7.2.1 Have ready sterile peptone water ( PW) or peptone/tween 80 (PT) diluent, nutrient agar

(NA) plates (if necessary), m-FC agar plates and enzyme solutions needed for food product category if required (see Appendix E of Volume 2). Use PT diluent if the Spreadfilter is to be used.

7.2.2 Clean the surface of the working area with a suitable disinfectant.

7.2.3 Mark clearly the duplicate Petri plates identifying sample, sample unit, dilution, and date of inoculation.

7.3.1 To ensure a truly representative analytical unit agitate liquids or free flowing materials until the contents are homogeneous. If the sample unit is a solid, obtain the analytical unit by taking a portion from several locations within the sample unit.

7.3.2 Prepare a 1:10 dilution of the food by aseptically adding 10 g or mL (the analytical unit) into 90 mL of the PW or PT diluent. If the Spreadfilter is to be used to inoculate the HGMF, use PT diluent. Stomaching is the preferred method of suspending the organisms, since it minimizes the quantity of suspended food debris. Transfer a representative portion from the 1:10 dilution for higher serial dilutions as needed. Consult Appendix E of Volume 2 to see if enzyme treatment is required. Enzyme treatment is not necessary for samples filtered at a dilution of 1:100 or greater.

7.3.3 Filtration through the HGMF will remove acids or other inhibitors; there is no need to check and adjust the pH of the suspension.

7.3.4 The HGMF will allow counts to be made from suspensions containing up to 5,000 organisms/mL. There normally should be no need to prepare further dilutions. If this is necessary, prepare succeeding decimal dilutions as required, using a separate sterile pipette for each transfer. Record the dilution (C) used for analysis.

7.3.5 If a low count is expected, filter more than 1.0 mL of the suspension. Filter the total volume to be filtered in one operation; do not attempt to filter successive 1 mL aliquots. Record the volume (V) filtered.

7.3.6 Shake all dilutions immediately prior to making transfers to ensure uniform distribution of the microorganisms present.

7.4.1 Agitate each stomacher bag or dilution bottle to resuspend material that may have settled out.

7.4.2 Handle HGMF with sterile forceps.

7.4.3 Following the manufacturer's instructions for use of the filtration apparatus and/or pipet-tip prefilter, aseptically pipette 1.0 mL of the required dilution and inoculate the HGMF. Open the filter valve until all liquid has passed through and aseptically remove the HGMF. Do in duplicate.