How To Prepare, Sterilize, AND Test Culture Media Preparation of Culture Media Important precautions: 1. Prepare the media from dehydrated products in a damp-free environment. 2. To prevent the risk of inhaling fine particles of culture media powder, wear a dust mask while handling media powder. 3. Wash the hands immediately after preparing media. 4. Once the ingredients are weighed, do not delay in making up the medium. 5. Use completely clean dry glass wares. 6. The container in which the medium is prepared should have a capacity of at least twice the volume of the medium being prepared. 7. Use distilled water, as the tape water contains substances (chlorine) harmful to bacteria and can alter the pH of a medium or cause a precipitate to form. Preparation: 1. Follow exactly the manufacturer’s instructions for preparation i.e. amount of media and volume to prepare. 2. Accurately weigh, add the media powder to the exact volume of distilled water. 3. Heat the media and stir by placing media flask on the hot plate with magnetic stirrer. 4. Do not shake vigorously, prevent overheating, boiling and foam formation, which can damage the medium. 5. Autoclave a medium only when the ingredients are completely dissolved. Add distilled water to make the right volume. Heat & stir (agar will burn if it is not stirred) until all of the ingredients go into solution. When the media boils, it is ready for sterilization. Sterilization of culture media:  The following methods are used to sterilize culture media: 1. Autoclaving. 2. Steaming at 100 0C. 3. Filtration.  This done in order to make the media completely free of microorganism.  For this purpose we use Autoclave machine which operates at following conditions i.e. 1. Temperature = 121oC 2. Time = 15 minutes and 3. Pressure = 15psi (Pounds per square inch). Overheating effects  Overheating is a common cause of darkening, precipitation, poor gel strength and reduced bacteriological performance.  All culture media should be in solution before sterilization.  Overheating effects will occur if agar media are allowed to gel in bottles and are later steamed to melt the agar. Preparation errors and possible causes Preparation errors Possible causes Poor bacterial growth. 1. Prolonged and excessive heating, incomplete solution. 2. Inhibitory substances in water or containers. 3. Darkening and pH drift. Turbidity, precipitation. 1. Poor quality water or containers. 2. Overheating or prolonged storage at 50°C. 3. pH value incorrect. 4. Incomplete solution. Preparation errors Possible causes Soft gel. 1. Agar not in solution, poor mixing, prolonged storage at 50°C. 2. Overheating at low pH values. 3. Error in weighing or overdilution with inoculum or media supplements. 4. pH too low for agar. Darkening. 1. Overheating, incomplete solution or pH drift. 2. Presence of phosphate in addition of glucose or other sugars and agar. Dispensing of culture media:  Dispensing means pouring of sterilized medium into suitable glass containers.  By using aseptic techniques, sterilized medium can be dispensed into sterile Petri dishes or Screw capped test tubes. Aseptic Techniques  Aseptic technique refers to a procedure that is performed under sterile conditions to prevent any contamination e.g. use of flame. Dispensing sterile media into petri dishes 1. Arrange the sterile petri dishes on a level surface e.g. a table or bench. 2. Mix the medium gently by rotating the flask or bottle. Avoid forming or air bubble formation. 3. Sterilize the neck of the flask on a flame and pour 15–20 ml of medium into each dish (90 – 100 mm diameter). 4. If air bubbles enter while pouring, rapidly flame the surface of the medium before gelling occurs. 5. Rotate the petri dish on the surface of the bench to ensure an even layer of agar. 6. When the medium has gelled and cooled, cover the plates and seal them in plastic bags to prevent loss of moisture and reduce the risk of contamination. 7. Do not leave the plates exposed to bright light especially sunlight. 8. Store at 2–8 0C. Dispensing and solidifying high protein media (Inspissation)  High protein media such as Dorset egg medium and Loeffler serum medium are dispensed aseptically in screw-capped bottles and solidified in a sloped position at a controlled temperature (75–80 0C) for 1–2 hours.  Solidification of protein media using heat to coagulate the protein, is called inspissation. Labeling and Storage: 1. All culture media must be clearly labeled. 2. Each batch of prepared medium should be given a number and the date of its preparation. 3. Should be stored at an even temperature in a cool dry place away from direct light. 4. Container tops must be tight-fitting. 5. Plates of culture media should be stored at 2-8oC, preferably in sealed plastic bags. Sterility Testing:  The simplest way to test for contamination is to incubate 5% of the batch at 35–37oC overnight.  Contamination by microorganisms capable of overnight growth, will be shown by a turbidity in a fluid medium and growth on a solid medium. Note: All media, even those that have been sterility tested at the time of preparation, should always be checked visually immediately before being inoculated, for any change in appearance that could indicate contamination.  This is particularly important during the hot season and when the humidity is high.