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A START-UP MANUAL FOR UNDERGRADUATE RESEARCH STUDENTS IN MICROBIOLOGY
Active Learning from the Very Beginning
Keri Law, Rachel Lamb, and Min-Ken Liao

LABORATORY SAFETY AND HOUSEKEEPING

The key to an accident free research experience is common sense. Of course everyone makes mistakes, but if you are conscientious about your work area and aware of your surroundings, you can prevent a harmful situation.

The microbiology lab is generally a safe place, and contrary to popular belief is not filled with deadly disease-causing agents. However, the microorganisms you work with should still be treated as pathogenic agents, a mind set that will encourage you to work extra carefully and maintain an exceptionally clean working environment.

Besides handling materials that are considered biohazards, several of the techniques described in this manual involve open flames, glass, and chemicals that should be handled with great care.

The safety of others who work in the lab is also a concern, so communicating by labeling things clearly, asking questions, and cautioning others is very important. Thorough cleaning is also essential to a safe lab.

Again, personal safety and the safety of others in the lab can be assured by exercising common sense and following the specific guidelines listed below:

PART 1: PREPARING FOR RESEARCH

1) Come to the lab wearing old clothing or a lab coat to avoid staining or singeing good clothes. Tie back long hair and do not wear a necktie that could come in contact with an open flame. Wear closed shoes with socks in case of broken glass.

2) Wash bench or work area before beginning with a disinfectant, and wash your hands to ensure a better aseptic technique (more in Section 4). 75% ethanol is an effective disinfectant because it evaporates at a slow enough rate and has a high enough concentration to ensure a near sterile working area. Soap and water is fine for hands.

3) Know where safety devices are located in the lab and in the building (fire extinguisher, safety shower, eye wash, etc...) as well as the location of the nearest phone in case of an emergency. You can dial 911 directly from campus phones, but you must dial an 8 before dialing any local number. You can also call Public Safety at x7770 for a non-emergency (getting into a locked room after hours). Also know where to find Dr. Liao's office and home phone numbers and Dr. Barney's (the Chair) office and home phone numbers.

4) Before beginning an experiment, make sure you can locate and understand the general facts in the lab. This includes charts on the walls describing concentrations, colors of plates, etc., as well as the contents of the strain book and the lab protocol book.

PART 2: WORKING IN THE LAB

1) Keep only your notebook and the materials you are working with on the bench: clutter can lead to spills and confusion.

2) Remember to treat the bacteria as if they were pathogenic and avoid contact with your mouth: no eating or drinking in the lab.

3) Be very careful around flames: watch sleeves and hair, also avoid contact with recently flamed loops, spreaders, pipettes, etc.

4) Do not touch any broken glass: sweep it into a dustpan and discard it in the broken glass box or a red "sharps" box on the bench, usually labeled "Biohazard."

[images of a sharps box]

5) When inserting a glass pipette into a Pipette pump, DO NOT PUSH ON THE TIP OF THE PIPETTE. The pipette might break and puncture your hand. Push the pipette in near the top, close to the Pipette pump.

[image of INCORRECT usage] [image of the CORRECT usage]

6) Do not ever "mouth pipette."

7) Carefully label everything: plates, solutions, frozen cells, etc. This is even more important to the organization and success of your research than to the safety of yourself and others. Labels should include your name or initials, the date, the page number in your lab notebook, and a brief description of what the plate, vial, etc. contains, including the name and concentration. Include any comments that would be helpful to a newcomer in the lab. Write legibly!

8) Be aware of warnings on the labels of some chemicals in the stockroom. The bases, stains, and culture mediums used in microbiology are generally harmless, but you may encounter more hazardous materials when taking inventory (see Appendix B for a description of the labels on chemicals).

9) You are strongly encouraged to work independently. However, if you do not know how to do something, ASK! You will look more foolish if you hurt yourself or break something by charging ahead than stopping to ask a simple question.

PART 3: LEAVING THE LAB

1) Dispose of biohazardous waste (any plates, slants, pipettes, gloves, etc. that came into contact with a living thing) in the large biohazard bags. Autoclave them and THEN throw them away. Liquid mediums containing bacterial cells must also be autoclaved before dumping the liquid down the sink. REMOVE ITEMS FROM AUTOCLAVE WITH CARE-THEY WILL BE HOT! (More on autoclaving in Section Four).

2) Glass or other sharp objects should be discarded in a sharps disposal box, (red, on the lab bench) or the larger broken glass box if it is not a biohazard.

3) When washing glassware, the key is thorough rinsing. Many a microbiologist has been frustrated by bacteria that refused to grow because a flask was still coated with unseen soap residue even after autoclaving. Therefore, use a moderate amount of dish detergent, rinse 3 times with tap water and 5 times with distilled water (dH2O).

4) Clean your work surface again with disinfectant (70% ethanol) and wash your hands.

REVIEW

1) Locate the nearest safety shower. Record location here:

2) Locate the nearest fire extinguisher. Record location here:

3) Is there a broken glass box in the lab? Where?

4)Are there a red sharps box and a large biohazard bag for disposing of plates? Where?

5) Find Dr. Liao's home and office phone numbers and write them here.

Home:____________ Office:_____________

6) Find Dr. Barney's home and office numbers and write them here.

Home:____________ Office:_____________

7) Find Public Safety's phone number and write them here:

8) Find the closest campus telephone to the research lab and write them here:

9) Interpret the labels given below:

10) In the following exercise, make a label for a vial that contains 0.5 grams of wet cells (E. coli), 10 mL of phosphate buffer, and 0.050 g of substrate.