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26.4: Materials and Procedures

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    • Water sample, at least 150ml, collected by student
    • Presumptive Testing-Multiple Tube Test (Most Probable Number-MPN):
      • 3 Double Strength Lactose Broths (DSLB)
      • 6 Single Strength Lactose Broths (SSLB)
      • 1-10ml sterile pipette
      • 1-1ml sterile pipette
      • (MPN Table needed for analysis)
    • Confirmed:
      • 1 EMB plate
    • Completed:
      • None, this will not be performed
    • Standard Plate Count:
      • 1 TGEA Pour
      • 1 Sterile Petri plate
      • 1-1ml Sterile pipette
    • Membrane Filter Method:
      • 1 Sterile Nalgene Membrane Filter Unit (0.45um)
      • 1 mEndo agar plate
      • 1 Sterile forceps
      • 1 Sterile scalpel


    Multiple Tube Test (MPN)

    Presumptive Test:

    1. Set up three DSLB and six SSLB tubes as shown by your instructor. Label each tube with the amount of water that is to be dispensed into it: 10ml, 1.0ml, and 0.1ml. (10ml in each DSLBs, 1.0ml in 3 SSLBs, 0.1ml in remaining 3 SSLBs as directed below)
    2. Shake/swirl water sample 25 times if possible.
    3. Inoculate Durham tubes as follows
    1. With a 10 ml pipette, transfer 10 ml of water to each of the DSLB tubes.
    2. With a 1.0 ml pipette, transfer0.1 ml of water to each of the lastset of SSLB tubes, and1.0ml to each of the middlethree SSLB tubes (this way only one pipette is used and prevents carry over from the lower volume to the higher volume).
    1. Incubate the tubes at 35C for 24h.
    2. Day Two: Examine the tubes and record the number of tubes in each set that have gas present.
    3. Determine the Most Probable Number of coliforms in your sample by referring to MPN Determination Table provided.

    Confirmed test:

    If a water sample is positive for gas then it is presumed that the sample contains coliforms. The confirmed test would then be performed via inoculation of a plate of EMB agar from a gas positive tube. In this lab you will instead conduct the confirmed test prior to getting results from the presumptive test.

    1. Inoculate an EMB plate with a loop of your original sample of water.
    2. Incubate at 35C for 24h.
    3. Day two: Observe plate for coliforms; purple colonies with dark centers. E. colimay exhibit a green sheen. You can compare this to your Membrane Filtration test.

    Completed test:

    Coliform colonies from EMB would be inoculated again into Lactose Broth with a Durham tube and checked for gas, and inoculated on NA or TSA and checked via Gram stain for GNRs. If these tests are positive it shows that coliforms (not another gas producer) are present and indicates that the water sample is contaminated. Due to time constraints you will not be performing the Completed Test.

    Standard Plate Count

    A standard plate count can be done to determine total numbers of bacteria in a sample, but is not specific for coliforms. Depending on the water source a dilution series might be appropriate. However, since we don’t suspect your water samples to have high numbers of bacteria you will plate directly from your water sample.

    1. Transfer 1ml of your sample to the empty petri dish and add the TGEA pour. Rotate the plate on the table in a slow figure-eight motion, then set aside to harden. Incubate at 35C for 24h.
    2. Day two: Count total colonies, determine #CFU/ml in your sample.

    Membrane Filter Technique

    The Membrane Filter Technique utilizes an apparatus containing a filter membrane with a 0.45um pore size that will trap microorganisms larger than that on the surface of the membrane as the water sample passes through the membrane. The membrane is then placed onto a plate of m-Endo agar, which is similar to EMB agar. It has lactose to indicate lactose fermenters and basic fuchsin, which causes the colonies to appear red and/or the green sheen typical of coliforms. The media also contains ingredients that inhibit Gram-positive bacteria. Nutrients in the media diffuse into the membrane filter to support growth of organisms on the surface. If coliforms are present, the number of coliforms/mL in the original sample can be calculated.

    1. Attach the Membrane Filter apparatus to the vacuum line.
    2. Remove the lid and pour the remaining water sample (optimally 100ml) into the filter and turn on the vacuum line.
    3. Once the water has been filtered turn off the vacuum line. Remove the filter by scoring around the edge of the filter with a sterile scalpel. Gently lift the filter with sterile forceps and place it onto themEndoagar plate, right side-up.
    4. Invert the plate and incubate at 35C for 24h.
    5. Day two: Evaluate for the presence of coliforms. If coliforms are present and countable, quantify the amount (CFU/ml) in your original sample.


    Water sample source: _____________________________________

    Presumptive test:


    LB with 10ml

    LB with 1.0 ml

    LB with 0.1ml

    # of tubes with gas

    Can you presume that there are coliforms present in your sample? ___________

    MPN (use the MPN chart): ____________________________

    Confirmed test:

    Draw and describe your observations on the EMB plate.


    If you had a positive presumptive test, did your EMB plate confirm coliforms? ________


    A. # of colonies onplate:_________________

    B. Calculate the number of bacteria in the original sample:

    Membrane Filter Technique

    1. Draw and describe what you observe on your membrane filter/m-Endo agar plate:
    1. B. Number of coliforms: ________________
    2. C. Number of coliforms in your original sample/mL: _________________

    Show your calculation:

    Class Results


    Water Source


    EMB +/-


    Mb Filter +/-







    Contributors and Attributions

    This page titled 26.4: Materials and Procedures is shared under a CC BY license and was authored, remixed, and/or curated by Kelly C. Burke.

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