The suc­cess of clean­ing and dis­in­fect­ing mea­sures needs to be con­trolled in health­care sec­tor and food in­dus­try on di­verse sur­faces. In these hy­gien­i­cal­ly de­mand­ing areas hy­giene stan­dards and mon­i­tor­ing re­quire­ments were in­creased con­stant­ly in re­cent years. There­fore, fast and easy self-con­trol meth­ods are re­quired to con­trol the biobur­den of sur­faces with­in mon­i­tor­ing sys­tems.

How­ev­er, sur­faces with a com­plex ge­om­e­try (e.g. con­vex or con­cave sur­faces or cor­ners) can­not ef­fec­tive­ly be sam­pled with stan­dard mi­cro­bial wip­ing meth­ods or con­tact slides. In ad­di­tion, stan­dard meth­ods like ATP-mon­i­tor­ing are not ca­pa­ble to de­tect small num­bers of bac­te­ria and can’t dis­tin­guish be­tween live and dead cells. Fur­ther more ATP oc­curs in all types of cell mak­ing a dif­fer­en­ti­a­tion of bac­te­ria and eu­kary­ot­ic cells im­pos­si­ble.

The aim of this re­search pro­ject was there­fore the de­vel­op­ment of an im­proved sam­pling method using a gela­tine ma­trix in com­bi­na­tion with flow cy­tom­e­try anal­y­sis as a tool for an easy, self-prac­ti­ca­ble and in­ex­pen­sive as­sess­ment of clean­ing and dis­in­fect­ing mea­sures. Flow cy­tom­e­try is a stan­dard tech­nique in bi­ol­o­gy for count­ing, analysing and sort­ing of par­ti­cles in re­la­tion to their size, inner com­plex­i­ty and flu­o­res­cence. In the flow cell of a flow cy­tome­ter a liq­uid stream car­ries and aligns stained cells that they pass in sin­gle file through a laser beam for sens­ing.

For sam­pling a gela­tine ma­trix was de­vel­oped to com­plete­ly re­move bac­te­ria from sur­faces. A sur­face with com­plex ge­om­e­try (e.g. milk pipe thread or per­fo­rat­ed metal plates) can for ex­am­ple be sam­pled with this liq­uid ma­trix. After hard­en­ing of gela­tine the mi­croor­gan­isms are in­clud­ed in the gel ma­trix and can be de­tached residue-free from the sur­face of in­ter­est. To anal­yse the gel ma­trix with the flow cy­tome­ter the gela­tine need to be liq­ue­fied by an en­zy­mat­ic treat­ment.

The cells are stained with two dif­fer­ent nu­cle­ic acid bind­ing dyes to be able to dis­tin­guish be­tween liv­ing and dead bac­te­ria when per­form­ing the flow cy­tom­e­try anal­y­sis. The de­vel­oped method takes less than 90 min­utes in­clu­sive prepa­ra­tion of gela­tine, en­zy­mat­ic treat­ment, stain­ing and anal­y­sis with the flow cy­tome­ter.

The IGF-pro­ject 276 ZN of the re­search as­so­ci­a­tion Europäische Forschungs­ge­mein­schaft Reini­gungs- und Hy­gien­etech­nolo­gie e.V., Cam­pus Ficht­en­hain 11, D-47807 Krefeld, was sup­port­ed via the AiF with­in the fund­ing pro­gramme „In­dus­trielle Gemein­schafts­forschung und –en­twick­lung (IGF)“ by the Fed­er­al Min­istry of Eco­nom­ic Af­fairs and Tech­nol­o­gy (BMWi) due to a de­ci­sion of the Ger­man Par­lia­ment.

The re­search re­port is avail­able on re­quest from FRT