Practical Medical MicrobiologyPHT313

By

Dr. Mohamed Al-AgamyAssistant Professor of Microbiology

Department of Pharmaceutics & Microbiology

College of Pharmacy

King Saud University

2010/1431

Classification of Bacteria

Staphylococci• Three important species of staphyloccoci have medical importance

– S. aureus : pathogenic and commensally found in nose

– S. epidermidis: non pathogenic and commensals in skin

– S. saprophyticus: causes UTI in sexually active women

• Rarely found in healthy humans but is commonly isolated from

animals and their carcasses

– S. haemolyticus: Frequently found as a commensal organism on

humans and animals

• It occurs infrequently as a cause of soft-tissue infections, usually in

immunocompromised patients

General characteristics

• General

characteristics– Gram Positive Cocci– Grape-like– Non Motile– Non Spore Forming– Non Fastidious– Facultative Anaerobes– Fermentative (O+/F+)– Halotolerant– Catalase positive

• Characteristics of S. aureus

– Production of coagulase

– Production of

phosphatase

– Production of DNase

– Ferment Mannitol

– Gelatin liquefied

– β-hemolysis on blood

agar

– Acidification & clotting of

litmus milk

Gram stain of Staphylococci

Virulence Factors

• Antigens– Protein A– Capsule– Adhesins

• Enzymes– Coagulase– Lipase– Hyaluronidase– Staphylokinase – Nuclease

• Toxins– α-Toxin– β-Toxin– δ-Toxin– P-V Leukocidin– Enterotoxin– Exfoliative Toxin– Toxic Shock Syndrome

Toxin

Laboratory Diagnosis

• I- Specimen:– Pus, Urine, Stool, Blood, CSF

• II- Gram Stain:– Gram positive cocci, arranged in cluster

• III- Culture:– A. Blood agar (Non-Selective Media)

• Colonies of S.aureus are golden yellow and -hemolytic• Colonies of S. epidermidis are non-pigmented and non-hemolytic

Colonies of S. aureus showing beta hemolysis

Colonies of s. epidermidis (up) showing porcelin- white colonies as compared to S. aureus (down) the golden appearance of the colonies. This clear distinction in colony color is not seen at all times.

Mannitol Salt Agar

• 2. Mannitol Salt Agar (MSA)– MSA is selective and differential medium for

staphylococci– MSA contains– NaCl (7.5%), as selective agent– Mannitol as a differential agent– Phenol Red (pH indicators)

• turns yellow in acidic pH and turns red in alkaline pH– S. aureus ferment mannitol and give yellow colonies– S. epidermidis and S. saprophyticus do not ferment

mannitol and appear red on MSA.

• Catalase test is used to distinguished between staphylococci (positive) from streptococci (negative)

• Flood the culture with drops of 3% H2O2 • Catalase-positive cultures give air bubble at once

• The test should not be done on blood agar because blood itself will produce bubbles

H2O2 H2O + O2 (gas, ↑)Staphylococci

Catalase

IV- Biochemical tests1. Catalase test

2- Coagulase TestPrinciple:• This test is used to differentiate between S. aureus (CPS) &

other Staphylococcus species (CNS)• This test is done by tube method or slide method

Coagulase testCoagulase Positive

Staphylococus aureusCoagulase-NegativeS. epidermidis & S.

saprophyticus

Fibrinogen (Plasma)

Coagulase Fibrin (Clot)

• The tube coagulase test (Free):• Procedure:

– Mix 0.1 ml of culture + 0.5 ml of plasma– Incubate at 37C for 4 h– Observing the tube for clot formation– Any degree of clotting constitutes a positive test

• Advantage– More accurate

• Disadvantage– Time consumed

• The slide coagulase test• Procedure:

– Used to detect bound coagulase or clumping factor– Add one drop heavy bacterial suspension and one drop of plasma on slide– Mixing well and observing for clumping within 10 seconds

• Advantage– Rapid diagnosis

• Disadvantage– Less accurate

S. aureusS. epidermidis

3 -Deoxyribonuclease (DNAase) test

• Principle:– DNA is hydrolyzed into oligonucleotides by the action of DNase– S. aureus produces DNase while S. epidermidis and most staphylococci have not DNase– DNA is insoluble in acid– Nucleotides are soluble in acid

• Procedure & result:– Inoculate DNA agar plate with tested organism in circular motion (Spot)– Incubate at 37C for 24 h– Observe DNase activity by adding 1N HCl to the agar surface, a zone of clearing

indicates a positive test– The zone represents the absence of DNA– The medium around colonies not producing DNase remains opaque, which is a reflection

of the precipitation of DNA by the added acid.

4- Novobiocin Sensitivity

• Novobiocin resistance is intrinsic to S. saprophyticus but uncommon in other clinically important species.

• A simple disk diffusion test for estimating novobiocin susceptibility is used to distinguish S. saprophyticus from other clinically species

• Inoculated overnight culture on Mueller-Hinton agar or MSA• Add novobiocin disk on inoculated plate• Incubate at 37C overnight

Differentiation between Staphylococcus speciesS. aureus S. epidermidis S. saprophyticus

Gram stain Gram +ve cocci Gram +ve cocci Gram +ve cocci

Catalase Positive Positive Positive

Blood agar β-hemolysis Non-hemolytic Non-hemolytic

Mannitol fermentation )MSA(

Ferment mannitol

Does not ferment

Does not ferment

Coagulase Positive Negative Negative

DNAase Positive Negative Negative

Novobiocin sensitivity

Sensitive Sensitive Resistant

Preparation of Smear and Staining

• Preparation of smear– Solid culture– Liquid culture– Distribute culture in slide– Air dry– Heat fix– Ready to stain

• Gram Stain– Primary Dye (C.V.) – Mordant (iodine)– Decolorizer (Alcohol)– Counterstain (Safranin)– All applied for 1 min– After each step wash with water– Blot dry– Add one drop of immersion oil– Examine under oil immersion lens

Practical Work• Gram stain

• Catalase test

• Mannitol fermentation on MSA

• DNAase Test

• Tube Coagulase Test (Demo)

• Novobiocin sensitivity (Demo)

Streptococci• General Characteristics of Streptococci

– Gram positive cocci– Chains or pairs– Usually capsulated– Non motile– Non spore forming– Facultative anaerobes– Fastidious– Fermentative (O+/F+)– Catalase negative (Staphylococci are catalase positive)

Classification of Streptococci

• Streptococci can be classified according to:

– Oxygen requirements

• Anaerobic (Peptostreptococcus)

• Aerobic or facultative anaerobic (Streptococcus)

– Hemolysis on Blood Agar (BA)

– Serology (Lanciefield Classification)

Classification Based on Hemolysis•Hemolysis on blood agar

–-hemolysis• Partial hemolysis• Green discoloration around the colonies• e.g. non-groupable streptococci

