This check list can help
you avoid ruining your new kit by a simple error,
or suffering an accident. The links are to information pages.
!! Beginners must not work with disease producing bacteria !!
Think Sterile. Everything must be sterile (autoclaved). That means cooked 15 minutes at 15 pounds pressure. Many plastic items can't be autoclaved. Always have a rack in the bottom of the pressure cooker to reduce breakage. TGY broth is a liquid. TGY agar is the same with 10 grams of agar per liter of final medium. Be sure to use the recommended amount of tap water or other specified liquid.
__ 1. Read our safety pages. Have a safety buddy watch your work.
__ 2. Mix each medium correctly. Use agar for solid medium. Make notes before you begin.
__ 3. Heat to boiling to dissolve the agar, if medium contains agar. Be careful to avoid boil over.
__ 4. Dispense into tubes, jars, or bottles. Insert cotton stoppers or apply loose caps.
__ 5. Place tubes in wire baskets or in metal cans with lots of holes in bottom
__ 6. Autoclave 15 minutes at 15 pounds. Apply pressure regulator after full steam is flowing. Be sure to use 2 to 4 glasses of water in the bottom of the pressure cooker.
__ 7. Let autoclave cool until no steam remains before you remove the regulator and open lid. When autoclaving liquids, never remove the regulator until all the steam is gone, else much of the liquid will explode out of the tubes and bottles and if a lid is tight that container may explode. Hot bottles may break and soak you with hot liquid. You must use a rack in bottom of cooker, bottles placed directly on bottom will break due to unequal expansion.
__ 8. Letting pressure cooker cool overnight is the best way to avoid burns and contamination.
__ 9. If making slants, you must remove tubes and lay them on a stick after cooling but before they gel.
__ 10. Store your media in clean boxes to avoid dust--if possible. Boxes will rduce contamination.
__ 0. Agar has no food value, be sure to add the food your bacteria needs. TGY agar already has food in the powder. Add any special ingredients that you want, such as phenol red, iron, inverted gas tubes, etc. Usually you do need to add anything to prepared agar mixes.
__ 1. Plates must be sterile. Dishes from unopened packages are sterile. If reusing plastic dishes, they can't be autoclaved. Used dishes can be sterilized in 1 volume Chlorox plus 9 volumes of tap water for 20 minutes. Then you must remove Chlorox by dipping plate in sterile tap water using sterile forceps (tweezers). It is OK to use the plates wet. If you do not use all the plates keep the remainder sterile by bending over top and taping it in place.
__ 2. Autoclave the medium in jars or bottles with loose caps for 15 minutes at 15 pounds. Then allow to cool until steam is gone before opening. Then tighten the loose caps; not too tight. Such bottles of sterile agar will keep for weeks, months, or years. To reheat cold agar, loosen cap and put in pressure cooker and heat at 15 pounds pressure for a couple minutes. Then allow to cool as before to pouring temperature; 55C is ideal. (Estimate tempature; do not stick thermometer into bottle).
__ 3. When sterile agar is cool but not gelled, pour 20 to 25 mL into each plate. You should practice first with water. Most beginners use too much agar and that wastes agar. Pour the agar about 5 mm deep. Most dishes are 15 mm deep and filled 1/3 deep is enough agar. Hold the lid in the left hand and hold above the plate to shield falling dust while pour agar from bottle with the right hand. Do not be overly concerned; very few plates will be contaminated with falling dust. Some steaming of the plate cover will occur, but that moisture will disappear in day or so. You can wrap plates in paper and store them in refrigerator for days or a few weeks. If you cooled the agar in a 55C water bath, do not let any of the water drip into the sterile plate. You may catch the drips with a clean towel.
Before autoclaving, fill the tube about 2.5 to 3 cm deep for slants and stabs; about 6 cm deep for deeps.
Slants. While still warm and liquid, lay tubes on a stick so that the slant comes down about half way--or as you desire.
Deeps and stabs let these cool more or less vertically. Since the cooker is dust-free, letting deeps and stabs cool overnite inside the cooker is the best method. If you need the cooker, let the tubes cool in a draft free, dust-free place. If the table is dusty wipe it with a clean wet rag and the wet surface will help trap dust.
Don't worry if you have minor problems. It is wise to leave a few tubes unused as sterilization controls. These tubes should last weeks without growth of mold or other contaminants. You must know whether your tubes were sterile. You may wrap tubes with paper or plastic wrap as a precaution against dust if you are going to keep them longer than a few days.
While you have the tube open, hold it almost horizontal so dust can't fall into the tube. When using cotton stoppers, it is customary to flame the mouth of the tube to burn away contaminated fibers, but I oppose open flames in beginner labs due to the risk of burns and setting home on fire. I do not flame the mouth of tubes and have had no problems.
Touch a sterile loop or stick to a colony or pure culture. Push the tool to the bottom of a stab. Inoculate slants along the slant, and also by stabing to the bottom of the tube if you wish. TSI (triple sugar iron) slants must be inoculated to the bottom of the butt with minimum disturbances (no air visible).
Inoculate broths by insert the tool in the broth and rolling the stick between thumb and fore finger rapidly.
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