Professional Documents
Culture Documents
Objectives
1. Isolate, identify and analyze the bacteria that cause disease in humans.
2. Prediction and interpretation of antimicrobial susceptibility patterns
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i. Exotoxins: Secreted by the organism into the environment. The organism must possess the gene ii.
Endotoxins : Lipid A of the outer membrane. Released upon lysis of the organism
Characteristic Exotoxins Endotoxins
Organism Type G(+) / G(-) G(-)
Chemical Nature Simple protein Lipid A
Stability at 100°C Labile Stable
Ab neutralization Detoxified Not detoxified
Biologic Activity Individual to toxin Same for all toxins
1c. Control of microorganism
A. Sterilization Versus Disinfection
1. Disinfectants - chemical agents applied to inanimate objects
2. Antiseptic - a substance applied to the skin for reducing the number of bacteria
B. Methods of Disinfection and Sterilization
1. Physical Methods
a. Heat (°C) Time Required Applications
Boiling Water 100 15 mins.
Disinfects
Pasteurization 63 (72) 30 mins. (15 secs)
Oven (Dry Heat) 160-180 1.5 – 3 hrs.
Autoclave (Moist Heat) 121 15 min. at 15 psi Sterilizes
132 30-60 min. at 15 psi
b. Filtration Applications
Plastic polymers or cellulose esters (0.22 μm) parenteral and antibiotic solutions & vaccines.
HEPA filters (0.3 μm) biological safety cabinets
c. Radiation
X rays, gamma rays and UV disposables: syringes, catheters or gloves
2. Chemical Methods
a. Antiseptics b. Disinfectant (Sterilizers)
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3. Ingestion - failure to wash hands after work, eating, drinking and mouth pipetting (Salmonella and Shigella)
4. Direct Inoculation - puncture by contaminated needles and broken glass (Hepatitis virus)
B. Safety from Infectious Agents
1. Safety Program
a. Standard Precaution: Blood and body fluids from all patients should be considered infectious b. Work and
Environmental Practice Controls
• No mouth pipetting, eating, drinking, smoking or applying cosmetics
• No recapping of needle, dispose needles to sharps container
• Disinfect workstations, wash hands frequently and minimize generation of aerosols
• Wear personal protective equipment (PPE) and work in BSC
2. Biologic Safety Cabinets (BSCs)
• Containment barrier that protects the worker from aerosolized organism. Air is sterilized by UV and HEPA filter
• Cabinet Classification:
a. Class I - Room air pass into the cabinet sterilizing only the air to be exhausted
b. Class II - Sterilize air that flows over the work surface and the air to be exhausted
c. Class III - Self contained ventilated system. Closed front contain attached gloves
3. Biosafety Levels
a. Biosafety Level-1 - For handling organisms not known to consistently cause disease in healthy adults. Work done
in open bench tops with adherence to standard precautions. Limited access, presence of hazard warning signs,
decontamination of infectious waste (autoclave).
b. Biosafety Level-2 - For handling common or likely encountered pathogens in a routine clinical laboratory. Use BSC
I or II. Trained personnel, presence of biosafety manual and sharps containers.
c. Biosafety Level-3 - For handling organisms that can be transmitted by aerosols. Controlled access. Ducted air
ventilation, special lab clothing and personal respirator.
d. Biosafety Level-4. Research facilities handling exotic viruses and potential bioterrorist agents. Personnel and all
materials are decontaminated before leaving the facility. Non Circulating ventilation system. Maximum
containment and use of class II or III BSCs.
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ii. Deep - aspirate with needle and syringe and place in an anaerobic transport system f.
Lower Respiratory Tract
i. Sputum - gargle with water, cough deeply into sterile, screw cap container (AFB, Legionella and Nocardia) g.
Upper Respiratory Tract
i. Collected using swab moistened with transport medium
ii. Nasopharynx: Whooping cough - B. pertussis
iii. Pharynx (Throat): Strep Throat - S. pyogenes; Epiglotitis - H. influenzae; Oral gonorrhea – N.
gonorrhoeae h. Urine
i. Clean Catch Mid Stream, Catheter, Suprapubic aspirate
ii. Placed directly in a sterile, screw-cap container
iii. For diagnosis of lower UTI (cystitis, urethritis) and upper UTI (glomerulonephritis)
3. Labeling and Requisitions
a. Specimen Label: Patients name, age and gender; identification nu er; patie t’s roo u er or location;
requesting physician; culture site; date and time of collection
b. Requisition Form: Patie t’s a e, age a d ge der; patie t’s roo u er or lo atio ; physi ia ’s a e;
address;
culture site; date and time of collection; clinical diagnosis or patient history; name of individual transcribing orders
B. Preservation, Storage & Transport
1. Specimen Storage
Refrigerator Temp. (4°C) Room Temp. (22°C) Body Temp. (37°C)
