Identification and Characterization of Neisseria Meningitidis
N. meningitidis
are gram-negative, coffee-bean shaped diplococci that may occur
intracellularly or extracellularly in PMN leukocytes. N. meningitidis is a
fastidious organism, which grows best at 35-37°C with ~5% CO2 (or in a candle-jar). It can grow on both a blood
agar plate (BAP) and a chocolate agar plate (CAP). Colonies of N. meningitidis are grey
and unpigmented on a BAP and appear round, smooth, moist, glistening, and convex, with a clearly
defined edge.
N.meningitidis
appear as large, colorless-to-grey, opaque colonies on a CAP. 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 BAP
(Figure 1) or a CAP (Figure 2) 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 N. meningitidis (Figure 3). N. meningitidis
can be identified using Kovac’s oxidase test and carbohydrate utilization. If the oxidase test is positive,
carbohydrate utilization testing should be performed. If the carbohydrate utilization test indicates that
the isolate may be N. meningitidis
, serological tests to identify the serogroup should be performed. This
sequence of testing is an efficient way to save costly antisera and time. Additional
methods for identification and characterization of N. meningitidis using molecular tools are
described in the next article.
Biosafety Level 2 (BSL-2) practices are required for work involving
isolates of N. meningitidis, as this organism presents a potential hazard to laboratory personnel and
the surrounding working environment. Please refer to Chapter 4: Biosafety in order to follow the
guidelines that have been established for laboratorians working in BSL-2 facilities as many of
the tests described in this chapter require opening plates with live cultures and are often
performed outside of a biosafety cabinet (BSC).
I. 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 N. meningitidis, but other members of the genus Neisseria, 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.
A. 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.
Read Also : Microbiology Laboratory : MacConkey Agar Media
B. P erforming Kovac’s oxidase test
Filter paper method
1. Grow the isolate(s) to be tested for 18-24 hours on a BAP 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 BAP and rub it onto the treated filter paper (Figure 4).
-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 Positive Result
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 N. meningitidis .
· Negative reactions will not produce a color change on the treated filter paper.
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