Practical Medical Microbiology
PHT313
By
Dr. Mohamed Al-Agamy
Assistant Professor of Microbiology
Department of Pharmaceutics & Microbiology
College of Pharmacy
King Saud University
2010/1431
Classification of Bacteria
Bacteria
Gram-Positive
Cocci
Acid fast bacteria
Gram-negative
Bacilli
Staphylococci
Streptococci
Spore forming
Aerobic
Bacillus species
Anaerobic
Clostridium species
Non-spore forming
Other bacteria e.g. Mycoplasma,
Spirochetes
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
• Characteristics of S. aureus
– Gram Positive Cocci
– Production of coagulase
– Grape-like
– Production of phosphatase
– Non Motile
– Non Spore Forming
– Non Fastidious
– Production of DNase
– Ferment Mannitol
– Facultative Anaerobes
– Gelatin liquefied
– Fermentative (O+/F+)
– β-hemolysis on blood agar
– Halotolerant
– Acidification & clotting of
– Catalase positive
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
porcelinwhite
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.
IV- Biochemical tests
1. Catalase test
• 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
Catalase
H2O + O2 (gas, ↑)
H2O2
Staphylococci
• The test should not be done on blood agar because blood itself will
produce bubbles
Catalase test
Positive
Negative
Microcococcaceae
Staphylococci
Streptococcaceae
Streptococci
2- Coagulase Test
Principle:
• This test is used to differentiate between S. aureus (CPS) &
other Staphylococcus species (CNS)
• This test is done by tube method or slide method
Fibrinogen
(Plasma)
Coagulase
Fibrin
(Clot)
Coagulase test
Coagulase Positive
Staphylococus aureus
Coagulase-Negative
S. epidermidis & S. saprophyticus
• 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
•
•
•
S. aureus
Procedure:
S. epidermidis
– 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
3- Deoxyribonuclease (DNAase) test
DNase test
Positive
Staphylococus aureus
•
•
Negative
S. epidermidis & S. saprophyticus
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 test
Sensitive
Resistant
S. aureus, S. epidermidis
S. saprophyticus
• 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 species
S. 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
-hemolysis
-hemolysis
– 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
Serology: Lancefield Classification
Streptococci
Lanciefield classification
Group A
S. pyogenes
Group B
S. agalactiae
Group C
S. equisimitis
Group D
Enterococcus
• 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
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 resistant
S. viridans
Optochin susceptible
S. pneumoniae
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
whereas S. viridans insoluble
in
bile
Differentiation between -hemolytic streptococci
Hemolysis
Bacitracin
sensitivity
CAMP test
S. pyogenes

Susceptible
Negative
S. agalactiae

Resistant
Positive
Differentiation between -hemolytic streptococci
Hemolysis Optochin
Bile
sensitivity solubility
Inulin
Fermentation
S. pneumoniae

Sensitive
(≥ 14 mm)
Soluble
Not ferment
Viridans strep

Resistant
(≤13 mm)
Insoluble
Ferment
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
Bacteria
Gram-Positive
Cocci
Acid fast bacteria
Gram-negative
Bacilli
Staphylococci
Streptococci
Spore forming
Aerobic
Bacillus species
Anaerobic
Clostridium species
Non-spore forming
Corynebacterium
Listeria
Other bacteria e.g. Mycoplasma,
Spirochetes
Aerobic Spore Forming Bacillus spp
Bacillus species
Pathogenic
Bacillus anthracis
Bacillus cereus
Non-pathogenic
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.
Motility
B. anthracis
Non-motile
B. cereus
motile
Capsule
Hemolysis
capsulated
Non-hemolytic
Non-encapsulated
β-hemolytic
Resistance to Penicillin
S
R (produce β-lactamase)
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 hemolytic
B. cereus colonies are β-hemolytic
B. 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.
Biochemical tests
1- Catalase Test
Broth Cultutre & H2O2 on the slide
H2O2 added on culture grown on nutrient agar
• All Bacillus species are catalase positive
• Remember: staphylococci are catalase positive
2- Starch Hydrolysis (Amylase Activity)
• Principle
– Starch + Iodine
– Glucose + Iodine
•
blue color
No reaction
Nutrient Agar containing 1% Starch + M.O
Amylase
Glucose
Iodine
Appearance of colorless zone around the growth
• 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
Spore Stain Procedure
1. 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
Tetanus
Cl. tetanii
Gas gangrene
Sacchrolytic
Cl. perfringens
Mixed
Cl. histolyticus
Botulism
Cl. botulinum
Proteolytic
Cl. sporogenes
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 gangrene
Saccharolytic
organisms
Cl. perfringens
Ferment
carbohydrates
Acid and gas are
produced
Proteolytic organisms
Cl. sporogenes
Digest proteins with
blackening bad smell
production
Mixed saccharolytic
& proteolytic
Cl. 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. perfringens
Causing
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 Toxins
A
B
C
D
E