Foreign Devices Abscess, lesion, wounds CSF
Feces Body Fluids
Urine Genital Samples
Sputum Nasopharynx, Throat
2. Anticoagulant: 0.025% Sodium polyanethol sulfonate (SPS) and Heparin
3. Holding or Transport Medium: Stuarts & A ie’s (Dacron, Rayon or Calcium Alginate swabs) or JEMBEC System
A. Introduction
1. Confirm that the specimen is representative
2. Identify agents using direct visual detection using stains
3. To guide the workup of specimen for culture.
B. Preparation of Samples
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General Morphology
Wright- Sample with cellular
Giemsa background
D. Microscopes
Microscope Magnification Application
Bright Field 10-1000 Stained cells
Dark Field 10-400 For cells not readily stained
Phase-contrast 10-400 Living or unstained cells
Fluorescence 10-400 Cells stained w/ fluorochromes
Review Questions
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2. The biosafety level practice for handling blood samples suspected of containing HIV and HBV:
A) BSL I B) BSL II C) BSL III D) BSL IV
3. Which of the following specimen preparation procedure is commonly applied to pus discharge? A) Homogenization
B) Concentration C) Decontamination D) None of these
E. Terminology for Direct Examinations
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Spiral Borrelia, Leptospira, Treponema
F. Examination of Prepared Material
Cells & structures Associations
Amorphous debris Necrosis, heavy protein fluid
Epithelial cells Cell surface in collection site
Mononuclear cells Chronic inflammation
Mucus Irritation of glandular surfaces
G. Examples of Sample Observations Purulence Acute inflammation, exudation
Red blood Cells Trauma, hemorrhage
A. Introduction
1. To grow (cultivate) and isolate all bacteria in the specimen
2. To determine which bacteria that grow are most likely causing the infection
3. To obtain sufficient growth of clinically important bacteria and allow identification
B. Nutritional Requirements
1. Types of Bacteria by Nutrient Requirements
a. Fastidious - Complex nutritional requirement b. Non fastidious - Basic nutritional requirement
2. Media Classifications
a. Supportive (general isolation) media - support growth of most non fastidious organisms
b. Enriched (nonselective) media - supplement added to supportive media for growth of fastidious
microbes
i. Sheeps Blood Agar - trypticase soy agar w/ 5% sheep's blood
• Enriched & differential. Determines hemolytic patterns
• Beta- complete clearing of RBC, Alpha - greenish discoloration around the colony, Gama - no effect ii.
Chocolate Agar - blood are lysed when added to molten base releasing hemin & NAD
• Can support for N. gonorrhoeae & Haemophilus spp.
c. Enrichment Broth - permits growth of certain bacteria while inhibitory to others
d. Selective Media - permits growth of certain bacteria while inhibitory to others.
e. Differential Media - provides a distinct cultural appearance of microorganisms.
f. Antibiotic Media - Selective for a certain group of bacteria through addition of antibiotics
g. Back-up Broth - Broth w/ agar (0.075% ) & thioglycolic acid (reducing agent) creating anaerobiosis C.
Environmental Requirements
1. O2 and CO2 Availability
a. Obligate aerobe - requires oxygen for growth
b. Facultative anaerobe - can grow either with or without oxygen
c. Obligate anaerobe - cannot grow in the presence of oxygen
d. Aerotolerant anaerobe - can survive in the presence of oxygen but do not use oxygen for metabolism
e. Microaerophile - requires a reduced level of oxygen for growth
f. Capnophilic - requires extra carbon dioxide (5% to 10%)
2. Temperature, pH & Moisture
a. Temperature: 35-37 C (42 C for C. jejuni, 0 C for L. monocytogenes & Y. enterocolitica)
b. pH: neutral (6.5-7.5)
c. Moisture: humidified incubators and sealing of agar plates
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1. Microscopic Morphology & Gram Reaction 5. Nutritional Requirements and Metabolic Capabilities
2. Macroscopic Morphology (Colony appearance) a. Enzyme capabilities (Enzyme based test)
3. Environmental Requirements b. Presence of Metabolic Pathways
4. Susceptibility to Antimicrobial Agents
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2. Clinical Infections
a. Skin and wound infections – i. folliculitis & furuncles; ii. boils & carbuncles; iii. bullous impetigo
b. Scalded skin syndrome; c. Toxic shock syndrome; d. Food poisoning
B. Staphylococcus epidermidis
1. Virulence Factor: Exopolysaccharide sli e or biofilms
2. Infections: Hospital acquired UTI, prosthetic valve endocarditis
C. Staphylococcus saprophyticus
1. Virulence Factor: Adherent to Urogenital tract epithelium
2. Infections: UTI in sexually active young females and in older women with indwelling catheters
D. Staphylococcus lugdunensis; E. Staphylococcus haemolyticus
III. Laboratory Diagnosis
A. Isolation
1. Specimen: Aspirate or Swabs
2. Culture Media:
a. Sheeps Blood Agar (SBA): Enriched and Differential (5% Sheeps blood)
• S. aureus: Medium to large. Pigmented yellow. β-hemolytic
• S. epidermidis: Small to medium. Gray-white. ɣ-hemolytic
• S. saprophyticus: Small to medium. White-yellow or orange. ɣ-hemolytic
b. Mannitol Salt Agar (MSA): Selective (7.5% NaCl) and Differential (D-mannitol, phenol red)
• S. aureus: Growth w/ fermentation (yellow halos)
• S. epidermidis: Growth w/o fermentation (remains pink ) c. Laboratory
Diagnosis
Organism Slide Coagulase Tube Coagulase DNase MSA Fermentation Novobiocin (5μg)
S. aureus + + + +
S. epidermidis ̶ ̶ ̶ ̶ S
S. saprophyticus ̶ ̶ ̶ ̶ R
a. Coagulase Test- Test for the ability to convert fibrinogen into fibrin. Differentiate S. aureus from Coagulase (-)
Staphylococci. 2 types: Bound and Free coagulase a1. Coagulase
Slide Test- Dete ts ou d oagulase lu pi g fa tor .
