A Novel Double Subculture Method and Its Theory for the Enumeration of Injured Cells in Stressed Microbial Population

Abstract

A novel double subculture method, termed DiVSaL Differential Viabilities between Solid and Liquid media method, for the enumeration of injured cell population of a microorganism, which occurs after some sublethal to lethal treatment, was proposed. In this method injured cells were enumerated as the differential value between viabilities determined with two different techniques, the conventional plate counting using a solid agar medium and the growth delay analysis using a liquid medium. In the former technique, the viable cell number is obtained as colony forming unit CFU formed on an agar medium where sublethally injured cells are as much rescued as possible. In the latter technique, on the other hand, the integrated viability defined by Takano and Tsuchido 1982 is introduced and is calculated from the growth delay of a stressed population, referred to unstressed one. For the growth delay analysis, in this paper, not only the original theoretical model, where the specific growth rate and therefore the defined G 10 value does not change after the exposure to a stress treatment, but also a novel modified theory, where the parameter changes, is proposed. On the theoretical background, this DiVSaL method as a double subculture method can be used to enumerate the injured cells without selection by addition of some inhibitor or by nutritional shortage.

A Modified Double Subculture Method for the Two-Mode Injuries Evaluation in a Stressed Fungal Spore Population

“…Healthy Dead functions of fraction of each differently injured subpopulation and λ, as reported previously (Takano and Tsuchido, 1982;Tsuchido, 2017) .…”

“…We discuss this below in close relation to the μ injury. From this result, in order to estimate the overall injury of the stressed population, we determined to adopt the DiVSaL method with the variable μ model rather than the constant μ model (Tsuchido, 2017) .…”

“…Following the method reported previously (Tsuchido, 2017) , the overall injured population size for the above heat-stressed conidia of C. cladosporioides was evaluated comprehensively by the differential viabilities obtained between plate count method and GDA method. The kinetic model for this method is described as the reduced μ model with the following Eq.…”

“…The kinetic model for this method is described as the reduced μ model with the following Eq. (1) (Tsuchido, 2017) .…”

“…In the previous paper (Tsuchido, 2017) , the double subculture (DS) method as a combination of the conventional plate count method using a proper agar medium and the growth delay analysis (GDA) method using a liquid medium (Takano and Tsuchido, 1982) , which is called DiVSaL (differential viabilities between solid and liquid media) method, has been proposed for the comprehensive evaluation of overall injury of a bacterial stressed population. However, the effect of injury sometimes appear seems not only as a prolongation of lag period but also as a reduced growth rate of regrowth phase after a prolonged lag period resulting from exposure to a stress is decreased.…”