Medical Oral Microbiology Topic A ­ 06
Name: Date:
A6, p. 1
Diagnostics of some other gram-negative bacteria (Neisseria, Moraxella,
Bordetella, Legionella, Francisella.....)
Table for major results of Task 1 to Task 4:
Strain K L M N P
Gram stain of a strain ­ Task 1b
"Poor" BA ("KA") Growth Y/N
"Rich" BA+ ("KA+") Growth Y/N
Chocolate agar ("ČA") Growth Y/N
Culture­Task2
Description of colonies on BA+*
a) Oxidase test (+/­)Task
3 b) Indoxylacetate (INAC) test (+/­)
FINAL CONCLUSION (result of Task 4
­ NeisseriaTest, or result of Task 1 for
the strain proven not to be G­ cocci)
*Use ChA (Chocolate agar) for bacteria not growing on BA+
Task 1: Microscopy of a clinical specimen and microscopy of strain
a) Observation of a urethral smear in gonorrhea
Observe and Gram stained smear.
Pay attention not only for bacteria, but also for the cells of
macroorganism, especially leucocytes. Mention, that cocci
are not present in all white blood cells. Draw your result
and draw lines connecting the description with the objects
in your picture.
Note: Very similarly looks also a smear of purulent CSF
(cerebrospinal fluid) in meningococcal meningitis.
b) Microscopy of suspicious strains ­ search for gram-negative
cocci
There are letter-labelled strains on the table. Gram-stain them and write your results to the table. Observe them
and write your results to the table. Strain that is NOT G­ coccus should not be used in tasks 3 and 4 but in Task
2 it should be described, for comparison.
Task 2: Cultivation on agar media
Mark in the table, what bacteria grow on ,,nutritionally poor blood agar BA-", ,, rich blood agar BA+" and
chocolate agar. Oral species of Neisseri as well as Moraxella and majority of G+ cocci are also able to grow on
nutritionally poor BA . Neisseria meningitidis (,,meningococcus") can only grow on ,,rich" blood agar. Neisseria
gonorrhoeae (gonococcus) is not able to grow on blood agar at all, chocolate agar is needed. After that, describe
the colonies of all strains on BA+. If the strain does not grow it should be described on chocolate agar. Write all
your results to the table.
white blood cells
G­ diplococci
________________
________________
________________
Medical Oral Microbiology Topic A ­ 06
Name: Date:
A6, p. 2
Task 3: Basic biochemical tests for gram-negative cocci
Both tests will be done as a demonstration at a side table. Write your results to the table.
a) Oxidase test
Your teacher will touch several colonies of strains identified as G- cocci by the oxidase diagnostic strip. Blue
colour should appear in several seconds, when positive. Draw a positive and a negative result.
b) Indoxylacetate test
The procedure is similar as that of oxidase test, but the strip should be moistened in advance, the colour is rather
blue-green instead blue and it is not visible immediately, but it is necessary to wait several minutes. Draw a
positive and a negative result.
Task 4: Species diagnostics of Neisseria and Moraxella using
biochemical tests
In strains, found to be gram-negative cocci, read the biochemical microtest (NEISSERIAtest by Lachema)
inoculated one day before. Read it according to the scheme. The first well contains negative control, so the
proper test starts in the SECOND well! Dropping of Lugol solution was already done, you should not do it
yourselves. Remark low biochemical activity of some Neisseria. Compare the result with grow requirements (the
strain, found to be N. gonorrhoeae, should grow on chocolate agar only; the strain, found to be N. meningitidis,
on chocolate and modified blood agar only).
Strain: H G F E D C B A
NEC Code: Identification:
× 1 2 4 1 2 4 1
×
Strain: H G F E D C B A
NEC Code: Identification:
× 1 2 4 1 2 4 1
×
Strain: H G F E D C B A
NEC Code: Identification:
× 1 2 4 1 2 4 1
×
Strain: H G F E D C B A
NEC Code: Identification:
× 1 2 4 1 2 4 1
×
Medical Oral Microbiology Topic A ­ 06
Name: Date:
A6, p. 3
Task No. 5 Susceptibility tests of G- cocci 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. Zone 
(mm)
Interpr. Zone 
(mm)
Interpr.
Task No. 6 Culture Diagnostics of Bordetella
There is a special medium for Bordetella pertussis, and a special way of inoculation is used here. Unlike many
other bacteria, Bordetella is resistant to penicillin; so we start by making a drop of penicillin solution in the
middle of the agar plate. The swab is mixed with the drop, and inoculated in a spiral form. Then the loop is used
to make radial rays. Write down the name of the medium, and re-draw the way of its inoculation from your
slideshow.
Name of the medium: ____________________
Check-up questions:
1. What specimens are recommended to take for gonorrhoea diagnostics? And how should they be transported to
the laboratory?
2. When taking CSF specimen, can you see any differences between healthy person and a person with purulent
meningitis? (Just at the patient, not in the laboratory.)
3. Neisseria and Moraxella are both gram-negative. Does this mean that they grow on Endo agar?
Medical Oral Microbiology Topic A ­ 06
Name: Date:
A6, p. 4
4. What species of Neisseria are the less biochemically active? What sugars do they split?
5. What causative agent of meningitis is the most common one in pre-scholar age, what in teens, what in elder
people?
6. What is the most typical source of tularaemia infection?