Molecular biology and bacteriology use agar plates to culture microorganisms such as bacteria for experimentation. The agar is the agent that solidifies, or gels, a liquid nutrient medium required by the microorganism to survive and grow. Once transferred to an agar plate, the bacteria grow into distinct colonies (if grown at low concentrations) or smears (if grown at high concentrations) and can be harvested for analysis. Biologists prepare agar plates, as the process requires access to laboratory-grade reagents and machines and a good understanding of molecular biology and sterile techniques.
- Skill level:
Things you need
- Lab coat
- Face mask
- Bunsen burner
- Laminar flow hood
- Sterile petri dishes
- 10g bacto-tryptone
- 5g yeast extract
- 15g agarose
- 1mL 1N NaOH
- Sterile distilled water
- Glass bottle
- Autoclave tape
- Supplements, e.g. antibiotics such as ampicillin
Put on proper protective equipment, including gloves, a lab coat and a face mask, and decontaminate all surfaces. Work next to a Bunsen burner or, preferably, in a sterile laminar flow hood. This prevents airborne contaminants from falling into the agar.
Remove the stack of petri dishes from their packaging but save for repacking the agar plates. Label the external surface of the dish (not the lid) with the type of agar and the date of preparation. Lay the plates out as a single layer with the lids removed to make pouring of the agar easier.
Mix the bacto-tryptone, yeast extract and agarose together and then add the sodium hydroxide (NaOH) solution. Add sterile distilled water to obtain 1L of nutrient media. Transfer this solution to a glass bottle with at least 1.5L volume. Loosely screw on the lid of the bottle and label the date of preparation and the name of the media. Place a strip of autoclave tape on the bottle and autoclave it for 25 minutes. Let the bottle cool to no lower than 45 degrees Celsius (or it will harden prematurely) but no higher than 50 degrees Celsius. You can store bottles of cooled and solidified agar at 4 degrees Celsius and then warm until liquefied. If required for your particular experiment or strain of bacteria, add the appropriate type and amount of supplement such as antibiotics. If the media is too hot, the supplements will be destroyed, so carefully check the temperature of the media before proceeding.
Pour a sufficient volume of agar to half-fill the dish while the agar is still warm and liquefied. Ensure that the surface area is completely covered. Do not pour an excessive amount or let the agar reach the top of the dish. Keep the plates in a sterile atmosphere by allowing the Bunsen burner or laminar flow hood to run continuously. Allow the plates to set and then replace the lids.
Dry the agar plates for about 30 minutes to an hour in an oven set no higher than 37 degrees Celsius. Alternatively, you can leave them in the laminar flow hood for three minutes or on a decontaminated laboratory bench in a low-traffic area that is not draughty for a day or two. Drying the plates stops any bacterial solution placed on the agar surface from simply diluting out and sliding off, instead of forming colonies that stick to the agar.
Carefully store the plates in the dark and at 4 degrees Celsius. Stack the dried agar plates with their bottoms up to allow you to clearly see the labels and to prevent microorganisms and debris from falling onto the agar surface and contaminating them. Put the dishes back into their packaging, cover with aluminium foil and tape the packaging shut. Label the packed plates with their content and date of preparation. You can store the plates for up to two months and should discard them after that.
Tips and warnings
- Maintain the highest standards of sterility at all times. Even the tiniest quantities, such as nanograms, of contaminants or unwanted bacteria will result in unusable plates.
- Be careful when using an autoclave, as it operates at high temperatures and pressures. Incorrect use can result in expensive damage or injury.
- All items should be laboratory-grade to prevent contamination with fungi, bacteria or other unwanted debris.
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