1. The document discusses laboratory diagnosis of clostridiosis caused by Clostridium bacteria. It describes the morphology, staining properties, cultivation techniques and toxin production of key Clostridium species like C. perfringens, C. tetani, and C. botulinum that cause diseases like gas gangrene, tetanus, and botulism. 2. Students are expected to learn laboratory techniques for identifying Clostridium bacteria under the microscope, inoculating specimens into different media, and observing changes in media that can help diagnose infections. 3. The document provides diagrams summarizing the pathogenic mechanisms and epidemiology of diseases caused by different Clostridium species.

1. Lesson N11: LABORATORY DIAGNOSIS of clostridiosis
1.Scientifically methodical ground of theme
Clostridium is genus of gram-positive, spore –forming rods, that is widely distributed in nature.
The large genus (over 120 species) is extremely varied in its habitats. Saprobic members reside
in soil, sewage, vegetation and organic debris and commensals inhabit the bodies of humans and
other animals. Infection caused by pathogenic speciesare not normally communicable but occur
when spores are introduced into injured skin. Clostridial diseases can be divided into (1) wound
and tissue infections, including myonecroses (gas gangrene), antibiotic-associated colitis, and
tetanus and food intoxication of perfringens and botulism varieties. Most of these diseases are
caused when exotoxins some of which are highly potent, act on specific cellular targets
2.Educational purpose
STUDENTS MUST KNOW:
1. Structure, staining properties and cultivation of gas gangrene Clostridia ,C. tetani and C.
botulinum.
2. Antigenic structure of Clostridia.
3. Fermentative properties and toxin production of Clostridia.
4. Ecology of gas gangrene Clostridia,C. tetani and C. botulinum. Resistance. Pathogenicity
for animals.
5. Epidemiology and pathogenesis of gas gangrene, tetanus and botulism.
6. Methods of laboratory diagnosis of gas gangrene tetanus and botulism, main methods of
prophylaxis and treatment
STUDENTS SHOULD BE ABLE TO:
• stain the smears by Gram’s technique;
• examine the smears under the microscope;
• value the growth of Clostridia on different nutrient media;
• inoculate spesimen into Kitt–Tarozzi medium or Vrublevsky medium
• draw the scheme of laboratory diagnosis of tetanus and boptulism.
3.Chart of topic content.
FigN1. Clostridia as human pathogens
A. The gas gangrene group:
1.Established pathogens Cl. perfringens
Cl.novyi
Cl.septicum

2. 2. Less pathogenic Cl. hystolyticum
Cl.fallax
3. Doubtful pathogens Cl. bifermentans
Cl. sporogenes
B. Tetanus Cl. tetani
C. Food poisoning
1. Gastroenteritis Cl. perfringens(typeA)
2.Botulism Cl.botulinum
D. Acute colitis Cl.difficile
FigN2:Important species of clostridium
species Chief important to humans Description of role
Cl. perfringens pathogen Principal cause of gas gangrene and
myonecrosis; common agent in
enterotoxigenic food poisoning
Cl.novyi pathogen Second most cause of gas
gangrene
Cl.septicum pathogen Third most cause of gas gangrene
Cl. tetani pathogen Cause of tetanus
Cl.botulinum pathogen Cause of botulism
Cl.difficile opportunist Involved in antibiotic- associated
colitis
Cl. iodofilum industrial uses Produces organic acids and
alcohols for commertial use
Cl.acetobutylicum industrial uses Produces organic acids and
alcohols and benzene
Cl.butyricum industrial uses Produces butyric acid in butter and
cheese
Cl. cellobiovarum industrial uses Digest cellulose
FigN3: Circumstances and events that may lead to gas gangrene
Tissue damage and contamination with clostridial spores and pyogenic microorganisms
Presence of foreign bodies, including soil with damaging salts
Inflammatory reaction

3. Extravasation of fluid, oedema
Impaired tissue perfusion; stasis
Poor oxygenation, with reduction in local Eh
Impaired phagocytosis and intraleukocytic bactericidal mechanisms
Multiplication of facultative bacteria
Further reduction of Eh and pH
Germination and out growth with production of toxin
Multiplication of obligate anaerobes with production of toxins, aggressins and gas in affected
tissues
Further impairment of local blood supply and extension of area of tissue damage
Profound toxaemia and shock; severe hypotension; multiple organ failure; coma; death
FigN4: Mode of action Сl. perfringens exotoxin (enzymatic lysis)
Cl. perfringens
Alpha toxin
(phospholipase C)
hydrolyses phosphoryichlorine in cell membrane
Cell death
Cell membrane
Lysis
FigN5: Mode of action Сl.tetani exotoxin (effects on nerve-muscle
transmission)
Continuous stimulation by excitatory transmitter
Cl. tetani

4. Excitatory
transmitter
Tetanospasmin
Inhibitory transmitter
release blocked
Inhibitory transmitter
FigN6: Mode of action Сl. botulinum exotoxin (effects on nerve-muscle transmission)
Сl. botulinum
ACH- containing vesicles
Motor
end plate
Stimulation blocked
Toxin blocks release of ACH
from vesicles
Muscle fibre
4. Student’s independent study program
1. Structure, staining , biochemical properties of causative agent of tetanus, gas gangrene and
botulism. Cultivation.
2. Toxin production and virulence factors of these causative agents.
3. Ecology and resistance.
4. Epidemiology of tetanus, gas gangrene and botulism. Pathogenesis. Clinical pictures.
5. Main methods of laboratory diagnosis of clostridiosis.