– S. pneumoniae & S. viridans–-hemolysis

• Complete hemolysis• Clear zone of hemolysis around the colonies• e.g. Group A & B

– S. pyogenes & S. agalactiae)–-hemolysis

• No lysis• e.g. Group D

– Enterococcus spp

-hemolysis

-hemolysis-hemolysis

Serology: Lancefield Classification

• Streptococci classified into many groups from A-K & H-U• One or more species per group• Classification based on C- carbohydrate antigen of cell wall

– Groupable streptococci• A, B and D (more frequent)• C, G and F (Less frequent)

– Non-groupable streptococci• S. pneumoniae (pneumonia)• viridans streptococci

– e.g. S. mutans– Causing dental carries

StreptococciLanciefield classification

Group AS.

pyogenes

Group BS.

agalactiae

Group CS.

equisimitis

Group DEnterococcu

s

Other groups

(E-U)

Differentiation between -hemolytic streptococci

• The following tests can be used to differentiate

between -hemolytic streptococci

– Lancefield Classification

– Bacitracin susceptibility Test

• Specific for S. pyogenes (Group A)

– CAMP test

• Specific for S. agalactiae (Group B)

Bacitracin sensitivity Test• Principle:

– This test is used for presumptive identification of gp A

– To distinguish between S. pyogenes (susceptible to B)

& non group A such as S. agalactiae (Resistant to B)

– Bacitracin will inhibit the growth of gp A Strep. pyogenes

giving zone of inhibition around the disk

• Procedure:

– Inoculate BAP with heavy suspension of tested organism

– Bacitracin disk (0.04 U) is applied to inoculated BAP

– After incubation, any zone of inhibition around the disk is

considered as susceptible

CAMP test• Principle:

– Group B streptococci produce extracellular protein (CAMP factor)– CAMP act synergistically with staph. -lysin to cause lysis of RBCs

• Procedure:– Single streak of Streptococcus to be tested and a Staph. aureus are

made perpendicular to each other– 3-5 mm distance was left between two streaks– After incubation, a positive result appear as an arrowhead shaped

zone of complete hemolysis– S. agalactiae is CAMP test positive while non gp B streptococci are

negative

CAMP test

Differentiation between -hemolytic streptococci

• The following definitive tests used to differentiate

between S. pneumoniae & viridans streptococci

– Optochin Test

– Bile Solubility Test

– Inulin Fermentation

Optochin Susceptibility Test• Principle:

– Optochin (OP) test is presumptive test that is used to identify S. pneumoniae

– S. pneumoniae is inhibited by Optochin reagent (<5 µg/ml) giving a inhibition zone ≥14 mm in diameter.

• Procedure:– Blood agar plate is inoculated with organism to be tested– OP disk is placed on the center of inoculated BAP– After incubation at 37oC for 18 hrs, accurately measure the diameter

of the inhibition zone by the ruler– ≥14 mm zone of inhibition around the disk is considered as positive

and ≤13 mm is considered negative• S. pneumoniae is sensitive (S) while S. viridans is resistant (R)

Optochin Susceptibility Test

Optochin susceptibleS. pneumoniae

Optochin resistantS. viridans

Bile Solubility test• Principle:

– S. pneumoniae produce a self-lysing enzyme to inhibit the growth

– The presence of bile salt accelerate this process

• Procedure:

– Add 10 parts (10 ml) of the broth culture of the organism to be tested

to one part (1 ml) of 2% Na deoxycholate (bile) into the test tube

– Negative control is made by adding saline instead of bile

– Incubate at 37oC for 15 min

– Record the result after 15 min

Bile Solubility test

• Results:

– Positive test appears as clearing in

the presence of bile while negative

test appears as turbid

– S. pneumoniae soluble in bile

whereas S. viridans insoluble

Differentiation between -hemolytic streptococci

CAMP test Bacitracin sensitivity

Hemolysis

Negative Susceptible S. pyogenes

Positive Resistant S. agalactiae

Inulin Fermentation

Bile solubility

Optochin sensitivity

Hemolysis

Not ferment Soluble Sensitive (≥ 14 mm)

S. pneumoniae

Ferment Insoluble Resistant(≤13 mm)

Viridans strep

Differentiation between -hemolytic streptococci

Outline of differentiation between Gram-Positive cocci

e.g. S. epidermidis

Practical Work• Gram stain of Streptococcus species

• Hemolysis on blood agar (S. pyogenes, S. pneumoniae and

Enterococcus faecalis)

• Bacitracin susceptibility test (S. pyogenes and S. agalactiae)

• CAMP test (S. agalactiae and S. pyogenes )

• Optochin susceptibility test (S. pneumoniae and S. viridans)

• Bile solubility test (demo)

Aerobic Spore Forming Bacillus spp Classification of Bacteria

Aerobic Spore Forming Bacillus spp

Bacillus species

Pathogenic

Non-pathogenic

Bacillus anthracis Bacillus cereus

Bacillus subtilis

Bacillus species• General Characteristics• Very large Gram positive bacilli• 1-1.2 µm in width x 3-5µm in length• Arranged in long chains• Motile except B. anthracis• Spore forming (outside the host)

– Spores are central and oval• Capsulated (inside the host)• Non fastidious• Aerobic• Fermentative i.e. O+/F+• Catalase positive• Natural Habitats• It is found in soil habitats

Anthrax• Anthrax is caused by B. anthracis

• Types of Anthrax

– Cutanoues Anthrax (Malignant Pustule) (20% fatal)– Intestinal Anthrax– Pneumonic Anthrax (Woolsorters disease)

• Virulence factors• Poly-D-glutamyl Capsule

– Mediates the invasive stage of the infection– Antiphagocytic

• Anthrax Exotoxins– Mediates the toxigenic stage

Bacillus cereus B. cereus is a normal inhabitant of soil Isolated from foods (Grains and spices) B. cereus causes food poisoning B. cereus deposits its spores in food Bacteria germinates in food & begin releasing their exotoxins Spores are not killed during cooking The following table differentiates between Bacillus sp.

