LB Media is a generic rich media suitable for growing many aerotolerant species of bacteria, including E. coli, Bacillus subtilius, Staphylococcus auerus or Staphylococcus epidermidis and different yeasts including Saccharomyces and Candida species. Many kinds of molds will also grow (Aspergillus or Penicillium, for instance). However, the presence of these organisms on your plate generally represents contamination, and the plate should be tossed! LB agar will not necessarily support the growth of more fastidious organisms such as Streptococci, and pH media and conditions are less than ideal for culturing species of Lactobacilli from yogurt.
Ingredients (per Liter):
Adjust as needed to make 500, 250 or 100mL of media.
- 10g Tryptone: Tryptic digest of proteins to release small peptides and amino acids that bacteria can use for food and/or protein synthesis.
- 5g Yeast Extract: Source of additional proteins, vitamins and minerals; some sugars present, necessary for growth.
- 10g NaCl (table salt)
- 15g agar
- 1L distilled water - note tap water may work if you don't have access to a source of distilled water. However, note that the pH of municipal water supplies can sometimes drop to as low as pH=6, and there may be contaminants of ions or other compounds that could potentially impact the growth of bacterial species of interest. For growth of the bacteria listed above, however, tap water should work; else try a local drug store for gallon/liter jugs of distilled water.
- Erlenmeyer flask - the sloped sides of the flask are ideal for preparing and autoclaving media. A standard straight-sided flask should not be used. The volume of the flask should be at least twice the volume of the media to be prepared, so a 2L flask to prepare 1L of media. If the flask is the same volume as the media it will be very difficult to mix the components, and exceedingly awkward to handle the autoclaved flask.
- Scale and weighing papers or trays/"boats" for measuring ingredients.
- Autoclave or pressure cooker. In some instance sterilizing the media in an oven may work, but cooking in an oven will not always inactivate spores of thermophilic Bacillus or aerotolerant Clostridial species that could be contaminating the flask or other components of the media.
- Oven mitts - preferably water proof for handing the autoclaved (and hot!) flask.
- Empty Petrie plates, which should be sterile and packaged in a plastic bag/sleeve. Open the top of the sleeve to slide them out. Save the sleeve for later storage of your poured plates.
Preparing the media
- Measure all dry ingredients except the agar and place into a 2L Erlenmeyer flask.
- Add the water and shake/stir vigorously to get most of the dry material into solution.
- If you have a pH-meter or pH paper available, take a small aliquot of the solution and check the pH. If it falls well below pH 7, the pH should should be adjusted to 7-7.4.
- Add agar and continue mixing - the agar will not go into solution at this stage, but it's important that it not form a large clump on the bottom.
- Cover the opening of the flask with tin foil and place it in an autoclavable plastic or metal bin with some water in the bottom (~1cm deep).
- Autoclave the media for a minumum of 20' on the liquid cycle. Pressures typically range from 15-20psi.
- If you don't have access to an autoclave, smaller volumes of media (~250mL in an Erlenmeyer flask) can be "cooked" in standard pressure cookers for approximately 20-30 minutes with a maximum pressure of 20psi. Check your pressure cooker's operating manual to insure you do not exceed appropriate limits for its operation.
- In a worst case scenario, the media can be placed in a metal oven pan and "cooked" at 350*F for ~1 hour, gently swirling the flask every 20 minutes to get the agar in solution. Have an adult perform the mixing, using water-proof oven gloves to insure a firm grip upon the flask. Hot agar will rapidly burn the skin!. Note that the cooking in the oven may not completely inactivate spores of thermophilic soil bacteria such as Bacillus sterothermophilus, but it should kill most everything else.
- After autoclaving, gently swirly the flask while holding it in water-proof oven or heat-proof gloves. This action is necessary to insure even distribution of the agar in the media, else it often remains denser near the bottom.
- The media needs to temper before it is poured into plates. Place the flask on a heat-proof surface and let it cool. Large volumes (1L or more) should be swirled every 10-20 minutes to redistribute the media within the flask. Swirl while holding the flask in water and heat-proof mitts. Don't agitate the solution too much, to prevent the formation of bubbles near the surface.
- The media is sufficiently cooled when you can gently place your hand against the side of the flask for 1-2s without it being so hot that you withdraw your hand (have an adult perform this test, and only after the media has sat for at least 15 minutes).
- After the media has tempered, additives such as antibiotics, carbohydrates such as glucose or other carbon sources, may be added to the media. Be certain to gently swirl the media after adding components so materials are evenly distributed within the agar.
- Get your plates ready - if you're new to pouring plates, it's probably easiest to pour them individually. Otherwise, ~10 plates can be stacked and poured from bottom to top, lifting the lid of sequential plates (and those above it) to pour the media. Wear the oven-safe mitts while pouring.
- The bottom half of the plate should be ~1/2 full, approximately 25mL of media per plate. Plates poured singly generally solidify within 1/2 hour at room temperature.
- Stack your solidified plates right-side up and slide the original bag in which the empty Petrie plates came.
- Turn the plates over so the opening of the bag is on top -- the plates should now be facing agar-side up, exactly as how they should be stored.
- Tape the bag shut and label it with the type of media and date poured.
- Store plates in this position at 4*C in a refrigerator. If antibiotics have been added, cover the stack of plates in tin foil to prevent light-inactivation of the antibiotic.
- Standard LB plates will keep for a few weeks, or up to a month in this state. Be certain the bag is sealed to prevent loss of moisture.