Positive (+): Macroscopic clumping
Negative (-): No clumping; a2. Coagulase
Tube Test - Detects free coagulase.
Positive (+): Clot of any size
Negative (-): No clot
b. DNase Test - Test for DNA hydrolysis.
Positive (+): clear zone
Negative (-): No clearing
c. Novobiocin Susceptibility - Test for susceptibility to 5 μg Novobiocin. Susceptible(S): zone diameter >16 mm
Resistant (R): zo e dia eter
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a. Microscopy: Curved, G+ rods w/ pointed ends that becomes coccal after 48 hrs
b. Colony: “ all olo ies, β-hemolytic, pits agar, leaves black dot under a colony
c. Identification: Lipase and Lecithinase positive (EYA)
Exhibit CAMP inhibition reaction(phospholipase D inhibits β-lysin)
C. Gardnerella vaginalis
1. Clinical Infections: Bacterial vaginosis (Excessive vaginal discharge, pH > 4.5 and foul smell) associated in UTI
2. Laboratory Diagnosis
a. Microscopy: Pleo orphi Clue cells in vaginal fluid.
b. Colony: Ɣ-hemolytic (BAP) and β-hemolytic (HBT)
c. Other Test: Whiff Test (Vaginal secretion + 10% KOH fishy aminelike odor), urease positive
Characteristic Catalase Motility BEA Hipurate H2S (TSI) Hemolysis Urease
C. diptheriae + - - - - β -
L. monocytogenes + + + + - β -
E. rhusiopathiae - - - - + ɣ,α -
A. haemolyticum - - - - - β -
G. vaginalis - - - - - ɣ +
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B. Bacillus cereus
1. Virulence Factor and Clinical Infections:
Characteristics a) Diarrheal toxin b) Emetic toxin
Incubation period 8-16 hrs 1-5 hrs
Symptoms Diarrhea Vomiting
Duration of illness 24 hrs 9 hrs
Food Implicated Meat producers Fried & Boiled rice
Stability to heat Negative Positive
2. Laboratory Diagnosis:
a. Microscopy: Large, G+ bacilli
b. Colony: Large, feathery, spreading, wide zo e of β he olysis
c. Identification: Lecithinase Test, Motility test, Penicillin Susceptibility, Presensence of Parasporal crystals
Organism Lecithinase Motility Penicillin Sensitivity Parasporal Crystals
B. anthracis (+) (-) (+) (-)
B. cereus (+) (+) (-) (-)
B. thuringiensis (+) (+) (-) (+)
B. mycoides (+) (-) (-) (-)
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Medium Inhibitory Agents BACTERIOLOGY
Suppressed HANDOUTS
Organisms
Vancomycin G(+)
Thayer-Martin Colistin G(-) S small, tan, translucent and
raised
Nystatin Yeast
b. M. catarrhalis: Colony can be swept
Modified TM Trimethoprin Swarming of Proteus intact (hockey puck), resembles
Martin Lewis Anisomycin Yeast wagon wheel (48 hrs)
New York City Amphothericin B Yeast
F. Identification
Blood MTM, ML, Superoxol
Organisms Glucose Maltose Lactose DNase / TH
Agar NYC (30% H2O2 )
N. gonorrhoeae (-) (+) (+) (+) (-) (-) (-)
N. meningitidis (+) (+) (-) (+) (+) (-) (-)
M. catarrhalis (+) (-) (-) (-) (-) (-) (+)
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b. H. ducreyi - Small, flat, smooth, transparent to opaque; colonies can pushed intact; clumpy in saline
4. Identification
a. Neufeld Quellung Rxn: antisera is reacted with the antigens in the capsule making the capsule more prominent
