PHT 416 Lab 7

Steps

• Microscopic

Morphology

• Growth

• Biochemical Tests

Nutrient agar

Blood agar

Mannitol Salt Agar

MacConkey’s agar

StaphylococciStreptococci

NeisseriaCorynbacteriumClostridumBacillus

Enterobacteriaceae Pseudomonas.

Gm’s +ve

CocciStaphylococciStreptococci

BacilliCorynbacterium

ClostridumBacillus

Gm+ve Cocci

Staphylococci Streptococci

H2O2

Catalase enzymeH2o + O2

Air bubbles

Catalase test

Catalase –veCatalase +ve

• Gram positive cocci, arranged in grape like clusters, non-motile, non-spore forming.

Staphylococci

Staphylococci

Staph. saprophyticus Staph. aureus Staph. epidermidis

Coagulase Test

Staph. saprophyticus Staph. aureus Staph. epidermidisFibrinogen

Plasma

Coagulase enzymeFibrin

Visible Clot

(+ve) Formation of visible clot -ve

Staphylococci

Staph. aureus Staph. epidermidis

Staphylococci on Nutrient Agar

Golden Yellow colonies White colonies

Staphylococci

Staph. aureus Staph. epidermidis

Staphylococci on Blood Agar

Beta heamolysis(completehaemolysis)

Non hemolytic

Staphylococci

Staph. aureus Staph. epidermidis

Staphylococci on MSA

Yellow colonies Pink colonies

MSA is differential for S.aureus

Deoxyribonuclease (DNase) Test

Staphylococci

Staph. aureus Staph. epidermidis

DNADNase enzyme

NucleotidesInsoluble in acid soluble in acid

DNase activity is indicated by a clear zone around the growth after addition of Hcl

Clear zone around the growth while the rest of the plate appears cloudy

Cloudiness in all the plate

S.epidermidis

S.aureus

Gm+ve Cocci

Staphylococci Streptococci

H2O2

Catalase enzymeH2o + O2

Air bubbles

Catalase test

Catalase –veCatalase +ve

Gram positive cocci,arranged in chains or pairs, non-motile,

non-spore forming.

Streptococci

(Fastidious organism)

Complex nutritional requirements

Blood agar

Streptococci

β-hemolyticS. pyogenes

Non-hemolyticα-hemolyticS.pneumonia ,

viridans streptococci

Streptococci

Growth on Blood agar

α-Haemolysis β-Haemolysis

Optochin Test Bacitracin Test

S RS R

S.pneumoniaeS.pneumoniae Viridans Streptococci

CAMP+veS.agalactiae

S. agalactiaeS. agalactiae

Bile Solubility test CAMP test

1 1

2 2

S. pyogenesS. pyogenes

++veveS.pneumoniaeS.pneumoniae

-veViridans

Streptococci

Optochin Test

α-Haemolysis

Principle:S.pneumoniae is inhibited by less than 5µg/ml Optochin reagent giving a zone of inhibition more than 15 mm in diameter.

Positive test: any zone of inhibition around the disc.

SR

S.pneumoniaS.pneumoniaee Viridans

Streptococci

Streptococci

Growth on Blood agar

α-Haemolysis β-Haemolysis

Bacitracin Test

S RS R

S.pneumoniaeS.pneumoniae Viridans Streptococci

CAMP+veS.agalactiae

S. agalactiaeS. agalactiae

Bile Solubility test CAMP test

1 1

2 2

S. pyogenesS. pyogenes

++veveS.pneumoniaeS.pneumoniae

-veViridans

Streptococci

Bile Solubility testOptochin Test

α-Haemolysis

Bile Solubility testS.pneumoniae produces a self-lysing enzyme to depress the growth of old colonies. The presence of bile salt accelerate this process.

Principle:

Visible clearance

S.Pneumoniae

Remain turbid

Viridans Streptococci

Results

Streptococci

Growth on Blood agar

α-Haemolysis β-Haemolysis

Optochin Test Bacitracin Test

S RS R

S.pneumoniaeS.pneumoniae Viridans Streptococci

CAMP+veS.agalactiae

S. agalactiaeS. agalactiae

Bile Solubility test CAMP test

1 1

2 2

S. pyogenesS. pyogenes

++veveS.pneumoniaeS.pneumoniae

-veViridans

Streptococci

β-Haemolysis

Bacitracin TestBacitracin Test

Principle:A low conc. of Bacitracin (0.04 units) will selectively inhibit the growth of S.pyogenes giving a zone of inhibition around the disc

B

Positive test: any zone of inhibition around the disc.