+
+
+
+
+
Components of Toxins
 Epsilon Iota Enterotoxin
+
+
+
+
+
+
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 & sporulated
The spore is large, oval, central to sub-terminal & non
bulging (non swelling)
Spores are rarely observed
 Culture: Anaerobically at 37C
On Robertson's cooked meat medium → blackening of
meat will observed with the production of H2S and NH3
On 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
Contains
Skimmed Milk
(Without Fat)
Lactose
Sugar
Casein
Protein
Litmus indicator
Acid Base and Redox indicator
Reaction on Litmus Milk
1- Acidic Reaction
Fermentation
Lactose
Acid
Litmus Indicator
Pink Color
(Milk Sugar)
2- Basic Reaction
Digestion
Casein
(Milk Protein)
Litmus Indicator
Alkaline amines
Blue Color
Reaction on Litmus Milk
Stormy Clot Formation
Lactose
Milk Sugar
Fermentation
Acid + Gas
Stormy Clot
Coagulation
Casein
Milk Protein
Clot
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
Procedure of Nagler Reaction
Positive 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
• 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)
Anaerobic Jar
Candle Jar
• 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
Corynebacterium
Pathogenic
C. diphtheriae
Causative agent of
diphtheria
Commensal "Diphtheriods"
C. hofmannii, C. xerosisacne
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
Laboratory
Diagnosis
Diagnosis of
diphtheria
Clinical Diagnosis
Case
Symptomatic
patient
Carrier
Asymptomatic
patient
Clinical 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
Loeffler’s seum
Gram stain of
C. diphtheriae
C. diphtheriae on BTA
Biochemical Reaction
Catalase 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 hrs
Glucose 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
Glucose Maltose
+ve
Glucose
+ve
Maltose
C. xerosis
+ve
+ve
+ve
Sucrose
-ve
Sucrose
C. diphtheriae
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
Lines of precipitations
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
Inoculated M.O.
Positive Elek’s Test
Bacteria
Cocci
Acid fast bacteria
Gram-positive
Gram-negative
Bacilli
Neiserria gonorrhoeae
Oxidase negtaive
Neisseria meningitidisi
Enterobactericeae
Oxidase positive
Pseudomonadaceae
Vibrionaceae
Other bacteria e.g. Mycoplasma,
Spirochetes
Gram negative bacteria
O/F Test
Oxidative (O+/F-)
Pseudomonas
Fermentative (O+/F+)
Enterobacteriacae
Vibrioionaceae
Gram negative bacteria
Oxidase Test
Oxidase positive
Pseudomonas
Vibrioionaceae
Oxidase negative
Enterobacteriacae
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 Enterobacteriaceae
Enterobacteriaceae
Lactose fermenters
E. coli, Citrobacter,
Klebsiella, Enterobacter
Non-lactose fermenter
Salmonell, Shigella
Proteus, Yersinia
There are several selective and differential media used to isolate
distinguishes between LF & LNF
The most important media are:
MacConkey agar
Eosin Methylene Blue (EMB) agar
Salmonella Shigella (SS) agar
In 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 agar is selective & differential medium for Enterobacteriaceae
Contains
Bile salts
Crystal violet
Inhibit growth of G+ve bacteria
Cause of selectivity
Lactose feremnters
Pink colonies
Lactose
Cause of differential
Neutral red
pH indicator
Acidic: Pink
Lactose non feremnters
colorless colonies
Classification of Enterobacteriaceae according to lactose
fermentation (growth on MacConkey Agar)
Lactose Fermenters
Acid
Lactose Non-Fermenters
No acid
Neutral red
Pink colonies
Escherichia coli
Klebsiella spp
Enterobacter spp
Citrobacter spp
Colorless colonies
Salmonella spp
Shigella spp
Proteus spp
Yersinina spp
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 Agar
Lactose non ferementers
Salmonella, Shigella, Proteus
Lactose ferementers
E. 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 Enterobacteriaceae
by 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
Reaction on TSI
Result
Butt
color
Slant
color
H2 S
Red
Red
- ve
Alk/Alk/(No action on sugars)
- ve
A/Alk/(Glucose fermented
without H2S)
A/Alk/+
(Glucose fermented
with H2S)
Yellow
Yellow
Yellow
Red
Red
+ ve
black in
butt
Yellow
- ve
A/A/(All sugars are
fermented)
Non fermenter
e.g.
Pseudomonas
LNF
e.g. Shigella
LNF
e.g. Salmonella
& Proteus
LF
e.g. E. coli,
Klebsiella,
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 Enterobacteriaceae
Biochemical 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
Enterobacteriaceae.
the
various
members
of
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.
Tryptophane Tryptophanase
amino acids
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 IMViC
Indole test
Negative
Methyl Red test
Positive
Citrate utilization test
Positive
Negative
Voges-Proskauer test
IMViC test
Methyl Red-Voges Proskauer (MR-VP) Tests
Glucose
(MRVP medium)
Acidic pathway
(MR test)
Neutral pathway
(VP test)
Mixed Acids,
pH less than 4.4
Acetylmethylcarbinol
(Acetoin)
Acidity is detectd by adding
Methyl red indicator
Acetoin is detected by
adding Barrit’s reagent
 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
Principle:
Citrate
Citrate Utilization Test
Pyruvate
CO2 + Na + H2O
Na2CO3
Alkaline,↑pH
Simmone’s Citrate media
Contains Citrate as a sole of C source
The color of medium is green
 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
Positive
Negative
Klebsiella, Enterobacter E. coli
Urease Test
 Principal




