PINPOINT, transparent colonies

✓ ___________________________ will develop grayish white mucoid colonies, or semi-opaque coloniaes with organism concentrated at the
center appearing BULL’s EYE
✓ ___________________________media used to detect genital carriage of group B Streptococci during pregnancy
✓ LIM BROTH is for _______________________________________________
✓ Identification test MORE SPECIFIC to S. pyogenes ______________________
TEST (+) CONTROL (-) CONTROL

BACITRACIN DISK TETS S. pyogenes S. agalactiae
M. luteus S. aureus
CAMP TEST S. agalactiae S. pyogenes
PATHOGENESIS & SPECTRUM OF DISEASE DIAGNOSTIC TESTS for SCARLET FEVER

S. pyogenes 1. Bacterial pharyngitis / Strep throat DICK’S TEST Susceptibility test for scarlet fever
(+) redness at the site of injection
2. Pyodermal Infection – ERYSIPELAS SCHULTZ- PURPOSE:
CHARLTON
BLANCHE
3. scarlet fever – strawberry tongue PHENOMENON
(+) fading of rashes/gradual

disappearance of rashes
4. Necrotizing Fascitis
S. agalactiae Septicemia
POST STREPTOCOCCAL SEQUELAE Normal flora of # 1 cause of

female genital _____________________________
tract or lower
GIT In adults it can cause Postpartum
endometriosis

VIRULENCE FACTORS OF S. pyogenes GAMMA HEMOLYTIC
M protein Group D Streptococci
Enterococci
Protein F Promotes attachment to epithelial cells
Streptokinase Dissolution of clot
Screening Test: BILE ESCULIN TEST
Hyaluronidase Spreading factor
Erythrogenic or Group D Non-Enterococci & Enterococci = _______
Pyrogenic Toxin Media
STREPTOLYSIN Indicator
Streptolysin O Oxygen labile, Antigenic (+) Result
Can cause sub-surface hemolysis on CONTROLS (+) Control: E. faecalis
BAP
(-) Control: E. coli; S. pyogenes
Can cause hemolysis only when
incubated anaerobically
Streptolysin S Oxygen stable, non-antigenic
DIFFERENTIAL TESTS
Can cause surface hemolysis on BAP
PYR TEST SAL PENICILLIN
TOLERANCE
Can cause hemolysis when incubated
6.5% NaCl
aerobically
Group D
Non-
Enterococci
VIRULENCE FACTORS OF S.agalactiae
Enterococci
CAPSULE
ENTEROCOCCI
(E. faecalis; E. faecium, E. durans, E. avium)
GROUP C, F & G Normal flora: ✓ E. faecalis & E. faecium are normal GIT flora
Beta Hemolytic Skin, Nasopharynx ✓ Most common isolate ____________________
Streptococci Gastrointestinal tract ✓ Causes ___________________________
Genital tract ✓ Can tolerate o.o4% tellurite
GROUP D NON-ENTEROCOCCI / S. bovis group

S. equinus, S. gallolyticus, S. infantarius, S. alactolyticus
✓ Encountered in blood cultures of patients with bacteremia, septicemai & endocarditis
✓ Presence of S. gallolyticus subspecies gallolyticus, in blood cultures has HIGH CORRELATION with GIT Carcinoma/colon
cancer
MISCELLANEOUS
Tests Group A Group B S. pneumoniae Viridans Group D Enterococci

Bacitracin S S
SXT S S V
Optochin S
PYR
Bile Esculin
Growth in 6.5% NaCl
CAMP
LAP + + + + + +
Hippurate + -
Exceptions may occur
STREPTOCOCCUS LIKE ORGANISMS:

Abiotrophia & Granulicatella ✓ Previously NVS-nutritionally variant Streptococciotitis media
✓ A.k.a Thiol Requiring, Pyridoxal requiring, Satelliting Streptococci
✓ Can cause bacteremia, endocarditis &
✓ ______________________________________________________

NEISSERIA & MORAXELLA (M. catarrhalis)
• Non-motile, aerobic, capnophilic _______________________, use of _____________________ effective & economical way of providing
increased CO2
• specimens will require used of TRANSPORT media: Amies with charcoal, Transgrow, JEMBEC (commercial transport system)
• COLD SENSITIVE; non-hemolytic; Most species are CHO fermenters
• Develops non-pigmented colonies except: N. flava, N. flavescens, N. subflava
• All are Catalase test & Cytochomre Oxidase Positive except: N. elongata
• SCREENING TEST: Oxidase Test
Reagent: tetramethyl-p-phenylenediamine dihydrochloride (+) Result: ___________________________________________
• To speciate: CHO UTILIZATION TEST media: _____________________________________indicator___________________
In an acid pH (+) media will turn ___________________________________
Pseudomonas, Plesiomonas, Aeromonas, Moraxella, Vibrio, Campylobacter, Helicobacter, Neisseria
SELECTIVE MEDIA INHIBITORY AGENTS

FOR ISOLATION
Thayer Martin VCN Vancomycin ______________________
Colistin __________________________
Nystatin __________________________
Modified Thayer Martin VCN - T Same as Thayer Martin but with Trimetophrim -
Martin Lewis VCA - T Same as Modified Thayer Martin but Nystatin is replaced by Anisomycin -
New York City VCA – T Same as Martin Lewis but Anisomycin is replaced by Amphotericin B
GC-LECT medium VCA-T plus Same as New York City but with Lincomycin -
Lincomycin
N. gonorrhoea N. meningitidis
✓ Kidney, coffee bean shaped diplococci intracellular within PMN ✓ Bean shaped diplococcic; sensitive to SPS
✓ Primary Virulence Factor: _________________________ ✓ Primary Virulence factor: ___________________
✓ FERMENTS ___________________________________ ✓ Neufeld Quellung Test _____________________
✓ SUPEROXOL TEST (+) __________________________ ✓ FERMENTS ______________________________
_____________________________________________ ✓ Can be a normal flora, natural habitat is oro and
Weakly (+) N. meningitidis and N. lactamica nasopharynx. (Nonencapsulated strains)
✓ Specimen: pus secretions from urethra, cervix, prostate, throat and ✓ Specimen to detect carrier state
rectal mucosa _____________________________________
✓ Causes: ✓ Causes:
a) Gonorrhea __________________________________ a) Bacterial Meningitis __________________
b) Meningococcemia
b) Opthalmia neonatorum – a gonococcal eye infection acquired c) Waterhouse Friderichsen Syndrome – severe
by newborns when discharge from infected mother accumulates in form of meningococcemia characterized by
their conjunctiva bleeding of adrenal glands/DIC
c) PID which may cause sterility, perihepatitis or Fitz Hugh Curtis

Syndrome
✓ To collect specimen use ________________________________

Calcium alginate and cotton swab may be inhibitory to N.
gonorrhoea
Moraxella catarrhalis
✓ Morphologically and biochemically resembles Neisseria, Oxidase test _________________
✓ Catalase negative, on BAP ____________________________________________
✓ a normal flora of oro and nasopharynx but may cause otitis media (3rd most common cause)
✓ does not degrade sugar _______________________________________________
✓ BUTYRATE DISK TEST / Tributyrin test Positive ___________________________________
Uses substrate: bromo-chloro-indolyl-butyrate; (+) result ____________________________
_______________________________________________________________________________________
✓ __________________________________Colonies remaining intact when pushed across the plate using inoculating
loop; colonies with WAGON WHEEL APPEARANCE
✓ Compared with Neisseria, it is Tributyrin Test & DNase Test ____________
✓ (-) growth on media for Neisseria because of _________________

RESULTS in CHO UTILIZATION TEST ACID PRODUCED FROM:
Specie Glucose Maltose Lactose Sucrose Fructose
N. gonorrhea + - - - -
N. meningitidis + + - - -
N. lactamica + + + - -
ONPG _____
N. cinerea, N. flavescens - - - - -
N. elongata
N. sicca- bread crumb like, wrinkled + + - + +
colonies
N. mucosa
N. subflava + + - V V
MORAXELLA - - - - -
Agents of Pelvic Inflammatory Agents of Otitis Media: - (+) smear in SYMPTOMATIC male is
Disease/PID: _________________________________ considered diagnostic of gonorrhea
_________________________________ _________________________________ - N. elongate-clay like consistency of
_________________________________ _________________________________ colonies on BAP
_________________________________
BUTYRATE DISK TEST: (+) Control – M. catarrhalis; (-) Control – N. gonorrhoea
SPORE FORMERS – Bacillus(aerobic) & Clostridium(anaerobic)

GENUS BACILLUS
. Bacillus cereus Bacillus subtilis
Bacillus cereus & Bacillus subtilis are both ________________ and on BAP __________________
• Virulence factor: exotoxin/enterotoxin cholera like toxin; • Opportunistic pathogen; Source of bacitracin
Causes food poisoning (Diarrheal & Emetic Type) • Cause eye infection in heroin addicts
• Produces two types of toxins • Blood bank contaminant @ RT
• Best specimen for testing ________________________ • Penicillin ________________
• (+) gelatin hydrolysis; (+) growth on PEA • Selective media: Mannitol egg yolk polymyxin B agar
• Emetic – Cereulide
• Diarrheal – Nhe, cytK or Hemolysin IV and HBL
• Media: MEYP, PEMBA
Bacillus anthracis
✓ Characteristics that will differentiatie B. anthracis from other Bacillus spp : ________________________________
✓ forms the so called “disjointed bamboo fishing rod appearance”, Typically has ___________________
✓ Virulence Factor
a) polygama D-glutamic acid – demonstrate using M’Fadyean, India ink
b) Exotoxin with 3 components: edema factor, lethal factor, protective antigen- function of exotoxin
__________________________
✓ selective medium: PLET Polymyxin Lysozyme EDTA Thallous acetate
✓ colonies are so TENACIOUS that when lifted using a loop it will stand like beaten egg white
Colonies with ____________________________________________________
✓ colonies may show swirling projections forming the so-called ________________________or _________________
colonies with cut-glass appearance or __________________________
✓ Forms the string of Pearl appearance (MHA with 10 u penicillin) or. 05 u penicillin on BAP)
String of Pearl Pattern is due to _________________________________________
Inoculate on MHA + disk + coverslip incubate 3 -6 hrs 37 degC, remove colonies and place on slide
✓ Forms the so-called “inverted Fir tree or inverted pine tree appearance
✓ Lecithinase test Positive culture media __________________________ + result ______________________________
✓ Gelatin hydrolysis _______ and no growth on PEA agar
✓ Causes anthrax, Ascoli Test ____________________________________________________
Note: Clostridium septicum, w/c is associated with malignancies (colorectal cancer) – “medusa head colonies”, beta hemolytic, smoothly
Swarming

EXTERNAL INTERNAL
Cutaneous Anthrax Pulmonary Anthrax Intestinal anthrax Injectional anthrax
(+) Black Eschar ingestion of improperly Use of contaminated
Woolsorter’s disease cooked infected meat drugs of abuse like
Ragpicker’s disease Heroine
Hide porter’s disease
• Heat or ethanol shock test treatment = 70 degC 30 mins or 80 degC 10 mins, for B. anthracis 62-65 degC for 10-15 minutes =
PURPOSE _____________________________________________________________________
GENUS CLOSTRIDIUM
Strict anaerobes but some are aerotolerant, catalase negative
Histotoxic Neurotoxic Enteric
Clostridium Gas gangrene/ Clostridium Tetanus Clostridium Pseudomembranous
perfringens Myonecrosis tetani difficile colitis
Enteritis Antibiotic associated

necroticans diarrhea
Clostridium Foodborne Botulism
botulinum Infant Botulism
SID – sudden infant death
syndrome
Virulence Factors
C. perfringens Alpha toxin & enterotoxin
C. tetani TETANOSPASMIN – blocks the release of neurotransmitters causing
C. botulinum Botulinum Toxin
C. difficile Toxin A -Enterotoxin and Toxin B- Cytotoxin
C. perfringens (Frankel’s Bacillus; C. welchii) C. tetani

