Survival of Lactobacillus leichmannii in Relation to Vitamin B12 Assays

APPLIED MICROBIOLOGY, May, 1965 Copyright @ 1965 American Society for Microbiology Vol. 13, No. 3 Printed in U.S.A. Survival of Lactobacillus leichmannii in Relation to Vitamin B12 Assays JOSEPH A. VALUl Bacteriology Section, Good Samaritan Hospital, Phoeni, Arizona Received for publication 27 January 1965 ABSTRACT VALU, JOSEPH A. (Good Samaritan Hospital, Phoeni, Ariz.). Survival of Lactobacillus leichmannii in relation to vitamin B12 assays. Appl. Microbiol. 13:486-490. 1965.- Since washed cells of Lactobacillus leichmannii ATCC 7830 frequently showed erratic results in vitamin B12 assays, a depletion technique was used to stabilize the inoculum. The method consisted of incubating the twice-washed cells at 37 C for 45 min in distilled water. Survival and growth studies indicated that the stabilizing procedure (i) did not affect cell numbers, (ii) was optimal for depleting possible carry-over of vitamin B12 or other nutrilites and reserves, and (iii) brought about a phasing of the cells of the inoculum which underwent logarithmic growth immediately on inoculation. This minimal period (45 min) was the same when the cells were incubated in detrose-water. Survival of the cells in physiological saline was greater than in distilled water, but a longer time was required for stabilizing the inoculum in saline, which precluded its use in routine work. The prepared inoculum showed an improvement over the washed inoculum in that readings between duplicate and triplicate tubes agreed closely and the standard curves were reproducible whether acidimetric or turbidimetric methods were followed. No erratic results were noted in more than 1 year of testing. In the preparation of inoculum with Lactobacillus leichmannii ATCC 7830 for the vitamin B12 assay procedure, the bacteria are usually washed one to three times before use (Hoff- Jorgensen, 1954). In our eperience, such assays frequently showed erratic results in spite of strict precautions in the cleaning of glassware and setting up of the test. In an effort to improve the procedure, we considered the possibility that the mere washing of the cells was not sufficient to remove the nutrients which might be carried over on the surface of the cells or in intracellular pools. These nutrients might conceivably affect the physiological state of the inoculum and cause the erratic results. In addition, since the assay procedure is sensitive to micromicrogram amounts of the vitamin, etraneous sources of B12 introduced into the test via the inoculum would be objectionable for methodological reasons. This study was undertaken to determine whether vitamin B12 or interfering growth substances, or both, are carried over in the washed cells, and, if so, how best to ehaust them before utilizing the cells as an inoculum. Culture Collection, Washington, D.C. This strain is an auotroph requiring vitamin B12 for growth (Hoffmann et al., 1948), and is used in the USP method for assaying the vitamin. L. leichmannii has been found to respond to thymidine (Skeggs et al., 1948) as well as to other deoyribosides (Kitay, McNutt, and Snell, 1949). The refrigerated stock was subcultured every month and stored in tubes of Micro Assay Culture Agar (Difco). Lactobacilli were grown in tubes containing 10-ml amounts of Micro Inoculum Broth (Difco). After 15 to 24 hr at 37 C, the cells were centrifuged at 3,000 rev/min in an International centrifuge (model V or UV) and washed twice in sterile distilled water (deionized) or physiological saline (10-ml amounts) in identical fashion; the final pellet was suspended in 10 ml of the same or B12 Assay Broth (Difco) (B12-free broth) for the survival and growth studies, with or without prior incubation. Cells were also suspended in detrose-water, and their survival, as well as subsequent growth characteristics in B12- free broth, was studied. Finally, the washed and prepared inocula were seeded in Micro Inoculum Broth for the eperiments on