Practical) )Pharmaceutical Microbiology

PHT313

By

Elmutasim O. IbnoufLecturer of Microbiology

Department of Pharmaceutics & Microbiology

College of Pharmacy

Salman bin Abdulaziz University

Medical Microbiology

Infectious

Microorganisms

Bacteria

"Bacteriology"

Fungi

"Mycology"

Virus

"Virology"

Classification of BacteriaBacteria

Gram-Positive Gram-negative

Cocci Bacilli

Acid fast bacteriaOther bacteria e.g. Mycoplasma,

Spirochetes

Staphylococci

Streptococci

Spore forming

Non-spore forming

AerobicBacillus species

AnaerobicClostridium species

Staphylococci

Major pathogenic species

• Three important species of staphyloccoci have medical

importance

– S. aureus is the major pathogenic staphylococci & commensally

found in nose (nares)

– S. epidermidis is non pathogenic & common commensals in

nares & skin

– S. saprophyticus causes UTI in sexually active women in the

community and not in the hospital & occasionally commensally

found on the skin

General characteristics

• General characteristics

– Gram Positive Cocci

– Grape-like

– Non Motile

– Non Spore Forming

– Non Capsulated

– Non Fastidious

– Facultative Anaerobes

– Fermentative

– 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

Laboratory Diagnosis

• Specimen:

– Pus, Urine, Stool, Blood, CSF

• Gram Stain:

– Gram Positive cocci, arranged in cluster

• Culture:

– Blood agar (Non-Selective Media)

• Colonies of Coagulase Positive Staphylococci (CPS) are golden yellow &

-hemolytic

• Colonies of Coagulase Negative Staphylococci (CNS) are non-pigmented

& non-hemolytic

Laboratory Diagnosis• Mannitol Salt Agar (MSA)

– MSA is selective and differential medium for staphylococci

– It contains NaCl (7.5%), Mannitol, & Phenol Red

– The cause of selectivity due to presence of high salt concentration

– The cause of differential because contains mannitol (sugar) and phenol red (pH indicators turns yellow in acidic pH and turns red in alkaline pH).

Mannitol fermentation on MSA

Mannitol fermented

Yellow colonies:

S. aureus

Mannitol nonfermenter

Red colonies:

S. epidermidis& S. saprophyticus

• Catalase test is used to distinguished between staphylococci

(positive) from streptococci (negative)

• Flood the culture with drops of 3% H2O2

• Catalase-positive cultures bubble at once

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

produce bubbles

Catalase test

PositiveMicrocococcaceae

Staphylococci

NegativeStreptococcaceae

Streptococci

H2O2 H2O + O2 (gas, ↑)

Staphylococci

Catalase

Catalase test

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

Coagulase test

Coagulase Positive

Staphylococus aureus

Coagulase-Negative

S. epidermidis & S. saprophyticus

Fibrinogen

(Plasma)

CoagulaseFibrin

(Clot)

Coagulase Test• 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. aureus S. epidermidis

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 with tested organism in circular motion (Spot)

– Incubate at 37C for 24-48h

– 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.

DNase test

Positive

Staphylococus aureus

Negative

S. epidermidis & S. saprophyticus

Novobiocin Sensitivity

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

• Inoculated overnight culture on Mueller-Hinton agar

• Add novobiocin disk on inoculated plate

• Incubate at 370C overnight

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

Novobiocin test

SensitiveS. aureus, S. epidermidis

ResistantS. saprophyticus

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

• Coagulase Test by Tube and Slide Method

Streptococci

• General Characteristics of Streptococci

– Gram positive cocci

– 1µm in diameter

– Chains or pairs

– Usually capsulated

– Non motile

– Non spore forming

– Facultative anaerobes

– Fastidious

– Fermentative

– 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

Streptococci

Group A

S. pyogenes

Group B

S. agalactiae

Group C

S. equisimitis

Group D

Enterococcus

Lanciefield

classification

Other groups

(E-U)

Group A streptococci

• Include only S. pyogenes

• -hemolytic organism

• Group A streptococcal infections affect all ages peak

incidence at 5-15 years of age

• 90% of cases of pharyngitis

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:

– Bacitracin test is used for presumptive

identification of group 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:

– BAP 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 susceptible

S. pneumoniae

Optochin resistant

S. 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 ten 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 to the

culture

– 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 testBacitracin

sensitivity

Hemolysis

NegativeSusceptibleS. pyogenes

PositiveResistantS. agalactiae

Inulin

Fermentation

Bile

solubility

Optochin

sensitivity

Hemolysis

Not fermentSolubleSensitive

(≥ 14 mm)

S. pneumoniae

FermentInsolubleResistant

(≤13 mm)