b. Staphylococcus Streak: Haemophilus is streaked w/ S. aureus, S. pneumoniae, Neisseria and certain yeasts
• Positive: Satellitism dewdrop colonies surrounding S. aureus
c. X and V requirement; d. Hemolytic patterns
e. Porphyrin Test: Test for the ability to produce ALA (ALA porphobilinogen/porphyrin Hemin)
• Porphobilinogen (add Ko a ’s rgt a d Porphyri s emit with 360nm UV, red-orange)
Species X factor V factor Hemolysis ALA
H. influenzae (+) (+) (-) (-)
H. aegyptius (+) (+) (-) (-)
H. haemolyticus (+) (+) (+) (-)
H. parahaemolyticus (-) (+) (+) (+)
H. parainfluenzae (-) (+) (-) (+)
H. ducreyi (+) (-) (-) (-)
A. aphrophilus (-) (-) (-) (+)
4b. Other Fastidious Gram Negative Bacilli
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6. Enteroadherent E. coli
a. Diffusely adherent E. coli (DAEC) UTI & diarrhea
b.Enteroaggregative E. coli (EAEC) Diarrhea (watery)
Indole – + + +
H2S + – –
ODC + – – +
Motility S/+ +
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A. Salmonella enterica subsp. enterica: Reservoir - GI tracts of poultry, pets and domestic animals
1. Classification: 7 subspecies, clinical isolates are subgroup 1, E.g . Salmonalla Typhi, Paratyphi and Chloraesuis
2. Virulence Factors: Fimbria, enterotoxin, transverse intestinal mucosa
3. Clinical Infections: Acute gastroenteritis or food poisoning, Enteric Fever or Typhoid fever (S. Typhi & S.
Paratyphi). Isolated in blood (week 1-2), urine (3-4), stool (2-3). Bacteremia (other serotypes)
4. Other Characteristics: Metallic colonies w/ black ring in Bismuth sulfite agar, Diagnosed with Widal’s test.
B. Shigella: Reservoir - Humans only
1. Virulence Factors: neurotoxin & enterotoxins (S. dysenteriae), Other species: only enteroxin 2. Disease: Shigellosis
or bacillary dysentery
Test S. dysenteriae S. flexneri S. boydii S. sonnei
Mannitol – + + +
ONPG / ODC – – – +
Serogroup A B C D
TSI Yellow/Orange
Motile (25°C) – +
Sucrose – +
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B. Eosin Methylene Blue - Selects G(-) bacteria. Differentiates lactose from non-lactose fermenters
• Eosin and methylene blue and Lactose
C. Hektoen Enteric Agar (HEA) - Selects for stool pathogens. Differentiates lactose from non-lactose fermenters
• Bile salts (high amounts): inhibit G(+) bacteria, G(-) coliforms ; Lactose, Bromthymol blue
• Sodium thiosulfate: sulfur source ; Ferric ammonium citrate: H2S Indicator
D. Xylose-Lysine-Deoxychlate (XLD) Agar - Selects for stool pathogens. Differentiates lactose from non-lactose fermenters
• Bile salts (high amounts); Xylose, Lactose; Phenol Red; Sodium thiosulfate and Ferric ammonium citrate
E. Salmonella-Shigella Agar) - Selects for stool pathogens. Differentiates lactose from non-lactose fermenters
• Bile salts, Brilliant Green Agar, Lactose, Neutral Red, Sodium thiosulfate and Ferric ammonium citrate
F. Cefsulodin-irgasan-novobiocin (CIN) Agar - Selective media for Yersinia species
• Cefsulodin – Inh. G(+) bacteria & most G(-) bacilli ; Novobiocin inh. G(+) cocci ; Crystal violet – inh. G(-)