B. cereus B. anthracis

motile Non-motile MotilityNon-encapsulated capsulated Capsuleβ-hemolytic Non-hemolytic HemolysisR (produce β-lactamase) S Resistance to Penicillin

Identification of Bacillus Spp.• Specimen

– Pastular exudates in malignant pustule – Sputum in pneumonic anthrax – Stool in intestinal anthrax (also in food poisoning by B.

cereus)• Stool specimen is emulsified and heated to 80 C to kill

non spore forming microorganism• Morphology

– Macroscopical (Cultural characteristics)– Microscopical (Gram Stain, Spore Stain)

• Cultural Characteristics• Grow on nutrient Agar

• On ordinary medium• Grow aerobically at 37C with characteristic mucoid or

smooth colonies, which indicates the pathogencity of organism (presence of capsule)

• Rough colonies are relatively avirulent• Stab culture on gelatin medium results in inverted fire

tree appearance.• Growth on Blood Agar

Bacillus anthracis colonies are non hemolyticB. cereus colonies are β-hemolyticB. subtilis colonies are β-hemolytic

• Microscopical examination

• Gram Stain, Capsule stain and motility

–Gram positive bacilli

–Found in chains

–B. anthracis is not motile

–B. cereus is motile

–B. anthracis is capsulated inside the host

• Spore Stain

Bacillus spores are oval & central

By spore staining (Malachite green & safranin), the spore

appears green while the vegetative cells appear red.

• All Bacillus species are catalase positive

• Remember: staphylococci are catalase positive

Biochemical tests1- Catalase Test

Broth Cultutre & H2O2 on the slideH2O2 added on culture grown on nutrient agar

2- Starch Hydrolysis (Amylase Activity)• Principle

– Starch + Iodine blue color– Glucose + Iodine No reaction

• Nutrient Agar containing 1% Starch + M.O Glucose

• Procedure– Inoculate nutrient agar plate containing 1% Starch with the M.O.– Incubate the plate at 37 for overnight– After incubation, flood the plate with Iodine solution

• Result– Activity of amylase is indicated by a clear zone around the growth while the rest of the plate

gives blue color after addition of iodine solution

AmylaseIodine

Appearance of colorless zone around the growth

Spore Stain Procedure1. Make a heat fixed smear of Bacillus

2. Place the slide on the slide rack

3. Cover the smear with malachite green stain

4. Apply heat for 3-5 min without boiling and drying of the slide

5. Wash the slide gently in running water about 20 S

6. Counterstain with safranin for one minute

7. Gently rinse with water

8. Gently blot the slide dry, no rubbing, and let it air dry and examine with oil immersion optics.

9. Observe red vegetative cells and sporangia, and green endospores and free spores

Practical Work

• Gram Stain

• Spore Stain

• Catalase Test

• Starch hydrolysis

Clostridia• General Characteristics of Clostridia

– Large Gram positive

– Straight or slightly curved rods with slightly rounded ends

– Anaerobic

– Spore bearing

– Fermentative, or proteolytic or both

– Catalase and oxidase are negative

• Natural Habitats

– Their habitats are soils and animal & human gut which invade the

blood and tissue when host die and initiate the decomposition of

the corpse (dead body)

Clostridium

• Diseases– Their pathogenesis by producing potent exotoxins and

enzymes which attack the neurons pathways– Rapid diagnosis is crucial or patient will die

Clostridium causing

TetanusCl. tetanii

Gas gangrene

SacchrolyticCl.

perfringens

BotulismCl. botulinum

Antibiotic associated diarrhea

Cl. difficile

Clostridium tetani causing tetanus

• General characteristics of Cl. tetani

– Gram positive, straight, slender rod with rounded ends

– All species form endospore

– Spores are terminal

• drumstick with a large round end)

– Fermentative

– Obligate anaerobe

– Motile by peritrichous flagella

– Grows well in cooked meat broth and produces a thin spreading film

when grown on enriched blood agar

– Spores are highly resistant to adverse conditions

Clostridium tetani• Causative agent

• Cl. tetani is the causative agent of tetanus (Lockjaw)

• Virulence factors

• The patheogenis of Cl. tetani is due to potent exotoxins

• Cl. tetani produces two types of toxins:

• Tetanolysin, which causes lysis of RBCs

• Tetanospasmin is neurotoxin

Laboratory Diagnosis of Tetanus• The diagnosis of tetanus depends primarily upon the clinical

manifestation of tetanus including muscle spasm & rigidity.

• Specimen:

– Wound exudates using capillary tube

• Culture:

– On blood agar and incubated anaerobically

• Growth appears as a fine spreading film and β-hemolytic

• Gram stain is a good method for identifying Clostridium

– Cl. tetani is Gram positive rod, motile with a round terminal spore

giving a drumstick appearance

Clostridium Causing Gas Gangrene

Clostridia causing gas gangreneSaccharolytic

organisms Cl. perfringens

Ferment carbohydratesAcid and gas are produced

Proteolytic organisms

Cl. sporogenesDigest proteins with blackening

bad smell production

Mixed saccharolytic &

proteolyticCl. histolyticum

Clostridium perfringens• General characteristics

– Large Gram-positive bacilli with stubby (short) ends– Spore forming

• Spores are oval and subterminal and not bulging• Seldom to see

– Capsulated

– Non motile (Cl. tetani is motile)

– Anaerobic• Natural habitats

– Animal and human excreta– Soil

Cl. perfringensCausing

Gas gangrene Food poisoning(Enterotoxin)

Pathogenesis (Virulence factor)• Toxins of Cl. perfringens• There are five different toxin types (A-E)• Each type of toxin composed of different components• All types of toxin contain toxin

• Distribution of major toxins among types of Cl. perfringens

Types of ToxinsComponents of Toxins

Epsilon Iota Enterotoxin

A + - - - +B + + + -C + + - -D + - + -E + - - +

Laboratory Diagnosis of gas gangrene Specimen: Histological specimen or wound exudates

Specimens of exudates should be taken from the deeper areas of the wound

Microscopical examination (Gram, Spore stain etc)Gram-positive bacilli with blunt (not sharp) ends occurring

singly or in pairs, non motile, capsulated & sporulatedThe spore is large, oval, central to sub-terminal & non

bulging (non swelling)Spores are rarely observed

Culture: Anaerobically at 37COn Robertson's cooked meat medium → blackening of

meat will observed with the production of H2S and NH3On blood agar → double zones of β-hemolytic colonies

Biochemical Tests

Fermentation of many sugars with acid & gas

Saccharolytic organism

Acidification litmus milk with stormy clot production

Nagler reaction

1- Reaction on Litmus Milk

Litmus Milk

Skimmed Milk(Without Fat)

Litmus indicator

Acid Base and Redox indicatorLactoseSugar

CaseinProtein

Contains

Reaction on Litmus Milk

Lactose Acid Pink Color (Milk Sugar)

Fermentation Litmus Indicator

1- Acidic Reaction

2- Basic Reaction

Casein Alkaline amines Blue Color (Milk Protein)

Digestion Litmus Indicator

Reaction on Litmus Milk

Stormy Clot Formation

Fermentation

CaseinMilk Protein

Coagulation

Gas

Clot

Stormy Clot

Milk SugarLactose Acid +

Reaction on Litmus Milk

Nagler’s Reaction• This test is done to detect the lecithinase activity

– The M.O is inoculated on the medium containing human

serum or egg yolk (contains lecithin)

