Tubercle, Lond., (1965), 46, 65

AN IMPROVED NIACIN TEST

By J. MARKS

from/he Tttherc.losis Reference Laboratory (P.blic Health Laboratory Service). Cardiff

SUMMARY

A convenient technique is described for distinguishing between Myco. tuberculosis and other mycobacteria by the biological assay of niacin produced in subculture. Results are usually obtained in eight days. The method dispenses with the need for cyanogen bromide and enables results to be obtained with dysgonic strains.

R~SUM~

Une technique commode est d6crite pour distinguer Myco. tuberculosis des autres mycobac- t6ries par un essai biologique de production d'acide nicotinique en sub-culture. Les r6sultats sont habitueilement obtenus en 8 jours. La mfthode 6vite l'usage de brornure de cyanog6ne et permet d'obtenir des r6sultats avec les souches dysgoniques.

RESUMEN

Se describe una tdcnica adecuada para distinguir el Mycobaclerium tuberculosis de otros micobacterios empleando un mdtodo biol6gico de producciOn de niacina en el repique. Generalmente se tienen los resuitados al cabo de ocho dfas. El m&odo prescinde del uso de bromuro de cian6geno y permite obtener resultados con cepas disgbnicas.

All cultures of mycobacteria appear to liberate niacin, but exceptionally large amounts are produced by the human type of tubercle bacillus, Myco. tuberculosis (Pope & Smith, 1946; Konno, Kurzmann & Bird. 1957). in tests designed to identify M3,co. tuberculosis by this property, niacin is usually extracted with water fiom growth on solid medium and demonstrated by adding cyanogen bromide and aniline. The intensity of the yellow colour produced is not easily measured however, and cyanogen bromide is difficult to store and handle owing to its corrosive and poison- ous vapour. In addition, dysgon.ic strains of Myco. tuberculosis may not grow adequately on solid medium in a reasonable time, if at all. To avoid these difficulties, a convenient method of bio- logical assay has been devised of the niacin produced by subcultures in liquid medium.

Materials Glassware Containers should be new or reserved for assay work. Precautions are necessary against contamination with extraneous material containing niacin. Assa), medium Suitable medium is available commercially. Oxoid medium CM 21 i, 37-5 g./litre, can be recommended. Distribute it in convenient volumes, autoclave at 10 lb./10 rain. and store for up to 3 months.

66 T U il E R C L !-:

Test organism Grow LactobaciHus arab&osus 17-5, strain 8030 of the National Collection of Industrial Bacteria (Torry Research Station, Aberdeen) for 24 hours at 37~ in glucose broth. Stored at 4~ this culture can be used for at least 2 weeks. Withdraw a 2 ram. loop edgewise from it to inoculate 25 ml. of assay laaediuna. Larger inocula result in high blank readings in the test. t~Strichnlent medium--This medium has been described previously (Marks,1964). Alternatively, mix aseptically 100 ml. complete Dubos medium,* 0.2 nal. pyruvic acid (first neutralize), 0.3 ml. 0.4 ~o phenol red and 10 nal. citrated human plasma from out-dated transfusion blood. Dispense the medium in volumes of 1-5 ml. in sterile 7 ml. screw-capped bottles containing a few glass beads and incubate overnight to test sterility.

Niacin stamlards~Nicotinamide is preferable to niacin as it dissolves more easily. Autoclave a 0.1 ~>~i solution and dilute with sterile water to give standards containing 0.5 and 1.0 ~zg./ml. These may be stored at 0~ for many months.

Calibrated pipettes~Sterile Pasteur pipettes are used to measure and transfer 20 ~zl. of enrichnaent culture. Suitable marking allows their re-use. The pipettes may be calibrated with mercury ~ but a convenient alternative method is to attach translucent silicone rubber tubbing of 1 ram. internal diameter so that the free portion contains 20 ~I. (25.4 ram.).