Urea
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
Urease
H2O
CO2 + NH3
NH4 OH
↑ in pH
Phenol Red
Method
 Streak a urea agar tube with the organism
 incubate at 37°C for 24 h
Pink
Positive test
•
•
•
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
Summary of morphology, cultural characteristics, and biochemical
reactions of Enterobacteriaceae
Gram Oxidase
stain
Nitrate
reductase
O/F
MacCo
nkey
SS
EMB
E. coli
-ve
rod
-ve
+ve
O+/F+
LF
LF
Metallic
sheen
Citrobacter
-ve
rods
-ve
+ve
O+/F+
LF
LF
Dark
Klebsiella
-ve
rods
-ve
+ve
O+/F+
LF
LF
Dark
Enterobacter
-ve
rods
-ve
+ve
O+/F+
LF
LF
Dark
Salmonella
-ve
rods
-ve
+ve
O+/F+
NLF
NLF/
H2S
Colorles
s
Shigella
-ve
rods
-ve
+ve
O+/F+
NLF
NLF
Colorless
Proteus
-ve
rods
-ve
+ve
O+/F+
NLF
NLF/
H2S
Colorles
s
Summary of morphology, cultural characteristics, and biochemical
reactions of Enterobacteriaceae
TSI
Indole
MR
VP
Citrate
Urease
Motility
E. coli
A/A/-
+ve
+ve
-ve
-ve
-ve
Motile
Citrobacter
A/A/-
+ve
+ve
-ve
+ve
-ve
Motile
Klebsiella
A/A/-
-ve
-ve
+ve
+ve
+ve
Non
motile
Enterobacter A/A/-
-ve
-ve
+ve
+ve
+ve
Motile
Salmonella
A/Alk/
+
-ve
+ve
-ve
+ve
-ve
Motile
Shigella
A/Alk/- -ve
+ve
-ve
-ve
-ve
Non
motile
Proteus
A/Alk/
+
+ve
-ve
+ve
+ve
Motile
Swarwing
-ve
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+)
Enterobacteriacae
Vibrioionaceae
Gram negative bacteria
Oxidase Test
Oxidase positive
Pseudomonas
Vibrioionaceae
Oxidase negative
Enterobacteriacae
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
Ps. aeruginosa on cetrimide agar
Gram Stain of Pseudomonas
Ps. aeruginosa on Nutrient agar
Biochemical Reactions
• Oxidase positive
• Breakdown glucose oxdatively
• Nitrate Reductase positive (further reduction to N2)
• Gelatinase positive
• Utilize Citrate
Oxidase Test: Principal
Alternative substrate
for Cytochrome
Oxidize the reagent from
colorless to purple color
Oxidase Reagent
Indophenol
Cytochrome Oxidase
Tetramethyl-PPheneylenediamine
Colorless
Pseudomonas
Vibrio
Purple color
Play role in aerobic respiration
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 stick
Results
 Color change to purple within:
 10 seconds = positive
 10 - 60 seconds = delayed positive
 >60 seconds = negative
Positive
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
There are three types of reactions possible
Reaction 1
Non-Saccharolytic O-/F
Alcaligenes faecalis
Open & covered remain green
Reaction 2
Oxidative O+/FPseudomonas
Open turns yellow
Reaction 3
Fermentative O+/F+
Enterobacteriaceae
Both turn yellow
Gelatin Liquefaction Test:
Principle
 Certain bacteria are capable of producing a proteolytic exoenzyme called
gelatinase
 Gelatinase 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
microorganisms
 Pathogenic bacteria may breakdown tissue & spread to adjacent tissues
Pseudomonas
Gelatinase
Incubation at 37/overnight
Nutrient gelatin
Protein/Polypeptides
Solid
Gelatinase hydrolyze the
protein to aminoacids
Nutrient gelatin
Amino acids
