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Membrane Filtration 

Background: 

The Membrane Filtration method offers some advantages over other methods because it allows a much larger volume of water (for example, 100 mL) to be tested, and therefore it may detect lower levels of contamination.  It is also a quantitative test that determines the actual number of coliforms present per 100 mL volume. Water is filtered through a membrane of 0.45 mm porosity.  This membrane filter is then placed on a pad saturated with m-Endo MF broth, a selective and differential medium.  After a 24 hour incubation at 37˚C, colonies of coliforms, if present, will be dark pink/red and may exhibit a metallic sheen.  

Procedure

Students will work in groups of 4
Each group of 4 will filter 3 different volumes of their water sample: 1 mL, 10 mL, and 100 mL.

1. Label the sidewalls of the 3 Petri dishes with your initials, lab section number, water sample source, and volume of water filtered (1 mL, 10 mL, and 100 mL).

2. At the m-Endo broth station on the side bench place a sterile pad into the bottom of each dish and saturate each pad with 2 mL of m-Endo broth.

3. To optimize filtration, it is important to have a total volume of at least 100 mL. Therefore, it is necessary to add the 1 mL and 10 mL samples to 100 mL water blanks as described below:

a. Collect two 100 mL water blanks and label as 1mL and 10 mL.
b. Pipette 1 mL of the water sample into the first 100 mL water blank.
c. Pipette 10 mL of the water sample into the second 100 mL water blank.

4. For the third sample, simply measure 100 mL of the water directly into the 100 mL graduated cylinder.

a. At this point, every group should have 3 water samples — 2 diluted and 1 undiluted. All samples should be approximately 100 mL in volume.

5. Follow the procedure below to perform the filtrations:

a. Collect three membrane filters with grids. Assemble the filtration unit as follows:

i. Insert the funnel base into the side-arm flask

ii. Using flamed forceps, aseptically place a membrane filter (grid-side up) on the screen support. Be certain that only the filter, which is white in color, is placed on the screen. The filter is packaged between two blue sheets of paper, which, if placed on the grid, will stop all water from passing through the filter.

iii. Place the funnel securely on the base.

iv. Be sure all connections are firm!

b. Attach rubber tubing to the vacuum line.

c. Vigorously shake the water bottle containing the 1 mL sample (diluted to 100 mL in water) to disperse the bacteria. Pour the sample into the funnel. Slowly turn on the vacuum.

d. Without removing the filter, rinse the inside of the funnel with about 50 mL of sterile water. Turn the vacuum off. Release the vacuum by disconnecting the tubing.

e. Remove the funnel and set it on the foil wrapper. It will be used for the next sample.

f. With flamed forceps, transfer the filter to the labeled dish. Keep the grid side up. Gently push the filter down onto the media-saturated pad so there are no air bubbles. g. Repeat the procedure for the 10 mL and 100 mL samples.

6. Incubate the plates with the lids up (do not invert the plates) in a humidified chamber at 37°C.

7. Place all used equipment on the designated tray. Pour the filtrate in the vacuum flask down the sink. This should be really clean water! Label the flask and leave it on the bench for the next class.

Results

coliform colonies on filtered membrane

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