ExerciseⅢ Basic Manipulation of MicroorganismⅡ（Pre...
Learning Objectives
Each student should be able to
1.Describe the different types of culture media and their composition，and give several examples of what each is used for.
2.Describe the various ways culture tubes are capped.
3.Describe how to prepare and transfer culture media.
4.Prepare defined and undefined media，and prepare agar plates.
5.Describe the concept of sterility.
6.Describe how various media，supplies，and equipment can be sterilized.
7.Correctly and safely use the autoclave.
Materials per Group
24 to 48 hour tryptic soy broth culture of Staphylococcus aureus autoclave for the entire class；petri plates；culture tubes；test-tube rack or wire basket；testtube caps；defined culture medium as in table 13.1；complex culture medium as in table 13.2；2 liter Erlenmeyer flask；10 ml pipettes with pipettor；weighing paper or boats；balance agar；heat-proof Zetex fabric gloves；water bath set at 48℃to 50℃；boiling water bath；aluminum foil；stirring rod or plate with stirring bar；Bunsen burner or hot plate
Principles
Microbiological Culture Media
The survival and growth of microorganisms depend on available nutrients and a favorable growth environment.In the laboratory，the nutrient preparations that are used for culturing microorganisms are called media（singular，medium）.Three physical forms are used：liquid，or broth，media；semisolid media；and solid media.The major difference among these media is that solid and semisolid media contain a solidifying agent（usually agar），whereas a liquid medium does not.Liquid media，such as nutrient broth，tryptic soy broth，or brain-heart infusion broth[Figure 1（a）]，can be used to propagate large numbers of microorganisms in fermentation studies and for various biochemical tests.Semisolid media can also be used in fermentation studies，in determining bacterial motility，and in promoting anaerobic growth.Solid media，such as nutrient agar or blood agar，are used（1）for the surface growth of microorganisms in order to observe colony appearance，（2）for pure culture isolations，（3）for storage of cultures，and（4）to observe specific biochemical reactions.
Figure 1 Culture Media
Different forms of culture media with the proper volume in each
While in the liquefied state，solid media can be poured into either a test tube or petri plate（dish）.If the medium in the test tube is allowed to harden in a slanted position，the tube is designated an agar slant[Figure 1（b），（c）]；if the tube is allowed to harden in an upright position，the tube is designated an agar deep tube[Figure 1（d）]；and if the agar is poured into a petri plate，the plate is designated an agar plate[Figure 1（e）].Agar pours（the same as Agar deeps）containing about 15 to 16 ml of media are often used to prepare agar plates.
Microorganisms may be cultured using two different types of media.Chemically defined，or synthetic，media are composed of known amounts of pure chemicals.Such media are often used in culturing autotrophic microorganisms such as algae or nonfastidious heterotrophs.In routine bacteriology laboratory exercises，complex，or nonsynthetic，media are employed（Table 1）.These are composed of complex materials that are rich in vitamins and nutrients.Three of the most commonly used components are beef extract，yeast extract，and peptones.
Table 1 A Complex（Undefined）Medium-Tryptic Soy Broth
If the medium lacks agar，the powder will usually dissolve without heating.If it contains agar，you must heat the medium until it starts to simmer or boil in order to completely dissolve the agar.Specific heating instructions are given for each type of medium.Most of the exercises you will be doing in this manual will involve the use of sterile media in culture tubes.Normally，18 mm×150 mm，16 mm×125 mm，or 13 mm×100 mm bacteriologic culture tubes will be used.
These tubes must be capped in order to maintain media sterility.This can be accomplished by using cotton plugs，plastic foam plugs，or plastic or metal caps（e.g.Morton closures or Bacti Capalls）.All of these caps keep cultures free from contamination while allowing air into the culture tube，and at the same time，minimize evaporation.It is sometimes desirable to use screw-cap culture tubes.This is especially true when the culture，such as in the case of slants，may be sealed and stored for long periods.
This approach is fast，convenient，and relatively accurate.After sterilization of slant tubes，the tubes are removed from the autoclave while the agar is still melted and carefully laid on a table with a piece of wood，vacuum tubing，or metal elevating the capped ends.Some test-tube racks are also specifically set up for this.The tubes are then allowed to remain undisturbed until the agar has cooled and hardened.Slants should be stored in a vertical position.
