TEMPERATURE-INDUCED RECOVERY OF VIBRIO-CHOLERAE FROM THE VIABLE BUT NONCULTURABLE STATE - GROWTH OR RESUSCITATION

Vibrio cholerae cells were incubated at 4 degrees C in nutrient-limite d artificial seawater (ASW) microcosms. Plate counts declined from 8 x 10(5) to less than 2 c.f.u. ml(-1) in about 23 d. When samples of mic rocosms were shifted to 30 degrees C, plate counts increased to 2.2 x 10(5) c.f.u. ml(-1) in 72 h. An experiment was performed to determine whether culturable cells obtained after temperature upshifts were the result of 'resuscitation', or outgrowth, of nonculturable cells or of cell division and growth of the few culturable cells that remained in samples. Prior to temperature upshift, samples from the microcosms wer e diluted 10- and 100-fold in filter-sterilized (0.1 mu m) ASW from th e microcosms. Undiluted, 1/10, and 1/100 diluted samples recovered cul turability to about 2.2 x 10(5) c.f.u. ml(-1) within 72 h of temperatu re upshift. If resuscitation of nonculturable cells had occurred, the resultant number of culturable cells in diluted samples would have bee n 1/10 and 1/100 that of undiluted samples, respectively. In microcosm s where plate counts had declined to less than 1 c.f.u. ml(-1), 1/100 diluted samples did not regain culturability, i.e. no culturable cells remained from which growth could occur. Our conclusions are that in t he experiments reported here, recovery of culturable cells on temperat ure upshifts resulted from growth and that there were no growth-inhibi ting factors in the spent growth medium, supported by the finding that about 10(2) recovered V. cholerae cells ml(-1) inoculated into filter -sterilized microcosm ASW grew to about 6.2 x 10(5) c.f.u. ml(-1) in 2 4 h. confirming that V. cholerae is capable of significant growth in A SW.