Microbial Growth Media & Culturing Protocol (Biotech Series) Microbes such as bacteria, fungi, and viruses are like microscopic factories in biotechnology. Bacteria are the key constituent to most biotechnological processes and have revolutionized science and medicine. As part of the DNA revolution, biotechnologies have been developed for introducing DNA (such as a gene) from one species into another. Bacteria are particularly good at accepting foreign DNA, and introducing genes into bacterial cells is now a routine procedure. This procedure is a first step to making bacteria that can do new and useful things for society. In the biotechnology pathway you will routinely be culturing, cloning and transforming bacteria. This protocol details the basic procedures for preparing growth media such as agar plates, nutrient broth and slants. This protocol must be used in conjunction with Aseptic Technique Protocol (click here) and the Autoclave Protocol (click here). Mastering these critical laboratory skills we help to ensure your success as we progress with more advanced biotechnological techniques. Please refer to textbook pages 63-66 for additional details. Media are classified on the basis of composition or application. Usually, a particular medium may fall into two or more categories, which leads to some confusion among students. Thus, for example, a "defined" medium might be also classified as "selective" or "differential" depending upon its specific properties and application in a particular situation. The four principal types of culture media are described below. COMPLEX MEDIA: Pasteur, Koch, and Tyndall typically prepared media by boiling animal or plant materials to extract nutritive molecules. Today, many modern complex media (such as Tryptic Soy Agar) contain extracts of beef (peptone), milk (tryptone), soybean meal (soytone), or yeast. The proteins in these extracts are broken down into small peptides and amino acids. Although the specific amount of these molecules is not precisely determined, their wide assortment allows complex media to support a wide range of bacterial types. Examples may include nutrient agar, lysogeny broth (LB) or Luria-Bertani agar, Miller and Lennox LB, sheep blood, etc. DEFINED MEDIA: Defined media are formulated from pure substances at predetermined concentrations. Thus, unlike complex media, the exact chemical composition of defined media is known precisely. Because the composition is precisely established, defined media are often used to determine the nutritional requirements of bacterial species. SELECTIVE MEDIA: Complex or defined media may also be classified as selective (or enrichment) media, which support the growth of only certain types of bacteria. Media can be made selective through the addition of substances that enhance or inhibit the growth of particular types of bacteria. Media have been developed that are selective for an astonishing diversity of bacteria, and we will be using many of these media throughout the semester. DIFFERENTIAL MEDIA: Any of the above types of media might also be formulated as a differential medium. A differential medium reveals specific metabolic or metabolic characteristics of bacteria grown on it. Differential media are among the most powerful tools available to biotechnologists, revealing a wide range of information about an organism very quickly. Some media are both selective and differential. For example, the medium called MacConkey Agar is selective for gram-negative bacteria and will indicate whether bacteria can ferment lactose. Nutrient Broth, Agar Plates and Slants: The immense amount of variation in recipes for growth media is beyond the scope of this protocol; however, all recipes that will be used in the Biotechnology pathway will be archived and maintained in a separate logbook. A few of the most basic recipes are detailed on the next pages along with several schematic diagrams for inoculating, streaking and culturing microbes. Nutrient Broth: 1. Weigh out 8.0 grams of nutrient broth powder. 2. Add to 1.0 liter of distilled or deionized water in a 2.0 liter flask. 3. Dissolve the powder completely in the water. 4. Dispense into tubes or flasks using a graduated cylinder or a pipette. 5. Sterilize at 121° C for 20-25 minutes. Nutrient Agar Plates: 1. Weigh out 23.0 grams of nutrient agar powder. 2. Add to 1.0 liter of distilled or deionized water in a 2.0 liter flask. 3. Sterilize at 121° C for 20-25 minutes. Figure 1: Streaking an agar plate 4. Cool to 50° C. is done in 3-4 sections. The goal is 5. Swirl thoroughly to mix agar and nutrients. that sections “D” and “E” will 6. Pour 25-35 ml per petri plate. yield isolated, individual colonies. 7. Yields about 35 plates. Nutrient Agar Slants: 1. Place screw cap test tubes in a test tube rack (without the caps). 2. Prepare a nutrient agar medium and boil it with stirring until all agar is melted. You must stir this very well so that the melted agar is distributed throughout the medium. 3. Use a pipette to transfer ~5 ml of molten agar to each test tube. 4. When all tubes contain hot agar, place caps loosely on the tubes. 5. Sterilize the tubes at 121° C for 15 minutes with caps loosely on. Figure 2: Streaking and 6. While the medium is still hot, tilt the rack onto a thick book or other inoculating a slant. solid surface so that the medium in the tubes is slanted. Allow the medium to harden in this position. 7. When the medium is cool, tighten the caps. 8. These tubes can be stored at room temperature or in the refrigerator. Figure 3: Always label the bottom of Figure 4: Technique for streaking a plate the petri before pouring media. in three sections using an inoculating needle. .