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Syllabus

• Agar slant tubes
• Aseptic technique
• Assays
• Assays Illustrated
• Colony morphology
• Gram stain
• Isolating colonies
• Making serial dilutions
• Culture media
• Preparing spread plates
• Relationship to oxygen
• Viewing Gram stains
• Viewing living bacteria

• Characterization writeup
• Lab responsibilities
• Project proposal guidelines
• Using Bergey's

Supplemental pdfs

• Inoculating sticks
• Microscopy procedures

Laboratory Responsibilities

When many people share the same laboratory it is essential that each of us makes a wholehearted effort to maintain the facility in good order. Treating the facility and equipment with respect includes leaving benches and common areas in good condition, shutting off equipment when not in use, reporting low stocks, etc. Please review the list of best practices in our microbiology laboratory (below), organized by area, and plan to implement them throughout the course.

In addition, each of you is asked to select one of the eight areas and take charge of ensuring that the corresponding responsibilities are carried out. Taking charge means encouraging the rest of the class to adhere to the rules and to set a good example yourself. Taking charge may mean diplomatically pointing out violations to individuals or groups of individuals. It may mean reporting repeat violations to an instructor. It does not mean relieving others of responsibility by carrying out their obligations yourself.

Assay hood

One fume hood will be dedicated to assay reagents and related materials, and a list of these items will be posted prominently in or near the hood. Students responsible for the assay hood will ensure that all items on the list kept organized inside the hood and that all items taken from the hood are returned. They are to report low stocks of any item and note any items that have gone bad. At the end of each day the light is to be turned off and sash lowered to the fully closed position.

Contaminated material disposal

All contaminated materials, primarily agar plates and tubes containing bacterial cultures, are to be placed in the designated fume hood. Students will ensure that plates to be destroyed are placed in an autoclave bag and that tubes are placed in racks, consolidated and organized by type, ready to be sterilized. For tubes with screw caps the caps should be loosened a half turn. When an autoclave bag is past half full it is to be placed in an autoclave tray, sterilized in the next available autoclave run (along with any full tube racks), and replaced with an empty bag. After sterilization the bag is to be discarded in the regular trash. Racks of used, sterilized tubes are to be labeled as sterilized. Our lab assistants will clean up and/or dispose of the tubes. At the end of each day the fume hood sash is to be lowered to the fully closed position and the light turned off.

Gram stain stations

Students responsible for the Gram stain stations will be given a list of supplies to be maintained. Each day, each station is to be straightened up and any missing supplies are to be tracked down and replaced. All trays are to be emptied and rinsed at least once a day. Refill Gram stain reagent bottles and squirt bottles when they reach 1/3 full (check the Gram crystal violet and safranin bottles by feel). Likewise, carefully consolidate boxes of clean microscope slides and coverslips that are less than 1/3 full and put out fresh boxes as needed.

Incubators

Students responsible for the incubators are to check the temperature each day. They are to check for agar plates and/or tubes that have been left for more than a few days and notify teams and/or individuals who are responsible for such materials to remove them. They are also asked to organize the anaerobic incubations so that the jars and sachets are used efficiently, and to dispose of used sachets, rinse the jars, replace labels, and return them to the appropriate bench.

Media bench

Students are to straighten up the supplies on the media bench each day and track down missing media bottles, etc. and return them to the bench. To keep powdered media and chemicals fresh they are to check that lids are secured on the containers. Each day, each balance is to be examined and cleaned if necessary. The bench is to be wiped down as needed and spatulas, scoops, flasks, and beakers returned after use.

Microscopes

Each microscope, when not in use, should remain with eyepiece tube in the working position, plugged in with light off, no slide on the stage, 4x objective lens in place, and dust jacket in place. Students responsible for the microscopes should check each assigned instrument at least every other day by examining a slide with two Gram stained smears, one of which should be kept oil-free. Examine the oil-free smear in bright field and check for a clean image with the 4x, 10x, and 40x objectives, respectively. Check that phase contrast at 400x is not compromised. Examine the other smear at 1000x, checking both 100x objectives if the instrument is so equipped. If the instrument has a 100x phase contrast objective, then check it with the Ph 4 setting as well as in bright field. Report any problems to an instructor.

Prep rooms

The prep rooms include ABL 141A and 139 (the back room with large hood and water baths). Each day, check that the water baths, if not in use, are switched off, and keep them filled to the appropriate level with deionized water. If the large sterile cabinet in 139 is not in use then the light and blower should be off and sash all the way down. If it is in use for drying plates, the responsible team should have put a dated note on the window. Any plates left in the hood for more than a day and a half should be removed to the small cabinet in 141A and responsible team(s) notified if possible. Occasionally clean up any spilled agar, etc. by spraying the surface with Lysol and wiping it down with paper towels.

Safety

Students signing up for this set of responsibilities must be well aware of the rules for safe conduct in any biology laboratory as well as the specific rules for this one. Students violating the rules for proper lab attire, including long pants, lab coats, safety glasses, and closed-toed shoes, should be discretely reminded of the rules. Likewise for students showing up with food or drink or chewing gum. Watch for unattended flames, unsafe handling of hot media, etc. See that the lights are turned off at the end of each day, check that there are no open gas jet, and if no one is using gas, turn off the main gas valve.