PHT 416 Lab 7
Steps
• Microscopic
Morphology
Nutrient agar
• Growth
Blood agar
Mannitol Salt Agar
MacConkey’s agar
• Biochemical Tests
Staphylococci
Streptococci
Corynbacterium
Clostridum
Bacillus
Neisseria
Enterobacteriaceae
Pseudomonas.
Gm’s +ve
Cocci
Staphylococci
Streptococci
Bacilli
Corynbacterium
Clostridum
Bacillus
Gm+ve Cocci
Staphylococci
Streptococci
Catalase test
Catalase enzyme
H2O2
Catalase +ve
H2o
+ O2
Air bubbles
Catalase –ve
Staphylococci
• Gram positive cocci, arranged
in grape like clusters, nonmotile, non-spore forming.
Staphylococci
Staph. aureus
Staph. saprophyticus
Staph. epidermidis
Coagulase Test
Coagulase enzyme
Fibrinogen
Fibrin
saprophyticus
Staph. aureus
Plasma Staph. epidermidis Staph.
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
MSA is differential for S.aureus
Yellow colonies
Pink colonies
Staphylococci
Staph. aureus
Staph. epidermidis
Deoxyribonuclease (DNase) Test
DNase enzyme
DNA
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
Catalase test
Catalase enzyme
H2O2
Catalase +ve
H2o
+ O2
Air bubbles
Catalase –ve
Streptococci
Gram positive cocci,arranged in
chains or pairs, non-motile,
non-spore forming.
Streptococci
(Fastidious organism)
Complex nutritional requirements
Blood agar
β-hemolytic
S. pyogenes
α-hemolytic
S.pneumonia,
viridans streptococci
Non-hemolytic
Streptococci
Growth on Blood agar
α-Haemolysis
1
β-Haemolysis
1
Optochin Test
R
S
S.pneumoniae
2
Viridans
Streptococci
-ve
Viridans
Streptococci
R
S
S. pyogenes
2
Bile Solubility test
+ve
S.pneumoniae
Bacitracin Test
S. agalactiae
CAMP test
CAMP+ve
S.agalactiae
Principle:
S.pneumoniae is inhibited by less than 5µg/ml Optochin reagent giving a
zone
of inhibition more than 15 mm in diameter.
α-Haemolysis
Positive test: any zone of inhibition around the disc.
Optochin Test
S
S.pneumoniae
R
Viridans
Streptococci
Streptococci
Growth on Blood agar
α-Haemolysis
1
β-Haemolysis
1
Bile
Optochin
Solubility
Test
test
R
S
S.pneumoniae
2
Viridans
Streptococci
-ve
Viridans
Streptococci
R
S
S. pyogenes
2
Bile Solubility test
+ve
S.pneumoniae
Bacitracin Test
S. agalactiae
CAMP test
CAMP+ve
S.agalactiae
Principle:
α-Haemolysis
S.pneumoniae produces a self-lysing enzyme to
Bile Solubility
depress
thetestgrowth of old colonies. The presence of
bile salt accelerate this process.
Results
Visible clearance
S.Pneumoniae
Remain turbid
Viridans Streptococci
Streptococci
Growth on Blood agar
α-Haemolysis
1
β-Haemolysis
1
Optochin Test
R
S
S.pneumoniae
2
Viridans
Streptococci
-ve
Viridans
Streptococci
R
S
S. pyogenes
2
Bile Solubility test
+ve
S.pneumoniae
Bacitracin Test
S. agalactiae
CAMP test
CAMP+ve
S.agalactiae
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 β-Haemolysis
Positive test: any zone of inhibition around the disc.
Bacitracin Test
S
S. pyogenes
B
B
R
S. agalactiae
Streptococci
Growth on Blood agar
α-Haemolysis
1
β-Haemolysis
1
Optochin Test
R
S
S.pneumoniae
2
Viridans
Streptococci
-ve
Viridans
Streptococci
R
S
S. pyogenes
2
Bile Solubility test
+ve
S.pneumoniae
Bacitracin Test
S. agalactiae
CAMP test
CAMP+ve
S.agalactiae
CAMP reaction: The hemolysis is enhanced in the vicinity of
the ß -lysin producing S.aureus (Arrow head-sheaped
β-Haemolysis
zone of ß-hemolysis).
Group B streptococci (S.agalactiae. ) produce CAMP
a factor
that
test
increases beta hemolysis of an S. aureus indicator strain.
CAMP+ve
S.agalactiae
CAMP-ve
Gm’s +ve
Cocci
Staphylococci
Streptococci
Bacilli
Corynbacterium
Clostridum
Bacillus
Gram +ve Bacilli
Spore forming
Non spore forming
Anaerobic
Aerobic
Clostridium
Bacillus
All Catalae +ve
Corynebacterium
Listeria
Lactobacilli
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"
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
Other
corynebacterium
species
C.diphtheriae
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 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
Anaerobic
Aerobic
Clostridium
Bacillus
All Catalae +ve
Corynebacterium
Listeria
Lactobacilli
Bacillus
Gram-positive non-motile rectangular large
bacilli, that occur singly, in pairs, or in
chains and spore forming
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).
Biochemical reactions:
Starch Hydrolysis Test:
Principle:
amylase enzyme
Starch
I2
Blue colour
glucose
I2
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.
Gram +ve Bacilli
Spore forming
Non spore forming
Anaerobic
Aerobic
Clostridium
Bacillus
All Catalae +ve
Corynebacterium
Listeria
Lactobacilli
Gram +ve Bacilli
Spore forming
Non spore forming
Anaerobic
Aerobic
Clostridium
Bacillus
Environment: Anaerobic
- Thioglycollate broth.
- Cooked meat medium.
- Deep agar.
- Anaerobic jar.
Corynebacterium
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.
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:
Lactose (milk
sugar)
Fermentation acid
Litmus indicator pink colour
Stormy Clot Formation:
(Lactose)
milk sugar
(Casine)
milk protein
Fermentation
Coagulation
acid
+
gas
clot
Stormy
clot
The C. Perfringens cause rapid fermentation of lactose
in litmus milk and the gas produced split the clot
(Stormy Clot Formation).