Aerobic Non-Spore Forming
Gram-Positive Bacilli
Corynebacterium
Gram Positive Bacilli
Gram positive rods
Spore forming
Aerobic
Bacillus spp
Non spore forming
Anaerobic
Clostridium spp
Corynebacterium
Corynebacterium spp
Gram positive bacilli, with characteristic
morphology (club shaped and beaded)
Non motile
Non spore forming
Non capsulated
Facultative anaerobic
Breakdown glucose by oxidative and
fermentative i.e. O+/F+
C. diphtheriae is fastidious while diphtheriods are
non-fastidious
Catalase positive
Oxidase negative
Species of Corynebacteria
Corynebacterium
It caused
diphtheria
Pathogenic
C. diphtheriae
C. diphtheriae is the only
pathogenic members of
this genus
Commensal
"Diphtheriods"
C. hofmannii, C. xerosis,
C. acne
Normal flora of RT, urethra,
vagina, Skin
Cased by C.
diphtheriae
Mycocarditis, neuroitis,
palate perforation
Acute, Toxin
mediated
Childhood disease affect
upper respiratory tract
Diphtheria
Transmitted by droplet infection
2-6 days I.P.
Sore throat, Pharyngitis
Recovery or
complication & death (if
more toxin absorbed)
Respiratory
obstruction due to
extensive membrane
formation
2-3 days, Bluish white adherent
pseudo membrane
Diagnosis of diphtheria
Clinical Diagnosis
Specific treatment
must be never delayed
for laboratory results
Laboratory Diagnosis
To confirm the clinical
manifestation
Diagnosis of diphtheria
Diagnosis of case
Symptomatic patient
Diagnosis of carrier
Asymptomatic
Laboratory diagnosis of case
– Specimen: A throat swap
– Culture: The swap is inoculated on
Loeffler's serum medium and/or on blood
tellurite agar aerobically at 37C for 24.
On Loeffler's serum medium:
Corynebacteria grow much more
readily than other respiratory pathogens
–Used to enhance the characteristic
microscopical appearance of
corynebacteria
–The colonies of C. diphtheriae are
small, granular, grey, smooth, and
creamy with irregular edges
Loefflers serum
Cultural characteristics
On blood tellurite agar (Mcloed’s blood agar)
– It is selective medium for isolation of C. diphtheriae
(Potassium tellurite)
– 3 biotypes of C. diphtheriae are characterized on BTA
– i.e. Gravis, mitis and intermedius biotypes
– The most severe disease is associated with the gravis
biotype
– Colony of gravis biotype is large, non-hemolytic & grey.
– Colonies of mitis biotype are small, hemolytic and black
– Colonies of intemedius biotype are intermediate in size, nonhemolytic with black center & grey margin.
Morphology
– Gram +ve, nonspore forming nonmotile bacilli
– Club-shaped (Coryne= club) arranged at acute angles
or parallel to each other (Chinese letters appearance)
– Beaded (metachromatic granules)
Stain
– Gram stain:
C. diphteriae are gram positive bacilli arranged in
Chinese letters form often club shaped
– Polychrome methylene blue stain:
C. diphteriae appears beaded due to the presence
of intercellular “Metachromatic or volutin"
granules
By stain, the granules appear red while the rest of
organism appears blue.
Gram stain of
C. diphtheriae
C. diphtheriae on
BTA
Biochemical Reaction
All Corynebacterium species are catalase
positive (Also, Staphylococcus and
Bacillus species are catalase positive)
2- Carbohydrate Fermentation Test:
Principle:
 Each species of corynebacteria has its specific
carbohydrate fermentation pattern
 C.diphtheriae can be differentiated from other
Corynebacterium species by fermentation of glucose
and maltose but not sucrose with production of acid
only
Procedure:
 Inoculate three tubes of
carbohydrate fermentation medium
(broth containing one type of sugar
and phenol red as the pH indicator)
with the test organism
 Incubate the tubes at 37oC for 24 hrs.
Glucose
Maltose
Sucrose
Results:
Sugar fermentation can be indicated by change of color of the
medium from red to yellow due to formation of acid which
decrease the pH
Glucose
+ve
Maltose
+ve
C. xerosis
Sucrose
+ve
Glucose
+ve
Maltose
+ve
Sucrose
- ve
C. diphtheriae
Diagnosis of Carrier
I- Isolation of organism
II- Detection of exotoxin
Test for toxigenicity
Swap from throat & nose
Inoculation on Loeffler’s
Or BTA for 24 h/37 C
Diphtheria like M.O.
Detection of exotoxin
I- In vivo
II- In vitro
Two guinea pigs are used
One is used as
Test
The second is
used as Control
Injected with
diphtheria antitoxin
Both test and control injected with isolated MO
If both GP live
Diphthrioids 0r non-toxigenic
C. diphtheriae
i.e. non pathogenic
If control live & test
die
C. diphtheriae
i.e. produce exotoxin
In Vitro: Elek’s Test
Principle:
– It is toxin/antitoxin reaction
– Toxin production by C.diphtheriae can be
demonstrated by a precipitation between exotoxin
and diphtheria antitoxin
Procedure:
A strip of filter paper impregnated with diphtheria
antitoxin is placed on the surface of serum agar
The organism is streaked at right angels to the filter
paper
Incubate the plate at 37C for 24 hrs
Filter paper saturated with diphtheria antitoxin
Lines of precipitations
Resuls:
After 48 hrs incubation,
the antitoxin diffusing
from filter paper strip
and the toxigenic strains
produce exotoxin, which
diffuses and resulted in
lines four precipitation
lines radiating from
intersection of the strip
and the growth of
Inoculated M.O.
organism
Positive Elek’s Test