✓ Microscopically forms ______________________________ ✓ Lollipop Bacillus, Tack head Bacillus, tennis racket
✓ Hemolysis on BAP bacillus, Drumstick bacillus
________________________________________________ ✓ SPASTIC PARALYSIS
✓ Diagnosis is often made by
________________________________________________
1) Observation of symptoms:
✓ (+) Stormy fermentation of milk – Litmus Milk Trismus – lock jaw
✓ (+ Lecithinase Test & Nagler’s test using ________________ Risus sardonicus (sardonic smile)
✓ (+) Reverse CAMP TEST ____________________________
2) Observation of terminal swollen spores
_________________________________________________
(+) result: enhanced hemolysis as shown by arrow head zone of
beta hemolysis
C. botulinum – Canned Good Bacillus C. difficile

Von Ermengen’s Bacillus Major cause of diarrhea in hospitals
✓ ____________________________________________ ✓ Normal flora of the colon
✓ Not cultured; on BAP __________________________ ✓ Diagnosis is usually thru ________________________
✓ Spores are __________________________________ ✓ Specimen:
Freshly passed stool- liquid /unformed stools for culture & toxin
✓ (+) lipase (iridescent sheen on EYA)
assay
✓ FLACCID PARALYSIS
Formed stool/rectal swab ________________________
✓ Infant Botulism – FLOPPY BABY SYNDROME
✓ Media:
Crib Death / Sudden Infant Death (SID)
a) CCFA – Cycloserine Cefoxitin Fructose Agar
✓ To confirm:
___________________________________________________
Demonstration of neurotoxin in serum, feces,
b) on BAP – it fluoresces CHARTREUSE
vomitus or gastric contents
✓ ELISA -detection of GDH glutamate dehydrogenase – confirms
presence of C. difficile
✓ NAAT – detection of tcdA & tcdB genes

Motility Lecithinase Lipase Lactose Glucose Gelatinase Indole Stormy Nagler

Fermentation of Reaction
milk
C. - + - + + + - + +
perfringens
C. + - + - + + - - -
botulinum
C. tetani + - - - - + + - -
C. difficile + - - - + + - - -
All are saccharolytic EXCEPT: C. tetani and C. septicum Swarming (+) Anaerobe: C. tetani & C. septicum
NON-BRANCHING – GRAM (+) BACILLI
CATALASE (+) Listeria, Corynebacterium;
CATALASE (-) Erysipelothrix, Lactobacillus & Gardnerella vaginalis (gram-V/ (-)
Listeria monocytogenes
• Primarily an animal pathogen; MOTILITY at RT _____________________________________________with amphitrichous flagella
• Presumptive ID is thru _____________________________On BAP ________________________________________
• On Semi solid Media at RT i.e., Gelatin Medium ______________________________________________________
• Can tolerate cold enrichment (growth at 4 degC) because of this it is known to cause __________________________
• Other diseases caused: __________________________________________________________________________
• Virulence factor: ___________________________ allows survival of organism within phagocytes
• (+) CAMP Test, result is more pronounced if the known organism is ___________________________
(+) result ___________________________________________
• Hippurate Hydrolysis & Bile Esculin ______________
• ________________virulence test. Culture inoculated on conjunctival sac of rabbit (+) result: purulent conjunctivitis
• Major source of infection is the contaminated food (fruits, dairy products, cabbage)
• Cultured on Mc Bride’s media
REMEMBER:
Neonatal meningitis __________________________________________________________________________________
Meningitis in children less than 5 years ___________________________________________________________________
5 years -29 years ____________________________________________________________________________________
Meningitis in more than 29 years old _____________________________________________________________________
Meningitis in elderly/ immunocompromised patients _________________________________________________________
Corynebacterium diphtheriae a.k.a Kleb Loeffler’s Bacillus

✓ Non-motile & highly pleomorphic; MOT – exposure to respiratory droplets
✓ One end typically swollen – club shaped appearance; palisade arrangement –side by side arrangement, picket fence
arrangement, x,y,v,l formation
✓ Appears beaded when stained with methylene blue because of _________________________metachromatic granules
✓ Hemolytic pattern:
✓ MEDIA for isolation
a) CTBA -cystine tellurite blood agar _________________________________________________________________
b) Tinsdale medium ______________________________________________________________________________
c) Loeffler’s serum & PAI’s coagulated egg ____________________________________________________________
✓ Colony types: gravis, mitis, belfanti and intermedius - based on the phenotypic characteristics of size, texture, color,
hemolysis and the presence of metachromatic granules
✓ Toxigenic C. diphtheriae causes DIPTHERIA – low grade fever, mild sore throat & body malaise.
Virulence Factor ___________________________characteristic symptom __________________________________
✓ (+) growth on media will require detection of toxin production
ADDITIONAL INFORMATIONS:
✓ SPECIMENS FOR C. diphtheriae: oropharyngeal swab, throat swab, nasopharyngeal swab, nasal swab
✓ To collect specimen, use CALCIUM ALGINATE or swabs made of dacon.

✓ WHO requirement: collect 2 specimens: nasal and oropharyngeal swab)
✓ C. diphtheriae var. GRAVIS – produces the largest colony type, MITIS – colonies with fried egg appearance, INTERMEDIUS produces
small colonies and BELFANTI – most commonly recovered
TOXIGENECITY TEST
Modified Elek’s Animal Inoculation / Guinea Pig Lethal Test
• On agar media, place a filter paper strip with diphtheria • Suspension of isolated strain of C. diphtheriae (0.3 ml) is
anti-toxin, streak + control at right angle to the strip of injected subcutaneously into thigh of two guinea pigs, one
anti-toxin, streak negative control in the same manner. protected intramuscularly with 500 units of diphtheria
Unknown culture suspected of C. diphtheriae is streaked antitoxin 18-24 hours before the test.
parallel to + and - control. Incubate at 35 degC for 24 –
48 hrs. • (+) result: The unprotected animal dies within 2-3 days
• (+) result ___________________________________ with evidence of hemorrhage in the adrenal glands
❖ SHICK’s test ______________________________________________________________________________

❖ Vero cell cytotoxicity Assay – GOLD STANDARD for Diptheria toxin production
Other species
C. ulcerans Bovine mastitis Nitrate Reduction (-); Urease (+);
Esculin Hydrolysis (-)
C. pseudotuberculosis lymphadenitis nonlipophilic Nitrate Reduction (V); Urease (+)
Esculin Hydrolysis (-)
C. Throat flora but may cause Nitrate Reduction & Urease (+)
pseudodiphtheriticum endocarditis Esculin hydrolysis (-)
a.k.a
Hoffman’s Bacillus
C. minutissimum erythrasma Nitrate Reduction, Urease & Esculin
hydrolysis (-)
C. urealyticum UTI Lipophilic Nitrate Reduction (-) Urease (+)
C. jeikium Gamma hemolytic Nitrate Reduction, Urease & Esculin

a.ka. Infect immunocompromised hydrolysis (-)
JK Bacillus Causes
Prosthetic heart Valve infection
MDR except vancomycin
Metallic sheen on BAP
Erysipelothrix rhusiopathiae Lactobacillus Gardnerella vaginalis

• Easily decolorized so it may appear A normal vaginal flora, GIT & • Pleomorphic organism
gram variable mouth flora • Gram (V) or Gram (-)
• H2S positive on TSI media, NM • Inhibited by SPS
catalase negative Cultured on tomato juice agar • Agent of Bacterial vaginosis
• Glucose & lactose fermenter • Diagnosis of BV:
• Causes ERYSIPELOID, a localized Can be noted in PAP’s-stained
skin infection an occupational smears a) Whiff test
hazard for those handling meat and (+) result: fishy amine like odor
poultry and fish
• On Gelatin stab:
________________________________ b) Cytology / PAP’s-stained smears
__________________- squamous ET cells studded
________________________________
with Bacilli
________________________________
c) Use of HBT – Human Blood Bilayer Tween

a CNA with amphotericin B; human blood is
layered on top to enhance __________ hemolysis
of the organism
NUGENT SCORING- to detect bacterial vaginosis
❖ Mobilincus, Peptostreptococcus, Porphyromonas,Prevotella & M. hominis _____________________________________

L. monocytogenes E. rhusiopathiae
Beta hemolysis + -
Growth at 4 degC + -
Catalase Test + -
Motility + -
Esculin Hydrolysis + -
H2S on TSI - +
VP test + -
Catalase Test Motility Esculin H2S on TSI

Corynebacterium + - V -
Listeria + + + -
Erysipelothrix - - - +
Gardnerella vaginalis - - - -
MOTILITY SALICIN CAMP ESCULIN HYDROLYSIS

Corynebacterium
Listeria
BRANCHING-AEROBIC ACTINOMYCETES (Fungus Like)

ACTINOMYCETES Partially AF AEROBIC ACTINOMYCETES: Nocardia, Rhodococcus, Gordonia, Tsukamurella
Non Acid Fast aerobic actinomycetes: Streptomyces, Actinomadura, Dermatophilus
Nocardia Aerobic, branched, beaded, gram + bacilli, partially AF and beta hemolytic, produces crumbly wrinkled colonies
resembling bread crumbs, colony odor _____________________________
N. asteroides ___________________________________________________________________
N. brasiliensis _________________________________________________________________
Actinomadura Agent of
Rhodococcus equi Key ID characteristic:
Tropheryma whipplei Facultative intracellular pathogen (presence of PAS staining macrophages), identified thru PCR
Agent of _______________________________ affecting middle aged men
❖ Tap water Agar – to detect aerial hyphae
ACID FAST BACILLI - MYCOBACTERIUM

• Aerobic, non-motile;
• Slow growers and requires whole egg for growth
• Difficult to stain but once stained difficult to decolorize due hydroxy methoxy acid / mycolic acid
• Screening procedure to detect the GENUS MYCOBACTERIUM _____________________________________________________
Ziehl Neelsen or Kinyoun’s
Prmary dye ____________________________ Decolotizer ____________________ usual counterstain _____________________________
Result: AFB _____________________________________________________ NAFB ____________________________________________
THREE GROUPS:
M. tuberculosis complex MOTT/ Mycobacterium other than M. leprae
tuberculosis/ Runyon’s Classification
✓ a.k.a Hansen’s disease
PHOTOCHROMOGENS ✓ Causes LEPROSY
M. tuberculosis _________________
SCOTOCHROMOGENS ✓ Non-culturable In vitro but viability can be
M. bovis ______________________ NON-PHOTOCHROMOGENS maintains using MICE FOOT PADS or FOOT
RAPID GROWERS PADS of ARMADILLO
M. africanum __________________
✓ Under the microscope it forms the so-called
M. microti & M. canetti – can infect those Cigar Packet arrangement
who are immunocompromised ✓ Diagnosis is often made based on clinical
symptoms
PROCESSING OF SPECIMENS FOR MYCOBACTERIUMDETECTION
1st STEP: 2ND STEP SEDIMENTS

specimens are If STERILE used for Do SCREENING TEST
classified as centrifuge
Smear We stain the smear using ACID
STERILE If NON-STERILE before FAST STAINING
preparation
or centrifugation do the ff:
&
NON- STERILE Digestion &
Decontamination Culture
✓ Purpose of Digestion & Decontamination: Decontamination must be carried out to remove normal flora and other contaminating
organisms. Digestion must be carried out to dissolve disulfide bond in mucus which could trap MTB
✓ ____________________________________________________________________________________________________
✓ Gold standard for Digestion & decontamination: NALC (N-acetyl-L cysteine and NaOH) ______________________________________
NALC or dithiotreitol or SPUTOLYSIN and NaOH
✓ NaOH (2,3 and 4%) __________________________________________________________________
✓ Zephiran (benzalkonium chloride) and Trisodium phosphate / Z-TSP
1% Cetylpyridium chloride – can prolong shelf life of sputum for 8 days
Oxalic acid 5%_____________________________________________________________________________
DOH REPORTING (National) CDC-REPORTING

