Step 1 Follow provided directions for sampling and cracking the ampoule. Be sure to shake ampoule well to get all components of test into solution.

Step 2 Remove contents (without rod) into a test tube. Contents can be removed aseptically from opening in top with a needle and syringe. Or the outside of the ampoule can be cleaned with alcohol and then the top cracked open with a sterilized tool.

Step 3 Make a small ink mark at the top of the test tube.

Step 4 Set spectrophotometer to 465nm.

Step 5 Insert test tube into spectrophotometer with ink mark toward front and take a "time zero" reading of % Transmittance. Record this result along with the time.

Step 6 Incubate at 35-37C.

Step 7 Take a spectrophotometric reading every half hour making sure ink mark is toward the front. Record results. When there is a 10% reduction in %T the test is positive. (ie-75%T and 5 hours 65%T)

Step 8 Add 1/2 hour to the time in Step 7 and use visual results timetable to determine cfu/ml in the sample.

**NOTE**
This method works for counts of 10 to 1,000,000 cfu/ml. For counts higher than 1,000,000 cfu/ml it is best to use the visual test procedure or perform serial dilutions of the sample. Serial dilutions lower the bacterial count in the sample tested and enable you to use the spectrophotometric procedure. To obtain a final result simply multiply by the dilution factor. (example- a 1/100 dilution yields a 10% reduction in %Transmittance after 5 hours; using the visual results timetable for 5.5 hours the count in sample tested is 100,000 cfu/ml; multiply this result by 100 to get a count of 10,000,000 cfu/ml in the actual sample) **Please note that floating particulates in test sample can interfere with spectrophotometric readings. The sample can be filtered before beginning the test to remove particulates. Only filter sample if the floating matter is not necessary for your specific test. (ie-containing a biologic you are testing for) If the sample can not be filtered please use the visual method.

STEP 1 Follow provided directions for sampling and cracking the ampoule.

STEP 2 Dry ampoule and make an ink mark where glass begins to narrow.

STEP 3 Set spectrophotometer to proper wavelength. EC ampoule~ 355-358nm CF ampoule~ 405nm

STEP 4 Insert ampoule with mark toward front and take a "time zero" reading of %Transmittance. Record this result along with the time.

STEP 5 Check %T every hour making sure the ink mark is always toward the front. Record results. When there is a 10% reduction in %T the test is positive. (ie- time zero 65%T and 2 hours 55%T)

STEP 6 Use results timetable to determine the cfu/ml in the sample.