Clean­rooms are closed sys­tem rooms in which the par­ti­cle con­tent of the air is con­trolled ac­cord­ing to the spe­cif­ic clean room class. Such rooms are used in spe­cif­ic man­u­fac­tur­ing pro­cess­es, e.g. in the asep­tic prepa­ra­tion of phar­ma­ceu­ti­cals, foods and in hos­pi­tals. These con­trolled areas are sub­ject to strict clean­li­ness and hy­giene re­quire­ments. A con­tin­u­ous and com­pre­hen­sive mi­cro­bial mon­i­tor­ing of clean rooms is of high im­por­tance and rep­re­sents a fun­da­men­tal key­stone with­in the qual­i­ty man­age­ment sys­tem. Cur­rent­ly, the as­sess­ment of the num­ber and type of mi­croor­gan­isms on sur­faces is per­formed using con­tact plates or the swab method. A major dis­ad­van­tage of such clas­si­cal mi­cro­bi­o­log­i­cal meth­ods is the in­cu­ba­tion time and the anal­y­sis of sam­ples by ex­ter­nal lab­o­ra­to­ries that usu­al­ly takes at least two days. Thus, cor­rec­tions and ad­just­ments to the clean­ing and dis­in­fec­tion pro­ce­dures can be made only after a con­sid­er­able delay.

The aim of the pro­ject was the de­vel­op­ment of a suit­able rapid method for the eval­u­a­tion of the hy­giene of cleaned and dis­in­fect­ed sur­faces in clean­rooms.

An ef­fi­cient method for the sam­pling of mi­croor­gan­isms was es­tab­lished by means of a sam­pling ma­trix based on a spe­cial clean room ap­proved wipe made out of polyester/cel­lu­lose, which was moist­ened with 0.85 % NaCl so­lu­tion. For the sam­pling of plane as well as ge­o­met­ri­cal­ly com­plex sur­faces, a sam­pling pen with an elas­tic tip was de­vel­oped that holds the sam­pling ma­trix.

The elu­tion of mi­croor­gan­isms from the sam­pling ma­trix was car­ried out by “stom­ach­ing”. For sub­se­quent quan­tifi­ca­tion of mi­croor­gan­isms, an an­a­lyt­i­cal method based on flow cy­tom­e­try was de­vel­oped. For this pur­pose, at first a 15 minute “one-step” stain­ing with two suit­able flu­o­res­cent dyes was es­tab­lished, which al­lowed the dis­crim­i­na­tion of live and dead cells. Fol­low­ing, the flu­o­res­cent cells were de­tect­ed and quan­ti­fied using a flow cy­tome­ter.

The de­vel­oped rapid method pro­vid­ed re­pro­ducible and re­li­able re­sults when ap­plied to sur­faces such as stain­less steel, glass or plas­tics. In ad­di­tion, rep­re­sen­ta­tives of dif­fer­ent groups of mi­croor­gan­isms (e.g., Gram-pos­i­tive and Gram-neg­a­tive bac­te­ria and yeasts) and bac­te­ri­al spores could be quan­ti­fied.

Any residues on sur­faces such as blood, sil­i­cone, wool grease, sur­fac­tants and dis­in­fec­tants showed no neg­a­tive in­flu­ence on the de­vel­oped meth­ods as demon­strat­ed in prac­tice-rel­e­vant con­cen­tra­tions.

The en­tire pro­cess, i.e., sur­face sam­pling, elu­tion of mi­croor­gan­isms, cell stain­ing and flow cy­to­met­ric anal­y­sis, re­quires less than 20 min. This al­lows a sim­ple, fast and cost-ef­fec­tive as­sess­ment of clean­ing and dis­in­fec­tion pro­ce­dures in clean­rooms and en­ables a sim­ple doc­u­men­ta­tion of re­sults. For the ser­vice com­pa­nies en­trust­ed with clean room clean­ing and for clean room op­er­a­tors the de­vel­oped method of­fers a valu­able mean for in­ter­nal self-con­trol and qual­i­ty as­sur­ance.

The IGF-pro­ject 60 EN 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.