5. 6. Active and passive prophylaxis of clostridiosis. Preparation, which are used for this purpose.
Methods of their obtaining and determination of activity.
5. Students’ practical activities:
1To study morphology of gas gangrene Clostridia, C. botulinum and C. tetani in smears from the
culture and impression preparations (touch smears) from the tissues.Draw and record you
observation.
Note forms and localization of spores of different Clostridia species, staining reaction by Gram. C.
tetani appears by microscopy as a straight, slender rod with rounded ends.The spores are
sperical, terminal and bulging, giving the bacteria characteristic “drum stickâ€a appearance.
Cl. botulinum with oval, subterminal and swelling the sporangium spore resembles tennis racquet.
Сl.tetani.
Cl. botulinum
2.To inoculate a specimen (wound exudate) into Kitt-Tarozzi (Vrublevskyi) medium, Wilson-
Blair medium and milk.
Tested material (wound exudate) introduce into into Kitt-Tarozzi (Vrublevskyi) medium, Wilson-
Blair which has been cooled to 450 C and milk by bacterial loop (stab culture)
3. To familiarize with changing of media ( Kitt-Tarozzi (Vrublevskyi) medium, Wilson-
Blair medium and milk) after cultivation Cl. perfringens.
Into the Kitt-Tarozzi medium Cl. perfringens grow with intensive gas formation. On litmus milk
one can observe characteristic fermentation with lightening of the serum and formation of a
sponge clot of brick colour (“stormy clotâ€i reaction). In the Wilson-Blair medium occurs
black growth with ruptures.
These changing may be observed after 3-6 hours of growth Cl. perfringens. Therefore this is
rapid method of determination of Cl. perfringens in the tested material.
4. To familiarize with biological preparations which are used for laboratory diagnosis, active
and passive prophylaxis and treatment of gas gangrene, tetanus and botulism.
7. Control questions and tests
Select the correct answers.
1. A patient injured in an accident has been suspected of probable development of anaerobic
wound infection (gas gangrene). What is the most appropriate treatment before making a specific
laboratory diagnosis?

6. A. Plasma.
B. Toxoid
C. Typo specific immune serum
D. Polyvalent specific serum
E. Placenta gamma globulin.
2. Patient developed symptoms of bulbar paralysis: ptosis, diplopia, aphonia, dysphagia after
eating of tinned mushrooms. Botulism was diagnosed previously. What test should be used to
determine toxin type?
A. Neutralization
B. Agglutination.
C. Precipitation.
D. Complement fixation test
E. Immunofluorescence testing
.
3. The fish of domestic preparation was a reason of food poisoning. The fish was examined at
bacteriological laboratory. After cultivation in anaerobe condition there were revealed rods with
subterminal situated spores. What diagnosis is possible?
A. Botulism.
B. Salmonellosis.
C. Cholera.
D. Shigellosis.
E. Enteric fever.
4. The causative agents of gas gangrene have such properties:
ÐT – C. perfringens is motile; b – C. perfringens has a capsule; c – C. novyÑ– forms
spores; d – C. novyі has a capsule; e – C. septicum is motile.

7. 5. Features of growth of gas gangrene Clostridia:
ÐF – grow on simple media; b – on blood agar form hemolysis zones; c – on sugar broth
form pellicle; d – grow on Kitt-Tarozzi medium; e –colonies form in agar stab cultures.
6. Methods of laboratory diagnosis of gas gangrene:
ÐM – allergic test; b – bacteriological; c – neutralization test; d – CFT; e – biological.
7. The causative agent of tetanus has such morphological and staining properties:
ÐT – rod; b – produces capsule; c – has flagella; d – produces spores; e – Gram-
negative; f – Gram-positive.
8. Cultivation of C. tetani:
ÐC – aerobe; b – anaerobe; c – grows on simple nutrient media; d – grows on Kitt-
Tarozzi medium; e – zones of hemolysis are produced around the colonies on blood agar.
9. Toxin production of C tetani:
ÐT – produces enterotoxins; b – produces tetanospasmin; c – has necrotic factor; d –
produces tetanolysin; e – produces virulence enzymes.
10. Bacteria preparations used for passive and active immunization:
ÐB – Pertussis-diphtheria-tetanus vaccine; b – chemical associated adsorbed vaccine
which contains O- and Vi-antigens of typhoid, paratyphoid B, and a concentrated purified and
sorbed tetanus anatoxin; c – antitoxic antitetanus serum; d – BCG vaccine; e – associated
diphtheria-tetanus toxoid.
11. The causative agent of botulism has such morphological and tinctorial properties:
ÐT – is motile; b – has a capsule; c – produces spores; d – in stained preparation
occurs in chains; e – Gram-negative.
12. Cultivation of C. botulinum:
ÐC
it produces zones of hemolysis around colonies; d – it grows on Kitt-Tarozzi medium; e – it
grows on Ploskirev’s medium.
13. Toxin production of C. botulinum:

