How to Make Nutrient Agar Plates
Nutrient agar plates allow you to culture and enumerate many organisms. The beef extract and peptone in nutrient agar provide a conducive growth environment for most culturable species of bacteria and fungi. Agar powder solidifies the mixture as it cools, forming the gel-like substance you see in petri plates. You can mix these ingredients yourself, but commercially prepared nutrient agar powder makes the job quicker.
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Things You'll Need
- Nutrient agar powder
- Lab scale
- Weigh boat
- Sterile scoop
- Autoclavable bottle with screw cap
- Deionized water
- Magnetic stir stick
- Hot plate with magnetic stirrer
- Hot pad or glove
- Autoclave
- Thermometer
- Petri plates
Instructions
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1
Calculate the amount of agar you need to make. Estimate 50 100-mm Petri plates or 100 60-mm Petri plates per L of agar. Read the label on the bottle of agar powder to find the amount of powder per L, and scale the amount of water and agar powder up or down based on how many plates you need.
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2
Place a weigh boat on a lab scale and zero out the scale reading. Use a sterile scoop to add nutrient agar powder to the weigh boat up to the correct weight.
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3
Add the powder to an autoclavable bottle, using a size which your agar mixture will fill only about halfway. Measure the appropriate amount of deionized water, using a graduated cylinder, and pour it into the bottle. Drop a magnetic stir stick into the bottle and place the cap on, but do not tighten it.
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4
Place the bottle on a hot plate with a magnetic stirrer. Turn the heat to medium and set the magnetic stirrer at a speed where you can see movement in the water but no vortex forms.
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5
Heat the mixture just to the boiling point. When you see bubbles at the top and the liquid is a transparent, yellowish color, immediately remove the bottle from the heat, using a glove or hot pad. Turn off the hot plate and stirrer.
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6
Autoclave the agar mixture, using the standard liquid cycle on your autoclave. Allow the autoclave to return to atmospheric pressure and below 90 degrees C before opening the autoclave. Remove the bottle, using a glove or hot pad, and screw the cap on tightly.
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7
Allow the agar to cool to between 55 and 60 degrees C. Place the bottle in a water bath at about 55 degrees C, or simply allow it to cool at room temperature. Place the bottle under a laminar flow hood before opening it to check the agar temperature.
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8
Place a sleeve of Petri plates under the laminar flow hood; open the sleeve and remove the plates. Hold the bottle of agar in your dominant hand and lift the lid off a Petri plate with your other hand. Pour enough agar into the plate to cover the bottom about 1/8 inch thick. Replace the lid immediately. Swirl the plate gently to ensure the agar spreads to cover the whole plate. Repeat this procedure for as many plates as you need.
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9
Dry the plates under the hood for several hours or overnight. You may stack the plates or spread them out to dry more quickly. If you will not use the plates immediately, put them back in the sleeve and securely tape it shut. Store plates at room temperature for a few days or in a refrigerator for longer-term storage.
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Tips & Warnings
Wear latex or other lab gloves throughout the procedure.
If you do not have a hot plate with a magnetic stirrer, use a regular hot plate and stir the mixture frequently with a sterile spoon or stir stick.
Watch the agar mixture closely as it heats; it can boil over very quickly.
You can pour plates after boiling if you do not have an autoclave, but use them immediately to prevent contamination.
