Reference, Pages: Standard Methods (pages 9-1, 9-2, 9-15 through 9-22, 9-34 through 9-41, 9-49 through 9-56, 9-59 through 9-68, and instructor notes.
Purpose, Significance of this Analysis: Check if pathogens are in water. Bacteriological testing can prevent waterborne diseases and help identify a water system’s source of problem. Ensure and provide safe potable water to public. We will test for Total Coliform and Fecal Coliform in our analysis using the Presence/Absence (chromogenic substrate) and the Membrane Filter (filter, culture, count) techniques.
Sampling Container: Use only sterilized bottles. Purchased or prepared (washed and autoclaved).
Sample Storage and Preservation: Sample should be analyzed immediately if possible. Maximum storage for drinking water should be less than 30 hours. Sample should be cooled at 1 – 40 C during storage or transport.
Sample Volume Needed: 125mL
Equipment:
Autoclave
Drying Oven Incubator
Water Bath Incubator
Analytical Balance
Vacuum Flask/Hose
Filter Funnel
Beaker
Volumetric Flask
Erlenmeyer Flask
Forceps
Whirly Bag
Petri Dish
Safety Goggles and Apron
Chemical and Reagent Preparation: Weigh 0.85g of KH2PO4 and dilute to 25mL. Weigh 2.03g of MgCl2 and dilute to 25mL. Then take 1.25mL of KH2PO4 and 5.0mL of MgCl2 and dilute it up to 1L to create the sterile buffered dilution water.
Procedure:
1) Collect sample for Presence/Absence test. Add chromogenic substrate and incubate for 24 hours at 350 C. If sample is a darker yellow than the standard after incubation Total Coliform is present.
2) For the Membrane Filter Test: sterilize vacuum flask, volumetric flasks, Erlenmeyer flasks, and the filter funnel by washing with hot soapy water and rinsing with DI water. Then autoclave this lab equipment for 15 minutes. Add Sodium Thiosulfate to volumetric flasks to stabilize any chlorine that may be in sample.
3) Label one Petri dish for Total Coliform and one Petri dish for Fecal Coliform to include Name, Sample Location, Date of Sample, Test, and Volume. Place an absorbent pad in each Petri dish and the appropriate media on the pad (MENDO for TC, MFC for FC).
4) Sterilize forceps in alcohol and burn clean. Use sterile forceps to place filter in the filter funnel with the grid lines up.
5) Pour measured sample for TC of 0.01mL (1mL of sample to 100mL dilution water) into vacuum flask and rinse with dilution water. Then place the filter in Petri dish and close. Place the Petri dish with filter sample in drying oven incubator inverted for 24 hours at 350 C.
6) Repeat steps #3 and #4 for FC using a measured sample of 0.0001mL (1mL of sample to 100mL dilution water, then 1mL of this to 100mL more dilution water). Place FC Petri dish in water bath incubator for 24 hours at 44.50 C.
7) After the 24 hours incubation time count the appropriate colonies for TC (reddish with green metallic sheen) and FC (Blue).
