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Preparation of Culture Media
Any medium for the cultivation of bacteria or yeast must provide certain basic nutritional requirements, which include the following: (1) a carbon source that may also serve as an energy source, (2) water, (3) a nitrogen source, (4) a phosphorus source, (5) a sulfur source, and (6) various mineral nutrients, such as iron and magnesium. Escherichia coli and Saccharomyces cerevisiae are capable of growth on a medium consisting of a single carbon source, such as the carbohydrate glucose; a simple nitrogen source, such as ammonium chloride or ammonium sulfate; and other inorganic salts providing phosphorus, sulfur, and minerals. Saccharomyces cerevisiae requires some vitamins as well. This kind of medium is termed defined or synthetic because its exact chemical composition is known. For routine laboratory work, however, complex media are often employed. The basic nutrients in these media are provided by plant and animal extracts of which the exact composition is not known. For example, yeast extract and peptones (hydrolyzed protein) are the basic ingredients of the Luria-Bertani (LB) broth commonly used for E. coli and the yeast extract-peptone-dextrose (YEPD or YPD) broth commonly used for S. cerevisiae. These materials supply a variety of carbon sources; nitrogen-, phosphorus-, and sulfur-containing compounds in the form of peptides and amino acids; and a mixture of cofactors, such as vitamins. A broth medium is one in which the components
In this exercise. cerevisiae and how to sterilize the medium. these include exposure to elevated temperatures or radiation levels to kill microorganisms. a temperature of 121~ [achieved by using steam at 15 psi (lb/in2)] for 20 minutes is used to heat-sterilize microbiological media. Medium preparation requires an autoclave for sterilization. Generally. and instruments used in pure culture procedures. transfer loops.18 Exercise 2 are simply dissolved in distilled water. you will prepare both a defined and a complex medium. Solid agar melts at 90-100~ liquid agar solidifies at about 42~ Sterilization procedures eliminate all viable microorganisms. Reagents/Supplies Adenine sulfate Agar Beakers (1 liter) Dextrose (glucose) Erlenmeyer flasks (500 ml and 1 liter) Graduated cylinders (250 and 500 ml) Heat-resistant gloves Histidine Lysine . You will learn how to mix the proper constituents to support the growth of S. containers. flasks. and media must be free of viable microorganisms before they can be used for establishing pure cultures. There are various ways of sterilizing the liquids. which permits exposure to high temperatures for a specific period of time. Sterilization Methods). Culture dishes. The addition of agar (a complex carbohydrate extracted from seaweed) results in a solid medium. The culture vessels must be sealed or capped with sterile plugs to prevent contamination. pipets. and filtration to remove microorganisms from suspension (see Appendix 9. Agar is an ideal solidifying agent for a microbiological medium because of its melting properties and because it has no nutritive value for the vast majority of bacteria and fungi. test tubes.
we have found it important for the students to prepare their own media rather than having this demonstrated to them. and 10 g of glucose .Preparation of Culture Media 19 Peptone Petri plates. You may wish to divide preparation of various media among groups of students. Preparation of YEPD. a Complex Medium for Yeast [Yeast Extract (1%. Dextrose (2%. Peptone (2%. 10 g of peptone. w/v). w/v)] 1. Add 500 ml of distilled water to a l-liter Erlenmeyer flask. However. w/v). without amino acids (Difco) Equipment Autoclave Filter sterilization unit (for 100-ml volume) Incubator at 37~ Magnetic stirrer pH meter Top-loading balance Water bath (50~ Instructor's Note Proper budgeting of time is essential for completion of this exercise within the laboratory period. Procedure Part A. Weigh out 5 g of yeast extract. sterile Plastic bags Screw-cap bottle (100 ml) Test tube rack Test tubes (18 x 150 mm) with caps Tryptophan Uracil Yeast extract Yeast nitrogen base (YNB) with ammonium sulfate.
In the automatic mode. or during the autoclaving period. Slow venting is required to prevent liquids from boiling. The sterilization cycle involves filling the jacket. do not autoclave two of the test tubes. Dispense the broth medium (Flask A) into 15 test tubes. Swirl to disperse the agar. Before starting Step 6. Place Flask B and Flask A (with 175 ml of YEPD remaining) into the autoclave for autoclaving as well. (Note: Heating may be necessary. 6. and close the autoclave door. adding 5 ml of broth to each tube. make up YNB medium components (see Part B. below). In manual operation. Most autoclaves have an automatic cycle. Flask B: Add 5 g of agar.20 Exercise 2 (dextrose) on a top-loading balance. simply label and leave them on the benchtop until the next laboratory period. 4. you must move the selection lever to the vent position after the chamber has been at 121~ for 20 minutes. the chamber will begin to fill with steam after the jacket pressure reaches 15-20 psi. use of the pH meter may be demonstrated (see Exercise 2A in Appendix 1). allowing steam to enter the chamber. you must move the selection lever to the fill chamber position when the jacket pressure reaches this level. 3. holding the temperature for the amount of time that you have set. in the manual mode. At this time. To demonstrate the necessity of the sterilization step. 5. set the timer for 20 minutes on the slow exhaust mode. dissolve these in distilled water by stirring with a magnetic stirrer. Divide this broth solution into two equal parts by adding 250 ml each to two 500-ml Erlenmeyer flasks: Flask A: Make no further additions. Cap each test tube (do not tighten) and put aside for autoclaving later. Start the autoclave cycle by pushing the start button in the automatic cycle mode or in the manual mode by turning the selection lever to the fill position. . and venting the chamber.) 2. Load the autoclave with all solutions from Parts A and B that require autoclaving.
