Medium Setup and Creation 1

PRACTICE MEDIUM SETUP AND CREATION 1 1.1 Media Functions Media is a substrate needed to grow and reproduce microorganisms. Before being used in an experiment, this media needs to be sterilized first, so that unwanted microorganisms (contaminants) do not grow. In order for the microbes that we culture to grow well, the requirements that must be met by a medium are: a. It must contain the ingredients needed by the microbes to be grown. These ingredients include macro elements, micro elements, and trace elements as well as growth regulators. b. The media must have osmotic pressure, surface tension and pH that suit the needs of the microbes to be cultured. c. The media must be sterile before being used to grow the necessary microbes. 1.2. Media Type 1.2.1. According to the materials used in its manufacture, media can be classified into: a. Natural media: Media whose constituent components consist of natural ingredients, such as potatoes, bean sprouts, meat, rice, and so on. b. Semi-synthetic media: Media whose ingredients consist of a mixture of natural and synthetic materials. Examples: Bean Sprout Agar, Potato Dextrose Agar, etc. c. Synthetic media: Media whose entire constituent material is made from synthetic materials. Examples: Sabouraud Agar, Endo Agar, Czapex Dox Agar, etc. 1.2.2. According to its form, media can be classified into: a. Liquid media: Media that does not have solidifying agents (agar) added, so this media is in a dilute (liquid) state. Example: Lactose Broth, Nutrient Broth. b. Semi-solid media: Media that contains the same ingredients as liquid media, but adds a little agar (half the concentration of agar), so that it becomes slightly dense. This media is used to grow microbes that require a lot of water and live in an anaerobic or facultative anaerobic environment. This media is also used to test the motility of bacteria. c. Solid media: Liquid media that is added with agar so that it becomes solid. Examples: Nutrient Agar (NA), Potato Dextrose Agar (PDA), etc. 1.2.3. According to its use, media is classified into: a. General media: Media used to grow one or more groups of microbes in general. Examples: Nutrient Agar (media for growing groups of bacteria, Potato Dextrose Agar (media used for growing groups of fungi, etc. b. Enrichment media: Media used to fertilize certain microbes before they are grown on the media used in research. Example: Selenite Broth (to fertilize bacterial growthSalmonella. c. Selective media: Media used to grow certain species of microbes, by inhibiting the growth of other undesirable species. Example: SS Agar media (Salmonella and Shigella Agar) for Salmonella and Shigella bacteria. d. Counting media: Media used to count the number of microbes in a material. This media can be in the form of general media and selective media. 1.3. Media Creation In this practicum, you will make several types of media, namely Nutrient Agar media, Malt extract, Lactose Broth, Brilliant Green 2% Bile Broth, and Endo Agar media. All of these media are synthetic media which are widely sold in instant form, so when making them, you just need to pay attention to the instructions listed on each media. 1.3.1. How to make Nutrient Agar. To make 1 liter of this medium, do the following procedure: a. Weigh out 23.5 grams of instant medium. b. Suspension in distilled water and final volume made 1000 ml. c. Heat this suspension until the gelatin becomes cooked. d. Put it in a test tube (- 5ml for tilt media, and - 10 ml for upright media). e. Sterilize in an autoclave at 121oC and a pressure of 15 lbs. For 15 minutes. f. Store in a cool and dry place (in the refrigerator). 1.3.2. Making Malt Extract To make 1 liter of this medium, do the following steps: a. Weigh out as many grams of instant media. b. Suspend in distilled water until the final volume is 1000 ml. c. Put it in a test tube (approximately 7 ml per tube) d. sterilize with an autoclave at a temperature of 121 oC and a pressure of 15 lbs, for 15 minutes. 1.3.3. Making Lactose Broth medium In this practicum, lactose broth medium will be made with two concentrations (single strength and double strength). To make one liter of single strength lactose broth, do the following procedure: a. Weigh out 13 grams of instant lactose broth medium. b. Suspend in distilled water until the final volume reaches 1000 ml. c. Put it in a test tube equipped with a Durham tube. d. Sterilize in an autoclave at 121 oC at 15 lbs pressure for 15 minutes. e. Store in a cool and dry place (in the refrigerator). f. The same procedure is used to make double strength lactose broth, but the concentration is doubled. g. 1.3.3. Preparation of BGBB medium (Brilliant Green Bile 2% Broth) To make one liter of this medium, do the following procedure: a. Weigh out as many grams of instant medium. b. Suspension in distilled water and final volume made to 1000 ml. c. Put it in a small test tube that has been equipped with a Durham tube. d. Sterilize in an autoclave at 121oC at 15 lbs pressure, for 15 minutes. e. Store in a cool and dry place. 1.3.4. Making Endo Agar medium To make 1 liter of this medium, do the following procedure: a. Weigh out as many grams of instant medium. b. Suspend in distilled water and make the final volume 1000 ml. c. Heat until all components dissolve. d. Sterilize in an autoclave at 121 oC at 15 lbs pressure, for 15 minutes. e. Store in a cool and dry place (in the refrigerator). 1.3.5. Making Palte Count Agar medium To make 1 liter of this medium, do the following procedure: a. Weigh 23.5 grams of PCA instant medium, Pronadisa). Notes : Different brands have different compositions so the gram/lt weight may be different. So, pay attention to the guidelines written on the packaging/bottle of the media used. b. Suspend in distilled water and make the final volume 1000 ml. c. Heat until all components dissolve (this step may not be done if you do not prepare tega agar/mirin agar in a test tube). d. Sterilize in an autoclave at 121oC at 15 lbs pressure, for 15 minutes. e. The media can be poured into a petri dish that has been autovlaved (see image below). For one Petri dish with a diameter of 9.9 mm, a thickness of 1.5 cm, the volume of agar required is 17.5-20 ml for a good thickness of agar media. Bone agar was placed in a petri dish at medium temperature+ 65-75oC. At temperatures below that so that it will give me. f. Allow the agar in the petri dish to freeze, open the lid of the dish until the agar hardens (20-30 minutes in laminar flow). g. Store in a cool and dry place (in the refrigerator) wrapped in a plastic bag and placed upside down (petri dish lid at the bottom and agar at the top).