ZIEHL NEELSEN – KINYOUN’S AF STAINING-REPORTING Number of AFB REPORT
Number of AFB REPORT seen
seen 0 per 300 fields negative
0 No AFB seen in 300 visual fields
1-2 per 300 fields (+)/(-) report on 2nd slide
+n 1-9 /100 fields
1-9 per 100 fields 1+
1+ 10-99 /100 fields
1-9 per 10 fields 2+
2+ 1-10/OIF in at least 50 visual fields
1-9 per field 3+
3+ More than 10 AFB /OIF in at least 20 visual fields
More than 9 per field 4+
If AFB SMEAR is (+), we do CULTURE using the FOLLOWING CULTURE MEDIA

NON-SELECTIVE AGAR BASED MEDIA SELECTIVE LIQUID
egg based media with malachite green, inhibits clear media, easy examination of colonies with antibiotics these are non-
contaminating organism; growth in 6-10 weeks growth in 10-12 days conventional media
Common Disadvantage:
Lowenstein Jensen Media: Middlebrook 7H10 with dextrose Gruft Modified Bactec 12B,
___________________________________ Middlebrook 7H11 with casein Lowenstein Jensen Bactec 13A
Petragnani- more inhibitory: hydrolysate Selective Septi-chek
___________________________________ Mitchison 7H11 Middlebrook 7H11 Middlebrook 7H9
American Thoracic Society /ATS: Mitchison Selective 7H12
___________________________________ 7H11
Dorset Egg Media
Wallenstein medium: - recommended for M. avium

IF THERE IS (+) GROWTH IN ANY OF THESE MEDIA, NEXT STEP IS TO DO SERIES OF BIOCHEMICAL TESTS TO IDENTIFY SPECIES
BIOCHEMICAL TEST REQUIRED REAGENT (+) RESULT (-) RESULT POSITIVE NEGATIVE
MEDIA CONTROL CONTROL
NIACIN TEST Lowenstein Strip impregnated with YELLOW No color M. tuberculosis M.
Jensen Media CYANOGEN BROMIDE change intracellulare
NITRATE REDUCTION Sodium Nitrate After incubation we add: Initial (+) RED M. tuberculosis M.
Broth n-n -dimethyl-l- (-) no color change intracellulare
naphthylamine
sulfanilic acid After adding zinc powder:
(+) no color change
to confirm a (-) result we
(-) RED
add:
zinc dust/zinc powder Use of Strip (+) Blue
IRON UPTAKE TEST Lowenstein No color x
Jensen Media change
ARYLSULFATASE Phenolphthalein 2N Sodium carbonate . M. fortuitum M.
TEST media intracellulare
Detection of rapid
growers
PYRAZINAMIDASE Pyrazinamidase 1% ferrous ammonium (+) red
TEST agar sulfate
HEAT STABLE Tween 80 Heating of colonies at 68 QUALITATIVE M. fortuitum M.
CATALASE TEST Media degC prior to the (+) ____________________ M. gordonae tuberculosis
To detect heat labile addition of reagent is (-) ______________________
catalase required
Mycobacterium like M. QUANTITATIVE
tuberculosis (+) height of bubbling more
than 45 mm
(-) less than 45 mm
TWEEN 80 Useful in the identification of M. kansasii ____________________ M. kansasii M.
HYDROLYSIS TEST M. tuberculosis (+) in 10-20 days intracellulare
(+) result: _____________________________ (-) result: no color change
Indicator: _____________________________
TELLURITE For the identification of M. avium M. avium M.
REDUCTION TEST (+) result: smooth, fine black precipitate (smoke like action) tuiberculosis
(-) result:gray clumps (no smoke like action)
SAL TOLERANCE For identification of rapid growers & M. triviale M. fortuitum M. gordonae
TEST 5% NaCl (+) growth while (-) no growth
T2H/ TCH May be done to differentiate M. tuberculosis and M. bovis M. bovis M.
susceptibility (+) growth inhibition (no growth) tuberculosis
Thiopene-2- (-) growth
carboxylic hydrazide
Growth in Mac ✓ To identify M. fortuitum -chelonae complex
Conkey agar without
crystal violet
Mycobacterium tuberculosis
✓ a.k.a Koch’s Bacillus or Tubercle Bacillus; obligate aerobe, gram (+) or gram neutral
✓ Virulence Factors: (1) CORD FACTOR and (2) SULFATIDES _____________________________________________
✓ With MUCH GRANULES, length is 1-4 u while diameter is 0.2 – 0.6
✓ Produces “cauli flower like growth at 35 – 37 degC;
✓ Killed by boiling-10 mins; direct sunlight – 20-30 hrs exposure; exposure to 5% phenol for 24 hrs., autoclaving (moist heat)
Organisms from culture are killed within 2 hrs exposure to sunlight
Note: can remain viable for 6-8 months in dried sputum since it is resistant to drying, droplets of dried sputum in the air may be
infectious for 8-10 days
✓ Causes Pulmonary TB; BCG vaccine / Bacillus Calmette Guerin __________________________________
Types of TB: (Primary, Secondary, MDR-TB – shows resistance to PRIMARY TB DRUGS), XDR-TB- shows resistance not only to
primary but also to some 2 nd line TB drugs
POTT’s disease – TB of spine while MILIARY – disseminated TB, extrapulmonary
PRIMARY TB DRUGS – RIPES – Rifampin-Isoniazid- Pyrazinamide-Ethambutol- Streptomycin
2ND LINE TB DRUGS – ofloxacin, kanamycin, ciprofloxacin

DIAGNOSIS
1. Skin Testing – MANTOUX TEST – involves intradermal injection of PPD on the skin
PPD ________________________________________________________________________
Von Pirquet _____________ (old tuberculin mixed with lanolin to produce ointment w/c is scratch onto the skin)
Volmer’s Patch
DOTS ______________________________________________________________
2. Chest X-Ray
3. Sputum exam – collect _______________in a day (1st ______________________, 2 nd ______________________)
4. Culture – maintained for 8 wks / 2 months
✓ CAULI FLOWER LIKE GROWTH – tan to buff, dry, rough, warty, granular
5. QUANTIFERON TB (ELISA) rapid and requires heparinized whole blood
6. GeneXpert – PCR Based NAAT, rapid and sensitive
DIRECT SPUTUM SMEAR MICROSCOPY/DSSM Symptoms same with TB
• Smear size ___________________ N. asteroides ______________
• Dry prior to heat fixation – to prevent aerosol P. westermani _____________
• Examine 300 fields before reporting negative result H. capsulatum ______________
Mycobacterium bovis
✓ _____________________________________________________________________________________________
✓ ______________________________________________________________________________________________
✓ Nitrate and Niacin Test __________________
Mycobacterium leprae a.k.a __________________________
✓ Forms the so-called ____________________________________________________________
✓ Non culturable on artificial media, but can be maintained in the lab using ____________________________
✓ Causes Leprosy MOT – prolong skin to skin contact with infected person, droplets from nose & mouth (inhalation)
✓ Clin manifestation LEONINE FACE and it has tropism for ____________________
✓ Tuberculoid type /paucibacillary_____________________________________________________________________
✓ Lepromatous type /multibacillary ____________________________________________________________________
✓ Basis of diagnosis: _______________________________________________________________________________
✓ Lepromin Test___________________________________________________________________________________
Fernandez reaction ______________________________
Mitsuda Reaction ________________________________
✓ Specimen: TISSUE JUICE (earlobes, nasal scrapings)
NON-TUBERCULOUS MYCOBACTERIA
PHOTOCHROMOGENS
✓ Mycobacterium kansasii – regarded as the # 2 NTM to cause TB in those with AIDS; a.k.a Cross Barred Bacillus
✓ Mycobacterium marinum- can cause Fish Tank granuloma or _________________________________
✓ Mycobacterium asiaticum, M. simiae , M. intermedium & M. novocastrense
SCOTOCHROMOGENS
✓ Mycobacterium szulgai - pulmonary disease, predominantly in middle-aged men; cervical adenitis; bursitis
✓ Mycobacterium scrofulaceum - Cervical adenitis in children, bacteremia, pulmonary disease, skin infections
✓ Mycobacterium gordonae / M. aquae __________________________________
✓ M. xenopi - Primarily pulmonary infections in adults. ______________________colonies on CORN MEAL AGAR
✓ M. flavescens , M. thermoresistible
✓ M. interjectum - Unknown Potentially pathogenic Chronic lymphadenitis, pulmonary disease
✓ Others: M. heckeshornense, M. tusciae, M. kubicae, M. cookie, M. hiberniae
NON-PHOTOCHROMOGENS
✓ M. avium complex - pulmonary infections in patients with preexisting pulmonary disease; cervical lymphadenitis; and
disseminated disease (M. avium pathogenic – LADY WINDERMERE SYNDROME while M. intracellulare = Battey Bacillus)
✓ M. terrae _____________________________ M. triviale - ___________________________
✓ M. gastri , M. ulcerans - mycobacteriosis, also referred to as Buruli ulcer, biochemically inert
✓ M. malmoense M. genovense -Disseminated disease in patients with AIDS
✓ , M. haemophilum , Others: M. heidelbergense; M. simiae; M. shimoidei
RAPID GROWERS
✓ M. abscessus subsp. Abscessus - Disseminated disease, primarily in immunocompromised individuals; skin and soft tissue
infections; pulmonary infections; postoperative infections
✓ M. fortuitum - Postoperative infections in breast augmentation and median sternotomy; skin and soft tissue infections; pulmonary
infections, usually single. localized lesions. Central nervous system (CNS) disease is rare but has high morbidity and mortal ity
✓ M. chelonei - Skin and soft tissue infections, postoperative wound infections, keratitis
✓ M. phlei – breadcrumbs growth on LJ medium
✓ Other: M. smegmatis – Urine AFB +

Tests Mycobacterium tuberculosis Mycobacterium bovis
Niacin test + -
Nitrate Reduction Test + -
Heat Stable Catalase test - -
Tween 80 Hydrolysis Test V -
Arylsulfatase Test (3days) - -
Iron Uptake Test - -
Pyrazinamidase Test + -
Iron Uptake Test - -
Pyrazinamidase Test + -
FAMILY ENTEROBACTERIACEAE
GENERAL CHARACTERISTICS
✓ Gram (-) bacilli or coccobacilli; facultative anaerobe, non-spore former
Yersinia spp are
✓ Motile except: _____________________________________________
✓ Some are with pili or fimbriae; Grows well on Mc Conkey agar
motile at _______; non-motile at _____
✓ All Ferments glucose and often with gas production aerogenic except: Except: ___________________
____________________________________________
✓ Catalase ______except S. dysenteriae type 1 and Cytochrome oxidase ___________except: Plesiomonas
✓ Based on Clinical Infections produced there are 2 types of
1) Primary Intestinal Pathogens ___________________________________________________________________________
2) Opportunistic pathogens ______________________________________________________________________________
✓ May possess 3 types of antigenic determinants which can be used in the serologic identification of a particulate organism. These antigens
are particularly useful in the characterization of E. coli, Klebsiella, Shigella and Salmonella
O or somatic antigen K or envelope antigen H flagellar antigen

For E. coli there are 164 types of O antigen & specific Consist of capsular polysaccharide Protein in nature
types maybe associated with a particular disease Heat labile Heat labile
With K antigens
Serotype 0111 – ___________________________________ ✓ Klebsiella
✓ E. coli
Serotype 0157 – ___________________________________ ✓ Salmonella
Vi antigen = S. typhi
STEPS in IDENTIFICATION (Manual)
Use colonies for SMEAR DO A SERIES OF

INOCULATE on BIOCHEMICAL TESTS to
STOOL PREPARATION
PLATED MEDIA i.e., identify the Genera/Species
Mac Conkey Agar/EMB i.e., ONPG test; IMViC test
Use the colonies to do
CYTOCHROME OXIDASE TEST; etc.
result must be (-) to consider it as
gm (-) bacilli
CULTURE MEDIA CLASSIFICATION INHIBITOR FERMENTABLE INDICATOR FERMENTER NON-

CHO FERMENTER
EMB/ Eosin Y Eosin Y &
EOSIN and Methylene
METHYLENE Methylene Blue Blue
BLUE
MAC CONKEY SELECTIVE Crystal violet
AGAR & and
DIFFERENTIAL Bile salts
XLD MEDIA Bile salts Xylose yellow Red/colorless
Xylose Lysine Lactose
deoxycholate Sucrose
HEKTOEN Bile Salts Salicin Yellow – Green/colorless
ENTERIC AGAR Lactose w/ or w/o black W or w/o black
Sucrose w/black:

