Experiment 1a - Lab Protocol Basic Bacterial Techniques I

1. Pour plates Based upon the recipe in your lab manual, combine the necessary ingredients into a 500ml flask to pour 300ml of LB plates. Remember that water need to be added first. You can use tap water. It is not necessary to add NaOH. Add a magnetic stir bar, cover the flask opening with aluminum foil, label it with your initials on the foil. After your media has cooled, pour about 25ml of media into each of sterile Petri dishes enough to cover the bottom of the Petri dish. You will need at least 12 plates for the next laboratory. Please pay attention to the instructors demonstration. Use a stirring plate to mix the media prior to pouring do not stir so fast that you introduce bubbles into the media! 2. Listen to the Safety Briefing 3. Observe sterile technique The instructor or TA will demonstrate basic sterile bacterial techniques for 1) transferring liquid from one container to another 2) inoculating liquid media from a solid media culture using inoculating loops and applicator sticks. Practice inoculating a 5ml overnight culture Obtain and label a 50 ml conical tube that contains ~20ml of sterile LB media. This is your LB stock for this experimental procedure. Aseptically transfer 5ml of liquid LB media into two culture tubes. Label both tubes with your initials using a permanent marker (culture tubes are disposable, do not use tape). Mark your tubes very close to the cap or you risk the chance of rubbing off your label during incubation. Do not write on the caps! Inoculate one of the culture tubes with bacteria from a master plate provided. Set up a negative control overnight culture by inoculating the other culture tube, using the same inoculation procedure, but do not place bacteria on the loop/applicator stick. (This negative control is not normally performed in a research laboratory, however, it is a good check to see if you are aseptically performing the procedure.)

Your overnight cultures and LB stock bottle will be placed in the 37oC shaking incubator. Next week you can observe your two cultures and stock tube for growth. 4. Practice streaking for singles The instructor or TA will demonstrate basic sterile bacterial techniques for streaking for singles from a solid media culture using inoculating loops, applicator sticks, and toothpicks. Properly label a nutrient agar plate on the media half (the bottom) of the Petri dishes. (The lids tend to become mixed-up when working with multiple plates.) Divide the plate bottom into three sectors using a marker. Streak each section using a different applicator. Return the plate to the tray. Plates will be placed in the 37oC incubator for overnight. Be sure to lay the plates with the lids down (media upside down). Overnight, condensation forms on the top of the petri dishes. If this condensation drops onto media then it will splatter the bacteria. The result is a large smear of bacterial growth instead of single colony formation.

5. Phenotype testing of bacteria by patching. You will be given master plates with six bacterial strains that have different genotypes. You will work in pairs and will obtain a set of plates with selective media to do the phenotyping. You and your partner will need to patch all six unknown bacterial strains onto each of selective media plates. You can use any applicator that you like the most: a loop, a wooden applicator or a toothpick. Make sure you decide what pattern you will use for patching . Label the plates appropriately. When done, tape all six plates together, label the tape and return the plates to the tray. The plates will be kept at 37oC incubator overnight. You will look at your results at the next week lab.