PHT 416 Lab 7

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PHT 416 Lab 7
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Steps

• Microscopic
• Morphology
• Growth
• Biochemical Tests

Nutrient agar Blood agar Mannitol Salt
Agar MacConkeys agar
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Corynbacterium Clostridum Bacillus
Enterobacteriaceae Pseudomonas.
Neisseria
Staphylococci Streptococci
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Gms ve
Bacilli Corynbacterium Clostridum Bacillus

• Cocci
• Staphylococci
• Streptococci

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• Gmve Cocci

Streptococci
Staphylococci
Catalase test
Catalase ve
Catalase ve
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Staphylococci

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

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Staphylococci
Staph. saprophyticus
Staph. aureus
Staph. epidermidis
Coagulase Test

Staph. saprophyticus
Staph. aureus
Staph. epidermidis
(ve) Formation of visible clot
-ve
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Staphylococci
Staph. aureus
Staph. epidermidis
Staphylococci on Nutrient Agar
Golden Yellow colonies
White colonies
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Staphylococci
Staph. aureus
Staph. epidermidis
Staphylococci on Blood Agar
Beta heamolysis (completehaemolysis)
Non hemolytic
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Staphylococci
Staph. aureus
Staph. epidermidis
Staphylococci on MSA
MSA is differential for S.aureus
Yellow colonies
Pink colonies
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Staphylococci
Staph. aureus
Staph. epidermidis
Deoxyribonuclease (DNase) Test
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
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• Gmve Cocci

Streptococci
Staphylococci
Catalase test
Catalase ve
Catalase ve
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Streptococci

• Gram positive cocci,arranged in chains or pairs,
  non-motile, non-spore forming.

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Streptococci
(Fastidious organism)
Complex nutritional requirements
Blood agar
a-hemolytic S.pneumonia, viridans streptococci
ß-hemolytic S. pyogenes
Non-hemolytic
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Streptococci
Growth on Blood agar
a-Haemolysis
ß-Haemolysis
Optochin Test
Bacitracin Test
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1
S
R
R
S
Viridans Streptococci
S.pneumoniae
S. agalactiae
S. pyogenes
CAMP test
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Bile Solubility test
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-ve Viridans Streptococci
ve S.pneumoniae
CAMPve S.agalactiae
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Principle S.pneumoniae is inhibited by less than
5µg/ml Optochin reagent giving a zone of
inhibition more than 15 mm in diameter.
a-Haemolysis
Positive test any zone of inhibition around the
disc.
Optochin Test
S
R
S.pneumoniae
Viridans Streptococci
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Streptococci
Growth on Blood agar
a-Haemolysis
ß-Haemolysis
Bacitracin Test
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1
Bile Solubility test
Optochin Test
S
R
R
S
Viridans Streptococci
S.pneumoniae
S. agalactiae
S. pyogenes
CAMP test
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Bile Solubility test
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-ve Viridans Streptococci
ve S.pneumoniae
CAMPve S.agalactiae
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Principle
a-Haemolysis
S.pneumoniae produces a self-lysing enzyme to
depress the growth of old colonies. The presence
of bile salt accelerate this process.
Bile Solubility test
Results
Visible clearance
Remain turbid
S.Pneumoniae
Viridans Streptococci
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Streptococci
Growth on Blood agar
a-Haemolysis
ß-Haemolysis
Optochin Test
Bacitracin Test
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1
S
R
R
S
Viridans Streptococci
S.pneumoniae
S. agalactiae
S. pyogenes
CAMP test
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Bile Solubility test
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-ve Viridans Streptococci
ve S.pneumoniae
CAMPve S.agalactiae
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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
ß-Haemolysis
Positive test any zone of inhibition around the
disc.
Bacitracin Test
S
R
S. pyogenes
S. agalactiae
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Streptococci
Growth on Blood agar
a-Haemolysis
ß-Haemolysis
Optochin Test
Bacitracin Test
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1
S
R
R
S
Viridans Streptococci
S.pneumoniae
S. agalactiae
S. pyogenes
CAMP test
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Bile Solubility test
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-ve Viridans Streptococci
ve S.pneumoniae
CAMPve S.agalactiae
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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. ) produce a factor
that increases beta hemolysis of an S. aureus
indicator strain.
ß-Haemolysis
CAMP test
CAMPve S.agalactiae
CAMP-ve
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Gms ve
Bacilli Corynbacterium Clostridum Bacillus

• Cocci
• Staphylococci
• Streptococci

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Classification of Gram's ve Bacilli
Gram ve Bacilli
Spore forming
Non spore forming
Aerobic
Anaerobic
Corynebacterium Listeria Lactobacilli
Bacillus
Clostridium
All Catalae ve
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C.diphtheriae
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"
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Corynebacterium
.
Growth occurs on media containing blood or serum
-On blood tellurite medium (selective
differential medium) colonies appear grey to
black. -On Loefflers serum
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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.
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Results
Sugar fermentation can be indicated by change of
color of the medium from red to yellow.
C. xerosis
C. diphtheriae
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Toxigenicity testing of C.diphtheriae strains
Eleks 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.
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Classification of Gram's ve Bacilli
Gram ve Bacilli
Spore forming
Non spore forming
Aerobic
Anaerobic
Corynebacterium Listeria Lactobacilli
Bacillus
Clostridium
All Catalae ve
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Bacillus
Gram-positive non-motile rectangular large
bacilli, that occur singly, in pairs, or in
chains and spore forming
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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.
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• Media
• Grow on simple nutrient media.
• Bacillus species grow well on blood agar showing
  a double zone of hemolysis Except B. anthracis
  (No hemolysis).

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Biochemical reactions
Starch Hydrolysis Test
Principle
amylase enzyme
Starch
glucose
I2
I2
Blue colour
No colour
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.
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Classification of Gram's ve Bacilli
Gram ve Bacilli
Spore forming
Non spore forming
Aerobic
Anaerobic
Corynebacterium Listeria Lactobacilli
Bacillus
Clostridium
All Catalae ve
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Gram ve Bacilli
Spore forming
Non spore forming
Aerobic
Anaerobic
Corynebacterium
Clostridium
Bacillus

• Environment Anaerobic
• - Thioglycollate broth.
• - Cooked meat medium.
• - Deep agar.
• - Anaerobic jar.

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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.
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Clostridium
Biochemical reaction
Litmus milk medium

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

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