By Ms. Kostiuk from Microbiology department

1. Lesson N9: LABORATORY DIAGNOSIS of diphtheria
1.Scientifically methodical ground of theme
Diphtheria was formally an important pediatric disease all over the world but following the
development of effective prophylactics and mass immunization, the disease has been virtually
eradicated from most advanced countries. In those developing countries, where childhood
immunization programs have been implemented effectively diphtheria has become rare but in
others it still continues to be a serious problem.
2.Educational purpose
STUDENTS MUST KNOW:
1. Structure, staining properties and cultivation of Corynebacterium diphtheriaе
2. Fermentative properties and toxin production of Corynebacterium diphtheriaе
3. Epidemiology and pathogenesis of diphtheria.
4. Methods of laboratory diagnosis of disease, the main methods of prevention and
treatment.
STUDENTS SHOULD BE ABLE TO:
– prepare the smears from tested material;stain the smears by Gram’s and Loeffler
technique; make microscopical examination of the smears and make up conclusion
• value the growth of Corynebacterium diphtheriaе on different media;
– choose biological preparation used for laboratory diagnosis and specific prophylaxis of
diphtheria ; create scheme of diphtheria diagnosis.
3.Chart of topic content.
Fig. N 1: Usual biochemical reaction of C.diphtheriae
Catalase Nitrat reduction Urease Helatin Glucose Maltose Sucrose
hydrolisis fermentation
fermentation fermentation
+ + - - + + -
FigN2:Differential-diagnostic signs of corynebacteria diphtheria and non-pathogenic
corynebacteria

2. Type of Fermenta-tion Toxigenic Additional signs
corynebacteri sucrouseeeeeeeeeeeeeeeeeeee glucos starc ity cystina ureas Agglutinati
a eeeeee e h se test e test on with
antiserum
Diphtheria
corynebacteri
a
gravis – + + + + – +
mitis – + – – + – +
Diphtheroids + + – – + + –
Pseudodiphth + – – – – – –
eria bacteria
Fig. N3: Mode action of diphtheria toxin
C.diphtheriae
Diphtheria toxin
Cell membrane
AB
AB
Cell death
A
Prevents protein
synthesis
Inactivates by ribosome
elongation
factor-2
ribosome
Fig. N 4:Main effects of C.diphtheriae exotoxin

3. Local Systemic
Target: pharynx Targets: cardiac, nervous tissues and others
Inflammatory response with
pseudomembrane
Fig. N 5: Microscopic examination
taking
specimen
swab
Throat, nose
Prepared smears stain by Gram’s and
examine Loeffler technique
under microscope
Club-shaping, non-sporing, Gram positive rods, that show
methachromatic granules. Organisms arranged in
“Chinese letterâ€u patterns, or side- by- side in “palisadesâ€
Fig. N 6: Prevention of diphtheria
Prevention of diphtheria
Non specific
Specific(immunization)
Isolation of a patient, detection and
Active immunization

4. Passive immunisation
isolation of carriers
DPT-vaccine ADS-Antitoxin
(antidiphtheritic serum)
4. Student’s independent study program
1. Structure and staining properties of diphtheria causative agent
2. Cultivation of Corynebacterium diphtheriaе. Main nutrient media.
3. Biochemical properties of Corynebacterium diphtheriaе.
4. classification of Corynebacterium diphtheriaе.
5. Toxin formation of Corynebacterium diphtheriaе.
6. Epidemiology of diphtheria :
a – source of infectious agents;
b – mechanism of transmission;
c – factors of transmission.
8. Pathogenesis and clinical findings of diphtheria.
9. Laboratory diagnosis of diphtheria.
10. Treatment and prophylaxis of diphtheria.
a– specific prophylaxis of diphtheria.
c – urgent prophylaxis of diphtheria.
d – treatment
5. Students’ practical activities:
1. Stain ready smears of Corynebacterium diphtheriaе by Loeffler technique. Examine
under microscope; make up conclusion. Draw and record you observation. (See Fig.N5)

