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Background

• Local info
• Water issues
• Projects
• Characterization
• Sample article (pdf)
• How to use Bergey's Manuals

Methods

• Rules
• Media/culture
• Aseptic technique
• Staining/interpretation
• Isolation strategies
• Assays/characterization
• Microscopy
• Cultural characteristics

Media preparation
and training

Laboratory Safety and Conduct

With some exceptions the potential hazards in a typical biology or biochemistry laboratory are no different than hazards you face in your own kitchen or garage. For the most part, simply exercising common sense in the laboratory is sufficient. The general guidelines below spell out the 'common sense' of behavior in the laboratory. Immediately below this introduction we outline specific hazards associated with laboratory studies in microbiology and precautions that we must take to mitigate them. Remember, you are ultimately responsible for your own behavior – be careful.

Specific hazards and safety guidelines for Bioc 318

Specific hazards, required apparel, and how to avoid injury will be discussed at the introductory meeting and during the traning sessions. NOTE: Because of the specific hazards associated with microbiology, we must stress the importance of having at least one other person in the laboratory with you. You must not work alone in the laboratory.

Hazards

• Bacteria of unknown origin
• Open flames
• Hot liquids
• Explosion hazard from liquids under pressure
• Strong acids, bases, corrosives
• Toxic reagents

Required safety apparel

• Closed-toed shoes (always)
• Long pants or skirts/dresses to the knee or below
• Lab coat
• Safety glasses

Specifics

• Each student will be provided a disposable lab coat to be worn when in the laboratory and to be kept in the laboratory at all times
• Each student will be responsible for obtaining a pair of laboratory safety glasses or goggles; approved apparel must include side protection as required by chemistry labs; eyeglasses do not provide sufficient protection; approved safety glasses are available through the department at a small cost
• Eye protection is to be worn at all times in the laboratory except when working at a microscope; this requirement supercedes the recommendations for safety glasses in the general guidelines
• Disposable gloves will be provided to protect the hands during Gram staining and/or other "dirty" procedures; gloves are not to be worn when working near an open flame because contact with flame may cause the material to melt and burn the skin
• Long hair must be tied back to avoid injury when working near an open flame

General safety guidelines

• The teaching laboratory is a classroom and your full attention is required. For both safety reasons and to avoid disrupting classroom activities the use of cell phones and pagers in the laboratory is prohibited. During training and other formal classroom activities all such devices are to be turned off. At other times, such devices can be left on; users must step outside of the laboratory to answer or make calls.
• All persons in laboratories, including students, staff, and visitors, shall wear safety glasses, goggles, or face shields at all times where potential eye hazards exist. Goggles are recommended where chemical splashes are possible. If contact lenses are to be worn, the eyes should be protected by goggles when potential eye hazards exist. NOTE: prescription eyeglasses do not provide adequate protection.
• Eating, drinking, chewing gum, and applying cosmetics are prohibited in all teaching laboratories. Food and drink are permitted in areas that are safely separated from laboratory work areas, such as the basement laboratory classroom and picnic tables outside of the first floor laboratory.
• Do not store food or beverages in the same refrigerators or freezers with chemicals, biohazards, or radioactive materials.
• Appropriate gloves are essential when working with hazardous substances. All glove materials are not equally effective in protection from hazardous substances; consult a chemical resistance chart, a glove manufacturer, or EH&S for appropriate selection.
• Never conduct unauthorized experiments or engage in horseplay in a laboratory. Please immediately report any unsafe behavior to the instructor.
• Wear appropriate clothing. In particular, you must wear closed-toed shoes (i.e., NO sandals or flip-flops!) in the laboratory. Avoid wearing your best clothes, and if you have long hair tie it back. Avoid wearing dangling jewelry.
• Wearing an iPod, Bluetooth, or any other device that inteferes with hearing is not allowed.
• Never pipet anything by mouth.
• The work area must be kept clean and uncluttered. All chemicals should be labeled and stored properly.
• Never work alone in the laboratory.
• The hazards of chemicals used should be known (e.g., corrosiveness, flammability, reactivity, stability, and toxicity). Material Safety Dat Sheets (MSDSs) should be available for all non-routine or acutely toxic chemicals used and stored in the laboratory.
• Always pay attention to your surroundings and be aware of what others are doing. Always be courteous.
• Remove contaminated gloves before touching common use devices (door knobs, faucets, equipment); discard gloves before leaving the laboratory.
• Always wash hands and arms with soap and water before leaving the laboratory.
• No smoking!