– The plate is incubated anaerobically at 37 C for 24 h

– Colonies of Cl. perfringens are surrounded by zones of

turbidity due to lecithinase activity and the effect is

specifically inhibited if Cl. perfringens antiserum containing

antitoxin is present on the medium

Nagler Reaction

Positive Nagler ReactionProcedure of Nagler Reaction

Clostridium botulinum• General Characteristics

– Gram positive bacillus– Spore forming

• Spores are oval and sub-terminal – Motile with peritrichous flagella – Strict anaerobic– Formidable pathogen due to;

• Production of a potent neurotoxin in food • Resistance of its spores to inactivation

• Natural habitats• It is widely distributed saprophyte occurring in soil,

vegetables, fruits etc

• Causative agent– Cl. botulinum is the causative agent of botulism– Botulism is a severe, often fatal, form of food poisoning– Botulinal toxins are among the most poisonous natural substances

known – During the growth of the microorganism, toxin is liberated into the food– Toxins is classified into seven antigenic types (A-G) with types A, B and

E most frequently associated with human disease• Mode of infection

– Botulism results from ingestion of preformed toxin in the food– Insufficient heating in the process of preserving foods is an important

factor in the causation of botulism and great care must be taken in canning factories to ensure that adequate heating is achieved in all parts of the can contents

Laboratory diagnosis• The diagnosis must be suspected on clinical manifestation• The diagnosis may be confirmed by demonstration of

– Organism and/or its toxin may be detected in the patient's stool or gastric contents

– Organism and/or its toxin may be detected in the suspected food– Toxin may be demonstrated in the patient's blood

• Samples of vomit or feces may also yield such evidence • Food or stool specimens are emulsified, heated at 80 C & inoculated on

blood agar• Gram stain of the suspected colonies revealed that the organism is gram

positive bacilli, motile, and sporulated– The spores are oval and sub-terminal

• Toxin is detected in either food or blood by toxin-antitoxin neutralization test in mice

Clostridium difficile

• Cl. difficile is part of the normal intestinal flora in a small proportion of healthy persons & hospitalized patients

• Exposure to antibiotics alerts the normal enteric flora, permeating overgrowth of Cl. difficile or making the patient more susceptible to exogenous acquisition of Cl. difficile.

• Proliferation of Cl. difficile with localized production of

their toxins in the colon leads to disease

Clostridium difficile• General characteristics

– Gram positive rod – Oval spores– Motile– Quite commonly in the faces of neonates, but is not

generally regarded as a normal commensals of adults• Toxins

– Toxin A causes diarrhea– Toxin B is cytotoxic

• Disease– Antibiotic associated diarrhea– Pseudomembranous colitis

Laboratory diagnosis

• When a patient develops while antibiotics, Cl. difficile must be

considered as a possible cause

• Cl. difficile can be isolated from faces on selective media (CCFA)

• Toxin can be detected in the patient's faces by immunological

methods such as ELISA

• Culture without demonstration of toxin has little diagnostic value

• Observation of colonic pseudomembranes (white exudates on the

surface of large intestine) by colonoscopy is diagnostic for

pseudomembranous colitis, in which case laboratory confirmation is

unnecessary

Anaerobic Cultivation• 1- Anaerobic Jar

• Most frequently used system for creating anaerobic atmosphere

• Removal of oxygen & replacing it with inert gas

• It is especially plastic jar with a tightly fitted lid

• Anaerobic condition can be set up by use a commercially

available H2 and CO2 generators envelop that is activated by

adding water

• Hydrogen and carbon dioxide will release and react with oxygen

in the presence of catalyst to form water droplet

Anaerobic Jar Candle Jar

• Production of heat within few minutes (detected by touching

the top of the jar) and subsequent development of moisture on

the wall of the jar are indications that the catalyst and

generators envelop are functioning properly

• Anaerobic indicator (Methylene blue) is placed in the jar

• Methylene blue is blue in oxidized state (Aerobic condition)

while turns colorless in reduced state (Anaerobic condition)

• 2. Culture Media (containing reducing agent)– Thioglycollate broth

• Nonselective for cultivation of anaerobic bacteria as well as facultative anaerobes and aerobes

• It contains– Pancreatic digest of casein, soy broth and glucose that enrich

growth of bacteria– Sodium thioglycollate (Reducing agent)– Low percentage of agar to increase viscosity of medium– Thioglycollate and agar reduce Eh– Resazurin (redox indicator)

– Cooked Meat Medium• It contains

– Meat particles (prepared from heart muscles) which contain hematin & glutathione that act as reducing agent

Growth on Fluid Thioglycolate

Clostridium sporogenes Growing in Thioglycolate Medium

Reducing agents in the medium absorb oxygen

and allow obligate anaerobes to grow

Reaction on Cooked Meat Medium• Saccharolytic reaction

– It causes fermentation of glycogen of muscles

– Production of acid and gas

– Meat particles remain intact

– e.g. Cl. perfergines

• Proteolytic Reaction

– It causes digestion of meat particles

– Formation of black, foul smelling due to sulfur compounds

Corynebacterium spp• General Characteristics

– Gram positive bacilli, with pleomorphic, characteristic morphology (club shaped and beaded) & Chinese letters arrangement

– Non motile – Non spore forming– Non capsulated– Facultative anaerobic– Breakdown glucose by oxidative and fermentative i.e. O+/F+– C. diphtheriae is fastidious while diphtheriods are non-fastidious– Catalase positive– Oxidase negative

• Habitats– C. diphtheriae inhabits nasopharynx but only on carrier state– Isolation from health human is not common– C. xerosis is normal flora of human conjuctiva, skin & nasopharynx

Species of Corynebacterium

CorynebacteriumPathogenic

C. diphtheriaeCommensal "Diphtheriods"

C. hofmannii, C. xerosisacne

Causative agent of diphtheria

Normal flora of RT, urethra, vagina, Skin

Corynebacterium diphtheriae

• Diphtheria toxin

• C. diphtheriae produce powerful exotoxin

• The toxin inhibits protein synthesis which results in cell death

• Diphtheria toxin consists of 2 subunits

• The cells more affected are cardiac and nerve cells

Diagnosis of diphtheria

Laboratory DiagnosisCase

Symptomatic patient

CarrierAsymptomatic patient

Clinical DiagnosisClinical symptoms

Diagnosis by Physician

Laboratory diagnosis of case– Specimen:

• A throat swap by gentle touch the membrane to avoid bleeding

– Culture: • The swap is inoculated on

– Loeffler's serum medium (serum +glucose 3:1) broth)

– Blood Tellurite Agar [(BTA)(Blood + Potassium tellurite)]

• The inoculated plate incubated aerobically at 37C for 24.

