practical medical microbiology pht313 by dr. mohamed al-agamy assistant professor of microbiology...
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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 +
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.