456 Analyst, July, 1968, Vol. 93, pp. 456457 The Use of Depleted Cells as Inocula in Vitamin Assays BY L. GARE (Beecham Research Laboratories, Vitamins Research Station, Walton Oaks, Dorking Road, Tadworth, Surrey) Preparation of assay inocula in such a way that the cells contain a reduced level of the vitamin to be assayed gives low background growth in plates, and the greater contrast enhances zone definition. Steps required to be taken to find the optimum conditions for preparation of inocula are described. The assays of nicotinamide, pantothenic acid, folic acid and vitamin B, can be improved in this way. A FAMILIAR problem in microbiological assays is high “blank” growth. In our early attempts to assay folic acid by a plate (agar-diffusion) method, in which Lactobacillus helveticus was used, there was insufficient contrast between background growth and response zone.The inoculum was prepared by growing cells in “enriched culture medium”l for 18 hours and then washing twice with 0-85 per cent. saline. (The levels of the relevant B vitamins in the “complete” medium used were established by microbiological assay and are shown in Table I.) Further washing of the cells with 0.85 per cent. saline reduced the background growth, but only slowly and incompletely, as if the cells could store folic acid. In our first attempt at depletion, the cells were washed, re-suspended and incubated in a volume of folic acid free medium equal to that of folic acid rich medium (“enriched culture medium”) in which they were grown; this treatment gave no reduction in background growth.When cells grown in folic acid rich medium were diluted and incubated in a volume of folic acid free medium such that considerable increase in cell numbers could take place, background growth in plates was reduced, and the contrast was sufficiently great for zones to be well defined. The depleted cells are prepared by growing in “enriched culture medium” at 37” C for 18 hours, and a small volume is diluted fifty times in folic acid free medium, incubated for 7 hours and used without washing. It appears that cells growing in vitamin-rich media can accumulate vitamins to such an extent that, on being diluted and incubated in a medium deficient of one vitamin, they can divide until they are depleted of the vitamin absent in the medium.Because this treatment of L. helveticus cells in folic acid assay improved zone quality, attempts were made to improve other assays in the same way. The plate assays of vitamin B, with Saccharomyces carlsbergensis (with an inoculum grown initially on malt extract - agar), and pantothenic acid and nicotinamide with LactobaciZZus arabinosus (with an inoculum grown initially on “enriched culture medium”), have been improved in this way (see Table I). The suspensions can be stored in the refrigerator (about +5” C) for at least 1 week and are suitable for use in tube assays. The details of techniques for producing depleted inocula would probably differ between organisms, strains, media and laboratories. The steps required to be taken to find the optimum conditions for depletion are as follows.PROCEDURE- (1) Dilute a small volume of culture grown in a vitamin-rich medium with a medium free from the appropriate vitamin, incubate and determine, by taking viable counts, at intervals, when multiplication has ceased. (2) Dilute graded volumes of culture grown in a vitamin-rich medium with a medium free from the appropriate vitamin, incubate for the length of time indicated in (1) and measure the extent of multiplication by taking viable counts. This will indicate the minimum volume of culture which, on dilution and incubation, gives the maximum number of cells, These cells will be fully depleted. (3) Determine the optimum concentration of depleted cells for use in an assay plate. 0 SAC and the author. This is the depletion time.GARE TABLE I VITAMIN LEVELS IN MEDIA FOR INOCULA Amounts per ml of medium 457 r Pantothenic acid Nicotinamide Folic acid B‘, 2.3 ng - - 0.14 pg “Enriched culture medium” 0.27 p g 1.5 rg Malt extract - agar* ..- - * Difco malt extract 4-6 per cent.; agar 1-5 per cent. That micro-organisms can accumulate vitamins and then multiply in the absence of one of them has been known for some time,2s3s4 and the incubation of cells in media low in, or free from, vitamin has been advocated for assay inocula. Gibbons and E ~ ~ g l e , ~ for example, to demonstrate menadione qualitatively, prepared menadione-deficient Bacteroides melanino- genicus cells by incubating the organism in broth free from menadione “where limited growth occurred.” Simpson6 recommends growing L.helveticus for 16 to 18 hours in 100ml of single-strength assay medium containing riboflavin, washing and re-suspending the cells in the same volume of single-strength assay medium, without riboflavin, and incubating for 16 to 18 hours. In our experience, the cells must be able to multiply in order to deplete, and to effect this the cells should be diluted before they are incubated in the vitamin-free medium. Strohecker and Henning’ put forward the idea of depletion, but in no instance do they incubate in the absence of the vitamin to be assayed, and they describe no plate assays. The mechanism of the accumulation and depletion of vitamins is not yet known in detail or fully understood. Work has begun towards a better understanding of this phenomenon, and it is hoped to publish the results in due course. I thank Mr. S. A. Price for helpful criticism of the text. REFERENCES 1 . 2. 3. Meissel, M. N., Nature, 1947, 160, 269. 4. 5. 6. 7 . The Association of Vitamin Chemists Inc., Editors, “Methods of Vitamin Assay,” Third Edition, Knight, B. C. J. G., Vitam. Horm., 1945, 3, 139. Toennies, G., Das, D. N., and Feng, F., J . Bact., 1966, 92, 707. Gibbons, R. J., and Engle, Lois P., Science, N.Y., 1964, 146, 1307. Simpson, J. S., in Kavanagh, F., Editor, “Analytical Microbiology,” Academic Press Inc., New Strohecker, R., and Henning, H. M., “Vitamin Assay; Tested Methods,” Verlag Chemie, GmbH, Received September 19th, 1967 Interscience Publishers, New York, London and Sydney, 1966, p. 52. York and London, 1963, p. 115. Weinheim, 1965, p. 22.