Arseny S. Kaprelyants

Viability and activity in readily culturable bacteria: a review and discussion of the practical issues

In microbiology the terms ‘viability’ and ‘culturability’ are often equated. However, in recent years the apparently self-contradictory expression ‘viable-but-nonculturable’ (‘VBNC’) has been applied to cells with various and often poorly defined physiological attributes but which, nonetheless, could not be cultured by methods normally appropriate to the organism concerned. These attributes include apparent cell integrity, the possession of some form of measurable cellular activity and the apparent capacity to regain culturability. We review the evidence relating to putative VBNC cells and stress our view that most of the reports claiming a return to culturability have failed to exclude the regrowth of a limited number of cells which had never lost culturability. We argue that failure to differentiate clearly between use of the terms ‘viability’ and ‘culturability’ in an operational versus a conceptual sense is fuelling the current debate, and conclude with a number of proposals that are designed to help clarify the major issues involved. In particular, we suggest an alternative operational terminology that replaces ‘VBNC’ with expressions that are internally consistent.

Formation and resuscitation of 'non- culturable' cells of Rhodococcus rhodochrous and Mycobacterium tuberculosis in prolonged stationary phase

After growth of Rhodococcus rhodochrous in Sautons medium, and further incubation for about 60 h in stationary phase, there was a transient (up to 5 log) decrease in the c.f.u. count, whereas the total count remained similar to its initial value. At the point of minimal viability, the most probable number (MPN) count was 10 times greater than the c.f.u. count. This difference was further magnified by 3-4 logs (giving values close to the total count) by incorporating supernatant taken from growing cultures. A small protein similar to Rpf (resuscitation-promoting factor of Micrococcus luteus) appeared to be responsible for some of the activity in the culture supernatant. The formation of non-culturable cells of the Academia strain of Mycobacterium tuberculosis was similarly observed following growth in Sautons medium containing Tween 80 in sealed culture vessels, and further incubation for an extended stationary phase. This resulted in the formation, 4-5 months post-inoculation, of a homogeneous population of ostensibly non-culturable cells (zero c.f.u.). Remarkably, the MPN count for these cultures was 10(5) organisms ml(-1), and this value was further increased by one log using supernatant from an actively growing culture. Populations of non-culturable cells of Mycobacterium tuberculosis were also obtained by the filtration of clumpy cultures, which were grown in the absence of Tween 80. These small cells could only be grown in liquid medium (MPN) and their viability was enhanced by the addition of culture supernatant or Rpf. The non-culturable cells that accumulated during prolonged stationary phase in both the R. rhodochrous and the Mycobacterium tuberculosis cultures were small ovoid and coccoid forms with an intact permeability barrier, but with undetectable respiratory activity. The authors consider these non-culturable bacteria to be dormant. The observed activity of culture supernatants and Rpf with non-culturable bacterial suspensions invites the speculation that one, or more, of the cognate Mycobacterium tuberculosis Rpf-like molecule(s) could be involved in mechanisms of latency and reactivation of tuberculosis in vivo.

The use of 5-cyano-2,3-ditolyl tetrazolium chloride and flow cytometry for the visualisation of respiratory activity in individual cells of Micrococcus luteus

Abstract 5-cyano-2,3-ditolyl tetrazolium chloride is a redox dye which may be reduced to a fluorescent formazan derivative. We describe its use, together with flow cytometry, for the visualisation of respiratory activity in individual cells of Micrococcus luteus , and in combination with exogenous NADH for the distinction in a frozen/thawed population of ‘injured’ cells which have an impaired permeability barrier to the pyridine nucleotide.

Flow Cytometric Analysis, Using Rhodamine 123, of Micrococcus luteus at Low Growth Rate in Chemostat Culture

In microbiology it is often necessary to determine the number of viable cells in a sample or culture of interest. This is usually achieved by plating out the sample (diluted as required) on to an agar plate (Postgate 1969; Hattori 1988). There are several problems associated with this technique, the greatest of which is the length of time required to obtain the results. For some slowly growing organisms (e.g. Mycobacteria) it may take in excess of a week to determine how many cells were “viable” in the original sample, and even when the sample contains fast-growing organisms and the plates are incubated under optimal growth conditions a minimum of overnight growth is usually required before the resulting colonies can be counted. For some clinical specimens even an overnight incubation may be too long to be of use and consequently many alternatives to plate counts have been proposed in order to decrease the time required to determine numbers of viable cells (Harris and Kell 1985).