synchrony. Viable counts. In the standard protocol used in every instance, 1-ml amounts of the test substance were diluted by 10-6 in dilution bottles containing 99 ml of sterile distilled MATERIALS AND METHODS water; 0.5 ml of the final dilution was assayed in duplicate for colonyforming ability by inoculation into 10 ml of fluid Organism. L. leichmannii ATCC 7830 (USDA strain 313) was obtained from the American Type 1.5% Micro Inoculum agar which was tubed and kept in a water bath (55 C). The inoculated agar I Present address: Department of Microbiology, was immediately poured into petri plates (disposable) and allowed to gel; another tube of the Scientific Associates, Inc., St. Louis, Mo. 486

VOL. 13, 1965 VOL. 1, 1965L. LEICHMANNII FOR VITAMIN B12 ASSAYS48 487 same medium was used as a top layer. Since L. 10 leichmannii is a micro aerophile, thioglycolate 9 agar was not necessary. Growth was registered by well-defined isolated colonies in 48 hr under the 8 top layer, but less distinctly without it. Colony 7 counts on duplicate plates seldom differed more than -i-4%; counts differing more than -47% were e not included in the results. All incubations were at 37 C. - Preparation of the inoculum. Cells were washed 2 twice in sterile distilled water as described above. The final pellet of cells was then suspended in 10 ml of sterile distilled water and incubated in a A water bath (37 C) for 45 min; 0.2 ml was then B added to 80 ml of sterile distilled water, and two 3 drops (no. 18 needle) of the diluted prepared >- inoculum were added to each assay tube by use of 3 C 0 a 20-ml acid-washed sterile glass syringe. RESULTS2 To determine the survival of twice-washed cells in distilled water at 37 C, the final pellet was suspended in 10 ml of sterile distilled water in a tube, and the number of cells was assayed at intervals. As shown in Fig. 1, there was no significant change in cell numbers for about 45 mriin, after which time there was a precipitous ' 0 45 90 135 180 225 270 315 drop to approimately half the initial count. At FIG. 2. Survival of twice-wa-shed cell-s in B12-free B 7 li, 10 ~~~~~~~~~~~broth at 87 C. Symbols: 0 and A indicate two sur- 9 ~~~~~~~~~~~vival eperiments which show an almost doubling of - cell numbers; 0 indicates the same increase in cell numbers even after si washings. about 90 min, the numbers leveled off or slightly 6 -increased, only to drop to an even lower level II C after 270 min (not shown). Z- B The survival of twice-washed cells in B12-free broth at 37 C was studied by suspending the A cells in the broth and assaying the number of ~~~~~~~~~~~survivors at intervals. Figure 2A and B show z ~~~~~~~~~~~~that the twice-washed cells increased in numbers 03 to almost double the initial count when they >_ ~~~~~~~~~~were incubated in vitamin B12-free broth. As o-j shown in Fig. 2C, cells had the same ability to 0 ~~ ~ ~ ~ ~ ~ ~ ~~nraeee inraein nubrsee a4fte i' i washings.iti ~ i noteworthy that the curves differed with different 2 culture growths. In another eperiment, the cells were washed as above, but were incubated for various periods of time at 37 C in sterile distilled water before being suspended in B12-free broth at 37 C and assayed at intervals. When the washed cells were incubated for 45 min in distilled water prior to I I I I I I ~~~~seeding in vitamin B12-free broth, there was a 0 45 90 135 180 225 270 315 lihreua Slgh 1eida division after which the numbers FIG. 1. Survival of twice-washed cells in distilled leveled off for as long as 11.5 hr (Fig. 3A); 30-mmn water at 37 C. Results of three survival eperiments prior incubation (Fig. 3B) was not sufficient to are -shown. Similar results were obtained when 0.25 deplete the reserve nutrients; and 60 min (Fig. mg per 100 ml of detrose-water was used. 3C) proved deleterious and brought about an

