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

5.3.3 Preparation - Day Before Sample Collection
1. Make sure there is an adequate supply of sterile 1 mL, 10 mL and 25 mL pipettes and
PBS filled sterile squirt bottles AS WELL AS FILTER ASSEMBLIES. If not, autoclave
them.
a. Pipettes are sterilized in the pipette pouches that are laid on an autoclavable
tray.
b. Squirt bottles are sterilized empty, with foil over the squirt caps. Caps should
only be loosely placed in the bottle. Bottles should be placed on an autoclavable
tray when placed into the autoclave. Once cooled, sterile squirt bottles can be
filled with cool sterile PBS (refer to SOP 004 – General Autoclave Operation).
c. Filter assemblies are sterilized in autoclavable bags that are laid on an
autoclavable tray.
2. Create data sheets from the template found on the WW computers. It is very helpful to
include any known dilution information on the data sheet. An example data sheet is
located in Section 8.0 Documentation.
3. Prepare the TSB tube for the positive plate using the procedure outlined in Section
5.2.4.1.
5.3.4 Procedure - Day of Sample Collection
5.3.4.1 Initial Preparation
1. Take the appropriate number of mTEC media plates out of the fridge so that they can
begin to warm up a little. Allowing the media plates to come to room temperature
reduces condensation and ensures that the labels will not rub off.
2. Wipe down the bench in room 019 with Envirocide or Conflict; allow the bench tops to
dry.
3. Connect the side arm flasks to the vacuum manifold.
4. Set up the alcohol lamp, beaker with alcohol and filter forceps, membrane filters, Sharpie
marker, PBS squirt bottle, etc.
5. Remember to use basic hygienic practices when handling samples. Latex gloves,
laboratory coats and eye protection are required.
5.3.4.2 When Samples Arrive At The Laboratory
1. After logging in samples, store them in a cooler with ice packs or the refrigerator located
in room 019.
2. Sterilize the filter funnels and filtration bases in the UV box for at least two minutes.
a. The cardboard has to be over the button in order for the unit to turn on when the
door is closed.
b. The latch must be engaged to keep the door closed. Peek in the side to be sure
it is on – don’t stare at the light!
3. Retrieve samples in batches of 4 from the cooler.
4. Label the bottom (half holding the media) of mTEC media plates with the Sharpie. The
label should include all the information present on the sample bottle, as well as any
necessary sample dilution information.
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Ambient Waters Microbiological Procedure SOP 007
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