Medical Oral Microbiology Topic A - 03
Name: Date:
A3, p. 1
Diagnostic of some more Gram-positive bacteria (enterococci,
listeriae, corynebacteriae, bacilli)
Table for major results of Task 1 to Task 5 (to be filled step by step):
Kmen K L M N P Q R S
Gram staining ­
Task1
Size
Colour
Shape
Profile
Changes
in agar
Culti-
vation
(Blood
Agar)
Task 2
Other
Catalase test
Task 3a
Slanetz-Bartley
Task 3b
Bile-aesculin agar
Task 3c
Arabinose test
Task 4a
EnCoccus test
Task 4b
Growth in 4°C
Task 5a
Result*
* In G+ bacilli, write genus name only. Species level diagnostic would require more tests, that could not be
performed in our practital.
Task 1: Microscopy of microbial cultures
Gram-stain them and write your results to the table. Do not forget to write important details (,,rods in palisades",
,,robust, spore forming rods" etc.). The bacteria not being gram-negative are to be excluded from all remaining
tasks.
Task 2: Morphology of colonies of G+ cocci and bacilli
Describe the colonies as usually. Do not describe collonies of bacteria proven not to be G+ cocci or bacilli.
In strains, microscopically found to be gram-positive rods, try to guess, to which genus the bacterium might
belong, according to followindg description:
Medical Oral Microbiology Topic A - 03
Name: Date:
A3, p. 2
Bacillus ­ large, flat, dry, felt-like colonies, ,,spreading" through the agar surface, sometimes with a massive
haemolysis, sometimes with no haemolysisat all. Microscopically very robust rods, sometimes with finding of
central or subterminal spores, that may, but must not be larger then the diameter of the rod.
Listeria ­ colourless to greyish colonies, very simillar to those of Enterococcus, with or without haemolysis,
microscopically tinier than Bacillus, not arranged in pallisades, rather in short chains.
Corynebacterium (and related genera) ­ greyish or whitish colonies simillar to those of Staphylococcus, but less
or more smaller, usually ahaemolytical, in microscopy rather smaller than previous, but club-shaped and
arranged in palisades.
Task 3: Several common biochemical and culture tests
a) Catalase test
Perform catalase test for all strains proven to be G+. Mention, that Listeria, Corynebacterium and Bacillus are
positive, but some of conyneforms other than Corynebacterium (e. g. Arcanobacterium) are catalase negative.
b) Growth on Slanetz-Bartley medium
On your plate, the same strains as in Task 1 are cultivated in sectors. Positive strains should be not only growing,
but also maroon colour of colonies. Enterococcus is the only G+ bacterium growing on this medium. Write your
result to the table.
c) Growth on Bile-aesculin medium
Unlike Slanetz-Bartley medium, Bile-aesculin medium enables not only growth of Enterococcus (diagnostic for
this genus among G+ cocci), but also Listeria (diagnostic among G+ bacilli). Write your result to the table.
Task 4: Differentiation of enterococci
a) Arabinose test for species determination of two most common enterococci
Examine two strains proven to be enterococci in previous tasks. Observe the test tubes with the result of
arabinose test. Yellow colour means positivity (typical for Enterococcus faecium) and green colour means
negativity (typical for Enterococcus fecalis).
b) Biochemical test for species determination of enterococci from important
clinical materials (able to find more then two most importand species)
In important cases, we use rather a better species determination method than the arabinose test. We use a
biochemical tests in a microtitre plate. Notice, that the arabinose test takes part in this test, too. Mention, that
EN-COCCUS test is simple in comparison with Staphytest 16 and Streptotest 16. Read the results of the ENCOCCUStest
according to the instruction sheet in both strains from the previous task. Fill in the table bellow and
write your result to the main table.
Strain: H G F E D C B A Code:
Identification:
1 2 4 1 2 4 1 2 % of probability:
T index:
Strain: H G F E D C B A Code:
Identification:
1 2 4 1 2 4 1 2 % of probability:
T index:
Medical Oral Microbiology Topic A - 03
Name: Date:
A3, p. 3
Task 5: More methods for diagnostics of Listeria - growth of Listeria
at 4°C
Observe a plate with blood agar where the strains of gram-positive rods were inoculated, and the plates then
cultivated at refrigerator temperature. Write the results to the main table.
Task 6: Susceptibility tests of enterococci and gram-positive rods
to antibiotics
On your table, you will find diffusion disc tests for strains found to be Enterococcus faecalis, Enterococcus
faecium, Listeria sp. and Corynebacterium sp. There is no test for Bacillus sp.­ the findings of this genus is
usually interpreted as environmental contamination and thus not tested.
Write abbreviations of antibiotics according to the card and measure susceptibility zones for all tested strains.
Borderline zones are written on the cards; using them, interprete the strains as susceptible (S) resistant (R) and
dubious (D).
Strain
Antibiotic
(full name)
Zone 
(mm)
Interpr. Zone 
(mm)
Interpr. Antibiotic
(full
name)
Zone 
(mm)
Interpr. Zone 
(mm)
Check-up questions:
1. Which gram-positive rods are spore forming? Are the spores allways visible?
2. Why there are no cephalosporins in the set of antibiotics? (The set is primarilly used for enterococci.)
Medical Oral Microbiology Topic A - 03
Name: Date:
A3, p. 4
3. What are the VRE and what is their importance?
4. Which bacteria (other than Listeria) grow at the refrigerator temperature?
6. What is the use of Bacillus stearothermophilus and Bacillus subtilis in medical microbiology? (See J05.)