CULTURE MEDIA CLASSIFICATION INHIBITOR FERMENTABLE INDICATOR FERMENTER NON-
CHO FERMENTER
SSA Bile Salts Red Colorless with

Salmonella Black center:
Shigella Agar
Colorless without
SELECTIVE MEDIA black center:
BSA Brilliant green Bismuth Salmonella = Black

BISMUTH Sulfite
SULFITE AGAR
✓ Klebsiella pneumoniae develops pink-purple _____________COLONIES

✓ E. coli develops pink to purple colonies with GREENISH METALLIC SHEEN
✓ Enterobacter develops pink-purple colonies with ________________ giving it a FISH-EYE appearance
OTHER MEDIA
a) GN Broth ___________________________
b) Selenite broth –enrichment broth for Salmonella and Shigella
c) Tetrathionate broth –enrichment broth for Salmonella
d) CIN (Cefsulodin Irgasan Novobiocin _________________________________________________________________________
e) BGA for other Salmonella except S. typhi; inhibitory agent ____________________________-pH indicator _______________
Salmonella colonies growing on BG agar may appear like _____________________________
BIOCHEMICAL TESTS
1) CARBOHYDRATE FERMENTATION TEST

✓ Media: TSI/Triple sugar Iron dispensed as Butt & Slant
✓ Composition:
Fermentable CHO pH indicator H2S indicator
Possible results:
In an acid pH __________________ In an alkaline pH ___________
H2S production ______________________________
Gas production: Splitting of media; pulling away of media; cracks on media
Color: Slant / Butt Report result Interpretation Classification of Inoculated

as organism
Red / Red NON-FERMENTER

Red / Yellow NON-LACTOSE FERMENTER
Yellow/ Yellow LACTOSE FERMENTER
Lactose Fermenters Late Lactose Fermenters Non-Lactose Fermenter
Enterobacter Citrobacter Proteus – Providencia - Morganella

Escherichia Serratia Edwardsiela
Klebsiella S. arizonae All Salmonella except S. Arizonae
S. sonnei All Shigella except S. sonnei
Y. enterocolitica All Yersinia except Y. enterocolitica
Erwinia – plant pathogen
✓ RAPID LACTOSE FERMENTERS- with the ff: enzymes: beta -galactosidase & lactose permease
✓ LATE LACTOSE FERMENTERS – beta galactosidase

TSI REACTIONS
K/K H2S (-) Pseudomonas, Burkholderia
K/A H2S (-) Providencia, Morganella, Shigella
K/A H2S & Gas (+) Salmonella, Proteus, Arizona C. freundii, E. tarda
A/A H2S (-) & Gas (+) Klebsiella, Escherichia, Enterobacter
(2) ONPG TEST O-nitrophenyl-beta-d-galactopyranoside)

✓ To detect late lactose fermenters
✓ To differentiate Citrobacter which are ONPG (+) from Salmonella which are ONPG (-)
✓ Salmonella are ONPG (-) except S. arizonae
✓ May be carried out using ONPG broth/disk (+) result: ____________
ONPG Positive ONPG Negative
Escherichia coli - Klebsiella spp – Enterobacter spp Proteus spp- Providencia - Morganella
Y. enterocolitica - Citrobacter spp Salmonella spp. - Shigella spp.
S. sonnei - H. alvei - S. arizonae Y. pestis - Y. pseudotuberculosis
(3) GELATIN HYDROLYSIS TEST
✓ Useful in the detection of Serratia
✓ Organism is inoculated on Nutrient Gelatin Media & incubated for 14 days at 35-37 degC. Within the incubation period, tubes are
removed from the incubator daily & it is placed inside the ref for 30 minutes or until the media had gelled
✓ (+) RESULT _________________________________ (-) RESULT _____________________________________________
(4) UREASE TEST
✓ Useful in the Identification of PPM -Proteus-Providencia-Morganella
✓ Based on the ability of the organism to convert urea to ammonia thru the action of the enzyme urease
✓ PPM are regarded as the __________________________________
✓ Slow Urease (+); (+) after 4 hours are the following (KEY-SC) = Klebsiella; Enterobacter; Yersinia; Serratia; Citrobacter
✓ Media is Christensen Urea Agar /Urea broth
✓ Indicator ____________________________________________ RAPID UREASE (+)
✓ (+) result: pink; red (-) yellow; (+) result after 4 hrs - orange
(5) DEAMINASE TEST PPM, H. pylori, Ureaplasma,
✓ Useful in the identification of PPM, these are the only deaminase (+) enterics Nocardia, C. neoformans, T.
✓ Uses lysine, tryptophan or phenylalanine mentagrophytes
✓ PAD + overnight culture + 10% Ferric chloride
✓ (+) result with Phenylalanine agar __________________________
✓ (+) result with tryptophan agar ______________________________
6) IMViC TEST (Indole-Methyl Red-VoguesProskauer-Citrate)
INDOLE TEST METHYL RED TEST VOGUES PROSKAUER TEST CITRATE UTILIZATION
Based on the ability of the Detects acetoin or acetylmethyl Based on the ability of the
organism to produce indole from carbinol organism to utilize citrate as a
tryptophan sole source of carbon
Detects Tryptophanase
SIM MEDIA MRVP BROTH MRVP BROTH SIMMON CITRATE AGAR
TRYPTOPHAN BROTH OR OR
CLARK LUBBS BROTH CLARK LUBBS BROTH
KOVAC’S METHYL RED INDICATOR ALPHA NAPHTOL No reagent will be added after
OR & incubation since the media has
EHRLICH’S REAGENT KOH incorporated indicator which is
_________________________
(+) red ring (+) Distinct red color pH less (+) Pink to red color (+) Blue
(-) No color development than 4.5 (-) No color change (-) Green
(-) No color change
✓ KOVAC’S – paradimethyl aminobenzaldehyde

✓ MRVP reactions are ______________________________________
✓ MR tubes must be incubated for 48 hrs before reading the result, RESULTS read before 48 hrs __________________________
✓ VP (+) KEESH = Klebsiella, Enterobacter, Ewingella, Serratia, Hafnia
✓ CITRATE (+) PS- CHEEKS = Providencia, Serratia, Citrobacter, Hafnia, Enterobacter, Ewingella, Klebsiella, S. enteritidis

Species I M Vi C Species I M Vi C
Escherichia coli + + - - Citrobacter freundii - + - +
Yersinia enterocolitica + + - - Serratia marcescens - V + +
Hafnia alvei
Proteus vulgaris + + - - Shigella sonnei - + - -
Edwardsiella tarda + + - - Salmonella typhi - + - -
Shigella dysenteriae - + - - Klebsiella oxytoca + - + +
Morganella morganii + + - - Proteus mirabilis - + - +
Yersinia pestis - + - - Providencia rettgeri + + - +
Providencia stuartii
Klebsiella pneumoniae - - + + Enterobacter spp. - - + +
Klebsiella ozaenae
TESTS THAT WILL SEPARATE SALMONELLA & SHIGELLA

(1) MOTILITY TEST
✓ Very useful in detecting non-motile enterics Klebsiella & Shigella
✓ May be carried out using SIM MEDIA
✓ On a tube media, it is motile if growth is outside the line of streak, NM if growth is @ the line of streak
✓ Use of TTC-triphenyl tetrazolium chloride – colorless dye that turns red with organisms’ growth
(2) MALONATE UTILIZATION TEST
✓ Based on the ability of the organism to use sodium malonate as the only carbon source
✓ Indicator: Bromthymol Blue (+) result ___________________ (-) result _____________________
(3) TEST TO DETECT H2S PRODUCTION
✓ May be carried using any media provided that the media contains an indicator for H2S detection
✓ Examples of media with H2S indicator: TSI, LIA, SIM, HEA (+) H2S ________________________________________
✓ H2S indicator in HEA, SSA and LIA __________________________
✓ H2S POSITIVE: Salmonella; Edwardsiella tarda, Citrobacter freundii ; Proteus vulgaris and Proteus mirabilis
(4) DECARBOXYLASE TEST
✓ Based on the ability of the organism to remove carboxyl group from specific amino acid thru the release of the enzyme decarbo xylase
✓ There are 3 amino acids that can be used:
AMINO ACID CARBOXYL MOELLER’S BROTH:
BROMCRESOL PURPLE + AMINO ACID + 1%GLUCOSE
LYSINE --------------------- CADAVERINE ✓ Tubes are overlaid with Mineral Oil
ORNITHINE----------------- PUTRESCINE ✓ (+) result: ___________________
ARGININE ------------------ CITRULLINE ✓ (-) result ____________________
✓ Media: Moeller’s Broth ✓ USE OF MINERAL OIL
Indicator: ___________________________________________ ________________________________
LYSINE ORNITHINE ARGININE

Enterobacter - + + LYSINE IRON AGAR:
cloacae Media which can be used to detect deamination & decarboxylation
Indicator: ferric ammonium citrate
Enterobacter + + -
It is a media dispensed as slant & butt
aerogenes
SLANT is observed for _________________________________
Escherichia + + -
BUTT is observed for __________________________________
coli
(+) result in DECARBOXYLASE TEST _____________________
Citrobacter - + -
(+) result in DEAMINATION______________________________
Klebsiella + - -
pneumoniae
LIA – INTERPRETATION OF RESULTS Reported as Reported as Reported

K/K K/A as R/A
Color of Report it Interpretation Possible Organism Escherichia coli Enterobacter Proteus
Slant/Butt as Klebsiella spp. cloacae Providencia
E. Morganella
aerogenes Citrobacter
Serratia spp. spp.
E. gergoviae Yersinia spp.
Hafnia spp. Shigella spp.
E. tarda
Salmonella spp.

PROTEUS -PROVIDENCIA-MORGANELLA
Proteus mirabilis
✓ Biochemically similar; rapid urease producers & deaminase (+)
Most frequently isolated may cause pneumonia &
✓ Non-lactose fermenters
septicemia
GENUS PROTEUS
Proteus vulgaris
✓ use as source of antigens used in Weil Felix Test
Causes nosocomial UTI
Proteus vulgaris source of ________________ & indole (+)
Proteus mirabilis source of ________________ & indole (-)
✓ associated with development of renal stones / Struvite stones (made up of magnesium ammonium phosphate)
✓ Colonies on Mac Conkey agar with burnt chocolate/burnt gun powder odor
✓ Shows SWARMING on BAP
GENUS PROVIDENCIA
✓ No swarming on BAP; Normal intestinal flora, often resistant to antibiotic therapy
✓ Species: P. rettgeri (urease +) -causes UTI & P. stuartii (urease Variable) – UTI, wound infection in burn patients
GENUS MORGANELLA Species: Morganella morganii
✓ Can cause UTI’s & RTIs
GENUS YERSINIA Motile at room temperature but non-motile at 37 degC except Yersinia pestis
Yersinia pestis Causative agent of PLAGUE- Bubonic, Septicemic & Pneumonic A bioterrorism agent
MODE of Transmission: flea bite (Xenopsylla cheopis
Assumes a safety pin appearance because of Bipolar staining which can be demonstrated using Wayson stain
On liquid media/ broth it produces stalactite growth
On BAP it can develop Hammered copper colonies
Yersinia enterocolitica A late lactose fermenter
Can tolerate cold enrichment
Can cause enterocolitis in which symptoms may be mistaken for appendicitis
Can produce Bull’s eye colonies on CIN media
Yersinia Agent of mesenteric lymphadenitis
pseudotuberculosis
Y.pestis Y. Y. GENUS SALMONELLA

enterocolitica pseudotuberculosis ✓ Species can cause a number of infections
Motility at ✓ S. typhi causes typhoid fever
RT ✓ Site of long term of S. typhi is the gall bladder
Motility at (recovered patients may harbor the organism)
37 degC ✓ Selective media is
Urease - + + ____________________________________\
TSI ✓ Species: S. enterica and S. bongori
Sucrose - + -
SALMONELLA- SHIGELLA Gastroenteritis S. serotype typhimurium,
✓ Intestinal pathogens S. enteritidis

