〈Original Papers〉塩素系薬剤によるリステリア損傷菌のThin agar layer 法とFlow cytometry 法による検出比較

[要旨]損傷菌の菌数測定には培養法が用いられることが多いが、細胞計測法のFlow cytometry (FCM) では、生菌、死菌、および損傷菌の迅速評価が可能である。本研究では、Listeria monocytogenesの代替株であるL.innocua (毒素陰性株)の薬剤損傷菌数測定について、培養法のThin agar layer (TAL)法とFCM法の比較 を行った。有効塩素謹度0～6ppmの強酸性電解水で処理したとき、TAL法では1ppmおよび2ppmの強酸性電解水処理において、選択培地よりもTAL培地で0.6log菌数が多く検出され、それぞれ59%および67%の損傷薗が生残したことが示唆された。 一方FCM法では、生菌を染色する膜透過型DNA染色剤 (thiazole orange:TO) と、死菌を染色する膜非透過型DNA染色剤 (propidium iodide:PI) での二重染色の結果、TOとPIの両方に染色された損傷菌の割合は、有効塩素濃度 1～3ppmでそれぞれ59%～78%となった。薬剤損傷L. innocuaの回復においては、TAL法では2時間後には損傷菌が未検出となったが、FCM法では4時間後まで損傷菌が生残していた。この結果より回復期の薬剤損傷菌の検出においては、二つの方法で違いがあることが示された。[Abstract]Culture-based methods are often used for the enumeration of sublethally injured bacteria,while flow cytometry(FCM)can provide rapid quantitation of live,dead,and injured bacteria. In this study,the culutur-based thin agar layer(TAL)method was compared with a FCM assay to enumerate chlorine-injured Listeria.innocua,a surrogate for L.monocytogenes.Bacteria were injured by mixing them with diluted electrolyed water containing 0 to 6 ppm available chlorine. In the TAL method,counts of L.innocua exposed to 1 to 2 ppm available chlorine were 0.6log higher on the TAL medium than on the selective medium than on the selective medium,indicating that 59 to 67% of the viable cells were injured by a chlorine level of 1 to 2 ppm.In comparison,the available chlorine level of 1 to 3 ppm caused sublethal injury ranging from 59 to 78% as determined by the FCM assay based on the quantitation of staned cells with both permeant DNA dye (thiazole orange:TO)and impermeant DNA dye (propidium iodide:PI),relative to live and dead cells that were stained only with TO and PI,respectively.With regard to recovery of chlorine-injured L.innocua after treatment with electrolyzed water,injured cells were inactivated after two hours when enumerated with the TAL method,whereas injured cells remained injured for four hours after the treatment as determined by the FCM assay.This result indicated a difference in the detection of chlorine-injured L.innocua between the two methods during the recovery period of injuredv cells