paper for sterilization. The openings of graduated cylinders should be covered with
metal foil or kraft paper before sterilization.
(b) Sterilization in the autoclave at 121C (249.8F) for 15 minutes is
satisfactory for glassware.
(c) Pipettes and graduated cylinders to be used in the field should be
sterilized in the laboratory.
PREPARATION OF MATERIALS
a. Sterile Buffered Dilution Water.
(1) Stock phosphate buffer solution is prepared by dissolving 34.0 grams of
potassium dehydrogen phosphate (KH2PO4) in 500 milliliters (ml) of distilled water. The
pH is then adjusted to 7.2 with a solution of 40 grams per liter (1 N) sodium hydroxide
(NaOH). Distilled water is then added to increase the volume of the solution to
1 liter (1).
(2) Sterile buffered dilution water is made by adding 1.25 ml of the stock
phosphate buffer solution to 1Iiter of distilled water and autoclaving it at 121C (249.8F)
for 15 minutes.
b. Culture Media.
(1) Ampuled M-Endo medium. An ampule of M-Endo medium is sufficient
for one analysis. Ampuled medium, which has a limited shelf life, should be discarded
after the expiration date indicated on the ampule box. Furthermore, it should not be
used unless it is pink. Keeping ampuled medium refrigerated helps to reduce
(2) Laboratory-prepared M-Endo medium. This medium should be used
whenever possible. It may be prepared from dehydrated medium such as Difco
M--Endo MF or BBL M-Coliform MF. This medium must be prepared on the same day
that it is to be used in making the water analysis. The directions for preparation are on
the container of the dehydrated medium. In the field, based upon the number of water
samples to be analyzed, it is desirable to put proportional quantities of the dehydrated
medium in sterile screw cap vials. Then, on the day that the medium is to be used, the
proper amount of distilled water and ethanol can be added. The vials must then be
placed in a boiling water bath and heated until the medium reaches the boiling point.
The screw caps on the vials must be loosened during heating and tightened when the
vials are removed from the water bath. This medium must be allowed to cool to room
temperature before it is used
(3) Vials of dehydrated endo broth. Each vial contains 1.2 g of the
dehydrated endo broth that is reconstituted by mixing it thoroughly with 24.5 ml of water