Rapid Carbohydrate Utilization Test (RCUT) for Neisseria

Traditionally, cystine trypticase agar (CTA) supplemented with a 1% carbohydrate solution was used to determine acid production by Neisseria and related species. This test is no longer recommended now and has been replaced by the rapid carbohydrate utilization test (RCUT).  In this test, patterns of acid production from the carbohydrates – glucose, maltose, lactose, sucrose, (and fructose) – are used to identify Neisseria and related species. Production of acid from carbohydrates is indicated by a change in the color of the phenol red indicator from red to yellow.

Principle

Small (0.10 mL) aliquots of phosphate-buffered saline solution containing phenol red as the pH indicator are distributed in a series of tubes. These tubes are labelled as glucose, maltose, sucrose, and lactose.

Single drops of the individual carbohydrates (20% w/v) are added to individual tubes according to the labelling e.g. 20% w/v of glucose is added in the tube labelled as glucose.

A heavy suspension of the test isolate is prepared in the buffer without carbohydrates. One drop of this suspension is added to each of the labelled tubes.

Neisseria species utilize the carbohydrate source present in the tubes and produce acid by an oxidative pathway (which is in contrast to other organisms which produces acid by fermentative pathway). The acid thus produced when exceeds the buffering capacity of the solution, the color of the medium changes from red (alkaline) to yellow (acid).

Rapid carbohydrate utilization tests is also done for the identification of Aggregatibacter aphrophilus (formerly called Haemophilus aphrophilus, one of the HACEK organisms), A. actinomycetemcomitans and Gardnerella vaginalis.

Media and Reagents

A. Phosphate Balanced Salt Solution (BBS) (a 10× stock solution having the following formula)

• Dipotassium phosphate (K₂HPO₄): 0.40 g
• Monopotassium phosphate (KH₂PO₄): 0.10 g
• Potassium chloride (KCl): 8.0 g
• Distilled water: 100 mL

Filter-sterilize and store at 4°C–8°C

B. Working Solution

Add 10 mL of the 10× BSS to 90 mL of distilled water. After this, 0.5–0.8 mL of a 1% aqueous solution of phenol red is added to the solution, so that the final product is “cherry red.” This working solution is then filter-sterilized.

To ensure that the distilled water has the proper pH, the use of pharmacy-grade sterile distilled water is recommended.

C. Stock Carbohydrate Solutions

Weigh out 10 g each of glucose, maltose, sucrose, and lactose individually. Each is dissolved in 50 mL of distilled water. The solutions are filter-sterilized, dispensed into sterile vials, and frozen at 20°C.

It is important that “reagent-grade” carbohydrates be used, because the maltose from some bacteriologic media vendors may be contaminated with glucose.

Sample: Pure culture (18h-24h) of Gram-negative, oxidase-positive, catalase-positive diplococci grown on nonselective (chocolate agar or equivalent) medium.

Procedure

Carbohydrate tube preparation

1. Label a series of sterile 12 × 75-mm tubes with the carbohydrate to be tested (i.e. glucose, maltose, sucrose, and lactose) and add 0.10 mL of working BSS in each tube.
2. Add a single drop of each of the carbohydrates to the appropriately labeled tube with a Pasteur pipette.

Inoculum Preparation

1. Take a sterile tube and label it with the isolate or specimen number.
2. Dispense 0.30–0.40 mL of BSS in it.
3. Transfer few isolated colonies of the test organism (18-24 hour culture from chocolate agar) with a sterile bacteriologic loop.
4. Vortex the tube carefully to obtain a uniform, heavy suspension.

Mixing and Incubation

1. Add a single drop of inoculum suspension to each of the carbohydrate-containing tubes.
2. Agitate the tubes briefly to ensure thorough mixing.
3. Place the tubes in an aerobic incubator or water bath at 35°C for 4 hours.

Note: Carbohydrate tubes should NOT be incubated in an incubator with carbon dioxide-supplemented atmosphere. Carbon dioxide may diffuse into the medium and form carbonic acid which may cause a false-positive acid reaction.

Results and Interpretations

The development of yellow color in any of the carbohydrate-containing tubes indicates utilization of that carbohydrate by the organism. Negative carbohydrate-utilization tests remain red or red-orange.

Acid production patterns of Neisseria species and M. catarrhalis

Commercial Application

CarboFerm Neisseria Kit from Hardy Diagnostics is a rapid method for the identification of N. gonorrhoeae, N. meningitidis, N.sicca, N.lactamica and Moraxella catarrhalis isolates. CarboFerm Neisseria kit consists of an 8-well reagent cuvette that comes packaged as 12 strips snapped into a plastic frame. Based on the principle of the rapid carbohydrate utilization test, this kit gives results within 4 hours.

References and further reading:

1. Winn, W. C., & Koneman, E. W. (2006). Koneman’s color atlas and textbook of diagnostic microbiology. Philadelphia: Lippincott Williams & Wilkins.
2. Kellogg DS, Holmes KK, Hill GA. Cumitech 4. In: Marcus S, Sherris JC, eds. Laboratory Diagnosis of Gonorrhea. Washington, DC: American Society for Microbiology, 1976.
3. Acid detection test, Centers for Disease Control and Preventions.
4. Kulkarni, S., Bala, M., & Risbud, A. (2015). Performance of tests for identification of Neisseria gonorrhoeae. The Indian journal of medical research, 141(6), 833–835. doi:10.4103/0971-5916.160721
5. CarboFerm Neisseria kit. Hardy Diagnostics