Materials:
- Millipore@ filtration equipment and filters (available from Ward's Natural Science. catalog #36V5422).
- ampouled media for coliform bacteria. (Ward's #38V0062)
- sterile containers of either plastic or glass to collect samples.
- incubator
Procedure: The membrane filtration method involves filtering uniform sized portions of the sample using filters of a standard diameter and pore size, placing each filter on a selective nutrient medium in a petri plate, incubating the plates at a specific temperature for a specified amount of time, and then counting the colonies that have grown.
- Water samples are taken by holding the sample bottle upside down and plunging it below the water surface before the bottle is inverted. surface water film often contains higher numbers of bacteria and can bias the results.
- Label samples and place on ice for processing. (ideally, processing of sample should be done within 6 hours)
- Sanitize forceps by dipping into alcohol and burning the alcohol off with a flame. Using the sterile forceps, place a sterile pad into the pre-sterilized petri dish.
- Carefully break the neck of the ampouled media and drain the broth onto the pad.
- With grid side up, place a sterile filter on the millipore membrane.
- Screw on the top funnel to the bottom funnel on the filtration system.
- Pour standard amount of sample into the top funnel.
- Use suction to draw sample through the membrane.
- Using sterile forceps, transfer the filter, grid side up, onto the poured media in the petri dish.
- Incubate at 44.5oC for 24 hours.
- Inspect plates and count colonies that have a blue-green metallic sheen. These are coliform.
- Have someone else count colonies to further verify numbers.
Discussion: Although it is recommended that current EPA standards for acceptable levels of coliform bacteria be referenced, the following parameters may be used: fewer than 200 colonies/100ml. for swimming; fewer than 1000 colonies/100ml. for fishing and boating; and for domestic water supply, fewer than 2000 colonies/100ml.
The presence of fecal coliform bacteria in aquatic environments indicates that the water has been contaminated by humans or livestock. Although fecal coliform is a naturally occurring and necessary inhabitant of the vertebrate gut, the relative abundance of this bacteria may indicate a greater possibility of pathogens such as cholera that may negatively impact human health. Studies of this kind are useful in identifying point and non-point sources of pollution.
Extensions: To get a bigger profile of any aquatic community, students can measure other biotic and abiotic factors such as macroinvertebrates, dissolved oxygen, pH, temperature, water chemistry, etc. Students can use these skills to do independent research on a specific area of interest while relating their data to topographic and watershed maps. The possibilities are endless.
Genomic Connection: Over 1,000,000 million species of bacteria exist on this planet yet fewer than 4,500 have been described. A great genetic diversity exists within this group. New strains of bacteria are being sequenced and posted to various databases on a regular basis. Students can access this information and use it to formulate evolutionary pathways.
Links:
Environmental Protection Agency