Download Appendix A - Horry County Stormwater

Environmental Quality Lab
Microbiology Procedures
5.4
SOP No.: 502
Page 5 of 13
Rev. 5
10-15-09
x
To prepare buffered dilution water: dilute 1.25 mL stock phosphate
buffer solution and 5.0 mL magnesium chloride solution in 1 liter
deionized water.
x
Dispense in amount that will provide 99 r 2.0 mL in bottles or 9 r 0.2
mL in tubes after autoclaving for 15 minutes.
x
Alternatively purchase sterile buffered dilution water (e.g., Fisher or
IDEXX).
x
Store in dark at room temperature.
PROCEDURE
5.4.1
Locate samples from cooler according to the chain of custody and analysis
request forms.
5.4.2
Arrange fermentation tubes in three rows of five each for each sample
tested. The first row should contain double strength A-1 broth and the
next two rows should contain regular strength A-1 broth. At least one
tube of each set of tubes for a sample should be labeled by sample
number and dilution on the sample tube with a permanent marker
(usually double strength tube for each sample).
5.4.3
Sample analysis should begin immediately, preferably within 2 hours of
collection. The maximum transport time to the laboratory is within 6
hours of sample collection. Samples should be processed within 2 hours
of receipt at the laboratory. Immediately prior to sample analysis shake
sample vigorously 25 times before inoculating tubes.
5.4.4
Dispense with sterile 10 ml pipet, 10 ml of sample in each tube of the
first row (double strength A-1).
5.4.5
Dispense with sterile 1.0 ml pipet, 1.0 ml of sample in each tube of the
second row.
5.4.6
Dispense with sterile 1.0 ml pipet, 0.1 ml of sample per tube in the third
row.
5.4.7
If additional dilutions are required due to anticipated large bacterial
concentration, additional rows containing 0.01 sample per tube and 0.001
ml sample per tube can also be inoculated. The following guidelines are
for using volumes less than 0.1 mL of sample to inoculate tubes:
x
For a desired sample volume of 0.01 or 0.001 mL add 1.0 mL of sample
to 99 mL of sterile, buffered dilution water.
x
Shake diluted sample vigorously 25 times.
x
If 0.01 mL sample is desired, transfer 1 mL of diluted sample.
x
If 0.001 mL of sample is desired, transfer 0.1 mL of diluted sample.