• On Loeffler's serum medium (Non-selective media):

• This medium used to stimulate;

• The growth of C. diphtheriae

• Production of the metachromatic granules within the cells

• Cultural characteristics on BTA

– It is selective medium for isolation of C. diphtheriae

– 3 biotypes of C. diphtheriae are characterized on BTA

– i.e. Gravis, mitis and intermedius biotypes

– The most severe is the gravis biotype

• Colony of gravis biotype is large, grey, non-hemolytic

• Colonies of mitis biotype are small, black and hemolytic

• Colonies of intemedius biotype are intermediate in size,

non-hemolytic with black center & grey margin.

• Morphology– Gram-positive, nonspore forming, nonmotile bacilli– Club-shaped (Coryne= club) arranged at acute angles or parallel to

each other (Chinese letters appearance)– Beaded (metachromatic granules)

• Stain– Gram stain:

• C. diphteriae are gram positive bacilli arranged in Chinese letters form often club shaped

– Polychrome methylene blue stain: • C. diphteriae appears beaded due to the presence of intercellular

“Metachromatic or volutin" granules• By stain, the granules appear red while the rest of organism

appears blue

C. diphtheriae on BTAGram stain of C. diphtheriae

Loeffler’s seum

Biochemical ReactionCatalase test

• All Corynebacterium species are catalase positive (Also, Staphylococcus and Bacillus species are catalase positive)

2. Carbohydrate Fermentation Test• Principle Each species of corynebacteria has its specific carbohydrate

fermentation pattern C. diphtheriae can be differentiated from other

Corynebacterium species by fermentation of glucose and maltose (with production of acid only) but not ferment sucrose

• Procedure• Inoculate three tubes of carbohydrate fermentation medium

(broth containing one type of sugar and phenol red as the pH indicator) with the test organism

• Incubate the tubes at 37 C for 24 hrsGlucose Maltose Sucrose

• Result

Sugar fermentation can be indicated by change of color of

the medium from red to yellow due to formation of acid

which decrease the pH

C. diphtheriae can not ferment sucrose

C. xerosis can ferment sucrose

C. diphtheriaeC. xerosis

Glucose Maltose Sucrose

Glucose Maltose Sucrose+ve +ve +ve +ve +ve -ve

3. Test for detection of toxigenicity of C. diphtheriae In Vitro: Elek’s Test

• Principle:– It is toxin/antitoxin reaction– Toxin production can be demonstrated by a precipitation

of exotoxin with diphtheria antitoxin• Procedure:• A strip of filter paper impregnated with diphtheria antitoxin is

placed on the surface of serum agar• The organism is streaked at right angels to the filter paper• Incubate the plate at 37C for 24 hrs

• Results:

• After 48 hrs incubation, the

antitoxin diffusing from filter paper

strip and the toxigenic strains

produce exotoxin, which diffuses

and resulted in lines four

precipitation lines radiating from

intersection of the strip and the

growth of organism

Lines of precipitations

Inoculated M.O.

Positive Elek’s Test

Bacteria

Gram-negative Gram-positive

Cocci Bacilli

Acid fast bacteriaOther bacteria e.g. Mycoplasma,

Spirochetes

Neiserria gonorrhoeae

Neisseria meningitidisi

Oxidase negtaive

Oxidase positive

Enterobactericeae

Pseudomonadaceae

Vibrionaceae

Gram negative bacteria

O/F Test

Oxidative (O+/F-)

Pseudomonas

Fermentative (O+/F+)

EnterobacteriacaeVibrioionaceae

Gram negative bacteria

Oxidase Test

Oxidase positivePseudomonasVibrioionaceae

Oxidase negativeEnterobacteriacae

General Characteristics of Enterobacteriaceae

• All Enterobacteriaciae– Gram-negative rods– Ferment glucose with acid production – Reduce nitrates into nitrites– Oxidase negative– Catalase positive

• Facultative anaerobic• Motile except Shigellaand Klebsiella• Non-capsulated except Klebsiella• Non-fastidious• Grow on bile containing media (MacConkey agar)

Enterobacteriaceae

• Some Enterobacteriaceae are true pathogens

– Salmonella spp.

– Shigella spp.

– Yersinia spp.

– Certain strains of E. coli (ETEC, EPEC, EIEC, EHEC)

• Most members of the Enterobacteriaceae are opportunistic

or cause secondary infections of wounds, the urinary and

respiratory tracts, and the circulatory system e.g. E. coli.

Classification of EnterobacteriaceaeEnterobacteriaceaeLactose fermenters

E. coli, Citrobacter,Klebsiella, Enterobacter

Non-lactose fermenterSalmonell, ShigellaProteus, Yersinia

There are several selective and differential media used to isolate distinguishes between LF & LNF

The most important media are:MacConkey agarEosin Methylene Blue (EMB) agarSalmonella Shigella (SS) agarIn addition to Triple Sugar Iron (TSI) agar

Identification of Enterobacteriaceae

• Gram stain– All Enterobacteriaceae are Gram-negative rods– Arranged in single

Biochemical reactions• Oxidase test

– All members of Enterobacteriaceae are oxidase negative

– Pseudomonas is oxidase positive

– This test found in Pseudomonas Lab

• O/F test

– All members of Enterobacteriaceae are O+/F+

– Pseudomonas is O+/F-

– This test found in Pseudomonas Lab

Differentiation between LF & NLF by Growth on MacConkey agar

MacConkey AgarContains

Bile salts Crystal violet Lactose Neutral red

MacConkey agar is selective & differential medium for Enterobacteriaceae

Inhibit growth of G+ve bacteria

Cause of selectivity

Cause of differentialpH indicatorAcidic: Pink

Lactose feremntersPink colonies

Lactose non feremnterscolorless colonies

Classification of Enterobacteriaceae according to lactose fermentation (growth on MacConkey Agar)

Enterobacteriaceae

Lactose Fermenters Lactose Non-Fermenters

Escherichia coliKlebsiella spp

Enterobacter sppCitrobacter spp

Salmonella sppShigella sppProteus spp

Yersinina spp

Pink coloniesColorless colonies

AcidNeutral red

No acid

Identification of Enterobacteriaceae by Growth on MacConkey agar• Method:

– MacConkey agar is inoculated with tested organism using streak plate technique

– Incubate the plate in incubator at 37 C/24 hrs • Results:

– LF organism appears as pink colonies (e.g. E. coli)– NLF organism appears as colorless colonies (e.g. Shigella)

MacConkey AgarLactose non ferementers

Salmonella, Shigella, Proteus

Lactose ferementersE. coli, Klebsiella

Reaction on Salmonella Shigella (SS) agar• SS agar is a selective & differential medium for Salmonella and Shigella• It contains• Bile salts, and brilliant green dye as selective agents (inhibit G +ve) • Lactose as a differential agent• Neutral red as a pH indicator• The formation of acid on fermentation of lactose causes the neutral red

indicator to make pink colonies• Non lactose fermenting organisms are colorless on the medium• SS agar contains sodium thiosulfate and ferric ammonium citrate

allows the differentiation of organisms that produce H2S– Lactose fermenters, such as E. coli, have colonies which are pink– Shigella appears transparent or amber (NLF/H2S -ve)– Salmonella appears transparent with black centers (NLF/H2S +ve)

Identification of Enterobacteriaceaeby Growth on SS agar

• Method:

– SS agar is inoculated with

tested organism using

streak plate technique

– Incubate the plate in

incubator at 37 C/24 hrs A. Klebsiella pneumoniae (LF/H2S-ve)B. Escherichia coli (LF/H2S-ve) C: Salmonella sp. (LF/H2S+ve) D: Proteus mirabilis (NLF/H2S+ve) E: Ps. aeruginosa (NLF/H2S-ve) .