8. Р– produces exotoxins; b – does not produce hemolysin; c – produces neurotoxin; d
– produces enterotoxin; e – does not produce virulence enzymes.
14. Methods of laboratory diagnosis of botulism:
ÐM
15. What specific preparations are used for treatment and prophylaxis of botulism:
• anatoxin (toxoid); b – botulinuss vaccine; c – botulinum antitoxin; d – bacteriophage; e –
antibotulinum immunoglobulin.
16.The most important protective function of the antibody stimulated by tetanus immunization is:
A. to opsonize the pathogen (C.tetani)
B. to prevent growth the pathogen
C. to prevent adherence the pathogen
D. to neutralize the toxin of pathogen
17. Each of following statements concerning Clostridium perfringens is correct except:
A. It is an important cause of gas gangrene
B. It is an important cause of food poisoning
C. It produces an exotoxin that degrades lecithin and causes necrosis and hemolysis
D. It produces a toxin that inhibits the release of acetylcholine at the synapse
18. Each of following statements concerning Clostridium tetani is correct except:
A. It is a gram-positive, spore-forming rod
B. Pathogenesis is due to production of an exotoxin that blocs inhibitory neurotrasmitters
C. It is a facultative organism; it will grow on a blood agar plate of the presence of room air
D. Its natural habitat is primary the soil
19. Which one of following statements concerning immunization against diseases cased by
clostridia is correct?
A. Antitoxin against tetanus protects against botulism as well, because the two toxins share
antigenic sites
B. Vaccines containing alpha toxin (lecithinase) are effective in protecting against gas
gangrene

9. C. The toxoid vaccine against Clostridium difficile should be administered to
immunocompromised patients
D. Immunization with tetanus toxoid induces effective protection against tetanus toxin
20. Each of following statements concerning wound infection caused by Clostridium perfringens is
correct except:
A. An exotoxin plays a role in pathogenesis
B. Gram –positive rods are foud in the exudate
C. The organism grows only in human cell culture
D. Anaerobic culture of the wound site should be ordered
21. Each of following statements regarding tetanus is correct except:
A. The causative agent is gram-positive anaerobic rods
B. The antigen in the vaccine is tetanus toxoid, which is chemically modified toxin
C. The natural habitat of the organism is the soil.Infection typically occurs when spores enter
the body in contaminated wounds
D. The toxin lyses red blood cells, causing severe anemia and kidney failure
Real-life situation to be solved:
1. A patient injured the femur during transport accident. In the deep wound were left the pieces
of clothes and soil. In 2 hours after the accident the primary surgical treatment of the wound was
made and surgeon placed the sutures. In a day patient’s condition became grave. The femur
increased in its size, the skin became pale and lucid. Muscles look like boiled meat. There are
foamy discharge from the wound with unpleasant smell. During palpation of the tissues near the
wound the crepitaion was determined.
A. What disease may be in this patient?
B. What bacteria cause the disease?
C. What laboratory examinations can confirm the diagnosis?
D. What specific preparation is used for treatment?
2. A patient was injured during transport accident. After the primary surgical treatment of the
wound 3000 IU of antitetanic serum was injected to the patient.
A. How is it necessary to inject antitoxic serum to prevent complications.
B. How much time does this immunity last?
3. A patient with diagnosis of tetanus was admitted to the infectious diseases hospital. Two
months ago after the trauma he was injected with antitetanic serum. There were not any traumas
during last months.
A. Could tetanus develop in this patient?

10. B. What clinical findings testify about tetanus?
C. Why tetanus was developed in patient?
D. What mistakes did the doctor make?
E. What immune response does antitetanic serum create?
4. One family (3 persons) was hospitalized to the infectious disease department with
complaints of aphonia, disturbance of swallowing, double vision, ptosis of the upper lips, difficult
breathing, weakness in arms and legs. In a day they died. To determine the cause of intoxication
the examination of canned fish was perfumed.
A. What microbes can cause this disease?
B. What methods of diagnosis must be used for examination of food remains?
C. How canned fish was infected with these causative agents?
7. List of literature:
1.I. S. Gaidash, V.V. Flegontova, Microbiology, virology and immunology, Lugansk, 2004,
chapter18, p. 44-71.