Int. J. Environ. Res. Public Health 2012, 9 2.7.5. Mycetes 2767 An aliquot (100 mL) of the sample collected should be filtered through a 47 mm cellulose ester membrane with filtration characteristics corresponding to a nominal pore size of 0.45 µm. Place the membrane over a Sabouraud Dextrose Agar medium and incubate at 22–25 °C for 3 ± 5 days. Recognition of fungal species is a complex issue requiring lengthy observation and proven experience. The aspect of colonies varies according to the type of substrate (natural or artificial) and colonies develop on the basis of numerous factors. To identify yeasts sample from the centre of the colony and smear on to a glass slide in a drop of sterile distilled water. View under a microscope, preferably under contrast and at 20× or 40× magnification to identify pseudomycelium and spores; if necessary, perform commercially available miniaturised biochemical identification tests (UNI EN ISO 6222:2001). Use direct observation technique to view filamentous fungi under the microscope. Carefully attach a piece of transparent sticky tape to the surface of the colony to be examined; transfer the tape with the sticky part facing downwards, onto a glass slide containing a drop of lactophenol blue solution and cover with a glass cover slip; observe at 100X to enhance distinguishing of fructiferous bodies [20,21]. 2.7.6. Counting of Colonies 22 °C–37 °C Analytical procedures undertaken to assess the number of micro-organisms at 37 °C and 22 °C are identical for both parameters, comprising the agar inclusion technique. Seed 1 mL of the sample onto the bottom of a Petri dish. Pour approximately 15 mL of isolation substrate maintained in a liquid state at a temperature of 45 ± 1 °C on to the dishes containing the inoculum. Mix thoroughly by rotating dishes backwards and forwards to facilitate a complete mixing of culture medium and sample. Leave to set and incubate one dish at 36 ± 1 °C for 40–48 hours, and the other at 22 ± 1 °C for 64–72 hours (UNI EN ISO 6222:2001) [20]. 2.7.7. Counting at 22 °C (Mesophiles) (UNI EN ISO 6222:2001) An aliquot (100 mL) of the sample collected should be filtered through a 47 mm cellulose ester membrane with filtration characteristics corresponding to a nominal pore size of 0.22–0.45 µm. Place the membrane on the agar medium and incubate at 20–23 °C for 7 days (UNI EN ISO 6222:2001) ) [20]. 2.8. Endotoxin Tests Tests are carried out both on osmotic water as monitors are detached [22] and at random initial-intermediate and end points in the ring. Spectrophotometric methods are used (Endosafe®-PTS™, Charles River®) with gel-clotting at 37 °C. Acceptable limits of detection on ring detachment are <0.05, and <0.01 for online infusion liquid. A training course in use of this piece of equipment is required, the machine should be calibrated by the manufacturers on a yearly basis. Indispensable requisites for on-line hemodiafiltration are as follows: CFU: 0 colonies/mL bacteria and mycetes, endotoxins <0.03 EU/mL. This method should not be used if the requisites are lacking.