✓ Spread via fecal oral route Bacteremia S. cholerasuis
GENUS SHIGELLA
✓ Can cause shigellosis/bacillary dysentery Enteric fever
✓ Species can produce endotoxin
✓ S. dysenteriae can produce endotoxin & neurotoxin
Species Lactose Mannitol ODC MR ONPG Catalase
A Shigella dysenteriae/ - - - + - -
Shiga Bacillus
B Shigella flexneri / Strong’s - + - + - +
Bacillus
C Shigella boydii - + - + - +
New Castle Manchester Bacillus
D Shigella sonnei/ - + + + + +
Duval’s Bacillus LLF

Salmonella Shigella
Resembles
Motility
H2S
Malonate
Invasive
LIA K/K K/A
RAPID LACTOSE FERMENTERS
GENUS ENTEROBACTER
✓ Members of this genus are normal intestinal flora but may cause opportunistic infections
✓ Most predominant isolate is E. cloacae
✓ E. sakasakii is now Cronobacter sakasakii - This is known to produce yellow pigment that intensifies at room temperature
Can cause necrotizing colitis often due to powdered infant milk formula
GENUS KLEBSIELLA
✓ Significant species is Klebsiella pneumoniae
✓ Associated with plasmid mediated ESBL (extended spectrum beta lactamase)
Klebsiella pneumoniae
✓ Also known as Friedlander’s Bacillus
✓ Can cause community acquired pneumonia (currant jelly like sputum)
✓ (+) encapsulated -Neufeld Quellung _______________
✓ On plated media it can develop MUCOID COLONIES
✓ (+) String test – positive result is due to the mucoid colonies
✓ K. oxytoca biochemically resembles K. pneumoniae but can be differentiated from the latter for its being indole (+)
✓ K. ozaenae – purulent sinus infection while K. rhinoscleromatis – granuloma of the nose & nasopharynx
GENUS ESCHERICHIA
✓ Significant species is E. coli
Escherichia coli
✓ a.k.a Colon Bacillus
✓ # 1 cause of UTI
✓ Normal GIT flora so found in normal stools
✓ Use as the primary marker of fecal contamination
✓ IMViC reaction is: ++--
✓ It has 2 SEROTYPES
1) Nephropathogenic E. coli / Uropathogenic E. coli – this is the serotype that causes UTI
2) Diarrheagenic/Enterovirulent E. coli – these are the serotypes known to cause diarrhea
Diarrheagenic/Enterovirulent E. coli
ENTEROTOXIGENIC E. COLI Can cause PROFUSE/SEVERE watery diarrhea that is CHOLERA LIKE due to production of
ETEC toxin
ENTEROINVASIVE E. COLI Can cause bloody diarrhea that is Shigella like due to its ability to invade the bowel
EIEC mucosea
ENTEROPATHOGENIC E. COLI Non-toxigenic and non-invasive but can cause diarrhea in infants / infantile diarrhea
EPEC Watery diarrhea with mucus but no blood, nosocomial
ENTEROAGGREGATIVE E. COLI Can cause acute & persistent diarrhea in children and adults
EAEC Causes watery diarrhea, actual pathogenic mechanism is not known
DIFFUSELY ADHERENT E. COLI This constitutes the 6 th group in the diarrheagenic E. coli.
DAEC Can cause diarrhea in otherwise healthy patrients
ENTEROHEMORRHAGIC E. COLI Can cause SEVERE BLOOD DIARRHEA due to its ability to produce VEROTOXIN
VEROTOXIC E. COLI Specific example is E. coli 0157:H7
SHIGA TOXIN PRODUCING
✓ All E. coli serotypes are LACTOSE (+) so on Mac Conkey agar, all can develop Pink-purple colonies.
✓ To detect EHEC/E. coli 0157:H7 we use SORBITOL MAC CONKEY AGAR /SMAC. This is like the usual Mac Conkey agar but instead of
Lactose it contains SORBITOL. Inhibitor & indicator same as the usual Mac Conkey. Only Lactose was replaced.
✓ All E. coli serotypes are SORBITOL (+) except E. coli 0157:H7.
✓ (+) result on SMAC: Pink-purple colonies (-) result: colorless colonies
✓ All E. coli serotypes can develop pink-purple colonies on SMAC except E. coli 0157:H7

OTHER GENERA/SPECIES
Genus that resembles Salmonella but is ONPG (+) and LDC (-)
Red Pigment produced by Serratia marcescens
TRIPLE DECARBOXYLASE NEGATIVE
Genus known to produce a number of enzymes such as DNase, Gelatinase; Lipase
Triple Enzyme (+)
Considered as a late lactose fermenter, with delayed citrate activity
Can cause secretory diarrhea in AIDS patients with Inflammatory bowel disease P. shigelloides
Oxidase (+), new member of Family Enterobacteriaceae TRIPLE DECARBOXYLASE POSITIVE
Most clinically significant Serratia and known to cause UTI & RTI, septic arthritis S. marcescens
Colonies with dirty, musty odor like potatoes S. odorifera
Documented as a causw of outbreaks of neonatal meningitis C. koseri
VIBRIO – AEROMONAS – CAMPYLOBACTER – HELICOBACTER
GENUS VIBRIO
GENERAL CHARACTERISTICS:
✓ Gram negative , facultative anaerobes, curved or comma shaped rods
✓ Motile & Oxidase (+) except V. metschnikovii
✓ ALL species are HALOPHILIC except: _______________________________
✓ Found in Brackish or marine water & transmission to humans is by ingestion of contaminated water, fresh produce, meat, dairy products,
seafood or exposure of disrupted skin and mucosal surfaces to contaminated water.
✓ All are glucose (+) ; all are lactose (-) except: _______________________
DIAGNOSTICS:
✓ Specimens for diagnosis: _________________________________
✓ Transport media : ______________________________________
✓ Enrichment media _______________________________________
✓ Selective-differential media ________________________________
V. cholerae
✓ Most significant species since it causes the disease CHOLERA (asiatic cholera or epidemic cholera) w/c is characterized by the
production of _________________________________________
✓ Virulence factor : _________________________________________
✓ Motility : ________________________________________________
✓ String test (+) _______________________________________________________________________________
✓ MAJOR SUBGROUPS ( sharing a common H and O antigens)
V. cholerae 01 With 3 serogroups: BIOTYPES/BIOVARS
Ogawa or Variant F(India) , Inaba or Original J (Phil),
CLASSICAL EL TOR
Hikojima or Middle (Japan)
RBC hemolysis
causes epidemic cholera
V. cholerae 0139 Causes epidemic cholera along with V. cholerae 01 VP test
Polymyxin B S R
V. cholerae non 01 Resembles V. cholerae 01 but fails to agglutinate in Agglutination with
01 antisera Chicken RBC
Species Oxidase Indole Colony on

TCBS
V. alginolyticus + V
V. cholerae + +
V. fluvialis + V
V. furnissi + V
V. metschnikovii - V
V. mimicus + +
V. parahaemolyticus + +
V. vulnificus + +
GENUS AEROMONAS
✓ Although classified as enteric pathogens, may also cause extraintestinal infections (septicemia, meningitis & wound infection)
✓ oxidase (+); fermentative gram-negative bacilli, BETA HEMOLYTIC
✓ can grow on Mac Conkey, EMB, SSA and CIN (growth on this media indistinguishable to those of Yersinia enterocolitica)
✓ species: Aeromonas hydrophila water loving organism associated with GIT disease
V. cholerae OTHER VIBRIO AEROMONAS PLESIOMONAS

NaCl requirement 8-10% - + - -
Except V. mimicus
Growth in Broth without NaCl + - + +
Growth in Broth with 6.5% NaCl + + - -
Motility + + + +
Oxidase Test + + + +
Except V. metschnikovii
Mannitol + x + -
STRING TEST + + - -
Growth on TCBS + + - -
O129 susceptibility S R R V
LYSINE-ORNITHINE-ARGININE ++- x +-+ + ++
Gelatin Liquefaction + x + -
GENUS HELICOBACTER - Helicobacter pylori GENUS CAMPYLOBACTER - C. jejuni and C. coli

✓ Formerly called Campylobacter coli; habitat: human ✓ May appear like V. cholerae
gastric mucosa ✓ appears as S-shaped Bacilli resembling
✓ Causes: __________________________________ ________________________________________________
✓ Virulence factor: ___________________________ ✓ motile and shows ____________________________motility
Ability to produce urease creates an alkaline ✓ ______________________________________________
environment which is favorable for its growth ✓ Optimum temperature for growth 42-43 degC
✓ Most species are asaccharolytic
✓ Tests for Identification
✓ Guillain Barre + patients test positive for Campylobacter
antibodies
1) Culture of crushed tissue biopsy on urea
✓ Causes: febrile systemic syndrome, gastroenteritis &
broth/agar
periodontal disease
2) Urea breath test
✓ Media for Primary isolation
3) COPRO STOOL ANTIGEN TEST
1. Modified Skirrow’s Media
4) Tissue biopsy, stain is Warthin Starry
2. CAMPY-BAP
Can grow on the following media: CAP; Brucella agar with 5% 3. Modified Charcoal Cefoperazone Deoxycholate agar
sheep blood; Skirrow’s; Modified Thayer Martin 4. CAMPY – CVA
Campylobacter jejuni Campylobacter fetus Helicobacter pylori

Growth at 25 degC - + -
Growth at 42 deg C + - +
Hippurate hydrolysis + - -
Cephalotin R S S
Urease - - +
Nalidixic Acid S R R
NON-FERMENTING & MISCELLANEOUS GRAM-NEGATIVE BACILLI

✓ Organisms that can produce acid only under AEROBIC conditions are classified as OXIDIZERS or ________________________________
✓ Classified as non-Fermenters are those species under Family Pseudomonadaceae and those under Genus Acinetobacter
✓ To detect ability of non-fermenters to produce acid we use
OF MEDIA – OXIDATIVE FERMENTATIVE MEDIA of Hugh & Leifson FERMENTERS – can produce acid with or without air
i.e of FERMENTATIVE ________________________
to detect acid production of Enterobacteriaceae
COMPOSITION of OF- MEDIA of HUGH & LEIFSON
traditionally we use ______________
FERMENTABLE CHO
POSSIBLE RESULTS Inoculated organism Possible
pH indicator
may be classified as: organism/s
(+) in both OPEN & FERMENTTATIVE
____________carbohydrate & ____________peptone
CLOSED tubes
It is consist of 2 tubes: (+) in OPEN tube OXIDATIVE/OXIDIZER
OPEN or AEROBIC TUBE & CLOSED or ANAEROBIC TUBE (-) in CLOSED tube NON-FERMENTER
Will require use of MINERAL OIL in ANAEROBIC TUBE
(-) in both OPEN & NON-OXIDIZER
(+) result: yellow (acid pH)
CLOSED tubes NON- SACCHAROLYTIC/
(-) gree/ blue gree (alkaline pH)
ASACCHAROLYTIC

FAMILY PSEUDOMONADACEAE
What are non-fermenters?
✓ All are OXIDASE positive except _______________________ TSI _________
✓ All are MOTILE except: _______________________________
GENUS PSEUDOMONAS:
Pseudomonas aeruginosa Some VIRULENCE FACTORS of P. aeruginosa
✓ most frequently isolated non-fermenter, strict aerobe, TSI _________ Pili
✓ (+) growth at 37-42 degC; oxidase & catalase test _______________ Alginate
✓ Motile & On BAP _________________________________________ Exotoxin
✓ Can develop colonies with a unique odor, similar to an overripe grape or A
✓ ___________________________________________ endotoxin
✓ Can develop pigment:
PYOCYANIN
OTHER SPECIES
PYOVERDIN/ FLUORESCEIN
P. stutzeri SOIL DENITRIFIERS
P. mendocina
✓ Media used for initial isolation ________________________________ P. stutzeri Can develop wrinkled colonies
✓ Acetamide utilization test (+) P. fluorescens Associated with transfusion
✓ Can cause a number of infections P. putida associated septicemia
1) BLUE PUS __________________________________________ Gelatin hydrolysis Differential test for P.
2) SWIMMER’S /DIVER’S EAR/ OTITIS EXTERNA _____________ test fluorescens (+) & P. putida (-)
3) SKIN INFECTION – Ecthyma gangrenosum, jacuzzi or hot tub
syndrome, whirlpool dermatitis
4) # 1 cause of pneumonia in those with cystic fibrosis
GENUS BURKHOLDERIA Gram (-) MacConkey (+)