bacteria
G. Selinite, G(-) Broth - Enrichment broth for cultivation of GI pathogens. Selects for stool pathogens
5b. Biochemical Identification
I. Carbohydrate Utilization Test A.
Oxidation-Fermentation Tests
• Differentiating glucose fermenters from glucose oxidizers
• Hugh-Leifson O/F Basal Medium (OFBM)
• Contents: 1% Carbohydrates & 0.2% peptone
• Fermenter: Change in color in both tubes Enterobacteriaceae
• Oxidizer: Change in color in open tube Pseudomonad
• Nonoxidizer: No change in color in both tubes
B. Triple Sugar Iron Agar
• Test whether a G(-) rod utilizes glucose, lactose and sucrose
• Contents: 1% lactose, 1% sucrose & 0.1% glucose; Na thiosulfate & Ferrous sulfate (1st Bacteria + Na thiosulfate
H2S gas, 2nd H2S gas + Ferrous sulfate ferrous sulfide); Phenol red
Reactions Possible Organism(s)
K/K Pseudomonas
K/ A Serratia, Providencia, Morganella, Shigella
+
K/ A , H2S Citrobacter freundii, Proteus, Salmonella
A/ A Escherichia coli, Enterobacter, Klebsiella
A/A Citrobacter, Serratia
+
A/ A , H2S Citrobacter freundii
C. ONPG: Test capacity to produce β-galactosidase and hydrolyzes ONPG to form о-nitrophenol
• Positive: Yellow (Lactose and dLFs, S. sonnei ); Negative: Colorless (Non-lactose fermenters)
II. Glucose Metabolic Ends Products
A. MR-VP (Clark and Lubs)
• Determines the products of glucose fermentation
• 1st pathway produces mixed acid (MR - red)
• 2nd pathway produces acetoin (VP - pink-red)
a. Methyl Red Test: Glucose (fermentation) Mixed acid (acetic, lactic, succinic & formic) + MR
• Positive: Bright red color (E. coli) ; Negative: Yellow color (Klebsiella and Enterobacter)
b. Voges-Proskauer Test: Glucose (fermentation) Acetoin + VP (α-naphthol & 40% KOH)
• Positive: Red (pink-red) at the surface (Klebsiella and Enterobacter)
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I. Vibrio spp.
• Indications of Infection: consumption of raw seafood. Gastroenteritis (cholera-like). Contact with fresh, estuarine, or
marine water.
• Microscopy: Comma-shaped G(-) rods, polar/peritrichous flagella, exhibit darting or shooting star motility
• Physiology: Facultative anaerobe, reduces nitrate to nitrite , oxidase (+), most species are susceptible to O/129, most
species is positive to string test and halophilic (Except V. cholerae & V. mimicus)
A. Vibrio cholerae
1. Virulence Factor and Disease: Cholera enterotoxin (Choleragen) - profuse watery diarrhea (rice water stools).
Leads to dehydration and hypovolemic shock
• O Ag (01 & 0139): Markers for strains capable of epidemic and pandemic spread. Produce cholera toxin
Biogroups Eltor Classical
Hemmaglutination (chicken RBC)
β-hemolysis in SBA
Positive Negative
Voges-Proskauer Test
Resistance to Polymxin B (50 U)
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B. Vibrio parahaemolyticus
1. Disease: Gastroe teritis summer diarrhea ; 2. Other Characteristics: Kanagawa toxin positive: β-hemolytic
C. Vibrio vulnificus
1. Disease: Septicemia & wound infection ; 2. Other Characteristics: Lactose positive
D. Vibrio parahaemolyticus
1. Disease: Wound infection ; 2. Other Characteristics: Strict halophile
E. Laboratory Diagnosis
1. Specimen Collection & Transport: Body fluids, tissues, swabs, stool (alkaline peptone H2O, pH 8.5)
2. Culture Media: SBA (greenish hue, α or β hemolytic), Mac (NLF) & TCBS II. Aeromonas hydrophila ater lo i g
1. Virulence: Cytotoxic enterotoxins
2. Disease: Intestinal (five diarrheal syndrome), Extraintestinal (wound infections, septicemia & meningitis, etc.)
3. Lab Diagnosis: β-hemolytic (SBA),Ferments Lactose, pink-centered(CIN)
III. Plesiomonas shigelloides
1. Disease: Gastroenteritis, bacteremia, meningitis, etc.
2. Lab Diagnosis: Pink in Inositol brilliant green bile salt agar; LDC, ODC, ADH Positi e Trio
IV. Laboratory Diagnosis
A. Thiosulfate Citrate Bile Salt Sucrose Agar
Selective and differential media for Vibrio : Contents: Sucrose, bromthymol blue, Na Citrate and Oxgall
a. Growth w/ fermentation (yellow): V. cholerae, V. alginolyticus
b. Growth w/o fermentation (green): V. mimicus, V. parahaemolyticus, V. vulnificus
c. No growth: Aeromonas spp., Plesiomonas sp.
B. String Test: Reagent - 0.5% Na desoxycholate [Positive - Viscous stringing (Vibrio spp.)
Negative - No viscous stringing (Aeromonas, Plesiomonas)]
C. Vibriostatic test: Reagent: 0/129 disks [Susceptible - V. cholerae, P. shigelloides
Resistant - Aeromonas spp., Other Vibrio spp.]