Agar deep tubes can be stored after sterilization for use in preparing petri plates when needed.Some agar deeps may be stored at room temperature for several days before use.If longer periods of storage are required，they should be placed in the refrigerator in order to prevent drying of the agar.When petri plates are needed，the agar deeps are melted either in a boiling water bath[Figure 2（a）]or by bringing them to 121℃in an autoclave for 30 to 60 seconds and then releasing the steam under slow exhaust.After the agar has melted，the pours are transferred to a 48℃to 50℃water bath and kept there for at least 5 to 10 minutes before use[Figure 2（b）].The agar deeps should be cooled to about 50℃before they are used to minimize the amount of steam condensation on the petri plate lids after the agar has been poured.Agar does not solidify until its temperature drops to about 42℃.When the deeps have reached 50℃，one is taken from the bath and the outside is dried with a paper towel[Figure 2（c）].Its cap is removed and the top is briefly flamed using a Bunsen burner[Figure 2（d）].The agar is immediately poured into a sterile，dry petri plate while holding the top carefully above the petri plate bottom in order to avoid contamination[Figure 2（e）].Replace the top，allow the agar to cool and harden，and store the petri plates in an inverted position[Figure 2（f）].When storing petri plates，do not stack them more than three high，or use a special petri plate storage holder.
Sterilization of Media and Equipment(https://www.daowen.com)
Figure 2 Pouring Agar Plates
Sterilization is the process of rendering a medium or material free of all forms of life.There are three basic ways in which sterilization of media and supplies can be achieved.The most useful approach is autoclaving，in which items are sterilized by exposure to steam at 121℃and 15 lbs of pressure for 15 minutes or longer，depending on the nature of the item.Under these conditions，microorganisms，even endospores，will not survive longer than about 12 to 13 minutes.This method is rapid and dependable.Modern autoclaves are designed to ensure that all of the air has been expelled and only steam is present in the autoclave chamber.They are carefully temperature controlled as well.Almost all media and anything else that will resist 121℃temperatures and steam can be sterilized in this way.
Often，dry glassware such as pipettes and petri plates must be sterilized.Steam tends to etch glassware and also leaves it damp.Therefore，such items are generally dry-heat sterilized.The glassware is placed in an electric oven set to operate between 160℃and 170℃.Since dry heat is not as effective as wet heat，the glassware must be kept at this temperature for about 2 hours or longer.The oven temperature must not rise above 180℃or any cotton or paper present will char.
Two other sterilization techniques use ultraviolet radiation and ethylene oxide.Ultraviolet（UV）radiation around 260 nm is quite lethal to many microorganisms but does not penetrate glass，dirt films，water，and other substances very effectively.Because of this disadvantage，UV is used as a sterilizing agent only in a few particular situations.For example，UV lamps are sometimes placed on the ceilings of rooms or in biological safety cabinets to sterilize the air and any exposed surfaces.Many heat-sensitive items such as disposable plastic petri dishes and syringes，sutures，and catheters are now sterilized with ethylene oxide gas.Ethylene oxide is both microbicidal and sporicidal and kills by covalently attaching to cell proteins.It is a particularly effective sterilizing agent because it rapidly penetrates packing materials，even plastic wraps.
Procedure
Preparing a Complex Medium
1.Prepare 500 ml of tryptic soy broth according to the recipe outlined in table 1.1.
2.Add 375 ml of distilled water to a 1-liter Erlenmeyer flask and add the ingredients individually（use half the amounts given）；mix after each addition.
3.Add the remaining 125 ml of water to rinse the sides of the flask.
4.Adjust the p H to 7.3 by adding just enough HCl or NaOH dropwise.
5.Dispense 3 to 5 ml of the broth into each of 10 tubes and loosely cap them.Place the tubes in a test-tube rack or basket，and place in the autoclave.
6.To the remaining broth（450 ml），add 7.2 g of agar to give an agar concentration of approximately 1.6%.Heat the contents of the flask and gradually bring to a boil.Heat the agar until it is completely melted.Cover with aluminum foil and place in the autoclave.After autoclaving，cool the flask of sterile agar in a 48℃to 50℃water bath.Line up the desired number of sterile petri plates on the bench top.Remove the aluminum foil cap from the flask and briefly flame the flask's neck.Lift the top of each plate，pour about 15 ml of agar，and quickly replace the top（the agar should be approximately 3 to 5 mm deep in the plate）.Pour all plates without stopping.Alternatively，after dissolving the agar medium，dispense 15 ml portions into 18 mm×150 mm tubes，cap and autoclave the tubes.Cool them in a 48℃to 50℃water bath and pour the agar as shown in figure 2.
Procedure for Autoclaving
1.Instructor will demonstrate the use of the autoclave.
2.Load the autoclave with the freshly prepared culture media.
3.Close and lock the autoclave door.
4.Set the autoclave time for 15 minutes or longer and select a slow rate of exhaust.
5.Make certain that the autoclave temperature is set to 121℃.
6.Start the autoclave by pushing the start button or twisting the knob to the start position.
7.When the period of sterilization is completed and the pressure in the chamber reads 0，carefully open the door and remove the containers，using heat-proof gloves.
Preparing Agar Plates
1.As outlined previously and in Figure 2a-f，use some of the sterilized tryptic soy agar to prepare agar plates.
2.When the plates are cool（agar solidified），invert them to prevent condensing moisture from accumulating on the agar surfaces.
3.All plates and tubes should be incubated for at least 24 hours to ensure sterility before you use them.