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-limited 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 microcosms 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 were diluted 10- and 100-fold in filter-sterilized (0.1 microns) ASW from the microcosms. Undiluted, 1/10, and 1/100 diluted samples recovered culturability to about 2.2 x 10(5) c.f.u. ml-1 within 72 h of temperature upshift. If resuscitation of nonculturable cells had occurred, the resultant number of culturable cells in diluted samples would have been 1/10 and 1/100 that of undiluted samples, respectively. In microcosms 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 the experiments reported here, recovery of culturable cells on temperature upshifts resulted from growth and that there were no growth-inhibiting 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 24 h, confirming that V. cholerae is capable of significant growth in ASW.