Vibrio cholerae Other Vibrio spp. Aeromonas spp. Plesiomonas
TCBS + + - -
String Test + + - -
Broth w/ 6.5% NaCl + + - -
Broth w/o 6.5%NaCl + - + +
Vibriostatic Test (150 μg 0/129) + - - +
Inositol Fermentation - - - +
Comments LDC, ODC, ADH
I. General Characteristics
Introduction: Oxidase Positive, Small, curved, G(-) bacilli, most species are asaccharolytic, microaerophilic
II. Epidemiology and Disease
A. Campylobacter jejuni
1. Epidemiology: Exposure to animals and ingestion contaminated water and poultry
2. Disease: Most common cause of bacterial gastroenteritis worldwide, Guillain-Barre syndrome
B. Campylobacter fetus subsp. fetus: Bacteremia in Immunocompromised & elderly patient
1. Epidemiology: Immunocompromised & elderly patient
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2. Disease: Bacteremia
C. Helicobacter pylori
1. Epidemiology: Gastric ulcer patients
2. Virulence Factors: Urease, adhesin and cytotoxins
3. Disease: Gastric, peptic and duodenal ulcers; type B gastritis and GI carcinoma III. Laboratory
Diagnosis
A. Specimen
1. C. jejuni: Feces (Stuart, Cary-Blair & Campy Thio)
2. C. fetus subsp. fetus: Blood (Blood culture media)
3. H. pylori : Tissue biopsy “tuart’s , Cystei e-Brucella broth)
B. Culture Media and Incubation
1. Campylobacter spp: Butzler, “kirro ’s, ˚C or ˚C Mi roaerophili
2. H. pylori: “kirro ’s, Chocolate or Brucella agar with 5% horse blood, 35- ˚C Mi roaerophili
C. Microscopic Morphology
1. Campylobacter spp: S-shaped wings of seagulls a d produces darting motility
2. H. pylori: Multiple flagella at one pole
D. Colony Morphology: Moist ru y looki g C. jejuni), Convex & translucent (C. fetus), translucent & circular(H.
pylori)
E. Identification
Test C. jejuni C. fetus H. pylori
Hippurate hydrolysis + — —
Growth at 42°C + — —
Growth at 25°C — + —
Urease — — +
Nalidixic acid S R R
Cephalotin R S S
1. Urease Test: Tissue is place in Christensen medium for 37°C for 2 hours
2. Urea breath test : 13/14C labeled urea (oral dose) –Urease 13/14CO2 (Detected by scintillation counter )
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B. Acinetobacter spp.
1. Clinical significance: 2nd most common nonfermenter
2. Identifying characteristics: Plump, paired G(-) coccobacilli; A. baumanii (saccharolytic) - purplish (MAC) or
cornflower blue (EMB) ; A. iwoffi (assaccharolytic)
C. Stenotrophomonas maltophilia
1. Clinical significance: 3rd most common nonfermenter
2. Identifying characteristic: lavender green (BAP), Ammonia-like smell, oxidizes glucose W(+), oxidizes maltose
S(+) a. Burkholderia cepacia
1. Clinical significance: Pneumonia in patients with cystic fibrosis or chronic granulomatous dse.
2. Identifying characteristics: Dirtlike odor in BAP, ONPG +; dark pink in Mac in 4-7 d b.
Burkholderia pseudomallei
1. Clinical significance: Melioidosis
2. Identifying characteristics: Bipolar staining (G/S), wri kled & deep pi k i Ashdo edia, Earthy
odor c. Burkholderia glandioli: Associated w/ patients w/ CF & CGD,
d. Burkholderia mallei: Glanders, Nonmotile
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C. Exogenous
• Contamination of wound or puncture by objects. E.g. C. tetani (tetanus) and C. perfringens (gas gangrene)
• Ingestion of preformed toxins in vegetable or meat. E.g. C. botulinum (botulism), C. perfringens (food poisoning)
• Colonization of GI tract of toxin-producing organism. E.g. C. botulinum (infant botulism)
