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Abstract

It has been found previously that a significant number of Micrococcus luteus cells starved in a prolonged stationary phase (up to 2 months) and then held on the bench at room temperature without agitation for periods of up to a further 2-7 months can be resuscitated in liquid media which contained (statistically) no initially-viable (colony-forming) cells but which were fortified with sterile supernatant from the late logarithmic phase of batch growth. Here it was found that such resuscitation can be done only within a defined time period after taking the first sample from such cultures, necessarily involving agitation of the cells. The duration of this period depends on the age of the starved culture: cells kept on the bench for 3 months possess a 2 month period of resuscitability while cells starved for 6 months can be resuscitated only within 10 days after the beginning of sampling. It is suggested that the input of oxygen to the starved cultures while they are agitated may exert a negative influence on the cells, since cultures stored in anaerobic conditions (under nitrogen) had a more prolonged 'survival' time. The cells which experienced between 10 and 60 days of starvation on the bench could be resuscitated, although the number of resuscitable cells depended strongly on the concentration of yeast extract in the resuscitation medium. This concentration for cells stored on the bench for more than 2 months was 0.05% while '1-month-old' cells displayed a maximum resuscitability in the presence of 0.01% of yeast extract. Application of the fluorescent probe propidium iodide revealed the formation of cells with a damaged permeability barrier if resuscitation was performed by using concentrations of yeast extract of 0.1% and above. Thus the successful resuscitation of bacterial cultures under laboratory conditions may need rather strictly defined parameters if it is to be successfully performed for the majority of cells in a population.

Separation by dielectrophoresis of dormant and nondormant bacterial cells of Mycobacterium smegmatis

“…Cells in the dormant state have a much thicker cell wall compared to active cells. 16,25,26 The overall cell conductivity increases with an increase in the thickness of cell wall, 20,27,28 and thus the increase in the bacterial conductivity from active to dormant state can have been caused by an increase in wall thickness. The results obtained tie in with previous studies, in which the particle conductivity of M. luteus ͑but not E. coli͒ greatly increased when the cells went from exponential to stationary phase.…”

“…To obtain active cells M. smegmatis cells were cultured [12][13][14][15][16] in 100 ml glass flasks with loose caps for 24 h at 37°C on an orbital incubator shaker at 200 rpm in 20 ml of the rich medium nutrient broth E LabM, supplemented with 0.05 % ͑v/v͒ Tween-80. To obtain dead M. smegmatis cells, a 24-h-old active culture was autoclaved at 126°C for 30 min.…”

“…11 Mycobacterial cells at different physiological states have distinct morphological features including differences in cell size, the structure of cell walls, and the permeability of cell membranes. 4,[12][13][14] Such differences in the cell morphology, structure, and composition can be expected to lead to differences in cell electrical polarizabilities 15 and hence, their effective particle conductivities, which could form the basis for their dielectrophoretic separation.…”

Resuscitation of ‘non-culturable’ cells from aged cultures of Campylobacter jejuni

“…Kell and co-workers showed unequivocally that starved non-culturable cells of the Gram-positive organism Micrococcus luteus could be recovered in growth medium, provided that some filtered medium from a culture growing in late exponential phase was added (Kaprelyants et al, 1994) or that some viable cells were present (Votyakova et al, 1994). It was concluded that medium from exponentially growing cultures may promote resuscitation by overcoming the effect of an antibacterial factor that otherwise caused self-poisoning of dormant cells during resuscitation (Mukamolova et al, 1995).…”

Construction by dielectrophoresis of microbial aggregates for the study of bacterial cell dormancy

“…6 The "resuscitation promoting factor" ͑RPF͒ that is involved has been successfully isolated from supernatant of M. luteus and characterized by Mukamolova et al 7 RPFs have been found to be a family of proteins whose homologous genes are widespread among the guanine-cytosine ͑GC͒-rich Gram-positive bacteria, including streptomycetes, corynebacteria, and mycobacteria. 8 They stimulate cell growth and cell multiplication at very low concentrations and are active toward different strains of bacteria. 7 The analysis of the structure of the RPFs has revealed that they may be peptidoglycan hydrolases and may actively participate in the modification of the thickened cell wall of the NC cells during resuscitation.…”