place the flask in a 50~ water bath and equilibrate for 30 minutes. flame the mouth of the flask and. Allow the agar plates to cool. pour about 20 ml of agar into a plate (enough medium to cover the bottom of the plate). but carefully to prevent accidents. and medium. Never completely remove the lids of the Petri plates or the plates will become contaminated with bacteria and fungi from the air. 0 Part B. label the plates with your name.7 g of yeast nitrogen base (YNB) with ammonium sulfate. Autoclaving for 20 minutes actually takes about 40 minutes when you include the time required for heating and venting the chamber. without amino acids (Difco) to 100 ml of distilled water. use heat-resistant gloves: the material is still hot! 0 After removal from the autoclave. (The purpose of the cooling step is to prevent excessive water condensation on the Petri dish lid due to evaporation from agar at temperatures >50~ and to prevent solidification of agar at <50~ To dispense the medium into sterile Petri plates. Preparation of YNB Medium. a Defined Medium for Saccharomyces cerevisiae 1. Replace the lid and continue filling additional plates until all of the medium is dispensed. Add 6. The . Store the plates at room temperature or at 4~ in plastic bags for use in a later laboratory experiment. After the medium in Flask B has been sterilized.Preparation of Culture Media 21 Only after complete venting of the chamber can you open the autoclave and remove your material. and leave at room temperature or incubate at 37~ overnight to allow the agar to dry and to detect contaminated plates. Work quickly to minimize contamination. When removing material from the autoclave. You should periodically reflame the mouth of the flask to reduce contamination. allow the broth tubes and 175 ml of YEPD (Flask A) to cool before tightening the caps and storing for use in later exercises. After the agar has solidified. while carefully lifting the lid of a Petri plate. date.
as described below. being careful not to introduce excess bubbles. Also prepare 100-ml solutions of uracil and adenine sulfate (each 2 mg/ml). w/v) Distilled water Liquid (ml) 10 w 0.5 10 76. Table 2. Be sure to temperature equilibrate medium components to 55~ before addition of agar. (See Appendix 10.5 1.1 0.1 0. Prepare 100-ml stock solutions at 30 mg/ml of lysine. 2. tryptophan.5 10 26.5 1.1 1. Place the molten agar at 55~ to equilibrate for pouring of plates on addition of prewarmed YNB. Autoclave. and glucose (Table 2. which should be filter sterilized.1 0. w/v) Lysine (30 mg/ml) Tryptophan (30 mg/ml) Histidine (30 mg/ml) Adenine sulfate (2 mg/ml) Uracil (2 mg/ml) Glucose (20%. 4. Preparation of Stock Solutions for Culture Media. Mix well.1 Preparation of YNB Medium Medium Component YNB (10• Agar (45/0. Store in a refrigerator. add 4% agar as indicated below.1 0.22 Exercise 2 suspension may require slight heating to dissolve.1). All can be autoclaved except tryptophan and adenine sulfate. supplements (amino acids. Filter sterilize for a 10x stock solution.7 Plates (ml) 10 50 0. Prepare 100 ml of a 4% (w/v) agar solution.1 1. bases). For agar plates.) 3. 5. and histidine. Pour the plates. which should be autoclaved. Prepare minimal medium with YNB.7 . Prepare a 100-m120% (w/v) glucose solution.
Cold Spring Harbor. Cold Spring Harbor. New York. C." 2nd Ed." Cold Spring Harbor Laboratory. (1994). [This is an excellent source for most of the basic methods used for yeast genetics and physiology. E.. New York.] Sambrook. Cold Spring Harbor Laboratory. and histidine added to the YNB medium? . Michaelis. and Mitchell.. and Maniatis. (1989). Fritsch.Preparation of Culture Media 23 References Kaiser. Explain the usage of both defined and undefined media for the growth of bacteria and yeast. 3. Why were lysine.] Questions 1. "Methods in Yeast Genetics. T. "Molecular Cloning: A Laboratory Manual. Why is the requirement for amino acids much lower than that for glucose in defined medium? 2... [This was the first and remains among the most useful manuals for molecular biological techniques. adenine. A. T. S. F.
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