Oxidase (-)
GENUS
STENOTROPHOMONAS
B. cepacia B. mallei B. pseudomallei B. gladioli S. maltophilia
a.k.a a.k.a
GLANDER’S WHITMORE’S BACILLUS
BACILLUS
P. kingii or P. multivorans Causes Glander’s Causative agent of A plant pathogen Found in tap water &
are previous names disease which is a Melioidosis or Vietnam salads
respiratory zoonosis Time Bomb – glander’s like Catalase & urease (+)
causes ONION BULB ROT primarily affecting disease characterized by Infections are usually
in plants; FOOT ROT in livestocks severe pneumonia Generally, Oxidase (-) nosocomial
MAN but Causes catheter related
Colonies with EARTHY Some strains are bacteremia
an opportunistic pathogen ODOR on ASHDOWN weakly +
associated with medium
pneumoniae in those with Oxidase (-)
cystic fibrosis & CGD (+) wrinkled colonies Mannitol (+) Maltose loving
Decarboxylase (-) Produces brown pigment
on heart infusion agar
LAVENDER-LAVENDER
GRREN on BAP
MSICELLANEOUS ORGANISMS
Genus ACINETOBACTER ✓ Oxidase Negative & Catalase (+), NM,
✓ (+) growth on MAC CONKEY ____________ & EMB _______________On BAP ____________________
✓ Acinetobacter baumannii – IRAQIBACTER & Oxidizer (+) growth at 42 degC
✓ Acinetobacter iwoffi – non-oxidizer/ assaccharolytic
Alcaligenes faecalis ✓ Oxidase & catalase (+), Motile & Assacharolytic
✓ Colonies with apple fruity odor
Shewanella putrefaciens ✓ H2S (-) non-fermenter
Chryseobacterium ✓ Gelatin hydrolysis, Oxidase, Dnase Indole (+)
meningosepticum ✓ NM, (-) growth on Mac Conkey Agar, agent of meningitis
Chromobacterium violaceum ✓ Not a normal flora, found in patients with neutrophil deficit
✓ Produces violet pigment – VIOLACEIN, colony odor: ammonium cyanide odor, beta hemolytic

HAEMOPHILUS and OTHER FASTIDIOUS GRAM-NEGATIVE BACILLI &
ORGANISMS OPTIMALLY RECOVERED ON SPECIAL MEDIA
GENUS HAEMOPHILUS
Incubation
Most haemophilus - 5-10% CO2, 35-37 degC for 24-72 hrs
✓ Species are mostly normol respiratory & oral flora
H. ducreyi – 5-10% CO2, 33 degC high humidity up tp 7 days
✓ Requires X & V factors for growth
✓ Non-motile, capnophilic (5-10% CO2)
✓ Prefers incubation at 35-37 deg except for H. ducreyi ___________________________________________________
✓ Can grow on _________________________________________ ; Unable to grow on Sheep’s blood agar
✓ Mostly are catalase & oxidase (+),
Heat stable product released upon by CO-ENZYME I Heat labile , provided by some
the degradation of hemoglobin NAD species
V FACTOR S. aureus, S. pneumoniae
Neisseria, Yeast, potato extract
Haemophilus Haemophilus Haemophilus aphrophilus and Haemophilus aegypticus Haemophilus ducreyi

haemolyticus parainfluenzae Haemophilus paraphrophilus
An occasional Rarely infectious, Normal flora of the oral cavity, Causes Causes
normal flora of normal flora of the found in dental plaque, rarely __________________________ _____________________________
the upper upper respiratory associated with endocarditis and __________________ _____________________________
respiratory tract; tract pneumonia
beta hemolytic Causes Brazilian Purpuric Forms the so-called
Fever- affects children _____________________________
_____________________________
_____________________________
Haemophilus influenzae - A.k.a Pfeiffer’s Bacillus ; Most significant species

✓ Although a normal flora it is regarded as the # 1 cause of meningitis in children less than 5 yrs. Old
✓ Virulence factors : IgA protease, outer membrane LPS, pili & CAPSULE ___________________________________________
✓ H. influenzae strains : typeable & nontypeable. The nontypeable strains are not encapsulated & are usually normal flora. Typeable starins
are classified based on capsular characteristic. The capsule is cmposed of sugar alcohol phosphate complex. Differences in this complex
is the basis in separating the typeable strains into 6 groups ( type a,b,c,d,e,f). The most common serotype is ______________
✓ Hemolytic pattern _______________
✓ Can develop ___________________________colonies mousy odor
✓ Exhibits SATELLITISM – luxuriant growth near the source of the incorporated V factor.
Species X - FACTOR V - FACTOR D-ALA BETA GLUCOSE SUCROSE LACTOSE FRUCTOSE
HEMOLYSIS
Haemophilus + + - - + - - -
influenzae
Haemophilus - + + - + + - +
parainfluenzae
Haemophilus + + - + + - - WEAK +
haemolyticus
Haemophilus - + + + + + - +
parahaemolyticus
Haemophilus + + - - + - - -
aegypticus
Haemophilus - - + - + + + +
aphrophilus
Haemophilus - + + - + + + +
paraphrophilus
Haemophilus + - - - + - - -
ducreyi
PORPHYRIN TEST / D-ALA TEST:
1. _______________________________________________________________________--
2. To detect ability of the organism to convert the substrate D-ALA into porphyrin & porphobilinogen
Porphobilinogen – use of PDAB / KOVAC’s (+) result _____________
Porphyrin – use of UV (+) result – red fluorescence
GENUS BRUCELLA
✓ Non-motile, strict aerobes, stains poorly with conventional gram stain, intracellular
✓ Bioterrorism agents except ___________________________; Many requires supplemental CO2 (5-10%) for growth
✓ Catalase & oxidase ______; Hemolytic pattern: ___________; Growth is inhibited by dyes: basic fuchsin & thionine
✓ Species are normal animal flora but can cause UNDULANT FEVER/ MALTA FEVER/ GIBRALTAR FEVER. MEDITERRANEAN FEVER
✓ Modes of transmission: (1) inhalation of bacterial cells (2) direct contact with animals (3)ingestion of contaminated meat or dairy products
✓ Organism can survive for extended period in milk, ice cream (3 wks) and in cheese for several months
✓ Optimal specimen for diagnosis _______________________
✓ Rapid test to detect Brucellosis ________________________________________________; Significant titer_______________
Species NOT detected with this method _________________
✓ Media for isolation _________________________________
Brucella abortus Brucella melitensis Brucella suis Brucella canis
A.k.a.____________________
Animal host Sheep, goats
H2S production + - - -
Urease positive in 2 – 24 hrs 2 – 24 hrs 0 – 15 mins 0 – 15 mins
Growth on Media with Basic Fuchsin + + - -
Growth on media with Thionine - + + +
Need for CO2 - - -
OTHER GENERA / SPECIES

GENUS BORDETELLA Bordetella pertussis Bordetella Bordetella
Strict aerobe, replicates ✓ a.k.a Bordet gengou Bacillus bronchiseptica parapertussis
in ciliated respiratory ET ✓ Clinically significant, agent of Pertussis, whooping
cells cough Motile, most Causes pertussis like
✓ Virulence factor ________________________________ active and can symptoms
Non-motile except: ✓ Stages of Whooping cough grow on
B. bronchiseptica Catarrhal phase _______________________________ conventional
media
Paroxysmal stage _______________________________
Media:
Bordet Gengou media – Convalescence ________________________________ A rapid urease
Potato Blood Glycerol ✓ Specimen for detection: _________________________ producer; rare
agar ✓ In the collection of specimens thru swabbing never cause of
use cotton swab instead use dacron/rayon fibers or respiratory
Modified Jones Kendrick calcium alginate disease
Charcoal ✓ Can develop colonies appearing like ______________
Causes Kennel
Regan Lowe cough in Dogs
SPECIES Growth in Urease Motilit Nitrate Oxidase Citrate BAP

Regan Lowe y
Bordetella pertussis 3-6 days - + - -
Bordetella 2-3 days - - + +
parapertussis
Bordetella 1-2 days + + + +
bronchiseptica
GENUS ✓ Causes TULAREMIA, water rat trapper disease, Rabbit fever, deerfly fever, lemming’s disease, Market
FRANCISELLA men disease
Francisella tularensis ✓ Faintly staining, catalase (+) and oxidase (-) A BSL 3 pathogen
(Jellison Type A) ✓ Requires _______________________________________________________________
✓ Media __________________________________________________________________
GENUS LEGIONELLA ✓ Causes Legionnaire’s disease / Pontiac fever
Legionella pneumophila ✓ Isolated from airconditioning units, requires cysteine and iron salts
Legionella micdadei - ✓ MEDIA ___________________________________________________
causes Pitsburgh ✓ Catalase, oxidase and gelatinase (+), Motile and asaccharolytic
pneumonia ✓ Stain: Dieterle, Steiner
✓ Most common lab test________________________________________________________
Legionella bozemanii - ✓ Specimens: bronchial washings, sputum
Wiga’s agent of
pneumonia
GENUS PASTEURELLA ✓ agent of Shipping fever in cattles a form of hemorrhagic septicemia
Pasteurella multocida ✓ in Man it causes ____________________________________________
✓ Produces Musty odor ________________________________________
GENUS ✓ Capnophilic, normal flora of oral cavity
CAPNOCYTOPHAGA ✓ Exhibits ________________________________________________________
Capnocytophaga
gingivalis
Streptobacillus ✓ Gram negative, facultative anaerobe, non-motile & highly pleomorphic
moniliformis ✓ Human infection is thru (1) rat bite or possibly by direct contact with rats (2) ingestion of contaminated
food such as unpasteurized milk or milk products and, less frequently, water
✓ Can cause RAT BITE FEVER ____________________& HAVER HILL FEVER _________________________
✓ Forms ________________________________ on broth
✓ Forms L-forms colonies which can be demonstrated using _________________________
✓ Biochemically catalase, oxidase, indole & nitrate (-)
Spirillum minus ✓ Gram negative, helical, (gram negative spiral appearing with 2-3 coilsstrict aerobes
✓ Causes SODOKU _________________________________, Unable to grow on synthetic media
HACEK _____________________________________________________________________________________________________
Haemophilus parainfluenzae
AGGREGATIBACTER Aggregatibacter aphrophilus Aggregatibacter actinomycetemcomitans
✓ Grows together with A. israelii
✓ _________________________________
Cardiobacterium hominis
Eikenella corrodens ✓ Fastidious gram negative capnophilic rod; part of the gingival and bowel flora
✓ Non-saccharolytic
✓ ________________________________________________________ ; with typical bleach like odor
✓ Causes the so-called _________________________________________
Kingella kingae
Oxidase Catalase Indole LDC

A. aphrophilus + - - -
A. actinomycetemcomitans + + - -
Cardiobacterium hominis + - + -
Eikenella corrodens + - - +
Kingella kingae + - - -
ANAEROBES
A. SPECIMEN COLLECTION & TRANSPORT