Growth of Enterobacteriaceae on EMB agar

Coli-type colonies are very dark, almost black e.g. E. coli

Reaction on Triple Sugar Iron (TSI) Agar• TSI contains

– Three different types of sugars• Glucose (1 part)• Lactose (10 part)• Sucrose (10 part)

– Phenol red (acidic: Yellow)• TSI dispensed in tubes with equal butt & slant

• Principle – To determine the ability of an organism to attack a specific

carbohydrate incorporated into a basal growth medium, with or without the production of gas, along with the determination of possible hydrogen sulphide production.

• Method:– Inoculate TSI medium with an organism by inoculating

needle by stabbing the butt and streaking the slant– Incubate at 37°C for 24 hours

• Result:Example

ResultReaction on TSI

H2S Slant color

Butt color

Non fermenter e.g.

PseudomonasAlk/Alk/-

(No action on sugars) - ve Red Red

LNF e.g. Shigella

A/Alk/- (Glucose fermented

without H2S)

- veRed Yellow

LNF e.g. Salmonella

& Proteus

A/Alk/+ (Glucose fermented

with H2S)

+ veblack in

buttRed Yellow

LF e.g. E. coli, Klebsiella,

A/A/- (All sugars are

fermented)- ve Yellow Yellow

Practical Work• Gram stain

• Oxidase test

• O/F test

• Growth on MacConkey’s agar

• Growth on EMB agar

• Growth on SS agar

• Reaction on TSI

Identification of EnterobacteriaceaeBiochemical Reactions

• Indole, Methyl Red, Voges-Prosakaur, Citrate

(IMViC) Tests:

– The following four tests comprise a series of important

determinations that are collectively called the IMViC

series of reactions

– The IMViC series of reactions allows for the

differentiation of the various members of

Enterobacteriaceae.

IMViC: Indole test Principle

Certain microorganisms can metabolize tryptophan by tryptophanase

The enzymatic degradation leads to the formation of pyruvic acid, indole and ammonia

The presence of indole is detected by addition of Kovac's reagent.

Tryptophaneamino acids

Tryptophanase Indole + Pyurvic acid + NH3

Kovac’s Reagent

Red color in upper organic layer`

Method:

Inoculate tryptone water with the tested microorganism

Incubate at 37°C for 24 hours Result: A bright pink color in the top layer indicates the presence

of indole The absence of color means that indole was not

produced i.e. indole is negative

After incubation interval, add 1 ml Kovacs reagent,

shake the tube gently and read immediately

Results of IMViCMethyl Red test

Voges-Proskauer testCitrate utilization test

Indole test

Negative Positive

PositiveNegative

Glucose(MRVP medium)

Acidic pathway(MR test)

Mixed Acids,pH less than 4.4

Acidity is detectd by

adding Methyl red indicator

Neutral pathway(VP test)

Acetylmethylcarbinol (Acetoin)

Acetoin is detected by adding Barrit’s

reagent

IMViC testMethyl Red-Voges Proskauer (MR-VP) Tests

Method Inoculate the organism into One tube of MRVP broth Incubate the tubes at 37°C for 24 hours Pour 1/3 of the suspension into a clean tube Run MR test in the tube with 2/3 & VP test in the open tube with 1/3. Methyl red tube: Add 6-8 drops of methyl red reagent. Voges-Proskauer tube: Add 12 drops of Barritt's A (-naphthol), mix,

4 drops of Barritt's B (40% KOH) and mix Let sit, undisturbed, for at least 1hour Result

MR test: Red color indicates positive test (e.g. E. coli) Yellow or orange indicates negative test (e.g. Klebsiella)

VP test Appearance of crimson red color indicates positive test (Klebsiella) E. coli isolates give negative VP test

Citrate Utilization TestPrinciple:

Citrate Na2CO3

Alkaline,↑pH

Blue color (Positive)

Bromothymol blue

Simmone’s Citrate mediaContains Citrate as a sole of C source

The color of medium is green

CO2 + Na + H2OPyruvate

Methods

Streak a Simmon's Citrate agar

slant with the organism

Incubate at 37°C for 24 hours. Results Examine for growth (+) Growth on the medium is

accompanied by a rise in pH to change the medium from its initial green color to deep blue

PositiveKlebsiella, Enterobacter

NegativeE. coli

Urease Test Principal Urea agar contains urea and phenol red Urease is an enzyme that catalyzes the conversion of urea to

CO2 and NH3 Ammonia combines with water to produce ammonium hydroxide,

a strong base which ↑ pH of the medium. ↑ in the pH causes phenol red r to turn a deep pink. This is

indicative of a positive reaction for urease

UreaUrease

CO2 + NH3H2O

NH4 OH ↑ in pH

Phenol Red

PinkPositive test

Streak a urea agar tube with the organism

incubate at 37°C for 24 h

Method

• Result• If color of medium turns from

yellow to pink indicates positive test.

• Proteus gives positive reaction after 4 h while Kelebsiella and Enterobacter gave positive results after 24 h

Positive test Negative test

EMB SS MacConkey

O/F Nitrate reductase

Oxidase Gram stain

Metallic sheen

LF LF O+/F+ +ve -ve -ve rod

E. coli

Dark LF LF O+/F+ +ve -ve -ve rods

Citrobacter

Dark LF LF O+/F+ +ve -ve -ve rods

Klebsiella

Dark LF LF O+/F+ +ve -ve -ve rods

Enterobacter

Colorless

NLF/H2S

NLF O+/F+ +ve -ve -ve rods

Salmonella

Colorless NLF NLF O+/F+ +ve -ve -ve rods

Shigella

Colorless

NLF/H2S

NLF O+/F+ +ve -ve -ve rods

Proteus

Summary of morphology, cultural characteristics, and biochemical reactions of Enterobacteriaceae

