Supplement to MFLP-44

1. Application

The following information is offered as a supplement to the method, MFLP-44, dated April 1998, and should be used with this method. The purpose of the attached is to provide additional information (such as: source(s) of materials, critical steps, confirmation steps, or helpful hints etc.).

This information will become incorporated into the method when it has undergone review and editing (following Health Canada policy).

5. Materials and Special Equipment

Cooked Meat Media (CMM) (see MFHPB-16)
Tripticase (BBL) or Special Peptone (Oxoid)
Peptone (Difco)
McClung Toabe /Egg yolk/Yeast Extract agar plates ((MTEYE) (or C. botulinum Isolation Medium see MFHPB-16)
SM Media
Fortified Tryptone Glucose Extract Agar (FTGE).

8. Verification of the Method MFLP-44

The following methods may be used to obtain aerobic and anaerobic spore suspensions, which can be used to verify the ability of this method to enumerate both aerobic and anaerobic sporeformers.

8.1 Aerobic Sporeformers

A spore suspension of B. subtilis can be prepared as follows:

Use an ATCC strain or equivalent of B. subtilis. An 18 - 24 h culture of B. subtilis grown in nutrient broth in a water bath shaker at 37^oC is used to inoculate Fortified Tryptone Glucose Extract Agar (FTGE). FTGE is incubated at 37^oC for 48 h. Collect surface growth in sterile distilled water (~20 mL). The spore suspension is cleaned by repeated washing and differential centrifugation starting at 1,500 g for 20 min. and increasing the speed by 500 g up to 10,500 g. Before each centrifugation, pellets are observed for stratification. Strata is separated into individual centrifuge bottles and any stratum containing predominantly vegetative cells (as detected by phase contrast microscopy) is discarded. The final suspension should be made up a pellet showing a homogenous stratum and a high percentage of refractile spores. Final pellets are stored in distilled water at 4^oC until needed.

Alternately, spores can be frozen @-80^oC.