Summer Research Program for Science Teachers

Caren Cleckley

Bedford Stuyvesant Outreach

1999


General Microbiology Lab Skills



Objectives:

The students will:


Background and Procedure:


Sterile Technique

With any type of microbiology technique (i.e. working with and culturing bacteria), it is important not to introduce contaminating bacteria into the experiment. Because contaminating bacteria are ubiquitous and are found on fingertips, bench tops, etc., it is important to avoid these contaminating surfaces. When students are working with the inoculation loops, pipettes, and agar plates, you should stress that the round circle at the end of the loop, the tip of the pipetter, and the surface of the agar plate should not be touched or placed onto contaminating surfaces. While some contamination will not likely ruin the experiment, students would benefit from an introduction to the idea of sterile technique. Using sterile technique is also an issue of human cleanliness and safety.


Use of the Pipette

Before beginning the laboratory sessions, point out the graduations on the pipette to the students. Both the 100 and 250 l as well as the 1 milliliter marks will be used as units of measurement throughout the Labs. [Content Standard Unifying Concepts- Measurement]



Working with E. Coli

The host organism in this kit, an E. coli K-12 strain, the vector containing the recombinant GFP protein and the subsequent transformants created by their combination are notpathogenic organisms like the E. coli O157:H7 strain that has been in the news. However, handling of the E. coli K-12 entities of the Transformation Kit requires the use of Standard Microbiological Practices. These practices include but are not limited to the following. Work surfaces are decontaminated once a day and after any spill of viable material. All contaminated liquid or solid wastes are decontaminated before disposal. Persons wash their hands: (i) After they handle materials involving organisms containing recombinant DNA molecules, and (ii) before exiting the laboratory. All procedures are performed carefully to minimize the creation of aerosols. Mechanical pipetting devices are used; mouth pipetting is prohibited. Eating, drinking, smoking, and applying cosmetics are not permitted in the work area. [Teaching Standard D- Ensure a safe working environment]


Decontamination and Disposal

If an autoclave is not available, all solutions and components (loops and pipettes) that have come in contact with bacteria can be placed in a fresh 10% bleach solution for at least 20 minutes for sterilization. A shallow pan of this solution should be placed at every lab station. No matter what you choose, all used loops and pipettes should be collected for sterilization. Sterilize Petri dishes by covering the agar with 10% bleach solution. Let it stand for1 hour or more and then pour excess plate liquid down the drain. Once sterilized, the agar plates can be double bagged and treated as normal trash. Safety glasses are recommended when using bleach solutions.

Ultraviolet (UV) Lamps

Ultraviolet radiation can cause damage to eyes and skin. Short-wave UV is more damaging than long-wave UV light. The Bio-Rad UV lamp recommended for this module is long-wave. [9-12 Content Standard B- Interactions of matter and energy] If possible, use UV rated safety glasses or goggles.


Incubation

This guide is written to reflect the use of a 37 C incubator. The transformation experiment can be conducted without the use of an incubator, however, the number of days required to culture colonies to the optimum size depends on the ambient temperature. Best results are obtained if starter plate colonies are fresh (24-48 hours growth) and measure about 1-1.5 mm in diameter. Refrigeration of cultured plates will significantly lower transformation efficiency. 37 C (98.6 F) is the optimum temperature for growing E. coli and lower temperatures will result in a decreased growth rate. At 28 C (82 F) two days incubation is required to obtain optimum size. 21 C (70 F) requires three days incubation of plates to obtain optimum size.


Homework:

The experiments that we are going to do require that we incubate the cultured plates in a incubator at a specific temperature. If you do not have incubator, can you build one? If you could, what materials would we need and how would it look. You are to design an incubator to hold your groups' plates. (Question you need to ask are how can you regulate the temperature, what would be the best materials to build it) [9-12 Content Standard E- Abilities of technological design]

 

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