488 VALU APPL. MICROBIOL. In a final eperiment, the cells were washed twice in distilled water and were then either immediately seeded in Micro Inoculum broth (washed inoculum) or incubated in distilled water for 45 min prior to seeding (prepared inoculum). Figure 5A shows that, when the washed cells were immediately seeded in Micro Inoculum broth, the cell numbers increased for the first 60 min, decreased for the net 60 min, and then underwent logarithmic-phase growth. This log growth was achieved as soon as the incubated inoculum was seeded in Micro Inoculum broth (Fig. 5B), indicating that the cells were "phased" during the incubation period. DIscuSSION Our results show that vitamin B12 or other interfering nutrilites carried over on washed cells of the L. leichmannii inoculum or retained in intracellular pools alter the physiological state of the inoculum and cause the erratic results frequently eperienced with vitamin B12 assay. Since L. leichmannii ATCC 7830 is an auotroph FIG. 3. Survival of twice-washed cells in B12-free broth after incubation in distilled water at 37 C for various periods of time. Symbols: 0 indicates growth pattern after 45 min of prior incubation; A indicates pattern after 30 min of prior incubation; 0 indicates pattern after 60 min of prior incubation. Pattern after 45 min of prior incubation in 0.25 mg per 100 ml of detrose-water was somewhat similar to that obtained when distilled water was used for 45 min of incubation. erratic growth pattern. Neither 30- nor 60-min prior incubation stabilized the inoculum as well as 45 min. When the cells were washed twice in physiological saline and then suspended in physiological saline at 37 C, there was no significant change in cell numbers for 270 min (Fig. 4A). The cells evidenced a slight though definite increase in numbers after they were washed twice in physiological saline, incubated in physiological saline for 1 hr, and then suspended in vitamin B12-free broth at 37 C (Fig. 4B). In another similar eperiment, after 2 hr of incubation, the viable count taken over a period in vitamin B12-free broth showed the same slight but definite replication. When the cells were washed twice in distilled water, incubated in 0.25 mg per 100 ml of detrose-water for 45 min, and then resuspended in B12-free broth at 37 C, the curve resembled that obtained in plain distilled water (Fig. 3A). 9 _ 8 7 6 83 0. 0 u 0 45 90 135 ISO 225 270 315 FIG. 4. (A) Survival of cells washed twice in physiological saline, suspended in the same, and incubated at 37 C. (B) Growth of cells washed twice in physiological saline, incubated in the same for 1 hr, and then suspended in Ba-free broth at 37 C. Results of a 2-hr incubation in saline were similar to those for 1 hr. I

VOL. 13, 1965 L. LEICHMANNII FOR VITAMIN B12 ASSAYS 489 FIG. 5. Growth in Micro Inoculum broth of cells which were: (A) washed twice in distilled water; (B) washed in distilled water and incubated for 45 min in distilled water at 37 C. requiring vitamin BI, (Hoffmann et al., 1948), the ability of washed cells to replicate in vitamin B12-free broth (Fig. 2) indicated the presence in the inoculum of the vitamin or other growth factors (Skeggs et al., 1948; Kitay et al., 1949). To deplete these reserves under starvation conditions, a technique similar to that described by others (Aronson and Spiegelman, 1958; Horowitz, Saukkonen, and Chargaff, 1958; Harold, 1963) was used. Twice-washed cells were incubated in distilled water, detrose-water, and physiological saline; the optimal period for depletion was determined as judged by the inability of cells to replicate in vitamin B12-free broth. Survival studies of L. leichmannii in distilled water indicated that the cell numbers dropped to almost half the initial count after about 45 min (Fig. 1); thereafter, the numbers leveled off for some time, but the continuing deleterious effect brought about a further reduction to an even lower count at about 270 min. Few cells survived in distilled water; it was found, nevertheless, that for about 45 min there was no adverse effect on cell numbers. When such cells were seeded in vitamin B12-free broth, they showed an inability to replicate to any