Microorganism Disposal Requirements

You will work with a variety of microorganisms throughout this lab kit. Cautious safety measures have been put in place to ensure that your safety is maintained. Additionally, you are responsible for carrying out proper disposal techniques at the end of each lab. Generally, this requires you to flood any petri plates with a bleach solution and then sealing them with Parafilm™.

Bleach should be purchased as soon as possible to ensure that you have it available at all times. Bleach is available at most drug or grocery stores, and typically costs $2 - 4. You will need a minimum of 30 ounces of a 10% concentrated solution. If your solution has a higher bleach concentration, you can dilute the solution using water; just be sure to maintain a 10% concentration base. Be sure to rinse the bleach down the drain with running water to avoid any plumbing concerns.

After bleaching, you will seal the plates or containers with Parafilm™ and dispose of them in the trash. Standard trash receptacles are suitable for this, but please exercise caution if you live in an environment with animals or children and ensure that they do not re-open or crack the containers.

Hazardous Waste Disposal Requirements

The majority of the chemicals used in this kit can be safely disposed of down the drain. However, there are several chemicals required for staining microorganisms that must be disposed of with additional care. These chemicals include: Crystal Violet, Gram Decolorizer, and Safranin. These chemicals are all classified by the EPA under code D001: Ignitable hazardous waste. This is due to the alcohol concentration present in each solution. Local disposal requirements may vary. Therefore, please contact your state hazardous waste authority to determine what steps are needed to safely dispose of these chemicals. A library of each state's contact information can be found at the following website: http://www.epa.gov/osw/wyl/stateprograms.htm

Isopropyl Alcohol

A combination of alcohol immersion and candle flames are used for sterilization and staining purposes. Due to the high volume of alcohol required, you will need to purchase one bottle of isopropyl alcohol (rubbing alcohol) prior to starting your lab experiments.

Isopropyl alcohol can typically be purchased at a grocery or drug store. One 16 ounce bottle is sufficient. This will cost approximately $2.

Alcohol bottles should always be placed away from open flames. If you have any questions about the bleach or alcohol purchases please email eScience Labs at info@esciencelabs.com or call 888- ESL-KITS.

Sterile Technique

Microbiologists must be extra diligent about using sterile techniques to ensure that equipment does not become contaminated. In particular, petri dishe packages must be opened and closed with care to prevent airborne contaminants from settling onto a dish. Therefore, you must seal the petri dish packages with tape every time you close a package of dishes. More information about how to keep a petri dish package sterile can be found by logging on to the eScience Labs Student Portal at www.esciencelabs.com/portaland selecting the microbiology lab kit.

In addition to this, it is important to store your kit materials in the kit box. This further prevents any surface or airborne contaminants from compromising your equipment's sterility.

MSDS

The MSDS for every chemical in this kit is provided at www.esciencelabs.com/educators/msds. eScience Labs highly recommends that you download and print all of the MSDS prior to starting your experiments. By doing so, you will ensure that you have all of the safety and cleanup information you need should you spill or encounter an accident during an experiment.

Winogradsky Column

The Environmental Microbiology and Water Quality Lab includes an experiment in which a Winogradsky column is created. This task requires a 6-10 week incubation period; and, therefore, must be prepared in advance of the Environmental Microbiology unit so that results are available on time. A copy of this procedure has been provided for you on the following pages. The procedure can also be found in Lab 13: Experiment 3.

Thank you,

The eScience Labs Team

Lab 13: Environmental Microbiology and Water Quality

Experiment 3: Winogradsky Column

Sergei Winogradsky (1856-1953) was a Russian microbiologist who was one of the first scientists to study mixed populations of bacteria with differing metabolic abilities. In a Winogradsky column, two distinct gradients form in opposite directions: oxygen is high at the top of the column and non-existent at the bottom while hydrogen sulfide is high at bottom and non-existent at the top. Additionally, light facilitates the growth of aerobic photosynthetic bacteria. These generated gradients allow for growth of a large variety of bacteria that reproduce the biogeochemical cycles on which life depends.

Procedure:

1. Record the location where you obtained your sample in Table 2. Remove any large debris (sticks, rocks, etc.) from the dirt/mud.
2. Put approximately 2 C. of the dirt/mud into a large bowl or bucket and add the calcium carbonate and calcium sulfate.
3. Add enough water to make a thick, but somewhat fluid, mixture.
4. Add in the shredded newspaper and mix again.
5. Add this mixture to the soda bottle and tap the bottle on the ground or other hard surface to pack the mixture tightly to the bottom. It is important that no air pockets or bubbles should remain in this layer
6. Use a paint stirrer or handle of a long spoon to further pack the mud and remove any air.
7. Add approximately 2 - 3 C. of the remaining (unmodified) dirt/mud on top of the previous layer and tap again to pack it down.
8. Add water to a depth of approximately one inch above the last layer and make a small mark with a permanent marker on the column at the top of the water level.
9. Let the column sit undisturbed for 30 minutes and monitor the depth of the water. If the water level rises, remove some to return to the original level. If the water level decreases, add more to return to the original level. There should be ~1 inch of air space above the water.
10. Cover the top with Parafilm™ and secure with a rubber band.
11. Incubate for 6 - 10 weeks at room temperature in indirect sunlight (a south facing window) or approximately 2 ft. away from a lamp with a 40 - 60 W. bulb.
12. Observe the column weekly and record your observations in Table 3.