BS RS. S.

pyogenespyogenesS. S.

agalactiaeagalactiae

Streptococci

Growth on Blood agar

α-Haemolysis β-Haemolysis

Optochin Test Bacitracin Test

S RS R

S.pneumoniaeS.pneumoniae Viridans Streptococci

CAMP+veS.agalactiae

S. agalactiaeS. agalactiae

Bile Solubility test CAMP test

1 1

2 2

S. pyogenesS. pyogenes

++veveS.pneumoniaeS.pneumoniae

-veViridans

Streptococci

β-Haemolysis

CAMP test

CAMP reaction: The hemolysis is enhanced in the vicinity of the ß -lysin producing S.aureus (Arrow head-sheaped zone of ß-hemolysis).

Group B streptococci (S.agalactiae.S.agalactiae. ) produce a factor that increases beta hemolysis of an S. aureus indicator strain.

CAMP+veS.agalactS.agalactiaeiae

CAMP-ve

Gm’s +ve

CocciStaphylococciStreptococci

BacilliCorynbacterium

ClostridumBacillus

Gram +ve Bacilli

Spore forming Non spore forming

AerobicAnaerobic

BacillusClostridium

CorynebacteriumListeriaLactobacilli

All Catalae +ve

Small, irregularly staining pleomorphic Gram-positive

rods with club-shaped swelled ends.

It may be straight or slightly curved, non-motile and non

spore-forming.

"Chinese letters"

C.diphtheriae

Corynebacterium

.Growth occurs on media containing blood or serum

-On blood tellurite medium (selective & differential medium) colonies appear grey to black.

-On Loeffler’s serum

Corynebacterium

C.diphtheriaeOther

corynebacterium species

Carbohydrate Fermentation Test

Principle:

Each species of corynebacteria has its specific carbohydrate fermentation

pattaern.

C.diphtheriae can be differentiated from other corynebacterium species by

fermentation of glucose and maltose but not sucrose, with production of acid

without gas.

Glucose Maltose Sucrose

Results:

Sugar fermentation can be indicated by change of color of the medium from red to yellow.

Glucose Maltose Sucrose

C. xerosis

Glucose Maltose Sucrose

C. diphtheriae

Toxigenicity testing of C.diphtheriae strains

Elek’s Toxigenicity TestElek’s Toxigenicity Test

Results:Positive test: The antitoxin diffusing from the filter paper strip will form precipitation lines with the toxin diffusing from the toxigenic strain.

Absence of precipitation lines indicates that the strain is non-toxigenic.

Gram +ve Bacilli

Spore forming Non spore forming

AerobicAnaerobic

BacillusClostridium

CorynebacteriumListeriaLactobacilli

All Catalae +ve

Gram-positive non-motile rectangular large

bacilli, that occur singly, in pairs, or in

chains and spore forming

Bacillus

Spore Stain

It has oval central spores.Using the Spore stainingTechnique(Malachite green &safranin), the sporesappear GREEN while thevegetative cells appearRED.

Media: • Grow on simple nutrient media.• Bacillus species grow well on blood agar showing a

double zone of hemolysis Except B. anthracis (No hemolysis).

Starch Hydrolysis Test:

Principle:

Starchamylase enzyme

glucose

Blue colour No colour

I2 I2

Biochemical reactions:

Result:Amylase activity is indicated by a clear zone around the growth while the rest of the plate gives blue color after addition of iodine solution.

Gram +ve Bacilli

Spore forming Non spore forming

AerobicAnaerobic

BacillusClostridium

CorynebacteriumListeriaLactobacilli

All Catalae +ve

Gram +ve Bacilli

Spore forming Non spore forming

AerobicAnaerobic

BacillusClostridium

Corynebacterium

Environment: Anaerobic

- Thioglycollate broth.

- Cooked meat medium.

- Deep agar.

- Anaerobic jar.

Anaerobic Medium

Anaerobic jar

principle:

Removal of O2 & replacing it with an inert gas→ Blood agar plates in Anaerobic Jar.

Cooked meat mediumI

anaerobic medium due to presence of:

Meat particles (prepared from heart muscles) which contain hematin and glutathione that act as reducing agents.

Biochemical reaction

It Contains: • Skimmed milk (without fat) i.e: contains only sugar (Lactose) and protein (casine)• Litmus indicator (acid base and redox indicator).

Litmus milk medium:

Reactions:Acidic reaction:

Lactose (milk sugar)

Fermentation acid Litmus indicator pink colour

Clostridium

Stormy Clot Formation:

Fermentation(Lactose)

milk sugar

acid + gas

(Casine) milk

protein

Coagulation clot

Stormy clot

The C. Perfringens cause rapid fermentation of lactose in litmus milk and the gas produced split the clot (Stormy Clot Formation).