Motility Urease Citrate VP MR Indole TSI

Motile -ve -ve -ve +ve +ve A/A/- E. coli

Motile -ve +ve -ve +ve +ve A/A/- Citrobacter

Non motile

+ve +ve +ve -ve -ve A/A/- Klebsiella

Motile +ve +ve +ve -ve -ve A/A/- Enterobacter

Motile -ve +ve -ve +ve -ve A/Alk/+

Salmonella

Non motile

-ve -ve -ve +ve -ve A/Alk/- Shigella

MotileSwarwing

+ve +ve -ve +ve -ve A/Alk/+

Proteus

Summary of morphology, cultural characteristics, and biochemical reactions of Enterobacteriaceae

Practical Work

• Indole Test

• MR test

• VP test

• Citrate Utilization test

• Urease test

Gram negative bacteria

O/F Test

Oxidative (O+/F-)

Pseudomonas

Fermentative (O+/F+)

EnterobacteriacaeVibrioionaceae

Gram negative bacteria

Oxidase Test

Oxidase positivePseudomonasVibrioionaceae

Oxidase negativeEnterobacteriacae

Pseudomonas• Gram-negative bacilli belonging to Pseudomonadaceae

• Motile by means of a single polar flagellum. • Non spore forming• Capsulated "Polysaccharide capsule"• Aerobic• Breakdown glucose by oxidation i.e. Oxidative (O+/F-)• Oxidase and catalase positive• Non fastidious• The most important pathogenic organism is Ps. aeruginosa

• Optimum temperature is 37 C, and it is able to grow at 42 C• It is resistant to dyes, weak antiseptics, and many antibiotics• Common inhabitants of soil, water, GIT

• Ps. aeruginosa is opportunistic pathogen and associated with a variety of infections including:– Urinary tract infections– Wound and burn with blue green pus– Respiratory system infections (Pneumonia)– Eye infection and may lead to blindness– Ear infection (external ear or otitis media)– Meningitis– A variety of systemic infections

• Ps. aeruginosa produce two types of soluble pigments:– Pyoverdin or fluorscein: It is yellow-green pigment and fluorescent– Pyocyanin: It is a blue-green pigment and non-fluorescent

Identification of Ps. aeruginosa• Laboratory diagnosis

– Specimen:

• Urine, pus, sputum, CSF, blood, skin swap according

to the type of infection

– Microscopical Examination

• Gram Stain: Gram-negative rods

• Motility Test:– Hanging Drop Techniques

– Semisolid agar medium Motile

Cultural Characteristics• On Nutrient agar:

– Colonies are surrounded by bluish green coloration

• On selective media "Cetermide"

– Pigments are more obvious

• On Blood agar

– -hemolytic colonies

• On MacConkey agar

– Pale yellow colonies i.e. non lactose fermenters

• Ps. aeruginosa able to grow at 42 C for 3 days

Cultural Characteristics

Gram Stain of Pseudomonas

Ps. aeruginosa on Nutrient agar

Ps. aeruginosa on cetrimide agar

Biochemical Reactions

• Oxidase positive

• Breakdown glucose oxdatively

• Nitrate Reductase positive (further reduction to N2)

• Gelatinase positive

• Utilize Citrate

Oxidase Test: Principal

Oxidase ReagentCytochrome Oxidase

Indophenol

Play role in aerobic respirationPseudomonasVibrio

Alternative substrate for Cytochrome

Tetramethyl-P-Pheneylenediamine

Colorless

Purple color

Oxidize the reagent from colorless to purple color

Negative Positive

Method: hold a piece of the oxidase test paper with forceps and touch onto

an area of heavy growth Use platinum loop (not used nichrome) or wood stickResults Color change to purple within:

10 seconds = positive 10 - 60 seconds = delayed positive >60 seconds = negative

Oxidation/Fermentation (O/F) Test• Principle :

– To determine the ability of bacteria to breakdown glucose oxidative or fermentative

– O/F medium (Hugh and Leifson Medium) is formulated to detect weak acids produced from saccharolytic M.O.

– O/F medium contains• Sugar (glucose 1%)• Low percentage of Agar and Peptone• pH indicator (Bromothymol blue)

– Alkaline Blue– Neutral Green– Acidic Yellow

• O/F medium differs from carbohydrate fermentation medium

to be more sensitive to detect the small amount of weak

acids produced by M.O.

• O/F medium is more sensitive due to:

– Higher % of glucose to increase amount of acid produced

– Lower % of peptone to reduce formation of alkaline

amines which neutralize weak acids formed

– Lower % of agar making the medium semisolid to

facilitate diffusion of acid throughout the medium

• Procedure

• Each organism is inoculated into two tubes of glucose O/F medium

• Inoculation is carried out as a stab to within 1 cm of the bottom of the tube

• One of which is covered with mineral oil to exclude oxygen Incubate at 37°C for 24 hours.

O/F Test: Results

Non-Saccharolytic O-/FAlcaligenes faecalis

Open & covered remain green

Oxidative O+/F-Pseudomonas

Open turns yellow

Fermentative O+/F+Enterobacteriaceae

Both turn yellow

Reaction 1 Reaction 3Reaction 2

There are three types of reactions possible

Gelatin Liquefaction Test: PrincipleCertain bacteria are capable of producing a proteolytic exoenzyme called

gelatinaseGelatinase hydrolyze the protein (solid) to amino acids (liquid)At temperature below 25°C, gelatin will remain a gel, but if the

temperature rises about 25°C, the gelatin will be liquid.Gelatin hydrolysis has been correlated with pathogenicity of some

microorganismsPathogenic bacteria may breakdown tissue & spread to adjacent tissues

Nutrient gelatinProtein/Polypeptides

Solid

Gelatinase

Incubation at 37/overnight

Nutrient gelatinAmino acids

Liquid at > 25 C

Gelatinase hydrolyze the protein to aminoacids

Pseudomonas

Gelatinase Test: Procedure

Nutrient gelatin

Stab M.O.

Incubate at 37 C overnight

If tube remains solidNo change

-ve

E. coli

If tube liquefied at > 25 C

+ve

Ps. aeruginosa

Gelatin Liquifaction Test• Method

– Stab a nutrient gelatin tube with

inoculums of the tested organism

– Inoculated nutrient gelatin tube is

incubated at 37°C for 24 h

• Result – If a tube of gelatin liquefy indicates

positive test (Ps. aeruginosa)– If a tube of gelatin remains solid

indicates negative test (E. coli)

Positive test

Negative test

Nitrate Reductase Test• Principle

– To determine the ability of an organism to reduce nitrate to nitrites or free nitrogen gas

• Method – Inoculate a nitrate broth with tested M.O.– incubate for 24 hrs at 37°C.– Add 1 ml of sulphanilic acid and 1 ml of -naphtylamine to nitrate

broth tube • Result

– The production of a red color occurs in the presence of nitrite indicates the ability of the organism to reduce nitrate to nitrite.