appreciable etent for almost 12 hr (Fig. 3A). It is noteworthy that washing the cells as many as si times could not bring this about (Fig. 20). It appeared that 45 min was optimal for the depletion process; the cells still showed replication after 30-min starvation and 60-min treatment was deleterious as shown earlier (Fig. 1), with an erratic growth pattern (Fig. 30). An interesting finding was that 0.25 mg per 100 ml of detrose-water did not decrease the time at which the depletion process was completed; in fact, the result was identical to that obtained with distilled water (Fig. 3). This might indicate that an etracellular energy source was not necessary to the process. It was not surprising that the survival of cells in physiological saline was best, because none of the precipitous falls in cell numbers occurred (Fig. 4A). However, incubation for as long as 2 hr was not sufficient to stabilize the inoculum (Fig. 4B). This precluded the use of saline in routine work. Eperiments on synchrony indicated that the washed cells were not "in phase" and took about 120 min before they underwent logarithmic growth (Fig. 5A). When the prepared inoculum was used in an identical eperiment, the cells began logarithmic growth almost immediately, indicating that the stabilized cells were also phased in the process. With this method of inoculum preparation, the serum vitamin B12 assays for over 1 year have shown none of the erratic results previously noted with the usual washed inoculum. Acidimetric titrations between duplicate and triplicate tubes agreed closely, and standard curves were stable and reproducible. It should be pointed out that a limited number of observations indicated that a normal assay achieved with the washed inoculum showed little, if any, difference in the actual amount of the vitamin assayed. One other worker (P. B. Goins, personal communication) using the prepared inoculum in a turbidimetric method on pharmaceutical preparations also noted consistency in readings between duplicate tubes and growth curves. It is concluded that the method shows promise of achieving a consistent and, therefore, a more dependable assay of vitamin B12 in serum and commercial preparations. Similar techniques which might be of use with other microbiological inocula are under investigation. ACKNOWLEDGMENTS This investigation was supported by an assistantship in Bacteriology at Good Samaritan Hospital, for which the author wishes to thank James D. Barger and Joseph J. Likos, the past and present Director of Laboratories.

490 VALU APPL. MICROBIOL. Appreciation is epressed for the cooperation of Elizabeth S. Merritt, Chief Bacteriologist, as well as the many members of the staff, for volunteering serum samples. The constructive criticisms and interest of Ka Luomala, as well as the help of Palmer B. Goins and Scientific Associates, Inc., in the turbidimetric aspect of the study, is gratefully acknowledged. LITERATURE CITED ARONSON, A. I., AND S. SPIEGELMANN. 1958. On the use of chloramphenicol-inhibited systems for investigating RNA and protein synthesis. Biochim. Biophys. Acta 29:214-215. HAROLD, F. M. 1963. Accumulation of inorganic polyphosphate in Aerobacter aerogenes. J. Bacteriol. 86:216-221. HOFF-JORGENSEN, E. 1954. Microbiological assay of vitamin Br2. Methods Biochem. Anal. 1:81-113. HOFFMANN, C. E., E. L. R. STOKSTAD, A. L. FRANKLIN, AND T. H. JUKES. 1948. Response of Lactobacillus leichmannii 313 to the antipernicious anemia factor. J. Biol. Chem. 176:1465-66. HOROWITZ, J., J. J. SAUKKONEN, AND E. CHAR- GAFF. 1958. Effect of 5-fluorouracil on a uracilrequiring mutant of Escherichia coli. Biochim. Biophys. Acta 29:222-223. KITAY, E., W. S. McNuTT, AND E. E. SNELL. 1949. The nonspecificity of thymidine as a growth factor for lactic acid bacteria. J. Biol. Chem. 177:993-994. SKEGGS, H. R., J. W. HUFF, L. D. WRIGHT, AND D. K. BOSSHARDT. 1948. The use of Lactobacillus leichmannii in the microbiological assay of the "animal protein factor." J. Biol. Chem. 176: 1459-60.