D. Endogenous
• Gain access to normally sterile site
• Skin: Propionibacterium acnes
• RT: Prevotella, Porphyromonas, Fusobacterium, Anaerobic Cocci
• GIT: Bacteroides fragilis, Clostridium difficile
• GUT: Bacteroides, Prevotella, Fusobacterium
II. Specimen Selection, Collection, Transport and Processing
A. Specimen Quality (Selection)
1. Suitable specimens: Tissue biopsy (necrotic tissues), needle and syringe aspiration (exudates, abscess)
2. Unsuitable Specimens: Swabs, voided urine, feces, coughed sputum, bronchial washings.
3. Fecal specimens for Clostridial illness: Food poisoning (C. perfringens), botulism (C. botulinum),
pseudomembranous enterocolitis (C. difficile), neutropenic enterocolitis (C. septicum)
B. Specimen Transport
1. Rubber-stoppered collection vial: For liquid specimens (i.e. pus & body fluids)
2. Oxygen free transport tubes and anaerobic pouch: For swab specimens and tissue specimens, respectively
3. Contents: i. Reducing agent (thioglycolic acid, Na thioglycolate); ii. redox indicator (resazurin, methylene blue)
C. Processing Clinical Specimens
1. Macroscopic Examination: Foul odor (Anaerobic G- bacilli), brick-red fluorescence and necrotic tissue black
exudates (Porphyromonas, Prevotella), sulfur granules (anaerobic G+ bacilli)
2. Microscopic Examination of the Specimen: To determine a polymicrobic infection, guide for media selection,
provide a presumptive identification and reveal leukocytes and squamous epithelial cells
3. Inoculation to Plated or Tubed Media
a. Anaerobic blood agar: Enriched media
b. PEA & CNA blood Agar: Selective for G(+) anaerobes
c. Anaerobic broth: i . Thioglycollate broth ; ii. Cooked meat broth and iii. peptone-yeast extract glucose (PYG) -
analysis of metabolic end products by GLC
d. Kanamycin-vancomycin-laked blood (KVLB):Selective for Bacteroides & Prevotella
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e. Bacteroides bile esculin agar (BBE): Selective & differential for B. fragilis
f. Cycloserine cefoxitin fructose agar (CCFA): Selective & differential for C. difficile
g. Egg-yolk Agar (EYA): For determination of lecithinase and lipase production
• C. perfringens: Lecithinase + (white opaque zone) ; F. necrophorum, C. botulinum: Lipase + (iridescent sheen)
4. Anaerobic Incubation
a. Anaerobe Chamber: Storage & inoculation under anaerobic condition
b. Anaerobic Jars and Bags: Anaerobiosis produced by gas generator envelope
c. Holding Jars: During processing, for inoculated plates pending incubation & examination of cultures
• Contents: gas generator (85-90% N2 , 5% H2, 5-10% CO2), catalyst (palladium pellets, iron powder),
desiccant (silica gel, blue → pink), redox indicator (methylene blue and resazurin)
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II. Gram Positive Bacilli: Outline: (1) Disease, (2) Cellular Morphology, (3) Colony Morphology, ( 4) Other Characteristics
A. Actinomyces israelli
1. A ti o y osis si us tra ts, hi h erupt to the surfa e a d drai pus that ay o tai sulfur granules ),
2. Branching filamentous rods
3. White, opaque and rese le molar tooth
B. Propionibacterium acnes
1. Actinomycosis, acne, subcute bacterial endocarditis, contaminants of blood culture bottles
2. Anaerobic diphtheroids
3. Small & white to large & yellowish tan
C. Bifidobacterium
1. Actinomycosis, in mixed infections of abdomens and GUT
2. Diptheroid, e d of ells ay e spatulated or ifur ated dog bones
3. Small, white, convex, shiny with irregular edge
Branched bacilli Catalase/Indole Comments
A. israelii + - Molar tooth colony
P. acnes - + Diptheroid
Bifidobacterium - - Rods w/ forked ends
III. Gram Negative Bacilli
1. Virulence Factor: Polysaccharide capsules, adherence factors
2. Spectrum of Disease: In mixed infection, brain abscess and peritoneal infections
3. Cellular Morphology; 4. Colonial Characteristics; 5. Other Characteristics
A. Bacteroides fragilis
3. Coccobacili or pleomorphic
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IV. Cocci
A. Spectrum of Disease: In mixed infection, brain abscess and peritoneal infections
B. Cellular Morphology:
1. G(-) diplococci / in chains -> Veillonella parvula
2. G(+) cocci -> Peptococcus & Peptostreptococcus (Finegoldia magna) C. Colony
Morphology: Red fluorescence -> Veillonella parvula
V. Confirmatory Test
• Gas-liquid chromatography: Metabolic ends products from glucose metabolism
• Gas chromatography: Whole-cell long chain fatty acid methyl ester (FAME) analysis
9a. Spirochetes
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9b. Chlamydiaceae
I. Introduction: Obligate Intracellular Bacteria
A. General Characteristics
C. trachomatis C. pneumoniae C. psittaci
EB Morphology Round Pear-shaped Round
Glycogen in inclusions (+) (-) (-)
Sulfa drug sensitivity (+) (-) (-)
Natural Hosts Humans Humans Birds
B. Chlamydia trachomatis
1. Clinical Infections
Biovars Clinical Syndromes Route of Transmission
A,B,Ba,C Ocular (Endemic) Trachoma Hand to eye from fomites
L1,L2,L3 Lymphogranuloma venereum
Sexual
Urogenital Disease (PID , Reiter Syndrome)
D-K
Inclusion conjunctivitis Hand to eye
2. Laboratory Diagnosis
• Cell Culture, serology , molecular (PCR)
• Frei’s test (intradermal skin test of LGV Ag): Positive: erythema (redness) and induration (firmness) C.