– To broths showing a negative reaction add a few particles of zinc.– The appearance of a red color indicates that nitrate is still present

and hence has not been reduced by the organism. – If the solution does not change color the organism has reduced the

nitrate through nitrite to nitrogen gas.

Nitrate Reductase Test: Principal

Nitrate(NO3)

Nitrate reductaseNitrite(NO2)

α-naphthylamineSulfanilic acid

Red diazonium salt

Further reductionNitrogen gas

N2

Nitrate reductase

Nitrate Reductase Test: Procedure

Nitrate broth

M.O. 1m Sulfanilic acid

1m -naphthylamine

Red colorPositive

No red color

ZnIncubate at 37oC for 24 hrs

Red colorNegative

No red colorPositive

Nitrate Reductase Test: Results

Red color after addition of sulfanilic acid & -naphtylamine

Reduction of Nitrate to

nitrite

Red color after addition of zinc dust

-ve reductionNitrate

unreduced

No red color after addition of zinc dust

Nitrate reduced into nitrite and

further reduction to Nitrogen

Practical Work☺Gram stain

☺Growth on Cetrimide agar

☺Oxidase test

☺O/F test

☺Nitrate reductase test

☺Gelatinase test

☺Citrate Utilization Test

☺(See under Enterobacteriacea)

Vibrionaceae• General characteristics

– Gram negative, curved, comma shaped bacilli

– Motile by single polar flagella– Non spore forming– Non capsulated– Most vibrios have relatively simple

growth factor requirements and grow well in alkaline pH

– Facultative anaerobes– Fermentative i.e. O+/F+– Oxidase and catalase positive

• Natural inhabitants – Aquatic environment

Gram stain of Vibrio cholerae

Species of VibrioVibrios

V. parahaemolyticu

s

Vibrio choleraeCause Cholera

O1 V. cholorae

Allied vibriosSaprophytic

Species of Vibrio• V. cholerae

– V. cholerae divided serologically into 6 groups based on somatic O-antigens

– V. cholerae O1 and O139 are the most important agents that cause cholera

– V. El-Tor is O1 serotype that cause disease similar to cholera but milder

• V. parahaemolyticus – V. parahaemolyticus is the cause of acute gastroenteritis

following ingestion of contaminated sea-food such as raw fish

• Both V. cholerae & V. parahaemolyticus produce diarrhea, but in ways those are entirely different. – V. parahaemolyticus is an invasive organism affecting the

colon– V. cholerae is noninvasive, affecting the small intestine

through secretion of an enterotoxin.

• Allied Vibrios are a large group of organisms; some of them are saprophytic while others cause disease in animals

Vibrio cholerae• V. cholerae is the causative agent of

cholera

• Cholera is toxin mediated, a severe

diarrheal disease caused by V. cholerae O1

& 139 serotype and others

• Cholera is endemic in southern Asia (India,

Pakistan, and Bangladesh), Latin America.

• Transmission is by contaminated water or

food through oral-fecal routes.

• Incubation period of the disease is 1-4 days.

Pathogenesis• V. cholerae multiply in the small

intestine and cause the same disease as ETEC, but more severe

• V. cholerae attach to the intestinal mucosa without invading the blood

• V. cholerae secretes an enterotoxin (cholargen)that binds to a specific receptor on the intestinal mucosal cell

• The toxin stimulates the activity of cAMP, resulting in active secretion of chloride and secondary loss of Na and H2O

TCBS medium is selective because

Also, contains bile salts

High conc. of thiosulfate & citrate & strong alkalinity of this medium (pH9)

TCBS medium is differential because

It contains sucrose

It contains bromothymol blue

Some species ferment sucrose & others not ferment Sucrose fermenting Vibrio spp (V. cholerae) appears as yellow colonies

Sucrose non fermenting Vibrio spp (V. parahemolyticus) appears as blue to green colonies

Alkaline pH: blueNeutral pH: greenAcidic pH: yellow

Sucrose fermentation on TCBS is the gold standard in its identification

kills most intestinal commensals

Identification of V. choleraeGrowth on TCBSPrinciple

Identification of Vibrio Differentiation between SF & NSF by

Growth on TCBS• Method:– TCBS agar is inoculated with tested organism

recovered from alkaline peptone water using streak plate technique

– Incubate the plate in incubator at 37 C/24 hrs • Results:

– SF organism appears as yellow colonies (V. cholerae)

– NSF organism appears as blue to green colonies (V. parahaemolyticus)

Flame & Cool

Flame & Cool

Flame & Cool

1 23

45

Diagnosis of V. cholerae

• Gram stain– Any sucrose fermenting colonies were

subjected to Gram stain and oxidase test– Gram negative short rods, comma shaped,

motile• Biochemical reactions:

–Oxidase positive–O+/F+–Cholera red reaction

• M.O. is inoculated on nitrate peptone water and incubated at 37C overnight

• V. cholorae produces indole and reduce nitrate into nitrite

• Upon addition of sulfuric acid develops a red color of nitrosoindole

• Serology:–Diagnosis can be confirmed as well as serotyping done by agglutination with specific antisera (O1, O139 antisera)

Difference between O1 V. choleae

Vibrio El-Tor

V. cholerae

Hemolytic Non hemolytic

Hemolysis

Positive Negative Voges-Prosakaue

r

Resistant Sensitive Polymyxin B

resistance

Applied MicrobiologyFood Microbiology

Water Milk

Most important water contaminants:

Bacteriological Examination of water

Escherecia coli Enterococcus faecalis Clostridium welchii

Bacteriological Examination of water

Media used in bacteriological examination of water:

for Escherecia coli use MacConkey.for Enterococcus faecalis use Glucose azide broth.

for Clostridium welchii use reinforced anaerobic medium.

Methods used in bacteriological examination of water:

Membrane Filtration Method. Determination of Most Probable Number

(MPN) by dilution method.

Bacteriological Examination of water

Membrane Filtration Method Using Millipore Filter Apparatus

MacConkey’s agar

Determination of MPN of Coliforms by Dilution Method

Water Sample

50 mlDSMB

5 x 10 mlDSMB

5 x 5 mlSSMB

50 ml watersample 10 ml water

sample1 ml water

sample

• Results• Positive tubes are showing production of

acid or gas.• Acid production is indicated by change

color of tube from purple to yellow.• Gas production is detected in the

Durham’s tube in the 1st bottle.

Determination of MPN of Coliforms by Dilution Method

Determination of MPN of Coliforms by Dilution Method

Purple Yellow

Gas

• Determine no. of coliforms per 100 ml water sample (MPN) using the standard probability table.

Results:

Determination of MPN of Coliforms by Dilution Method

Results:

1 3 2MPN = 14

i.e: No. of coliform bacilli per 100 ml water sample is 14 cells.