Liquid at > 25 C
Gelatinase Test: Procedure
Stab M.O.
If tube remains solid
No change
-ve
E. coli
Incubate at 37 C overnight
If tube liquefied at > 25 C
+ve
Nutrient gelatin
Ps. aeruginosa
Gelatin Liquifaction Test
• Method
– Stab a nutrient gelatin tube with
Positive test
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)
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 reductase
Nitrate
(NO3)
Sulfanilic acid
Nitrite
(NO2)
Nitrogen gas
N2
α-naphthylamine
Red diazonium salt
Nitrate Reductase Test: Procedure
Red color
Positive
Nitrate broth
M.O.
1m Sulfanilic acid
Red color
Negative
1m -naphthylamine
Incubate at
37oC for 24 hrs
Zn
No red color
No red color
Positive
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 reduction
Nitrate
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
–
Aquatic environment
• Natural inhabitants
Gram stain of Vibrio cholerae
Species of Vibrio
Vibrios
V. parahaemolyticus
Vibrio cholerae
Cause Cholera
O1 V.
cholorae
Classical type
V. cholorae
Allied vibrios
Saprophytic
Non-O1 V.
cholorae
Non-classical type
V. El-tor
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 seafood 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
Principle
Identification of V. cholerae
Growth on TCBS
 TCBS medium is selective because
 High conc. of thiosulfate & citrate & strong alkalinity of this medium (pH9)
 Also, contains bile salts
kills most intestinal commensals
 TCBS medium is differential because
 It contains sucrose
 It contains bromothymol blue
Alkaline pH: blue
Neutral pH: green
Acidic pH: yellow
 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
 Sucrose fermentation on TCBS is the gold standard in its identification
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
2
1
3
5
4
Flame & Cool
Flame & Cool
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
V. cholerae
Vibrio El-Tor
Hemolysis
Non hemolytic
Hemolytic
VogesProsakauer
Negative
Positive
Polymyxin B
resistance
Sensitive
Resistant
Applied Microbiology
Food Microbiology


Water
Milk
Bacteriological Examination of
water
Most important water contaminants:
 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.
Bacteriological Examination of
water
 Methods used in bacteriological
examination of water:


Membrane Filtration Method.
Determination of Most Probable Number
(MPN) by dilution method.
Membrane Filtration Method
Using Millipore Filter Apparatus
MacConkey’s agar
Determination of MPN of
Coliforms by Dilution Method
50 ml water
sample
10 ml water
sample
1 ml water
sample
Water Sample
50 ml
DSMB
5 x 10 ml
DSMB
5 x 5 ml
SSMB
Determination of MPN of
Coliforms by Dilution Method
• 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
Results:
Gas
Purple
Yellow
• Determine no. of coliforms per 100 ml water
sample (MPN) using the standard probability table.
Determination of MPN of
Coliforms by Dilution Method
Results:
1
3
MPN = 14
2
i.e: No. of coliform bacilli per 100 ml water sample is 14 cells.