Medical Oral Microbiology Topic A ­ 07
Name: Date:
A7, p. 1
Topic P07: Diagnostics of anaerobic bacteria
To study: Clostridium; spore non-forming anaerobes (from textbooks, WWW etc.)
From spring term: Microscopy, cultivation, biochemical identification, animal experiment, neutralisation
Table for major results of Task 1 to Task 4 (to be filled step by step):
Strain A B C D
Gram stain of a strain ­ Task 1b
(including eventual information concerning
spore formation)
Blood agar ("BA") Growth Y/N
VL agar ("VLA") Growth Y/N
VL broth Growth Y/N
Cult.­task32
Description of colonies on BA/VLA*
FINAL CONCLUSION (result of Task 4
­ AnaeroTest, or result of previous tasks
for non-anaerobes)
*Use VLA (VL agar) for bacteria not growing on BA (blood agar)
Task 1: Microscopy of a clinical specimen and microscopy of strain
a) Observation of a clinical specimen
Observe Gram stained smear.
You will probably find a mixture of various bacteria, as it
is typical for anaerobic infections, that usually not one
microbe, but a mixture of them are responsible for an
infection. Besides bacteria, you might see leucocytes
(mostly polymorphonuclears), eventually epiteliae, tissue
detritus and so on.
Do not forget to describe your picture (use lines)!
b) Microscopy of suspicious strains
Anaerobic bacteria could be cocci or bacilli, Gram positive or Gram negative; so it is not different from other
bacteria. On the other hand, anaerobes are much more pleomorphic. In genus Clostridium, the endospores are
used as an important diagnostic sign. Try to find endospores in one of your strains (robust G+ rods).
Task 2: Anaerobic jar and anaerobic box
To obtain anaerobiose, three ways are used in the laboratory:
a) for liquid media, paraffin oil is used as a barrier between the medium and the atmosphere (nit ideal)
b) solid media are placed into an anaerobic jar, where oxygen is chemically replaced by a mixture of gases
c) solid media might be also placed into an anaerobic box; anaerobic atmosphere comes from a cylinder
Add your description to the pictures of an anaerobic jar and an anaerobic box (you will see a real anaerobic jar
and pictures of both anaerobic jar and anaerobic box in the slideshow)
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Medical Oral Microbiology Topic A ­ 07
Name: Date:
A7, p. 2
Task 3: Cultivation on agar media
Describe cultivation results of the given strains on both aerobic and anaerobic media
a) Aerobic culture on blood agar (BA)
Write down, whether bacteria do grow or not, and eventually describe the colonies
b) Anaerobic culture on VL agar (VL blood agar)
VL (blood) agar is similar to blood agar, but it has decreased redox potential and it is cultured either in anaerobic
jar or anaerobic box. Write down what strains do grow on it and describe those not growing on BA
c) Propagation of anaerobic bacteria in VL broth
VL broth is used especially for cultivation of rare anaerobic bacteria. Check the presence of turbidity (= growth)
in VL broth, write it to the table and compare with the results of part b)
Task 4: Species diagnostics of anaerobic bacteria using biochemical tests
In strains, found to be anaerobes, read the biochemical microtest (ANAEROtest 23 by Lachema)
inoculated one day before. Read it according to the scheme. Attention! The codebook has four parts, so
you have to find a proper part according to the microscopy. Results of ,,B" and ,,A" columns are NOT used
for code counting. So, you obtain 6 position code: only for results of tests in columns H to C.
Strain: H G F E D C B A Code:
1 Identification:
2 % of probability:
4 Typicity index:
Code
Strain: H G F E D C B A Code:
1 Identification:
2 % of probability:
4 Typicity index:
Code
Notes:
Task No. 5 Susceptibility tests of anaerobic bacteria to antibiotics
Evaluate the diffusion disc susceptibility tests to antibiotics in strains found to be gram-negative cocci and that
are pathogenous. Into the table, write the abbreviation of the antibiotics according to a card and for all tested
strains measure the susceptibility zones. On your card, you have limit zones ­ according to them, interpret the
zones as susceptible (S) resistant (R) and dubious (D).
Strain
Antibiotic
(full name)
Zone  (mm) Interpr. Zone  (mm) Interpr.
Medical Oral Microbiology Topic A ­ 07
Name: Date:
A7, p. 3
Check-up questions:
1. Where are anaerobes present in healthy organism under physiological conditions? How can they be
transported to other tissues where they cause diseases?
2. What are principles of sampling and specimen transportation in case of suspicion for anaerobic
infections?
3. What anaerobic bacteria could by typically found in oral cavity and what bacteria could by involved in
pathological processes.
4. Name at least three antibiotics suitable for anaerobic infections treatment.