Appendix A Standard Operating Procedures - University of Rhode ...

5.3.2 Preparation - At least 48 Hours Prior to Sampling Day (As Needed)
1. Autoclave an appropriate number of bottles for sampling. (Review SOP 005 - Bottle
Autoclaving Procedure). Put sterile labels and sample labels on the bottles.
2. Make up sterile phosphate buffered saline solution (PBS) as needed; 1L of PBS is
enough for approximately 25-50 samples. The solution must be room temperature when
used and will need 24 hours to cool. Instructions for preparation of the PBS are located
in Section 5.3.2.1.
3. Make sure the water bath and incubator are set to and holding the correct temperatures.
The flask with the thermometer in the white incubator should be full of DI water, with the
temperature reading 35 +/- 0.5 C. The water bath should be approximately ¾ full of DI
water, and set at 44.5 +/- 0.2 C.
4. Make sure there is enough urea substrate for plate counting. Approximately 3 mL of
urea substrate is needed per plate. Preparation of urea substrate is in Section 5.3.2.3.
5.3.2.1 Preparation of 1L Sterile Phosphate Buffered Saline Solution (PBS)
1. Add the following into a 4 L Erlenmeyer flask:
a. 1.25 mL Stock KH2PO4 solution (preparation information in Section 5.3.2.5)
b. 5 mL Stock MgCl2 solution (preparation information in Section 5.3.2.6)
c. 7 g NaCl
d. 1 L DI H2O
2. Stir the mixture using a stirring bar and magnetic stirrer until the NaCl is dissolved.
3. Cover the mouth of the flask with aluminum foil and place it on a metal tray.
4. Autoclave the tray and flask for 45 minutes at 121 C (refer to SOP 004 – General
Autoclave Operation).
5. Remove the flask and tray using insulated autoclave gloves as the PBS will be very hot.
6. Leave the flask covered and allow it to cool to room temperature before using.
5.3.2.2 Preparation of 4 L Sterile Phosphate Buffered Saline Solution (PBS)
This procedure will prepare enough PBS to fill the safe plastic carboy
1. Add the following to the 6 L plastic carboy
a. 5 mL Stock KH2PO4 solution (preparation information in Section 5.3.2.5)
b. 20 mL Stock MgCl2 solution (preparation information in Section 5.3.2.6)
c. 28 g NaCl
d. 4 L DI H2O
2. Stir the mixture using a stirring bar and magnetic stirrer until the NaCl is dissolved.
3. Set the carboy cap on the mouth of the carboy, but DO NOT thread. Place in a metal
tray.
4. Autoclave the tray and carboy for 60 minutes at 121 C. The carboy will have to be laid
on it side to fit into the autoclave. Use beakers or bottles on either side of the carboy
handle to brace it, preventing the carboy from rolling around on the tray (refer to SOP
004 – General Autoclave Operation).
11 of 23
Ambient Waters Microbiological Procedure SOP 007
S:\WW\awwword\LABPROC\all QAPPs\LABQAPPs\QAPP Rev5 -0609\SOPs\SOP 007AmbientMicrobes.doc