Viridans strep

Differentiation between -hemolytic streptococci

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 sppClassification of Bacteria

Bacteria

Gram-Positive Gram-negative

Cocci Bacilli

Acid fast bacteriaOther bacteria e.g. Mycoplasma,

Spirochetes

Staphylococci

Streptococci

Spore forming

Non-spore forming

AerobicBacillus species

AnaerobicClostridium species

Corynebacterium

Listeria

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 Habitates

• It is found in soil habitats

Bacillus cereus B. cereus is a normal inhabitant of soil

Also isolated from food such as grains and spices

It is different from of B. anthracis in that it is motile, non-encapsulated,

hemolytic and resistant to penicillin

B. cereusB. anthracis

motileNon-motileMotility

Non-encapsulatedcapsulatedCapsule

β-hemolyticNon-hemolyticHemolysis

R (produce β-lactamase)SResistance to Penicillin

B. cereus causes food poisoning

Food poisoning occurs when the B. cereus deposits its spores in food

Bacteria then germinates in food in the food & begin releasing their exotoxins

Spores are not killed during cooking & germinate in the unrefrigerated food

To inactivate the spores, the cooked food must be exposed to high temperature

and/or refrigeration

Identification of Bacillus Spp.• Cultural Characteristics

• Grow on nutrient Agar• On ordinary medium

• Grow aerobically at 37C with characteristic mucoid or smooth colonies, which

indicates the pathogensity 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 medium-large gray, irregular with swirling projection and

non hemolytic

B. cereus colonies are large, feathery and β-hemolytic

B. subtilis colonies are large, flat, dull, with ground glass appearance , may be

pigmented (pink, yellow, orange or brown) and may be β-hemolytic

• Special media used for isolation• Polymyxin-Lyzozyme-EDTA-Thallous acetate(PLET) medium

• Used for selection and isolation of B. anthracis from contaminated specimens

• Bicarbonate agar• Used to induce B. anthracis capsule

Cultural Characteristics

B. cereus B. anthracis

Nutrient Agar

Blood Agar

Identification of Bacillus anthracis

• Morphology

– Microscopical

• Stain

– Gram Stain

» Gram positive bacilli

» Found in chains

» Non motile

» Capsulated inside the host

» Sporulated outside the host

» Spore is central, oval and non-bulging

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

Identification of Bacillus Spp.

• Spore Stain

Bacillus spores are oval & central

By spore staining technique (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 tests

1- 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

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

– This group of bacteria is responsible for famous diseases such as gas gangrene, tetanus, botulism & pseudo-membranous colitis

– Their pathogenesis by producing potent exotoxins & enzymes which attack the neurons pathways

– Rapid diagnosis is crucial or patient will die

Clostridium causing

Tetanus

Cl. tetaniiGas gangrene

Botulism

Cl. botulinumAntibiotic associated diarrhea

Cl. difficile

Sacchrolytic

Cl. perfringens

Proteolytic

Cl. sporogenes

Mixed

Cl. histolyticus

Clostridium tetani causing tetanus

• General charcteristics 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 Causing Gas Gangrene

Clostridia causing gas gangrene

Saccharolytic organisms Cl. perfringens, Cl. septicum

Ferment carbohydrates

Acid and gas are produced

Proteolytic organisms Cl. sporogenes

Digest 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

Diseases caused by Clostridium perfringens

Cl. perfringens

Causing

Gas gangrene Food poisoning

(Enterotoxin)

Laboratory Diagnosis of gas gangarene

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 37COn 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

• Cl. perfringnes characterized by:

Fermentation of many sugars with acid & gas

Saccharolytic organism

Acidification litmus milk with stormy clot

production

Nagler reaction is positive

Reaction on Litmus Milk

Litmus Milk

Skimmed Milk

(Without Fat)

Litmus indicator

Acid Base and Redox indicatorLactose

SugarCasein

Protein

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

Casein

Milk Protein

Coagulation

Gas

Clot

Stormy Clot

Milk Sugar

Lactose Acid +

Reaction on Litmus Milk

Nagler’s Reaction

• This test is done to detect the lecithinase activity

– The M.O is inoculated on the medium containing

human serum or egg yolk (contains lecithin)

– The plate is incubated anaerobically at 37 C for

24 h

– Colonies of Cl. perfringens are surrounded by

zones of turbidity due to lecithinase activity and

the effect is specifically inhibited if Cl. perfringensantiserum containing antitoxin is present on the

medium

Nagler Reaction

Positive Nagler ReactionProcedure of Nagler Reaction

Anaerobic Cultivation

• Anaerobic Jar

• Removal of oxygen & replacing it with inert gas

• The most frequently used system for creating an anaerobic

atmosphere

• 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 JarCandle 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)

