Identification and Characterization of Haemophilus Influenzae
H. influenzae
are small, pleomorphic, gram-negative bacilli or coccobacilli with random arrangements. H. influenzae is a fastidious organism which grows
best at 35-37°C with ~5% CO2 (or in a candle-jar) and requires hemin (X factor) and
nicotinamide-adenine-dinucleotide (NAD, also known as V factor) for growth. The standard medium used for
growth of H. influenzae is a chocolate agar plate (CAP), which can
be prepared with heat-lysed horse blood, a good source of both hemin and NAD, although sheep blood can also be used.
Growth occurs on a CAP because NAD is released from the blood during the heating process of chocolate agar preparation (the heating process also inactivates growth inhibitors) and
hemin is available from non-hemolyzed as well as hemolyzed blood cells.
Alternatively, NAD can be included as a component of liquid H. influenzae growth media supplements, (available commercially or
prepared in the laboratory), which are incorporated into the chocolate
agar. H. influenzae appear as large, round, smooth, convex,
colorless-to-grey, opaque colonies on a CAP.
Encapsulated strains appear more mucoidal than non-encapsulated strains,
which appear as smaller, compact grey colonies. No hemolysis or discoloration of the CAP is
apparent. While H. influenzae
produce a pungent indol smell, plates should not be opened in order to
smell the cultures. H. influenzae cannot grow on an unsupplemented BAP.
Prior to identification and characterization testing procedures, isolates should always be inspected
for purity of growth and a single colony should be re-streaked, when necessary, to obtain a pure
culture. For the following identification and characterization procedures,
testing should be performed on 18-24 hour growth from a CAP at 35-37°C with
~5% CO2 (or in a candle-jar).
The following tests are recommended to confirm the identity of cultures
that morphologically appear to be H. influenzae (Figure 3). H. influenzae can be identified using Kovac’s oxidase test and determining the necessity of hemin and NAD as growth requirements. If
the oxidase test is positive, hemin and NAD growth factor requirement testing should be
performed. If the growth factor requirement test indicates that the isolate may be H. influenzae, serological tests to identify the serotype should be performed.
This sequence of testing is an efficient
way to save costly antisera and time. Additional methods for identification
and characterization of H. influenzae using molecular tools are described in Chapter 10: PCR Methods and Chapter
12: Molecular Method Biosafety Level 2 (BSL-2) practices are required for work involving
isolates of H. influenzae, as this organism presents a potential
hazard to laboratory personnel and the surrounding working environment.
Kovac’s oxidase test
Kovac’s oxidase test determines the presence of cytochrome oxidase. Kovac’s oxidase reagent, tetramethyl-p-phenylenediamine dihydrochloride, is turned into a purple compound by organisms containing cytochrome c as part of their respiratory chain. This test aids in the recognition of H. influenzae, but other members of the genus Haemophilus, as well as unrelated bacterial species, may also give a positive reaction. Positive and negative quality control (QC) strains should be tested along with the unknown isolates to ensure that the oxidase reagent is working properly.
D. Preparation of 1% oxidase reagent from oxidase powder
To prevent deterioration of stock oxidase powder, the powder should be stored in a tightly sealed
desiccator and kept in a cool, dark area. Kovac’s oxidase reagent is intended only for in vitro diagnostic use. Avoid contact with the eyes and skin as it can cause irritation. In case of accidental contact, immediately flush eyes or skin with water for at least 15 minutes.
1. Prepare a 1.0% Kovac’s oxidase reagent by dissolving 0.1 g of tetramethyl pphenylenediamine dihydrochloride into 10 ml of sterile distilled water.
2. Mix well and then let stand for 15 minutes The solution should be made fresh daily and the unused portion should be discarded. Alternatively, the reagent could be dispensed into 1 ml aliquots and stored frozen at -20°C.
The aliquots should be removed from the freezer and thawed before use. Discard the unused portion each day the reagent is thawed.
E. P erforming Kovac’s oxidase test
Filter paper method
1. Grow the isolate(s) to be tested for 18-24 hours on a CAP at 35-37°C with ~5% CO2 (or in a
candle-jar).
2. On a nonporous surface (i.e., Petri dish or glass plate), wet a strip of filter paper with a few drops of Kovac’s oxidase reagent.
3. Let the filter paper strip air dry before use.
4. Use a disposable plastic loop, a platinum inoculating loop, or a wooden applicator stick to pick a portion of a colony from overnight growth on the CAP and rub it onto the treated filter paper.
Do not use a nichrome loop, as it may produce a false-positive reaction.
5. Observe the filter paper for color change to purple.
6. Perform steps 3 and 4 with a positive and negative QC strain to ensure that the oxidase reagent is working properly.
Reading the oxidase test results
-Positive reactions will develop within 10 seconds in the form of a purple color where the bacteria were applied to the treated filter paper. Delayed reactions are unlikely with H. influenzae .
-Negative reactions will not produce a color change on the treated filter paper.
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