5. On fixed smear pour alkaline methylene blue to act for 3-5 minutes, wash with water, dry with
filter paper, and examine under the microscope. The cytoplasm of Corynebacterium diphtheriaе
is stained light-blue, while granules are dark-blue.
2. To familiarize with biological preparation used for laboratory diagnosis and specific
prophylaxis of diphtheria.
DPT-vaccine, that consists of diphtheria (D) and tetanus (T) toxoid, and pertussis(P) vaccine, all
three generally given together. toxoid is prepared by 0,4% formaline solution treatment from
diphtheria toxin at 400 C during 4 weeks. It is used for planned immunization against toxoid
administration results in the production of antibodies that specifically neutralize the diphtheria
toxin. Since the disease the results primarily from toxin adsorption rather than microbial invasion,
its control can be accomplished most effectively by immunization with toxoid. Unfortunately, these
immunizations have often been neglected, particularly among socioeconomically disadvantages
groups, and serious epidemics of the diseases have occurred periodically. Since the 1980s, there
has been an active campaign in the most of the United states, to ensure that children who are
entering school are immunized against diphtheria. As a result, only a few cases of diphtheria are
annually reported in the USA, as compared to the 30,000 cases reported in 1936.
ADS-Antitoxin ( antidiphtheritic serum) is used for treatment and prophylaxis.In1886 Bering
found antibodies in the blood of porpoises, immunized with sublethal doses of diphtherin. In 1896
Bering used sheep serum for diphtheria treatment. He was awarded Nobel Prize for creating of
new treatment method and for the successes of diphtheria treatment. Passive immunization is an
emergency measure to be employed when susceptibles are exposed to infection, as a case of
diphtheria is admitted to general pediatric ward.It is consist of the sucutaneous administration of
500-1000 unit of antitoxin As this is a horse serum, precaution against hypersensetivity should be
observe.
Antibiotics (erythromycin, penicillin) The bacteria are sensitive to these antibiotics, but such
treatmen stops only transmission of the disease; it has no effect on the toxin that has been
adsorbed already.
Effective treatment of diphtheria depends on giving antiserum against diphtheria toxin to the
patient as soon as possible. Administration of antiserum must be given if the disease is
suspected without waiting for proof of the diagnosis, since a delay of the several days need to
obtain confirmation from the culture results can be fatal.
3. Start diagnosis of diphtheria carrying. Take specimen from throat by cotton swab.
Inoculate it onto Loeffler medium
.
Throat Thermostat, at370 C
Cotton swab during 24-48 hours

6. Inoculate by swab onto Loeffler medium
6. Control questions and tests
1. It was necessary to make preventive inoculation of student's group because a case of
diphtheria. What preparation should be used to develop artificial active immunity?
A. Diphtheria toxoid
B. Antidiphtheritic serum.
C. A specific antibody.
D. Vaccine DPT (Triple vaccine.)
2. To determine the toxigenicity of causative agents of diphtheria obtained from patients,
cultures are inoculated into wells cooped round the central well with diphtheria serum into
nutritious agar of Petri dishes. After cultures incubation between central well and some wells with
cultures bands (lines of precipitation) are observed. Which serologic test is carried out?
A. Test precipitation in gel.
B. Tube precipitation test
C. Agglutination.
D. Floculation
E. Coombs test
3. Pure culture of corynebacteria diphtheria is obtained from a patient. What serologic test
should be used for determining toxigenity of culture?
A. Agglutinations.
B.Test. precipitation in agar
C. Complement fixation test
D. Hemagglutination inhibition.
E. Passive hemaglutination

