6.15.

MICROBIOLOGICAL DIAGNOSIS OF INFECTIONS

CAUSED BY ANAEROBIC BACTERIA.
The essence of the laboratory work:
A. Knowledge the main genera of anaerobic bacteria, their biological properties, ecology, ways of
spreading.
B. Knowledge the principles of anaerobic bacteria pure cultures isolation.
C. Knowledge the principles of microbiological diagnosis of anaerobic infections.
D. Knowledge the pathogenesis of anaerobic infections, immunity, treatment and prevention.
The plan of self-dependent work:
1. Biological properties of anaerobic bacteria, patterns of respiration.
2. Methods and schemes for isolation of pure cultures of anaerobic bacteria.
3. The main genera of anaerobic bacteria, their ecology.
4. Clostridia: morphological, biochemical, biological characteristics, ecology, pathogenesis,
species.
5. Pathogens of tetanus and gas gangrene. The main factors of pathogenesis.
6. Role of C.difficile in infectious pathology.
7. Microbiological diagnosis of gas gangrene.
8. Specific prophylaxis and therapy of tetanus, gas gangrene.
9. Bacterioides, Prevottela, Porphyromonas: classification, biological properties, ecology.
Microbiological diagnosis of caused diseases.
10. Fusobacteria, Veilonella, Propionibacteria: biological properties, ecology, caused diseases by
them and their microbiological diagnosis.
The tasks for the laboratory work:
Task 1. After extraction of a tooth, alveolus of patients tooth root suppurated. Pus was taken for
microbiological analysis and inoculated into the 2 Petri dishes with blood agar. The first (#1) plate
has been cultivated in anaerobic packets, and the second (# 2) - in aerobic conditions. Susceptibility
to Kanamycin, Vancomycin, Bile and metronidazole has been established. Biochemical activities
and antibiogram has been performed using an isolated bacterial culture.
Evaluate the results of microbiological analysis:
bacterias growth in Petri dishes (#1 and # 2), characterize grown colonies;
morphology and staining by Gram method;
identify isolated bacterial culture;
fill in the table No 1 and make a conclusion.

Results
Conclusion:

N-acetyl
glucosamine

Sucrose

Xylose

Salicin

Glucose

Eskulin

Indole

Catalase

Vancomycin

Bile

Kanamycin

Properties of
isolated culture

Gram stain

Table No 1. Identification of isolated microorganism

Morphology

Task 2. A patient is diagnosed with intraabdominal abscess. The abscess appeared after an
operation for colon cancer. Pus was taken for microbiological analysis and plated onto blood agar.
The first (#1) Petri dish has been cultivated in anaerobic packets. An aerobic condition was used for
the second (#2) bacterial culture. Susceptibility to Kanamycin, Vancomycin, Bile and
metronidazole has been established. Biochemical activities and antibiogram has been performed
using an isolated bacterial culture.
Evaluate the results of microbiological analysis:
bacterias growth in Petri dishes (#1 and #2), characterize grown colonies;
morphology and staining by Gram method;
identify isolated bacterial culture;
fill in the table No 2 and make a conclusion.

Rhamnose

Arabinose

Sucrose

Trehalose

Indole

Catalase

Vancomycin

Bile

Kanamycin

Pigment

Gram stain

Properties of
isolated culture

Morphology

Table No 2. Identification of isolated microorganism

Results
Conclusion:

Task 3. A man is diagnosed with a lung abcessus. Pus was taken for mocrobiological analysis and
inoculated into the 2 Petri dishes with blood agar. The first (#1) plate has been cultivated in
anaerobic packets, and the second (# 2) - in aerobic conditions. Susceptibility to Kanamycin,
Vancomycin, Bile and metronidazole has been established. Biochemical activities and antibiogram
has been performed using an isolated bacterial culture.
Evaluate the results of microbiological analysis:
bacterias growth in Petri dishes (#1 and # 2), characterize grown colonies;
morphology and staining by Gram method;
identify isolated bacterial culture;
fill in the table No 3 and make a conclusion.