Emergency

• Know where to find the nearest exit in case of fire or other emergency.
• Know the whereabouts of the nearest fire extinguisher, fire blanket, first aid kit, eye wash equipment, shower and telephone.
• For on-campus emergencies call x6000.
• In case of fire, clear out of the laboratory first, then call an emergency number.

Hazardous materials

• Both liquid and dry chemicals can be flammable, poisonous, carcinogenic, etc. Pay attention to special instructions, such as to work with a substance only in a fume hood.
• Biological hazards include bacteria and body fluids, such as blood. Handle with appropriate care, and dispose of biological hazards as instructed.
• Dispose of hazardous materials as instructed. Never put anything down the sink without checking with an instructor.
• Clean up spills and broken glass. Don't handle broken glass with your bare hands. Use a broom and dustpan, and throw away all broken glass and disposable glass pipets, coverslips, and other sharp or easily breakable glass in a container for glass disposal only. Notify the instructor immediately of all incidents.

Hazardous equipment

• If appropriate, turn off equipment that isn't being used.
• Do not use a Bunsen burner unless instructed to do so.
• Keep liquids and chemicals, especially flammable materials, well away from any heat source or electrical equipment.
• If any electrical equipment is malfunctioning, making strange noises, sparking, smoking, or smells "funny," do not attempt to shut it off or unplug it. Get an instructor immediately. It is imperative that the instructor know of any equipment problems.

Cleanup

• Clean spilled chemicals from equipment such as balances - they can corrode and ruin equipment.
• Mop any liquid spills - they are a slip hazard.
• Be especially careful around the ice machine - spilled ice quickly melts, creating a slip hazard.
• Clean up your bench area at the end of the day, or during your session in the lab if things are too messy.
• Always wash your hands before leaving the laboratory.

General conduct

• Keep coats, purses, backpacks, books, and similar materials away from your work area and out of the way of your colleagues. Please stash such items in an appropriate, perhaps designated location. We mustn't force people to step over a backpack placed in an aisle between benches, for example. Also, consider what will happen to such items if someone spills chemicals or biohazardous materials on them.
• Computer use in the laboratory should be limited to accessing course materials on the web or internet searches related to the projects; go outside to conduct personal business.
• On occasion, special equipment, e.g., stir plates and vortexers, will be used from the storeroom and this equipment should be returned complete, clean, and dry at the end of the experiment.
• When using equipment, be certain that you understand how to operate the device safely. Any equipment or areas you use must be cleaned after use. For example, the centrifuges should be wiped out and the rotors rinsed and allowed to drain.
• Do not waste materials, including disposable items and reagents. This practice is economically and ecologically important. Calculate how much you will need and take only a slightly greater amount (about 10% excess).
• Do not return excess reagent to the stock bottles. Discard properly at the end of lab period. Pay attention to the discussions prior to the experiment for special disposal instructions.
• Do not pipet directly from bottles or jugs containing buffers or reagents.
  Exception: you may use an automatic pipetor to obtain stock solutions of small volumes (less than 2 ml); be careful to use a clean pipet tip and do not contaminate these solutions.
• Put the lids on stock solutions and reagents after using them. Otherwise solutions can become contaminated or just evaporate. Many solid reagents are hygroscopic and pull water out of the air.
• Pay attention to lab "rules" for labeling containers, tubes, etc. Some labs require the use of tape, others expressly prohibit using lab tape for labels.
• If you don't find some of the required equipment or reagents, ask the instructor or a TA for help.