7. 4. Throat culture isolated from a 5- year- old child was identified as corynebacterium diphtheria
by morphological and fermentative properties, but exotoxin free. What process can make this
strain toxigenetic?
A. Chromosomal mutation.
B.Cultivation on telurite medium
C. Passing culture through organisms of sensitive animals.
D. Cultivation on medium with antitoxic serum.
E. Phage conversion.
5. Corynebacterium diphtheriae was obtained from a sick child with suspicion of diphtheria.
What research is necessary to use to be convinced, that this microbe causes diphtheria at the
child?
A. Agglutination test.
B. Staining a material using Giema’s method .
C. Culturing microbes on a blood agar.
D. To infect the rabbit.
E. To check up a toxin producing by microbes.
6. Among children there are cases of respiratory infection. The tonsil swab was stained by
Neisser technique. There were yellow rods with brown terminal granules, the rods were situated
like letters V, W, X. What infection can be suspected in this case?
A. Infectious mononucleosis.
B. Diphtheria.
C. Listeriasis.
D. Tonsillitis.
E. Scarlet fever.
7. Doctor noticed grayish patches “pseudomembraneâ€e on pharyngeal tonsils of 6- year-
old child at the examination. Bleeding was occurred when there was attempt of remove patches.
What symptoms can appear within the next few days without specific treatment?

8. A. Very strong attack-like cough.
B. Lungs edema.
C. Toxic lesion of a cardiac muscle, liver, kidneys.
D. Papular rash on skin.
E. Intermittent fever.
8. The two-year-old boy was not immunizated for diphtheria toxoid. On his pharyngeal tonsils
there were revealed microorganisms, which on morphological and biochemical properties are
identical to Corynebacterium diphtheriaе. But precipitation test in agar with antitoxic serum has
been negative. What form of infectious process can this agent cause in the child without
treatment?
A. Asymptomatic carriage of bacteria.
B. The easy nontoxic form of disease.
C. The toxic complicated form of disease.
D. Chronic disease with damage of the internal organs.
E. Persistent infection.
9. The girl, 7 years, with a high body temperature, complaints of sore throat, weakness has been
hospitalized to infectious clinic. The doctor has suspected diphtheria and has given the instruction
to take a material from pharynx for obtaining pure culture of the pathogen. Which one of the
following points is the most important for confirmation of the diagnosis "diphtheria"?
A. Revealing polymetaphosphate granules in agent.
B. Demonstrating toxin production.
C. Urease test.
D. Hemolytic activity of agent.
E. Bacteriophage typing.
10. In pediatric department of infectious clinic there was diagnosed diphtheria in the boy. Which
one of the following preparations should be injected first of all?
A. Diphtheritic antitoxic serum.

9. B. Diphtheritic toxoid.
C. BCG.
D. DPT.
E. ТABTe.
11. In smear from a tonsil of the patient with suspicion for diphtheria there have been revealed
blue rods with dark blue beads at the ends. What method of staining has been used?
A. Loeffler’s.
B. Giemsa.
C. Gins’.
D. Gram’s.
E. Neisser’s.
12. It is necessary to conduct specific prophylaxis of diphtheria in children at school. What
preparation should be used for this purpose?
A. Toxoid.
B. Antibiotics.
C. Probiotics.
D. A corpuscular vaccine.
E. Immune serum.
13. Each of following statements concerning Corynebacterium diphtheriaе is correct EXCEPT:
A. Corynebacterium diphtheriaе is gram positive rod that does not form spores
B. toxin production is dependent on the organism’s being lysogenized by bacteriophage
C. diphtheria toxoid should not be given to children under the age of 3 years because the
incidents of complications is too high
D. Antitoxin should be used to treat patients with diphtheria
14. diphtheria toxin is correctly described as:
A. Produced by Corynebacterium diphtheriaе both in vitro and in vivo

10. B. Immunogenic
C. The active agent in Schick test
D. Used to vaccinate infants
15. The primary virulence factor for Corynebacterium diphtheriaе is:
A. Enterotoxin
B. Capsule
C. Exotoxin
D. Endotoxin
16. For practical use of curative antitoxic serums, the patient is always given precisely defined
doses. What units is activity of diphtheria antitoxin measured in?
A. International.
B. Flocculation’s.
C. Antigenic.
D. Agglutination’s
E. Neutralization’s
7. List of literature: 1.I. S. Gaidash, V.V. Flegontova, Microbiology, virology and immunology,
Lugansk, 2004, chapterN17, p. 29-43.