Chlamydophilia pneumoniae:
• Pneumonia & pharyngitis
• Guillain-Barre syndrome D. Chlamydophilia psitacci:
• Psittacosis & ornithosis (parrot fever)
• acquired from birds by inhalation of aerosols
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C. Ehrlichia / Anaplasma
Ehrlichia chaffeenis • Monocytic ehrlichiosis
Ehrlichia ewingii • Granulocytic ehrlichiosis Ticks
A. phagocytophilum • Granulocytic anaplasmosis
D. Coxiella
Coxiella burnetii Q (query) fever Inhalation of dried birthing fluid
E. Laboratory Diagnosis Disease OX-19 OX-2 OX-K
1. Immunohistology and PCR Brill-Zinsser V V –
2. Embryonated eggs and tissue culture
Epidemic typhus + V –
3. Gie sa, Wright’s stai ed uffy oat
4. Weil-Felix rxn. (P. vulgaris OX-19, OX-2, P. mirabilis OX Murine typhus + V –
Rickettsialpox – – –
RMSF + + –
Scrub Typhus – – V
I. Characteristics
• Do not possess a cell wall
• Small cell, genome size and colonies
• Pleuropneumonia-like organisms (PPLOs)
A. Mycoplasma pneumoniae
1. Flora: Oropharnyx and upper RT tract
2. MOT: Contact with urine of animals (e.g. rodents) who carry the organism
3. Clinical infections: Asymptomatic infection, primary atypical or walking pneumonia
4. Others: Eaton Agent
B. Mycoplasma hominis and Ureaplasma spp.
1. Flora: GUT
2. MOT: sexual and vertical
3. Clinical infections: Urogenital tract infections (Non gonococcal urethritis, PID); systemic infections in neonates
and in immunosupressed patients
C. Laboratory Diagnosis
1. Specimen Collection and Transport
a. Body fluids, wound and blood
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10a. Mycobacteria
General Characteristics
Slender, slightly curved rods, high lipid content (mycolic acid), acid fast (stained by basic fuchsin dye but resist
decolorization by 3% HCl), slow growth (2-6 weeks)
1. Mycobacterium tuberculosis complex
A. Mycobacterium tuberculosis
a. Epidemiology: >1 billion persons are infected, 8-10 million new cases / year, 15-20% develops disease b.
Transmission: Inhalation of aerosols or close contact
c. Spectrum of disease: Tuberculosis
i. Primary / Reactivation: Tubercles or granuloma (granulomatous lesions from multinucleated cells) and
Caseation (cheese like masses from break down of tubercles) in the lungs ii.
Extrapulmonary (Miliary): Kidney, joints, CNS, GUT, body cavities, larynx, etc.
d. Culture: Raised, dry, rough, buff (2-3 weeks in LJ, 5-10 days in Middlebrook)
B. Mycobacterium bovis and BCG
a. Transmission: M. bovis (Ingestion of milk from infected cattle)
M. bovis / Bacille Calmette-Guerin (Immunization of immunocompromised individuals)
b. Culture: rese les water droplets i Middle rook
c. ScreeningTest: Tuberculin Skin Testing (Mantoux test, Pirquet test, PPD test)
Positive: erythema (redness) and induration (firmness) in 48-72 hours
2. No tu er ulosis y o a teria NTM’s
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3. Laboratory Diagnosis
A. Laboratory Safety Considerations
1. Specimen is collected in sterile, leak proof container.
2. BSL 2 - for preparing AFB smears and culture, BSL 3 - For propagation
3. Aerosol - generating procedures is performed in Class II or III BSC
B. Specimen Collection
1. Sputum and gastric lavage - early morning specimens in 3 consecutive days
2. Urine - early morning urine in 3 consecutive days, midstream clean catch
3. Stool - for isolation of M. avium in AIDS patients
4. Blood - for isolation of M. avium complex, can be recovered by BACTEC or by Isolator lysis centrifugation
5. Tissue and body Fluids - homogenized and concentrated), respectively
C. Specimen Preparation
1. n-Acetyl-L-cysteine (NALC) or dithioreitol: liquefy specimen (splits disulfide bonds)
2. 2-4% NaOH: both digestant (mucolytic) and decontaminating (antibacterial) agent
3. Trisodium phosphate & Benzalkonium chloride: acts as digestant and as decontaminating agent, respectively.
4. Oxalic Acid: for Pseudomonas contaminated samples
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I. Introduction
•
Variable Standard Note:
Inoculum Disk diffusion: 1.5 x 108 CFU/mL
Broth microdilution: 5 x 105 CFU/mL
Formulation Mueller-Hinton
25 mg/L Ca2+, 12.5 mg/L Mg2+ ↑ Concentration: ↓ a ti ity of
Ca2+, Mg2+ content
Aminoglycosides and Tetracyclines
Minimal or absent ↑ Concentration: ↑ a ti ity of
Thymidine content
Sulfonamide and SXT
7.2-7.4 ↑ pH : ↑ a ti ity of A i ogly osides,
pH
Erythromycin ; ↓ a ti ity of Tetra y li es
Agar depth 3-5mm (4 mm)
Determine the extent of its acquired resistance
II. Disk diffusion Testing Methods (Kirby-Bauer)
Incubation
Others
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