N-acetyl
glucosamine

Sucrose

Xylose

Salicin

Glucose

Eskulin

Indole

Catalase

Vancomycin

Bile

Kanamycin

Gram stain

Properties of
isolated culture

Morphology

Table No 3. Identification of isolated microorganism

Results
Conclusion:

Task 4. Suspecting gas gangrene in a gunshot wound, the wound discharge has been obtained and
plated onto blood agar and inoculated into thioglycolate medium. The blood agar plates incubated

anaerobically for 24 hours at 37. The growth inoculated onto the egg yolk agar and
lecitinase/lipase activity has been established. Biochemical activity has been performed.
Evaluate the results of microbiological analysis: morphology of bacteria and staining by Gram
method. The results fulfil in the table No4 and identify the species of bacteria.

Glucose

Esculin

Urease

Indole

Lipase

Lecithinase

Gram stain

Properties of isolated
culture

Morphology

Table No 4. Identification of isolated microorganism.

Results
Conclusion:

Appendix:
Table 5. Differential characteristics of anaerobic Gram negative rods.
Genus
Bacteroides
Prevotella
Pigmentation of
V
colonies
Kanamycin
R
R
Bile
R
S
Vancomycin
R
R
Colistin
R
V
Catalase
V
R resistant, S sensitive, V variable.

Porphyromonas
+

Fusobacterium
-

R
S
S
V
-

S
V
R
S
-

+
+

+
+
+
+
+

+
+
+

Catalase

Arabinose

+
+
+

Rhamnose

Sucrose

B. fragilis
B. vulgatus
B. distasonis
B.ovatus
B. uniformis
B. splanchnicus

Trehalose

Species

Indole

Table5a. B i o c h e m i c a l a c t i v i t y o f B a c t e r o i d e s s p p .

+
+
+
+
+

+
+
+
+

N-acethyl
glucosamine

Sucrose

+
+

Xylose

+
+
+

Salicin

Prevotella oralis
Prevotella buccalis
Prevotella intermedia
Prevotella melaninogenica
Prevotella denticola
Porphyromonas asacharolytica
Porphyromonas gingivalis
Porphyromonas endodentalis

Glucose

Indol

Species

Hydrolysis of
eskulin

Table 5b. Biochemical activity of Porphyromonas spp. and Prevotella spp.

+
+

Table6. B i o l o g i c a l p r o p e r t i e s o f
Species

Lecithinase

Lipas Indole
e
C. perfringens
+

C. novyi
+

+
C. septicum

C. histolyticum

C. tetani

C. difficile

Note: all Clostridum spp. are gram positive rods.

Glucose

+
+

Hydrolysis of
esculin

-hemolysis

F. neerophorum
F. nucleatum
F. ulcerans

Lipase

Species

Indol

Table5c. B i o c h e m i c a l a c t i v i t y o f F u s o b a c t e r i u m s p p .

Clostridium spp.
Urease

Esculin

Glucose

+
+
+

Questions for the self-control:

1. What microorganisms are called anaerobs?
2. How to isolate a pure culture of anaerobic bacteria?
3. What do we call clostridia? The main species of this genera?
4. Do clostridia belong to the normal human microflora?
5. Can clostridia form spores in the intestines of a healthy man?

6. Do clostridia produce exotoxins in the intenstines of a healthy man?

7. Is tetanus infectious disease?
8. Can a patient have bacteriaemia if he has tetanus?
9. What can be the sourse of tetanus bacteria?
10. Can we get infection with tetanus from a person having this disease?
11. Is microbiological diagnosis of tetanus necessary?
12. What factors of tetanus pathogenesis do you know?
13. What factors of pathogenesis do gas gangrenes pathogens secrete?
14. Is it possible that gas gangrene is monoinfection? Why?
15. What is the source of pathogens of gas gangrene?
16. How is anatoxin of tetanus obtained?
17. What is PDT vaccine?
18. How and why is tetanus seroprophylaxis done?
19. What are possible complications after injection of antitetanic serum? What is its
mechanism?
20. What diseases can C.perfringens and C.difficile cause? What are their mechanisms?
21. What is the difference between genera of Bacteroides, Prevotella, Porphyromonas and what
is the difference of them from Clostridia ?
22. Do genera of Bacteroides, Prevotella, Porphyromonas belong normal human microflora?
23. What factors of pathogenesis do Bacteroides spp., Prevotella spp. and Porphyromonas spp.
have, what infections do they cause ?
24. What is characteristic for bacteria of genera of Fusobacterium, Propionobacterium? Their
ecology, ways of spreading, caused infections.
25. Is specific active and passive prophylaxis of infections caused by genera of Bacteroides,
Prevotella, Porphyromonas possible ?