✓ In general, material for anaerobic culture is best obtained by tissue biopsy or by aspiration using a needle and syringe.
✓ SWAB –least favorable method for collection. (i.e THROAT SWAB for ANAEROBIC CULTURE = REJECT)
✓ Specimens on swabs can dry out during transportation and expose anaerobes to room air, not to be accepted unless transported
in proper anaerobic environment
GASPAK JAR containing 80% to 90% nitrogen, 5% to 10% hydrogen, and
Specimens ACCEPTABLE for UNACCEPTABLE for 5% to 10% CO2.
ANAEROBIC Culture ANAEROBIC Culture Many anaerobes require CO2 for maximal growth. The atmosphere in the
Blood, Body fluids, BM aspirate, Expectorated sputum, jars is monitored by including an indicator to check anaerobiosis.
Percutaneous lung aspirate or biopsy, feces, gastric juice, swabs, INDICATORS: ______________________________________________
suprapubic bladder aspirates, voided or catheterized ____________________________________________________________
transtracheal aspirate, Wound urine, bronchial washings
Anaerobic blood agar Non-selective media for anaerobes & Egg-yolk agar (EYA) Nonselective for determination of
facultative anaerobes lecithinase and lipase production by
clostridia and fusobacteria
Bacteroides bile esculin Selective and differential for Cycloserine cefoxitin Selective for Clostridium difficile
agar (BBE Bacteroides fragilis group fructose agar
Laked kanamycin- Selective for isolation of Prevotella Cooked meat or Non selective cultivation of anaerobes
Vancomycin blood agar and Bacteroides spp Chopped meat broth
Anaerobic PEA agar Selective for inhibition of enteric Thioglycollate broth Non selective for cultivation of
gram-negative rods and swarming by anaerobes, facultative anaerobes &
some Clostridia strict aerobes

APPROACH TO IDENTIFICATION OF ANAEROBES
1. Macroscopic Examination of 1) Foul odor – C. difficile, Fusobacterium, Porphyromonas
specimens 2) Fluorescence
Veilonella _____________________, Porphyromonas & Prevotella ________________
✓ The following characteristics must
3) Presence of sulfur granules: Actinomyces, C. acnes, E. nodatum, Propionibacterium
be noted since these strongly
suggest presence of anaerobes
2. Microscopy of gram-stained ✓ With Bifid ends __________________________________
smears ✓ With tapered ends _______________________________
✓ Safety pin ______________________________________
✓ Pallisade _______________________________________
3. Culture
4. Potency Disk test
A.israelii P. acnes Bifidobacterium Eubacterium Bacteroides F. Prevotella Prophyromonas
fragilis nucleatum
VANCOMYCIN S S S S R R R S
5 ug
KANAMYCIN S S S S R S R R
100 ug
COLISTIN R R R R R S S R
10 ug
CATALASE - + - - + - - -
INDOLE - + - - - + - +
5. Observation of other unique ✓ Swarming __________________________________________
features ✓ Colonies with molar tooth appearance
Gram (+) ANAEROBIC RODS Distinct Characteristic/s

Actinomyces israelii Branching, beaded, thin, filamentous rods
Causes Actinomycosis of Jaw called ______________________________________________
Propionibacterium acnes Club shaped to palisade, Forms LOG-JAM arrangement
Cutibacterium acnes Blood culture contaminant, sulfur granules seen MACROSCOPICALLY
Also called ANAEROBIC DIPHTHEROIDS
Bifidobacterium spp With Bifid / Bifurcated ends
Resembles bone shaped dog biscuit
Eubacterium alactolyticum Has a seagull wing shape similar to campylobacter
Gram (+) ANAEROBIC COCCI
Peptostreptococcus anaerobius Large colonies with sweet, fetid odor
(Anaerobic Streptococci) SPS ______________________
Peptococcus niger Anaerobic staphylococci
Produces black to olive green colonies, SPS -Resistant
Gram (-) ANAEROBIC RODS
Bacteroide fragilis Resembles safety pin, most abundant normal flora of colon
Shows RESISTANCE to all 3 disks (kanamycin, colistin and vancomycin)
Bacteroides urealyticus Requires formate & fumarate for growth in broth cultures
Same disk pattern as Fusobacterium
Fusobacterium nucleatum Smooth SPECKLED colonies and at times Irregular appearing like ___________________________
Shows greening of agar on exposure to air
Fluoresce chartreuse
Leptotrichia spp. Fusiformed rods with 1 pointed and 1 blunt end
Can develop ____________________likecolonies

Prevotella spp
Prophyromonas spp.
Gram (-) ANAEROBIC COCCI
Veilonella spp.

SPIROCHETES
GENERAL CHRACTERISTICS:
• Gram negative organisms appearing like corkscrews; moves in a unique spinning fashion via axial filaments/periplasmic flagella
• Dark field microscope, immunofluorescence, silver stains are required to view these organisms
• Silver stains: Levaditi’s, Fontana Tribondeau
GENUS TREPONEMA
Treponema pallidum
✓ ______________________________________________________________________________________________________________________
✓ Stages of Syphilis
Genital lesions called Diagnosis:

HUNTERIAN CHANCRE/HARD 1. Direct Visualization
CHANCRE 2. Serologic Tests
Extragenital lesions called Non-Treponemal Tests ________________________________
CONDYLOMA LATUM
Treponemal Tests ____________________________________
LATENT STAGE
GUMMAS
Treponema pallidum subspecies:

Treponema pallidum subspecies PERTENUE Treponema pallidum subspecies Treponema pallidum subspecies
CARATEUM ENDEMICUM
MODE OF TRANSMISSION:
GENUS LEPTOSPIRA
✓ Tightly coiled spirochetes with one or both ends bent to form a hook
✓ Definitive test: ___________________________________________
✓ Culture media___________________________________________; Ellinghausen Mc Cullough
Leptospira interrogans Leptospira interrogans LABORATORY DIAGNOSIS

serovar icterohemorrhagia
Darkfield Microscopy
Cause of human and animal leptospirosis, a zoonosis The organism is shed in the urine of animals
(dogs, rats etc.). Man acquires infection thru
WEIL’s disease _______________________________ direct contact with urine of animals carrying
____________________________________________ the organism Culture
Stages:
Anicteric _____________________________________ Blood, CSF – specimens on the
____________________________________________ ____________, urine during the immune
Icteric leptospirosis or Weil’s disease ______________ phase / 2nd week Serology
____________________________________________
GENUS BORRELIA
✓ Loosely coiled spirochetes, arthropod borne, blood spirochetes
✓ Transmitted thru arthropod vectors lice and ticks
✓ Diagnosis: ____________________________________________________________________________________________________
✓ culture of blood drawn on febrile state, culture media: Kelly’s
media Barber Stoenner Kelly media33 degC for 6 weeks
Borrelia burgdorferi Stage 1 ECM Erythema chronicum migrans
✓ Agent of ___________________________________
✓ Tick borne relapsing fever; transmitted by ticks / Ixodes (deer Stage 2 multiple smaller ECM disseminated thru
ticks)
blood may affect bones, heart and liver
✓ Best diagnostic method _________________________________
Stage 3

Borrelia recurrentis OTHER BORRELIA
Borrelia duttoni
louse borne relapsing fever or
famine fever Borrelia parkeri
Borrelia hermsii
Tick borne relapsing fever

Vector: Vector
Pediculus species Ticks of the genus ORNITHODOROS
(Soft ticks)
OBLIGATE INTRACELLULAR NON-CULTURABLE BACTERIAL AGENTS

GENUS CHLAMYDIA / CHLAMYDOPHILA
✓ Viewed as gram negative organism; obligate intracellular
✓ With 2 distinct forms: ELEMENTARY BODY infectious body and RETICULATED BODY –reproductive body
✓ Cultivated in tissue culture i.e., McCoy cells, HeLa cell line, monkey kidney cell
species Host Elementary Inclusion Glycogen Plasmid Susceptibility

range Body morphology containing DNA to
inclusions Sulfonamide
Chlamydia Causes TRIC- T Humans Round Yes Yes S
trachomatis Vacuolar
Causes LGV –
Lymphogranuloma
Venereum
Chlamydophila Parrot Fever / Psittacosis Birds Variable No Yes R

psittaci Ornithosis Lower Dense
mamals
Chlamydophila Previously known as humans Round No No R
pneumoniae TWAR dense
strain
CELL WALL DEFICIENT - Family Mycoplasmataceae

GENUS MYCOPLASMA
✓ Smallest free-living organism found in several animals and plants, requires _____________________for growth
✓ Pleomorphic and lacks cell wall; resistant to penicillin, since it lacks a cell wall where penicillin acts
✓ Culturable in artificial media producing tiny colonies which may have fried egg appearance
Mycoplasma pneumoniae
• Formerly known as EATON agent; causes primary atypical pneumonia or walking pneumonia
• Originally called PPLO – PLEURO PNEUMONIA LIKE ORGANISM
Genital Mycoplasma - Cause non-gonococcal urethritis in males and post partal fever in females; acquired thru sexual contact
Mycoplasma hominis - post abortal fever, post-partum fever, PID

Ureaplasma urealyticum - produces tiny fried egg colonies, causes NGU, can cross the placenta
Glucose Arginine Urease

M. hominis - + -
M. pneumonia + - -
U. urealyticum - - +
FAMILY RICKETTSIACEAE
✓ Arthropod-borne
✓ Intracellular
✓ Causes Rickettsial infections – FEVER, HEADACHE, RASH
Weil Felix Test
FAMILY RICKETTSIACEAE
GENUS RICKETTSIA, ORIENTIA, ANAPLASMA, EHRLICHIA
DISEASE VECTOR DIAGNOSTIC TEST
SPOTTED FEVER GROUP

Rickettsia rickettsi Tick SEROLOGY
Rickettsia conorii Meditteranean, Israel spotted fever / Tick PCR
Indian tick typhus / Kenya Tick typhus
fever
Rickettsia akari Rickettsial pox Mites
TYPHUS FEVER GROUP
Rickettsia prowazeki Epidemic typhus Lice SEROLOGY
Brill Zinsser None/recredescence PCR
Rickettsia typhi Murine typhus Fleas
SCRUB TYPHUS GROUP
Orientia tsutsugamushi Scrub typhus Chigger SEROLOGY
PCR
Ehrlichia, Anaplasma , Neorickketsia
Ehrlichia chaffeensis Human Monocytic Ehrlichiosis Lone StarTicks SEROLOGY
E. ewingii PCR
Anaplasma phagocytophilum Human granulocytic anaplasmosis Ticks (Ixodes)
Neorickettsia sennetsu Sennetsu fever Ticks
OTHER SPECIES: GENUS BARTONELLA

✓ Previously under Rickettsia, intracellular organisms
Coxiella burnetii Q fever an acute systemic infection ✓ Typically persist & multiplies in RBCs
that primarily affects the lung. ✓ Identification can be made thru histopathologic
examination of tissue biopsies stained with Warthin
Starry Stain, Molecular-PCR
smaller than Rickettsia spp
Species Disease
can survive extracellularly; however,
it can be grown only in lung cell Bartonella bacilliformis Orroya or verruga peruana
Transmitted by Carrion’s disease
Trophyrema whipplei Causes Whipple’s disease - found sandflies
primarily in middle-age men
Bartonella henselae
Patients develop diarrhea, weight
loss, arthralgia, lymphadenopathy, Peliosis hepatitis
hyperpigmentation, often a long Bartonella clarridgeaie Minor Cat scratch disease
history of joint pain, and a distended bacteremia
and tender abdomen. Neurologic
No endocarditis
and sensory changes often occur
Calymmatobacterium is the etiologic agent of granuloma Bartonella elizabethae Endocarditis, Bacteremia

