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

. If a particular location has a history of high levels of fecal coliforms or if there is
reason to suspect that high levels may be present due to events such as large
rain storms, several sets of diluted plates should be prepared.
Preparation of dilutions
i. Ambient water samples that must be diluted are generally diluted to 1, 10
and 100 mL.
ii. Prepared dilutions should be filtered sequentially in a single filter funnel in
the following order: 1 mL, 10 mL and 100 mL. This will ensure there is no
cross-contamination. Remember, only one filter and one dilution per
plate.
iii. A sterile 1 mL or 10 mL pipette is used to dispense the appropriate
volume. Approximately 10 mL of PBS should be added to the funnel prior
to adding sample volumes less than or equal to 10 mL to ensure
adequate dispersal of bacteria throughout the filter.
iv. See Elizabeth Herron for guidance on appropriate sample dilution values
as they may change over time.
5. Repeat steps 3 and 4 until all 4 filter funnels have sample in them.
6. Open the valve of each of the filter funnel setups, and turn on the vacuum pump.
7. After all the samples have filtered through, use the squirt bottle containing PBS to rinse
the inside of each funnel approximately three times to wash any stray bacteria onto the
filter. Do not touch the tip of the squirt bottle to the inside of any of the funnels. This
will contaminate other samples.
8. Turn off the vacuum pump.
9. Flame sterilize the forceps and shut off the valve for the first funnel.
10. Carefully remove the filter from the first funnel with the sterilized forceps.
11. Carefully place the filter onto the media of a labeled Petri dish, grid side up, so there are
no bubbles apparent. Touch the outer edge of the filter with the forceps until it is
completely flat. If it appears there are bubbles in the media pick up the filter and lay it
onto the media again.
12. Put the cover back on the plate, invert the plate and set it aside.
13. If additional dilutions or replicates will be completed on the sample, place a sterile filter
on the empty filter base and add the next dilution or replicate. The forceps do not have
to be re-sterilized for this step provided they have not made contact with anything but the
sterile media or a more dilute sample.
14. Repeat steps 9 through 13 for the rest of the filter funnels.
15. Once all dilutions and replicates for the first set of 4 samples have been filtered, place
the plates with filters into the 35 ºC incubator for 2 hours. Plates should be inverted and
stacked no higher than 2.
16. Remove the filter funnels and base replacing them with sterilized ones from the UV box
for the next set of samples. Put the non-sterile filter funnels into the UV box and turn the
UV lights on by closing the lid and depressing the button.
17. Repeat the procedure until all of the samples have been processed.
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Ambient Waters Microbiological Procedure SOP 007
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