Carbohydrate Utilization by N. meningitidis: Cystine
Trypticase Agar (CTA) Method
Carbohydrate utilization tests are used to validate the identification of a
strain as N. meningitidis. For this procedure, 4 different carbohydrates (glucose [also called
dextrose], maltose, lactose, and sucrose) are added to tubes containing a CTA base for a final
concentration of 1%. A phenol red indicator is also included in the medium. It is a sensitive indicator
that develops a yellow color in the presence of acid at a pH of 6.8 or less.
A panel of four tubes, each containing a different carbohydrate, is used to
test each isolate. Neisseria spp. produce acid from carbohydrates
by oxidation, not fermentation. N. meningitidis oxidizes glucose
and maltose, but not lactose or sucrose. While it is extremely rare,
strains of N. meningitidis have been reported to either utilize
glucose or maltose, but not both. Well-characterized QC strains should be
tested along with the unknown isolates to ensure that the CTA sugars are
working properly.
N. meningitidis and/or N. lactamica isolates should be used to QC the CTA sugar media.
Methods for preparation of the CTA sugar media are included in the Annex.
CTA sugars should be stored at 4°C and warmed to room temperature (25°C)
before use.
A. Performing CTA sugar testing
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. Allow the 4 CTA sugars, glucose (also called dextrose), maltose,
lactose, and sucrose, to warm to room temperature (25°C) and label the tubes with the lab ID.
3. Remove 3-5 colonies from overnight growth on the BAP using a 1 μl
disposable loop.
4. Stab the CTA sugar several times into the upper 10 mm of medium.
Approximately 8 stabs with the same loopful are sufficient. Use a separate disposable loop for
inoculating each carbohydrate to be tested.
5. Fasten the screw-cap of each tube loosely and place the tubes in a
35-37°C incubator without CO2.
Incubate the CTA sugars for at least 72 hours (and up to 5 days)
before discarding them as negative.
6. Observe the CTA sugars for development of visible turbidity and color
change to yellow.
7. Perform steps 1-5 using a N. meningitidis, N. lactamica, and a N. sicca QC strain to ensure that the CTA sugars are working properly.
B. Reading the CTA sugar results
Development of visible turbidity and a yellow color in the upper portion of
the medium indicates growth of bacteria and production of acid and is interpreted as a positive
test (Figure 5).
Although reactions may occur as early as 24 hours after inoculation, some
reactions are delayed and negative results should not be interpreted before 72 hours of
incubation. A color change to yellow without turbidity is usually not a positive reaction. Carbohydrate
utilization results for differentiating N. meningitidis from other Neisseria spp.
and bacteria are listed in Table 1.
Thanks For Visiting ! Keep Your Healthy !
0 comments:
Post a Comment