PHT 416 Lab 7

Steps Microscopic Morphology Growth Biochemical Tests Nutrient agar Blood agar Mannitol Salt Agar MacConkey’s agar

Staphylococci Streptococci Neisseria Corynbacterium Clostridum Bacillus Enterobacteriaceae Pseudomonas.

Gm’s +ve Cocci Staphylococci Streptococci Bacilli Corynbacterium Clostridum Bacillus

Gm+ve Cocci Staphylococci Streptococci H2O2H2O2 Catalase enzyme H 2 o + O 2 Air bubbles Catalase test Catalase –veCatalase +ve

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

Staph. saprophyticusStaph. aureusStaph. epidermidis Coagulase Test Staph. saprophyticusStaph. aureusStaph. epidermidis Fibrinogen Plasma Coagulase enzyme Fibrin Visible Clot (+ve) Formation of visible clot-ve

Staphylococci Staph. aureusStaph. epidermidis Staphylococci on Nutrient Agar Golden Yellow coloniesWhite colonies

Staphylococci Staph. aureusStaph. epidermidis Staphylococci on Blood Agar Beta heamolysis (completehaemolysis) Non hemolytic

Staphylococci Staph. aureusStaph. epidermidis Staphylococci on MSA Yellow colonies Pink colonies MSA is differential for S.aureus

Deoxyribonuclease (DNase) Test Staphylococci Staph. aureusStaph. epidermidis DNA DNase enzyme Nucleotides Insoluble 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 H2O2H2O2 Catalase enzyme H 2 o + O 2 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-hemolytic S. pyogenes Non-hemolytic α-hemolytic S.pneumonia, viridans streptococci

Streptococci Growth on Blood agar α-Haemolysisβ-Haemolysis Optochin Test Bacitracin Test S R S R S.pneumoniae Viridans Streptococci CAMP+ve S.agalactiae Bile Solubility test CAMP test S. pyogenes +veS.pneumoniae -ve Viridans 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. S R S.pneumoniae Viridans Streptococci

Streptococci Growth on Blood agar α-Haemolysisβ-Haemolysis Bacitracin Test S R S R S.pneumoniae Viridans Streptococci CAMP+ve S.agalactiae Bile Solubility test CAMP test S. pyogenes +veS.pneumoniae -ve Viridans Streptococci Bile Solubility testOptochin Test

α-Haemolysis Bile Solubility test S.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-Haemolysisβ-Haemolysis Optochin Test Bacitracin Test S R S R S.pneumoniae Viridans Streptococci CAMP+ve S.agalactiae Bile Solubility test CAMP test S. pyogenes +veS.pneumoniae -ve Viridans Streptococci

β-Haemolysis Bacitracin 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. B S R S. pyogenes S. agalactiae

Streptococci Growth on Blood agar α-Haemolysisβ-Haemolysis Optochin Test Bacitracin Test S R S R S.pneumoniae Viridans Streptococci CAMP+ve S.agalactiae Bile Solubility test CAMP test S. pyogenes +veS.pneumoniae -ve Viridans 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). S.agalactiae. Group B streptococci (S.agalactiae. ) produce a factor that increases beta hemolysis of an S. aureus indicator strain. CAMP+veS.agalactiae CAMP-ve

Gm’s +ve Cocci Staphylococci Streptococci Bacilli Corynbacterium Clostridum Bacillus

Gram +ve Bacilli Spore forming Non spore forming AerobicAnaerobic BacillusClostridium Corynebacterium Listeria Lactobacilli 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.diphtheriae Other 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.

GlucoseMaltose Sucrose Results: Sugar fermentation can be indicated by change of color of the medium from red to yellow. GlucoseMaltose Sucrose C. xerosis GlucoseMaltose Sucrose C. diphtheriae

Toxigenicity testing of C.diphtheriae strains Elek’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 Corynebacterium Listeria Lactobacilli 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 staining Technique (Malachite green & safranin), the spores appear GREEN while the vegetative cells appear RED.

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: Starch amylase enzyme glucose Blue colour No colour I2I2 I2I2 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 Corynebacterium Listeria Lactobacilli All Catalae +ve

Gram +ve Bacilli Spore forming Non spore forming AerobicAnaerobic Bacillus Clostridium 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).