granulomatis inguinale, or donovanosis, a sexually Bartonella Quintana /
transmitted disease Rochalimaea Quintana
QUALITY CONTROL FREQUENCY:
Autoclave Spore test weekly Gas pak Check for anaerobiosis with methylene blue
strip
Balances Check for accuracy annually Gram stain Control slides weekly ( E.coli/S. aureus)
BSCs Check airflow annually Temperatures Daily checks for incubators, water baths,
refrigerators and freezers
centrifuge Check rpm every 6 months Microscopes Clean and adjust 4 x/yr
CO2 Check CO2 and temperature daily
incubator
Bacteria Chlamydia Mycoplasma Rickettsiae Viruses
Size 0.4 – 2 um 0.3 – 0.5 um 300 nm 0.3 -0.5 um 25-300 nm
DNA & RNA Both both both both Either
Reproduce by binary Yes yes yes yes No
fission
Cell wall Yes yes no yes No
Growth on non-living Yes no yes no No
medium
Gram stain? Yes no no no no
CULTURE MEDIA
Culture Media Purpose
Amie’s Media Transport media, to maintain viability of the organisms from the source to the laboratory
Alkaline Peptone water Enrichment media for Vibrio
Selective and differential media. Selective for Bacteroides. Used to differentiate bile resistant from
Bacteroides Bile Esculiun (BBE)
bile sensitive Bacteroides
Nutrient agar base media with ferric citrate. Differential isolation & presumptive identification of
Bile Esculin Agar
Group D Streptococci and Enterococci
Selective & differential for cultivation of Vancomycin resistant enterococci from clinical and
Bile Esculin Azide agar with Vancomycin
surveillance specimens
Brain Heart Infusion (BHI) High protein media for growing a wide variety of bacteria
Used for isolation of Salmonella typhi. Produces black colonies. Not recommended for isolation of all
Bismuth Sulfite Agar
Salmonella
Blood Agar (BA) Cultivation of fastidious microorganisms, used in determining haemolytic reactions
Blood Cystine Dextrose Agar For growing Francisella tularensis
Bordet-Gengou Media Isolation of Bordetella pertussis
Selective media for isolation of Salmonella other than S. typhi.
Brilliant Green Agar (BGA)
Not recommended for Salmonella
Buffered Charcoal Yeast Extract Agar
Enrichment and selective for Legionella spp.
with Antibiotics
Chapman Stone agar Selective media for the isolation of Staphylococcus
Campy-Blood Agar Selective for Campylobacter spp.
Campylobacter Thioglycollate Broth Selective holding medium for recovery of Campylobacter spp.
Cary and Blair Media Transport media
Used for separating Nocardia & Streptomyces. Made from skimmed milk. Used as a media to test
Casein Media
for hydrolysis
Cefoperazone, Vancomycin,
Selective media for isolation of Campylobacter spp.
Amphotericin (CVA)
Cefsulodin-Irgasan-Novobiocin (CIN) Selective for Yersinia spp. Maybe useful for isolation of Aeromonas spp.
Chocolate Agar (CAP) Cultivation of Haemophilus spp. and pathogenic Neisseria
Chopped Meat Glucose Used for primary isolation of anaerobes. Used for Clostridium spp.
Columbia Colistin Nalidixic Acid Agar
Selective isolation of gram-positive cocci
(CNA)
Corn Meal Agar with 1% Tween 80 Used to stimulate production of Chlamydospores of Candida albicans
Cystine Tellurite Blood Agar Isolation of Corynebacterium diphtheria
CTA with carbohydrates added is excellent for determining the fermentation reactions of groups
Cystine Trypticase Agar (CTA)
such as Neisseria
Cycloserine cefoxitin Fructose agar
Selective media for C. Difficile
(CCFA)
Used for gram negative enteric bacilli differentiating lactose fermenters from non-lactose fermenting
Deoxycholate Agar
organisms
Deoxycholate Citrate For isolation of Salmonella and Shigella
Dieudonnes media Selective media for Vibrios
“E” agar and Edward Hayflick agar Selecxtive media for the isolation of Mycoplasma and Ureplasma
Egg Yolk Agar Used for the isolation and identification of Clostridium
Eosin Methylene Blue (EMB) Isolation & differentiation of lactose fermenting & non-lactose fermenting enteric bacilli

Ellinghausen, McCullough Johnson and
Selective media for Leptospira Interrogans
Harris Media (EMJH)
Fetal Bovine serum with Vancomycin Selective media for H. ducreyi
Fildes Enrichment Used for isolation of H. influenza
Gram Negative Broth (GN) Selective enrichment (liquid) medium for enteric pathogens
Differential, selective media for the isolation and differentiation of Salmonella and Shigella spp. from
Hektoen-Enteric (HEA)
other enteric bacilli
Human Blood Bilayer tween Selective media for the isolation of Gardnerella vaginalis
Leoffler's Coagulated Serum Used for C. Diphtheria
Used for the isolation of Mycobacterium tuberculosis and other Mycobacterium.
Lowenstein Jensen Media
Malachite green is used to inhibit all organisms except Mycobacterium
Peptone base media with lactose. Used for the isolation and differentiation of lactose and non-
Mac Conkey Agar
lactose fermenting enteric bacilli
A modified Mac Conkey that contains D-sorbitol instead of lactose as primary carbohydrate. For the
Mac Conkey Sorbitol Agar
selection and differentiation of E. coli O157:H7 in stool specimens
Mannitol Salt Agar Selective isolation of Staphylococci
Mc Bride Media For the isolation of Listeria monocytogens
Middelbrook 7H10 or 7H11 For the isolation of Mycobacterium tuberculosis
Modified Kelly’s medium or Barbour
Selective media for the isolation of Borrelia species
Stoenner Kelly
Used in Methyl Red and Vogues Proskauer tests. These are biochemical tests that help in the
MRVP broth
identification of enteric bacilli
New York City Agar Selective of Neisseria gonorrhea
Same use as Lowenstein Jensen with same ingredients except that this contains more malachite
Petragnani's Media
green
Phenylethyl Alcohol Agar (PEA) Selective isolation of gram-positive cocci and anaerobic gram-negative bacilli
Regan Lowe Enrichment and selective media for Bordetella pertussis
Sabouraud Dextrose or Maltose Agar Used for fungus culture
Salmonella-Shigella Agar (SSA) Selective for Salmonella and Shigella
Schaedler Agar Non-selective media for the recovery of anaerobes and aerobes
Selenite Broth Enrichment for the isolation of Salmonella spp.
Simmon Citrate Agar Green colored media used to test the organism's ability to use cirate as source of carbon
Skirrow Agar Selective for Campylobacter spp.
Streptococcal Selective Agar Selective for Streptococcus agalactiae in female genital specimens
Tetrathionate Broth Selective for Salmonella and Shigella spp.
Thayer Martin Agar Selective for Neisseria gonorrhoea and Neisseria meningitidis
Thioglycollate Broth Supports growth of anaerobes, aerobes, microaerophilic, and fastidious microorganisms
Thiosulfate Citrate Bile Salts (TCBS) Selective and differential for Vibrios
Todd Hewitt Broth supplemented with
Selective and enrichment for Streptococcus agalactiae in female genital specimens
antibiotics
Trypticase Soy Broth (TSB) Enrichment broth used for subculturing various bacteria from primary agar plates
Triple Sugar Iron Agar (TSI) Media used in biochemical tests to differentiate gram negative bacilli especially enteric bacilli
Xylose Lysine Desoxycholate Agar
Isolation and differentiation of Salmonella and Shigella spp. from other enteric bacilli
(XLD)
Urea Agar Used for Urease test to detect urease production which is ideal for identification of Proteus
PRAYER BEFORE STUDYING PRAYER AFTER STUDYING

Lord, true source of light & wisdom, give Lord, Pour out on us the spirit of
me keen sense of understanding, a understanding, truth and peace. Help me
retentive memory & a capacity to grasp to strive with all our hearts to know what is
things correctly. Grant me the grace to be pleasing to you, and when I know your will
accurate in my expositions and the skill to make me determined to do it. I ask this
express myself with thoroughness & clarity. through Christ our Lord Amen.
Be with me at the start of my work, guide
its progress and bring it to completion.
Grant this through Christ, our Lord Amen.
“All things happen in God’s time”



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REVIEW NOTES in BACTERIOLOGY # 2 – REVISED MAY 2023

STAPHYLOCOCCUS, MICROCOCCUS & RELATED ORGANISMS

GRAM (+) COCCI, CATALASE POSITIVE

Cocci in GRAPE LIKE CLUSTERS Cocci in TETRADS / CUBOIDAL PACKETS

IDENTIFICATION TESTS for REAGENT / MEDIA (+) RESULT

HCl precipitation method Clearing of agar around the colonies

Dye Method With Methyl green- clear zone around the

COAGULASE NEGATIVE STAPHYLOCOCCI

pg. 1 PREPARED BY : MA. CRISTINA SJ LIWANAG (MAM LIGHT)

IDENTIFICATION TEST for Modified Oxidase Test _____________________________________________________

PATHOGENESIS & SPECTRUM OF DISEASE – Staphylococcus aureus

Test for Beta Lactamase Production: Cephalosporinase test

uses __________________________, substrate ______________________Positive result __________________

pg. 2 PREPARED BY : MA. CRISTINA SJ LIWANAG (MAM LIGHT)

________________________________ 3) Oxacillin Screen Agar – use of MHA with 6 ug Oxacillin with

Penicillin RESISTANT (3) GOLD STANDARD for MRSA detection

Treatment for MRSA ___________________________________________

PYR VP TEST SLIDE COAGULASE TUBE COAGULASE

CATALASE TEST (+) Control – S. aureus (-) Control – S. Pyogenes

✓ Important marker of S. aureus ____________________

STREPTOCOCCUS, ENTEROCOCCUS & SIMILAR ORGANISMS

GRAM (+) COCCI, CATALASE NEGATIVE

Streptococci are non-motile & capnophilic

ALPHA HEMOLYTIC BETA HEMOLYTIC GAMMA HEMOLYTIC

pg. 3 PREPARED BY : MA. CRISTINA SJ LIWANAG (MAM LIGHT)

DIFFERENTIAL TESTS for ALPHA HEMOLYTIC STREPTOCOCCI – S. pneumoniae & VIRIDANS

S. pneumoniae VIRIDANS Streptococci

a _______________________________, ✓ Mouse Virulence test (-)

_______________________, colonies with Checker ✓ Classified as Viridians:

appearance. Doughnut (umbilicated)

BETA HEMOLYTIC S. pyogenes, S. agalactiae

IDENTIFICATION TEST for REAGENT RESULT

Purpose of Bacitracin Disk Test

pg. 4 PREPARED BY : MA. CRISTINA SJ LIWANAG (MAM LIGHT)

Group A Group B Group C, F, G

✓ ___________________________will develop small, PINPOINT, transparent colonies

TEST (+) CONTROL (-) CONTROL

PATHOGENESIS & SPECTRUM OF DISEASE DIAGNOSTIC TESTS for SCARLET FEVER

(+) fading of rashes/gradual

POST STREPTOCOCCAL SEQUELAE Normal flora of # 1 cause of

pg. 5 PREPARED BY : MA. CRISTINA SJ LIWANAG (MAM LIGHT)

GROUP D NON-ENTEROCOCCI / S. bovis group

Tests Group A Group B S. pneumoniae Viridans Group D Enterococci

STREPTOCOCCUS LIKE ORGANISMS:

pg. 6 PREPARED BY : MA. CRISTINA SJ LIWANAG (MAM LIGHT)

Pseudomonas, Plesiomonas, Aeromonas, Moraxella, Vibrio, Campylobacter, Helicobacter, Neisseria

SELECTIVE MEDIA INHIBITORY AGENTS

c) PID which may cause sterility, perihepatitis or Fitz Hugh Curtis

✓ To collect specimen use ________________________________

pg. 7 PREPARED BY : MA. CRISTINA SJ LIWANAG (MAM LIGHT)

BUTYRATE DISK TEST: (+) Control – M. catarrhalis; (-) Control – N. gonorrhoea

SPORE FORMERS – Bacillus(aerobic) & Clostridium(anaerobic)

pg. 8 PREPARED BY : MA. CRISTINA SJ LIWANAG (MAM LIGHT)

Enteritis Antibiotic associated

C. perfringens (Frankel’s Bacillus; C. welchii) C. tetani

C. botulinum – Canned Good Bacillus C. difficile

pg. 9 PREPARED BY : MA. CRISTINA SJ LIWANAG (MAM LIGHT)

Motility Lecithinase Lipase Lactose Glucose Gelatinase Indole Stormy Nagler

Corynebacterium diphtheriae a.k.a Kleb Loeffler’s Bacillus

pg. 10 PREPARED BY : MA. CRISTINA SJ LIWANAG (MAM LIGHT)

❖ SHICK’s test ______________________________________________________________________________

C. jeikium Gamma hemolytic Nitrate Reduction, Urease & Esculin

Erysipelothrix rhusiopathiae Lactobacillus Gardnerella vaginalis

uses ______, substrate Positive result