Use of ["4C]Lysine to Detect MicrobialContamination in - Applied and ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1978, p. 1211-1212 0099-2240/78/0035-1211$02.00/0 Copyright © 1978 American Society for Microbiology

Vol. 35, No. 6 Printed in U.S.A.

Use of ["4C]Lysine to Detect Microbial Contamination in Liquid Foods P. MAFART,'* C. BOURGEOIS,' B. DUTEURTRE,2 AND M. MOLL2 Centre de Recherche Agroalimentaire de Quimper, Universi de Bretagne Occidentale, 29191 Quimper, France,' and Centre de Recherches et Developpement Tepral, 54250 Champigneulles, France2 Received for publication 14 December 1977

A radiometric method for microbiological control in food industries is suggested. This method, based on the labeling of cells by ['4C]lysine, was tested by using nine species of yeast and two species of bacteria.

The best-known radiometric technique consists of providing microorganisms with a "Clabeled sugar and detecting metabolic "CO2. Another radiometric technique, proposed by MacLeod et al. (2, 3), consists of providing cells with a labeled compound and then collecting the cells on a membrane filter to measure their radioactivity. MacLeod et al. (2, 3) used 32Plabeled phosphate; the minimum that could be detected was 5 x 104 cells in 1 h. Our technique is based on a similar phenomenon, but we used "C-labeled amino acid compounds. Our method differs from that of MacLeod in several other respects. Of the labeled amino acids, lysine yielded the best results in previous studies (1); therefore, only amino acid was used in the present work. The liquid sample to be analyzed was filtered through a membrane filter (Millipore filter with hydrophobic edge; diameter, 47 mm; porosity, 0.8 ,um; Millipore Corp., Bedford, Mass.), which retains most of the cells. The membrane was then laid on an absorbing pad which was impregnated with 1 ml of a suitable culture medium to which ['4C]lysine (0.2 ,uCi/ml, 120 mCi/mmol) had been added. After an incubation period, the membrane was put back into a filter apparatus and rinsed with a detergent solution (DDN-50;

Hydro-Mesures, Asnieres, France) containing 0.02% lysine. This procedure results in the elimination of extracellular radioactivity. The membrane was dried, and its activity was measured with a scintillation spectrometer. The radioactivity fixed in a given time is an indication of the presence or absence of microorganisms and is thus a means of sterility control. At first the technique was used for testing the sterility of pasteurized beer and thus was adapted to the detection of Saccharomyces carlsbergensis (1). The present work was aimed at improving and achieving greater precision in detection of S. carlsbergensis as well as determining if the detection of other microorganisms was possible.

Wickerham's yeast carbon base (9) (pH 5.5) with added S04(NH4)2 and ['4C]lysine, was used as the yeast detection medium. For Escherichia coli, the same medium was used, but the pH was adjusted to 7. For Lactobacillus casei, the culture medium used was VLB L41 (8); to avoid dilution of ['4C]lysine by cold lysine of this complex medium, it was necessary to eliminate basic amino acids by chromatography (5) and then to restore arginine and histidine levels before adding ['4C]lysine. From the detection performance point of view, the technique is essentially characterized by the minimum detection time (MDT). The MDT is the time after which the radioactivity of a membrane which has retained one or more cells is significantly higher than that of a membrane devoid of cells (C0). After an incubation time of 9 h at 28°C and a rinsing volume of 500 ml, the mean value (22 assays) for Co was 253 cpm, and the experimental standard deviation a was 32 cpm. Beyond 9 h, CO and its a may be considered as constant. Therefore, the MDT of a microbial species in the course of a sterility control at a guarantee level of 95% is the incubation period needed by an initial cell and its daughter cells, emerging during incubation, to fix a radioactivity equal to 3.3 a (106 cpm). For S. carlbergensis the MDT was about 21 h. In our culture medium, S. carlsbergensis grew with a generation time of 3.2 h and, in 21 h, completed 6.6 generations, amounting to 102 cells. The MDT for eight other yeasts (S. anomala, S. chevalieri, S. diastaticus, S. italicus, S. pastorianus, and S. subpelliculosa and Candida lipolytica and C. tropicalis) ranged from 8 to 34 h. The MDT for E. coli was 9 h; for L. pastorianus the MDT was 3 days. Our method is considerably less sensitive for the detection of bacteria than for that of yeast. We are trying to balance this difference by improving culture media of bacteria to get higher growth rates. This technique thus seems suitable for the

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detection of various microorganisms. Compared with the '4CO2 technique, our technique has the advantage of measuring all the radioactivity that has been absorbed; in the former technique, only a part of the ["C]glucose radioactivity that the cells absorb is measured as '4CO2. The result is that for the same performance, a radioactivity level 10 times lower is sufficient. It is difficult to compare the performance of our technique with those in which 32P is used (2, 3). The apparent advantage of using '4C-labeled amino acids or nucleotides, rather than sugars, is that their growth-limiting concentrations are very low; this low concentration permits the use of high specific radioactivity with low total activity without limiting the growth rate. Among the amino acids and nucleotides, lysine gave the best results with S. carlsbergensis (1); this was due to the particular behavior of this yeast toward lysine (1, 6, 7). Lysine does not have the same advantages with all yeasts (4) or with bacteria, but the following factors favor its use: (i) most contaminating microorganisms in food are saprophytic and absorb amino acids actively; (ii) [4C]ilysine is used at a very low concentration; thus it does not act as a carbon or nitrogen source, is not toxic, is not catabolized, and remains in the cells; and (iii) lysine consti-

APPL. ENVIRON. MICROBIOL.

tutes a high proportion of the amino acids of microbial protein. LITERATURE CITED 1. Bourgeois, C. M., P. Mafart, and D. Thouvenot. 1973. Methode rapide de detection des contaminants dans la biere par marquage radioactif Eur. Brew. Conv. Proc. Cong. 14:219-230. 2. MacLeod, R. A., M. Light, L. A. White, and J. F. Currie. 1966. Sensitive rapid detection method for viable bacterial cells. Appl. Microbiol. 14:979-984. 3. MacLeod, R. A., L. A. White, and J. F. Currie. 1970. Detection of Aerobacter aerogenes by labeling with radioactive phosphorus. Appl. Microbiol. 19:701. 4. Mafart, P., C. M. Bourgeois, B. Duteurtre, and M. Moll. 1976. Radiometric method for control of filtration and pasteurization. Tech. Q. Master Brew. Assoc. Am. 13:157-160. 5. Nair, J. H. 1953. Chromatographic separation of some amino acids. Anal. Chem. 25:1912-1919. 6. Ramos, F., P. Thuriaux, J. M. Wiame, and J. Bechet. 1970. The participation of ornithine and citrulline in the regulation of arginine metabolism in Saccharomyces cerevisiae. Eur. J. Biochem. 12:40-47. 7. Romkes, S. C. E., and M. J. Lewis. 1971. Some factors which affect aminoacid uptake by Saccharomyces carlsbergensis. Appl. Microbiol. 21:799-805. 8. Wackerbauer, K., and C. C. Emeis. 1967. Methoden den Brauereibiologischen Betriebskontrolle. I. Nahrboden zum Spurennachweis der stabchenffirmigen Milchsaurebakterien. Monatsschr. Brau. 20:160-164. 9. Wickerham, L. J. 1951. Taxonomy of yeasts. U.S. Dep. Agric. Tech. Bull. 1029.