Medical Oral Microbiology Topic A ­ 04
Name: Date:
A4, p. 1
Topic P04: Diagnostics of enterobacteria and bacterial agents of
gastrointestinal infections
Table for major results of Task 1 to Task 5 (to be filled step by step):
Strain K L M N P Q R S
Gram stain ­ Task 1
Size on
BA
Colour
on BA
Other
on BA
Size on
Endo
Colour
on Endo
Culture
(blood
agar and
Endo
agar)
Task 2
Other
on Endo
Hajna medium
Task 3a
Oxidase test
Task 3b
PARTIAL
CONCLUSION
XLD
agar
MAL
agar
More
media
Task 4a
CIN
agar
EnteroTest 16
(Task 4b)
Antigen analysis
(Tasks 5a and 5b)
FINAL
CONCLUSION
Task 1: Microscopy of suspicious strains
There are letter-labelled strains on the table. Gram-stain them and write your results to the table. Strain that is
NOT G­ rod should not be used in tasks 3 to 5. Draw the results of your observation.
Task 2: Cultivation on blood agar and Endo agar
Using standard procedure, describe colonies of all strains on blood agar and Endo agar. If the strain on the
medium does not grow, cross the corresponding cell in the table. Bacteria, that do not grow on any of the media
and morphologically look like curved gram-negative rods, might be Campylobacter. A G­ rod, that does not
grow on any of the media, but is not curved, will be studied in the next lesson. For comparison describe also the
Medical Oral Microbiology Topic A ­ 04
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A4, p. 2
strain, that appeared morphologically as a gram-positive coccus. Stain just one strain and share the reset with
other student around the table. Using the dermograph, mark the slides with corresponding letter.
Task 3: Group diagnostics of the most important gram-negative
rods growing on Endo agar
a) Reading of an examination on oblique agar according to Hajna
Agar according to Hajna is a combined diagnostic medium. Nevertheless, in this task we will mostly search for
biochemically non-active, neither glucose nor lactose splitting and sulphan non forming rods ­ the gram-negative
non-fermenting bacteria (,,non-fermenters"). All strains, growing on Endo, were inoculated on Hajna medium.
Have a look to the result. Where the medium remained fully red, it is a biochemically non-active strain ­ very
likely, a gram-negative non-fermenter. This strain will not be used in Task 4 and Task 5.
b) Oxidase test
The teacher will do as a demonstration oxidase test for all Gram-negative, on Endo agar growing bacteria.
Oxidase-positive are members of family Vibrionaceae and some gram-negative non-fermenters; the
Enteobacteriaceae are (with exception of Plesiomonas) oxidase negative.
Make partial conclusion after tasks 1 to 3. What bacteria are enterobacteria? Tasks 4 and 5 will be only
performed with strains proven to be enterobacteria.
Task 4: Genus and species determination of enterobacteria using
cultivation and biochemical tests
a) Cultivation of enterobacteria on other media
You have already seen, how the colonies look like on BA and Endo agar. Exam and describe the appearance of
the enterobacterial colonies on CIN, XLD and MAL (fill the table).
b) Biochemical behaviour of enterobacteria
Evaluate given results of ENTEROtest 16, incubated a day before. Check, whether the results comply with
others, already done tests; e.g. strains with sulphan formation lead to black colour of Hajna medium, Yersinia has
tiny pink colonies, Salmonella pale transparent colonies with black centre on XLD and MAL medium...
For the strain found to be Salmonella, write Salmonella sp. only. Count % of probability as a total of all % of
probability of individual Salmonellas at the code; T index should be taken from the first Salmonella.
ONPG 1H 1G 1F 1E 1D 1C 1B 1A 2H 2G 2F 2E 2D 2C 2B 2A
1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2
Strain:
Code: Identification % of prob. T index
ONPG 1H 1G 1F 1E 1D 1C 1B 1A 2H 2G 2F 2E 2D 2C 2B 2A
1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2
Strain:
Code: Identification % of prob. T index
ONPG 1H 1G 1F 1E 1D 1C 1B 1A 2H 2G 2F 2E 2D 2C 2B 2A
1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2
Strain:
Medical Oral Microbiology Topic A ­ 04
Name: Date:
A4, p. 3
Code: Identification % of prob. T index
ONPG 1H 1G 1F 1E 1D 1C 1B 1A 2H 2G 2F 2E 2D 2C 2B 2A
1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2
Strain:
Code: Identification % of prob. T index
Task No. 5 Antigen analysis in diagnostics of enterobacteriae excluding
of EPEC
In strain identified as Escherichia coli, perform antigen analysis using slide agglutination with two polyvalent
sera. If both results will be negative, the strain does not belong into EPEC group.
Task No. 6: Susceptibility tests of enterobacteria to antibiotics
On your table, you will find diffusion disc tests for strains found to be Enterobacteriaceae. 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, interpret the strains as susceptible (S) resistant (R) and dubious (D).
Strain
Antibiotic
(full name)
Zone 
(mm)
Interpr. Zone 
(mm)
Interpr. Zone 
(mm)
Interpr. Zone 
(mm)
Interpr.
Task No. 7 Diagnostics of Campylobacter
Observe the cultivation appearance that did not grow neither on BA nor on Endo agar and which, according to
the morphology, is supposed to be a Campylobacter (because of being curved), on a special medium. Remember
four main conditions for cultivation of Campylobacter: (a) special medium with charcoal and addition of
antibiotics and antimycotics to prevent growth of other microbes, (b) microaerofilic conditions, (c) temperature
elevated to 42 °C, what correspons to body temperature of birds ­ natural hosts, and (d) prolongation of the
cultivation to 48 hours.
Describe the colonies write down the result of oxidase test (teacher will perform it as a demonstration). For
Campylobacter a retarded positivity is typical, e. g. the strip becomes blue, but not immediately, but after a
while.
Description of colonies Result of oxidase test More notes
Medical Oral Microbiology Topic A ­ 04
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Task No. 8: Urease test in diagnostics of Helicobacter
In diagnostics of helicobacters we use the urease test, performed directly with a bioptic specimen of gastric
mucosa. Excised tissue is placed on the surface of media contains urea and pH indicator. A positive result is red,
negative yellow. Among two specimens (X and Y) find the positive one.
Result: Positive urease test was found in specimen ____, negative in specimen ____
Task No. 9 Diagnostics of the family Vibrionaceae
Vibrionaceae is a bacterial family similar to Enterobacteriaceae, but oxidase-positive. We use special media to
culture Vibrionaceae. Mutual differentiation is possible through biochemical tests like for enterobacteria. Even
Enterotest 16 could be used, but a special codebook is required. Antigen analysis could be used, too. Draw here,
how a Vibrio looks like microscopically, and add some more properties according to the slideshow.
Most important solid medium for Vibrio:
Most important liquid medium for Vibrio:
The two most important serovars of V. cholerae
Microscopy:
The two most important biovars of V. cholerae O1
Check-up questions:
1. Do you know, what is the result of the catalase test in enterobacteria?
2. For practical reasons, one medium used in diagnostics of enterobacteria is missing: ­ selenite broth. What type
of medium is it and what is its use? (See practical J03)
3. Do you know at least some antigenic types of EPEC?
4. What pathogen is diagnosed by Widal reaction? Is it a direct, or indirect method? Which type of reaction is it?
5. Is it recommended to use antibiotic treatment for intestinal infection? Why?
6. In which clinical material it is more likelly to find Salmonella Typhi in typhoid fever rather than in the stool?