Conduct of the test

If the primary growth is on solid medium, inoculate a loopful into a bottle of enrichment medium, partially dispersing it by rubbing or shaking. Much less growth is needed as an inoculum when the primary culture is in liquid medium, the equivalent of 0.1 ml. of a well-grown culture in Dubos medium being sufficient.

The test must not be carried out until definite growth occurs in the subculture. A week's incubation at 37~ is usually sufficient and may be taken as the norm but the period can be extended for dysgonic strains. By this means standardization of growth is possible, which although crude works well in practice.

Cultures which are sufficiently grown may be stored at 4~ until testing is convenient.

Add 20 bd. of the culture under test to 3 ml. of inoculated assay medium and incubate at 37~ for 24 hours. The test is not affected by mycobacteria carried over. Each batch of tests requires a blank and two standard estimations. The blank is used to detect contamination or extraneous niacin and is made by adding 20 bd. of the enrichment medium to 3 ml. of inoculated assay medium. The two standards are prepared similarly with the addition of 20 p.l. of nicotinamide solution, 0.5 and 1.0 tzg./ml, respectively.

Completed assays are read more easily after the addition of 9 ml. water to each bottle. A nephelo- meter or absorptiometer is not essential but if one is used it is advisable to sterilize the cultures to be diluted by heating at 100~ for 5 minutes.

Interpretation

Growth in the assay equal to or greater than that supported by the higher nicotinamide standard (1-0 gg./ml.) is a positive finding which identifies the tested strain as M),co. tuberculos& with little risk of error. Growth equal to or less than that supported by the lower nicotinamide standard (0.5 [xg./ml.) is defined as a negative finding, which normally excludes Myco. tuberculos&. Inter- mediate results should be considered doubtful. They are not common and are mostly due to the use of inadequately grown cultures. In such cases a positive result is usual if the culture is re-tested after a further week's incubation. Only slight growth should occur in the blank test.

* The formula should not include niacin in any form.

IMPROVED I~IACIN TEST 67

Discussion

The growth of L. arabh~osus is conveniently dense and directly proportional to the niacin con- ccntration when this lies between 2-5 and 10 m ~.g./mI. in the assay medium. The technique described is designed to satisfy these conditions whcn the cultures tested contain niacin at concentrations near the critical diagnostic level. It is therefore inadvisable to make anyal terat ion without con- sidering its effect on the dose/response aspects of the assay.

In preliminary tests of the technique, three strains each of the seven anonymous mycobacteria groups defined by Marks and Richards (1962) were tested together with six unclassifiable anonymous strains, three strains of Myco. avium from animals and 13 strains of Myco. boris proved to be pathogenic for rabbits. These all gave negative results after both one and two weeks" incubation of their cultures in enrichment medium. A series of 100 cultures undergoing routine sensitivity tests were also tested. These had been obtained from patients with pulmonary tuberculosis and were taken without any selection other than the exclusion of known 'anonymous' or Myco. boris infec- tions. After one week's incubation, 87 gave a positive and 13 a doubtful result. The latter were all positive after a second week's incubation. This positive series included 25 strains resistant to isoniazid.

The biological niacin test described above has been used in this laboratory with completely satisfactory results for the past two years to help distinguish between Myco. tuberculosis and Myeo. boris. It is not normally used as a rneans of recognizing "anonymous" mycobacteria because simpler and quicker methods are available

The method of assay described can be adapted to give a result in six hours instead of 24, e.g. by using a heavy inoculum of washed lactobacilli, but the advantage gained is seldom worth pursuing.

REFERENCES KONNO, K.. KURZMANN. R., & BIRD, K. T. (1957). Amer. Rev. Tuberc., 75, 529. MARKS. J. (1964). Tubercle, Lond.. 45, 47. MARKS, J. & RiCHARDS, M. (1962). Month. Bull. Mhl. Hhh. and Pub. Hlth. Lab. Serv., 21,200. PoPZ, l-llLD,~, & SMvrH, D. T. (1946). Amer. Rev. Tuberc., 54, 559,