Special Equipment and Materials

A.

1. Temperature controlled room or refrigerator 2-8°C.
2. Blender or homogenizer.
3. Incubator 35-37°C.
4. Analytical balance and weigh boats.
5. Tray for dialysis.
6. pH meter. The pH during extraction and the pH of buffers used in the extraction are important. Make adjustments within ± 0.1 pH unit.
7. Refrigerated centrifuge 2-8°C.
8. Laboratory-ware in glass or polypropylene to avoid toxin adsorption.
9. Filter cloth is used to collect debris after centrifugation. Often, several layers of pre-wetted coarse cheesecloth are used to perform this task.
10. Separatory funnel.
11. Dialysis membrane MWCO 6,000-8,000 Daltons (e.g. Spectra/Por® with closures) flat width 23 ± 2 mm.
12. Vacuum conical tube filtration devices (0.22µm membrane) such as Steriflip (EMD Millipore) recommended for filtering liquid culture supernatants as a safety measure to avoid aerosolization of enterotoxins.
13. Biological safety cabinet.

Reagents

B.

Kit manufacturers may require different buffers or solvents.

1. Phosphate buffered saline (PBS solution) pH 7.3 + 0.2 (NaCl/Na2HPO4: 145 mM/10 mM) to prepare 1L PBS dissolve 9 g NaCl and 3.58 g Na2HPO4 in 1L distilled water. Adjust pH to 7.2 ± 0.2 using HCl.
2. Sodium chloride (NaCl).
3. Sodium phosphate (Na2HPO4).
4. Polyethylene glycol (PEG) (20,000 mol wt) Prepare 30% (w/v) PEG solution by adding 30g PEG for each 70 ml distilled water.
5. 1 N (or 0.1 N) NaOH.
6. 1 N (or 0.1 N) HCl.