PHT 313
Lab (3)
Bacteria Gram’s Stain
Gram’s +ve
Cocci
Staphylococci
Streptococci
Micrococci
Enterococci
Gram’s -ve
Bacilli
Cocci
Corynbacterium
Clostridum
Bacillus
Neisseria
Bacilli
Enterobacteriaceae
Pseudomonas.
Gram +ve Bacilli
Spore forming
Non spore forming
Anaerobic
Aerobic
Clostridium
Bacillus
Corynebacterium
Listeria
Lactobacilli
Non-spore forming bacilli
Corynebacterium
C. diphtheriae causes diphtheria.
Humans are the only known reservoir; carried
in the oropharynx or on the skin
Corynebacterium jeikeium: multiple antibiotic resistance
important in opportunistic infections of immunocompromised
patients
Corynebacterium urealyticum: urease hydrolyzes urea; release
of NH4+, increase in pH, alkaline urine, renal stones
Microscopical examination
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"
Palisade arrangement of cells in short chains ("V"
or "Y" configurations) or in clumps resembling
"Chinese letters"
(Cells tend to lie parallel to one another (palisades) or
at acute angles (coryneforms), due to their snapping
type of division)
Cultural characteristics
Environment: Facultative anaerobes
Temp.: 37 °C
pH: 7.2
Media: Growth occurs on media containing blood
or serum.
On blood tellurite medium (selective & differential
medium); colonies appear grey to black.
The four biotypes; gravis, mitis, intermedius and belfanti
are differentiated by colony morphology on blood tellurite
and biochemical reactions.
On Loeffler’s serum
Biochemical Reactions
Catalase test:
 All corynebacterium species are catalase +ve.
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.
Procedure:
1. Inoculate three tubes of sugar medium (broth
containing one type of sugar and phenol red as the pH
indicator) with the test organism
2. Incubate the tubes at 35oC for 24 hrs.
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
It is essential to demonstrate toxin production by
diphtheria isolates obtained from throat swabs of cases or
carriers, this is done by:
1)Elek’s test (or its modification)
2)PCR based methods are used for detection of
diphtheria toxin genes
3) ELISA can be used to detect diphtheria toxin
4) Immunochromographic strip assay allows
detection of diphtheria toxin in few hours
5) Tissue culture cytotoxicity assay
Elek’s Toxigenicity Test
Procedure:
1. Place a strip of filter paper saturated with
diphtheria antitoxin on a serum agar plate.
2. Streak the test organism across the plate at right
angle to the filter paper.
3. Incubate the plate at 35oC for 24 hrs.
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.
Non-toxigenic strain
Toxigenic strain
Spore forming bacilli
Aerobic
Anaerobic
Bacillus
Clostridium
B. anthracis
B. cereus
B. subtilis
Cl. tetani
Cl. perfringens
Cl. difficile
Cl. botulinum
Vegetative
cell division
Sporulation
Variations in endospore morphology
(1, 4) central endospore;
(2, 3, 5) terminal endospore;
(6) lateral endospore
Bacillus
Saprophytic organisms prevalent in soil, water,
and air.
Microscopic morphology:
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.

Cultural characteristics:
Environment: aerobic
PH: 7.2
Temperature: 37 ° C
Media:
• No special requirements for growth→ 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:
A) Catalase test: All bacillus species are catalase +ve.
B) Starch Hydrolysis Test:
Principle:
Starch
amylase enzyme
glucose
I2
Blue colour
I2
No colour
Procedure:
1. Inoculate starch agar plate with the test
organism.
2. Incubate the plate at 35oC for 24hrs.
3. Flood the plate with iodine solution.
Results:
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.
Key Characteristics to Distinguish between B.
anthracis & Other Species of Bacillus
Characteristic Bacillus anthracis
Hemolysis
Neg
Motility
Neg
Gelatin hydrolysis
Neg
Salicin fermentation Neg
Other Bacillus spp.
Pos
Pos (usually)
Pos
Pos
Bacillus stearothermophilus is thermophilic, so
its spores are used to test efficiency of killing in
autoclaves
Clostridium
Habitat: Some members of the genus Clostridium are
saprophytes in soil, sewage and water. Others are
commensals in GIT of animals and humans.
Cl. Tetani causing tetanus
Cl. Perfringens causing gas gangrene
Cl. Difficile causing enterocolitis
Cl. Botulinum causing food poisoning
Microscopic morphology:
Cl. Tetani : Gram +ve bacilli, swollen
at one end due to terminal spherical
projecting spores (Drum stick
appearance),
motile
with
peritrichous flagella.
Cl. Perfringens: Gram +ve bacilli,
rod-shaped, spores are oval,
subterminal and non projecting,
non-motile.
Cl. Botulinum: Gram +ve bacilli, motile, non-capsulated.
Spores are oval, central or subterminal.
Culture characteristic
•
•
•
•
Environment : anaerobic
Temp. 37 ° C
PH: 7.2
Media:
1- Removal of O2 & replacing it with an
inert gas→ Blood agar plates in
Anaerobic Jar.
2- Special anaerobic media containing a reducing agent.


Thioglycollate broth.
Cooked meat medium.
3. Deep agar stab cultures: a straight wire is charged with
inoculum and pluged into adeep column of nutrient agar
Thioglycollate broth:
The medium contains:
1- Sodium thioglycollate which acts as a reducing agent.
2- Small amount of agar to increase viscosity of the
medium.
It should be prepared in long tube
aerobic bacteria
anaerobic bacteria
Cooked meat medium:
It is anaerobic medium due to presence of:
Meat particles (prepared from heart muscles) which contain
hematin and glutathione that act as reducing agents.
Reactions on Cooked meat medium:
1.Saccharolytic reaction:
 Causes fermentation of the muscle glycogen
with production of acid and gas.
 The meat particles remain intact.
2. Proteolytic reaction:
 Causes digestion of the meat particles
leading to formation of black, foul smelling
sulphur compounds.
C. difficile colonies on a blood agar plate.
C. perfringens colonies on an egg
yolk agar plate showing a white
precipitate due to production of
Lecithinase (Nagler´ reaction)
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).