Anaerobic Cultivation• 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

Species of Corynebacterium

Corynebacterium

PathogenicC. diphtheriae

Commensal "Diphtheriods"C. hofmannii, C. xerosis, C. acne

Causative agent of diphtheria Normal flora of RT, urethra, vagina, Skin

Play role in acne vulgaris

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; not

considered part of normal flora

– Isolation from health human is not common

– C. xerosis (Diphtheriods) is normal flora of human conjuctiva, skin & nasopharynx

Diagnosis of diphtheria

Clinical Diagnosis Laboratory Diagnosis

To confirm the clinical

manifestation

Specific treatment must be never

delayed for laboratory results

Child with sore throat & fever

Pseudo-membrane more darker &

thicker than of strept membrane

Diagnosis of Diphtheria

Diagnosis of case Diagnosis of carrier

Asymptomatic patient Symptomatic patient

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 (3 parts of serum +1 part of glucose broth) and/or

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

– Modified Tinsdale’s agar

• 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 much more readily than other respiratory

pathogens

• Production of the metachromatic granules within the cells

– The colonies of C. diphtheriae are small, granular, grey, smooth, and creamy with

irregular edges

Laboratory diagnosis

• 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 disease is associated with the gravis biotype

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

• Colonies of mitis biotype are small, hemolytic and black

• 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 Reaction

• All Corynebacterium species are catalase

positive (Also, Staphylococcus and

Bacillus species are catalase positive)

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 but not sucrose with production of acid only

• 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

Carbohydrate Fermentation Test

• 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 SucroseGlucose Maltose Sucrose+ve +ve +ve +ve +ve -ve

Diagnosis of Carrier

I- Isolation of organismII- Detection of exotoxin

Test for toxigenicity

Swap from throat & nose

Inoculation on Loeffler’sOr BTA for 24 h/37 C Diphtheria like M.O.

Detection of exotoxin

I- In vivo II- In vitro

Two guinea pigs are used

One is used as

Test

The second is used

as ControlInjected with

diphtheria antitoxin

Both test and control injected with isolated MO

If both GP live If control live & test die

Diphthrioids or non-toxigenic C. diphtheriae

i.e. non pathogenic

C. diphtheriaei.e. produce exotoxin

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

Elek’S test

• Resuls:

• 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

How to test for susceptibility to diphtheria?

•Schick test

Diphtheria toxin produces marked local reaction when injected ID

A small dose of toxin injected ID in one forearm (Test)

Similar dose of heated toxin is injected ID in other forearm (Control)

Positive Schick test

Indicated by redness & swelling in control forearm

The reaction increases for several days & then slowly disappear

This means the person susceptible to diphtheria

Negative Schick test

No reaction appears in both forearms (Test & Control)

This means the person is immune

Pseudo-reactions

Reaction appears in both forearms & disappearing simultaneously after 2 days

Listeria

• L. monocytogenes is the most pathogenic species to human

• General Charcteristics

• This organism is

– Gram-positive short coccobacilli (0.5-2 by 0.4-.05 µm)

– Non-spore formers

– Facultative anaerobes

– Motile (tumbling motility at room temperature)

– Fermentative

– Beta-hemolytic on blood agar

– Catalase positive, nitrate reductase negative

– It grows under refrigeration temperatures

• Grow below 1ºC is slow

– It grows at pH values of between 4.4 and 9.6

Listeria monocytogenes• Habitats

– L. monocytogenes is widely distributed in nature

– It isolated from soil, water, vegetables, mammals, fish, & birds

– It may colonize human GIT

– 1-10% of humans may be intestinal carriers

• Mode of Transmission

– Usually by ingestion of contaminated food such as

• Meat (fermented raw-meat sausages, raw and cooked poultry, raw meats )

• Dairy products(raw milk, cheeses, ice cream)

• Unwashed raw vegetables

– L. monocytogenes is able to grow in a wide range of pH, as well as in cold temperatures

• Thus, refrigeration of contaminated foods permit the multiplication of the organism to an infectious dose

– Colonized mothers may pass organism onto fetus

– Portal of entry is from GIT to blood & from blood to menings

Diagnosis of Listeriosis

• Listeriosis can only be positively diagnosed by culturing the organism from blood, CSF, or stool (although the latter is difficult and of limited value).

• Specimen:– CSF (contain high number of neutrophils, a high protein level, a

low glucose level)

• Culture:– Listeria grow at low temperature

– Cold technique can be used to isolate Listeria selectively

– On 5% blood agar• Small, white, smooth, translucent, moist, β-hemolytic colonies

• Gram stain– Gram-positive bacilli, motile at 20 C etc