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

Abstract

In order to evaluate injury of a stressed fungal spore population, a modification of formerly presented double subculture method, which consists of both the conventional plate count method and the growth delay analysis method, was proposed. In this method, an apparent logarithmic growth kinetics was assumed and the previous kinetic model was improved to be able to estimate injured subpopulations in two different modes containing early occurring growth-independent and late occurring growth-dependent injuries, called the λ and μ injuries, respectively. Based on the kinetic theory developed here, this novel method was applied to heat-treated conidia of Cladosporium cladosporioides and these two mode injuries were evaluated.

Evaluation of distinct modes of oxidative secondary injury generated in heat-treated cells of Escherichia coli with solid/liquid and complex/semi-synthetic media sets

“…Based on our results, the following major points were discussed: (1) the difference in heat resistance of E. coli cells between the solid and liquid media, which affects the appearance of β o injury; (2) from the CFU data, the high survival rate among EM9‐EM9 cells rather than among LB‐LB cells; (3) the variations in the effects of oxidative scavengers on the survival rates of EM9‐EM9 and LB‐LB cells; (4) the effect of cross culturing using growth and recovery media on β o injury; (5) the degree of β o injury in the liquid culture or viability assay with the MPN and GDA methods and (6) the contribution of β o injury to the total injury, which was estimated as the difference between CFU or MPN and IV (Horikiri et al, 2020; Tsuchido, 2017).…”

“…media on β o injury; (5) the degree of β o injury in the liquid culture or viability assay with the MPN and GDA methods and ( 6) the contribution of β o injury to the total injury, which was estimated as the difference between CFU or MPN and IV (Horikiri et al, 2020;Tsuchido, 2017). First, the effects of solid and liquid cultures, as represented by the colony count and MPN methods, respectively, on the recovery of heat-injured cells have been discussed (Foegeding & Ray, 1992;Ray, 1979).…”

“…According to our results in Figure 5, in the case of DiVSaL, NaTS addition to EM9 agar and NaP addition to LB agar and in the case of DiVLaL either with or without either antioxidant in EM9 broth (but not available in LB) may be recommended for this purpose. Supposing that the population of the βo‐injured cells can be rescued, it may contribute to increasing the total viability, especially with CFU, for a more reliable evaluation of the total injury of the injured cells, including other modes of injured cells (Horikiri et al, 2020; Tsuchido, 2020). However, in our comparison of survival among the tested methods, the similar survival rates between IV and CFU in EM9 without antioxidants and between IV and MPN in LB with and without antioxidants might imply that the heat‐induced β o injury accounts for almost all of the total injury.…”

“…Although we have already proposed different microbial injury modes, including λ and μ injuries, in which repairs do not and do depend on growth, respectively, and the quantitative evaluation of injured cell populations (Horikiri et al, 2020; Tsuchido, 2017), β o injury can be regarded as another mode that is generated during post‐heating incubation or storage period. The detection and enumeration of the β o ‐injured cell population are significantly challenging during the viability assay, as well as the evaluation of the heat pasteurization process.…”

生死の間をさまよう損傷菌

“…文献 (15) から引用． 図 2 ■ 損傷菌の発生モデル 黒矢印は殺菌処理中，白矢印は処理後の反応．文献 (7,8) を改変引用． 凝集体と推測される．このとき，細胞内に常温で発現し ていた既存の熱ショックタンパク質(HSPs)の DnaK, GroE，低分子量 HSPs(sHSPs)などの分子シャペロン はすぐさま変性タンパク質に結合するとみられる．しか し，これらσ H 因子支配の HSPs (24) および 50 C 以上の加 熱で誘導されるσ E 因子支配の表層ストレスタンパク質 (ESPs) (25) (27,28) ．こ れらの大腸菌 sHSPs は citrate synthase などの細胞質酵 素・タンパク質に対して変性抑制作用をもつが，細胞内 では多量体を形成し，加熱損傷によって解離して変性タ ンパク質に結合する (29)(30)(31) ．また加熱によって細胞膜に 相互作用する可能性も示されている (29) ．ただ，これら の遺伝子の高発現株は細胞の熱耐性を上昇させるもの の，欠損株は親株と同程度の耐性であった (32) (18,26) を改変引用．図中の抗酸化系酵素群の 位置近傍の下方矢印は加熱損傷によるこれら の酵素の失活または活性低下を意味する． その後の研究 (33)(34)(35)(36) この方法の詳細は原著 (50)(51)(52) では，全細胞集団中の各損傷亜集団分布比( i , 0＜ i ＜1, (54,55) VBNG)状態 (7) の芽胞が生じる．またγ線照射のように DNA が主標的の処理の場合は，数世代間発育した後死滅 する増殖死・伸長死が修復と並行して発生するため，こ れらを識別して解析する工夫が必要となる (56) 2) 厚生労働省：https://www.mhlw.go.jp/stf/seisakunitsuite/ bunya/0000153364̲00001.html (2020). 3) 農 林 水 産 省：https://www.maff.go.jp/j/shokusan/sanki/ haccp/ (2020).…”