Medical Oral Microbiology Topic A - 05
Name: Date:
A5, p. 1
Diagnostics of Haemophilus and G­ non-fermenting rods
Table for major results of Task 1 to Task 5:
Strain K L M N P Q R S
Gram stain ­ Task 1
Growth on
BA (Y/N)
Growth
characteristics
on
BA (ChA*)
Endo agar
(­/L-/L+)
Cul-
ture
Task
2
MH agar
(colour)
Task 3a Satellite
phenomenon (+/­)
Task 3b Factor test
(X, V, X + V)
Glc fermentation
Task 4 (Hajna)
Oxidase test
Task 5a
NefermTest 24
(Task 5b)
FINAL
CONCLUSION
*Use ChA (Chocolate agar) for bacteria not growing on BA (blood agar)
Task 1: Microscopy of 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 (but in Task 2 it should be described, for comparison)
Task 2: Cultivation on agar media
First write down, what bacteria do grow on blood agar and what bacteria do not. Then, using standard procedure,
describe colonies of all strains on blood agar. In strains that did not grow on blood agar, describe their growth on
Chocolate agar instead. Then describe growth of bacteria on Endo agar (only ,,­" for not growing bacteria, ,,+"
for growing ones; lactose positivity/negativity cannot be seen, as the strains do not have isolated colonies) and
on MH agar (only ,,­" or ,,+", and eventually presence of specific colour).
Medical Oral Microbiology Topic A - 05
Name: Date:
A5, p. 2
Task 3: Identification of Haemophilus and their more precise
determination
a) Satellite phenomenon
Haemophili are typical by so named satellite
phenomenon. That means that they are not able to grow
on blood agar, unless a strain able to release growth factor
necessary for haemophili is present. Usually
Staphylococcus aureus is used for this purpose. Draw the
satellite phenomenon and connect the terms below with
the features on your picture
b) Identification of the hemophili on the basis of growth factors
necessity
Determine the given strains according to their requirements of growth factors. Draw the growth factor
tests for both strains.
Task 4: Hajna medium
Observe the results of culture of four strains on Hajna medium. Mark strain able to ferment glucose (yellow
colour) as ,,+", strains unable to ferment (red colour) as ,,­"
Task No. 5 Determination of G­ glucose non-fermenters
a) Oxidase test
Demonstration of oxidase test for three strains shown to be G­ non-fermenters. Write down your results to the
table. (Pseudomonas, should be always positive, Burkholderia is usually positive, too, but not necessarily; on the
other hand, Stenotrophomonas uses to be negative).
Staphylococcus aureus Colonies of haemophili
Medical Oral Microbiology Topic A - 05
Name: Date:
A5, p. 3
Oxidase positive bacteria with typical odour and pigmentation (mostly green, less often blue of maroon) is quite
sure Pseudomonas aeruginosa. In this bacterium it is not necessary to perform further biochemical testing,
described in Task 5a. In other two strains this biochemical testing is necessary.
b) Detailed biochemical testing
Evaluate given results of NEFERMtest 24, been prepared two days before (difference from other biochemical
tests) at 30 °C (another difference, other test require 37 °C). The way of code reading is different, too, as there
are three rows in the test. Always upper row is ,,1" when positive, medium row is ,,2" and lowest one ,,4". First
number is for oxidase test: write ,,0", when negative, and ,,1", when positive. Results of ,,B" and ,,A" columns
are NOT used for code reading. So, you obtain 7 position code: first number is ,,0" or "1", and six more positions
are for results of tests in columns H to C.
Strain: OX H G F E D C B A Code:
1 Identification:
2 % of probability:
4 Typicity index:
Code
Strain: OX H G F E D C B A Code:
1 Identification:
2 % of probability:
4 Typicity index:
Code
Notes:
Task No. 6: Susceptibility tests of pathogenic bacteria to antibiotics
Among your bacteria, there are five pathogens: two of Pasteurellaceae family, three G­ non-fermenters. Write
full names of antibiotics and measure susceptibility zones. Interpret the strains as susceptible (S) resistant (R)
and dubious (D). (Dubious are such stains that have the zone just the same as is the limit.)
Test for Haemophilus
Strain Strain
Antibiotic
(full name)
Zone  (mm) . Interpr. Antibiotic
(full name)
Zone  (mm) Interpr.
Test for Gram non-fermenters:
Strain
Antibiotic
(full name)
Zone 
(mm)
Interpr. Zone 
(mm)
Interpr. Zone 
(mm)
Interpr.
Medical Oral Microbiology Topic A - 05
Name: Date:
A5, p. 4
Task No. 7 Relations of bacteria to oxygen ­ comparison of
enterobacteria, G­ non-fermenters and anaerobes
Look at the broth cultivated under aerobic and anaerobic conditions (layer of paraffin oil on the surface),
evaluate bacterial growth and its character.
Strain
Growth in broth
Growth in VL-broth
Conclusion
Check-up questions:
1. Under what conditions are the hemophili able to growth on Blood agar? Why?
2. What is the most typical material for Pasteurella multocida findings?
3. Which hemophilus species is the most pathogenic? Which diseases it causes?
4. Why we have less newborn infections caused by Haemophilus in recent years?
5. Why it is usually not necessary to perform biochemical tests in Pseudomonas aeruginosa?
6. What are the most typical infections caused by G­ non-fermenters?
7. Make proposal of antibiotics suitable for treatment of infections caused by G­ non-fermenters.