Lab. No. 3
Bacteria Gram’s Stain
Gram’s +ve
Cocci
Bacilli
Gram’s -ve
Cocci
Rods
Gram’s +ve Bacilli
Spore forming
Anaerobic
Clostridium
Non spore forming
Aerobic
Bacillus
Corynebacterium
 Most
 C.
Important Species:
diphtheriae causes diphtheria.
Identification of Corynebacteria
(I) Morphology
(microscopical
examination):



Gram +ve, nonspore
forming nonmotile bacilli
club-shaped (Coryne= club)
arranged at acute angles or
parallel to each other→ a
Chinese letters appearance
Beaded (metachromatic
granules)
(II) Cultural characteristics:
Growth occurs best on media containing blood
or serum.
1. Loeffler’s serum agar:
 Used to enhance the characteristic
microscopical appearance of Corynebacteria.
2. Mcloed’s blood agar (blood tellurite medium):
 It is a selective medium (K tellurite) for
isolation of diphtheria bacilli from the pharynx
(The best isolation medium).
(III) Biochemical Reactions:
1- Catalase test:
 All corynebacterium species are
catalase +ve.
2- 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
+ve
Maltose
+ve
C. xerosis
Sucrose
+ve
Glucose
+ve
Maltose
+ve
Sucrose
- ve
C. diphtheriae
(IV) Toxigenicity testing of
C.diphtheriae strains:
 Elek’s
Toxigenicity Test:
Principle:
It is an Ag-Ab reaction, in which, toxin production by
C.diphtheriae can be demonstrated by a precipitation
between exotoxin and diphtheria antitoxin.
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:
formation of four radiating lines resulting from the
precipitation reaction between exotoxin and diphtheria
antitoxin.