Nutrient Agar Plates
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Making Nutrient Agar ("NA") Plates
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There are several ways to make NA plates, all depending on what you have to start with. The well equipped person has clean and sterile empty petri plates, and a bottle of NA powder sitting on the shelf. That person needs only to follow the directions on the bottle. As it is the purpose of this website to assist those who, being mostly young students, have neither NA powder nor even petri plates. What are they to do? Here's what!
PETRI PLATES FROM SCRATCH
(click this to find ready-made plates)
First, you must find something that simulates a petri plate. Rather shallow glass custard cups covered by a double thickness of aluminum foil works well.
Second, you must have the nutrient agar to put in them. What might you use as a alternative to the commercially available microbiological stuff? The answer is canned beef or chicken broth (supermarket) and agar (health foods store; sometimes called "agar agar").
Third, you must sterilize these things in appropriate combinations. Included in the following steps will be the way to use a pressure cooker. There is NO substitute for a pressure cooker or autoclave, which is an automated pressure cooker. Boiling is not a sterilizing condition. Boiling doesn't work! The only commonly employed sterilizing conditions are wet heat at 120°c (250°F) for 20 minutes (pressure cooker), or dry heat in excess of 190°c (375°F) or more for an hour or more (oven).
- Measure and record the amount of water that half-fills a custard cup.
- Determine the number of plates (custard cups) you will need to use, and from that the amount of medium you need to make to half-fill all the cups.
- Empty a can of fatfree beef or chicken broth into a large measuring cup and dilute, if necessary, with water as per the instructions on the side of the can. Mix. Discard the excess beyond what you need for your cups. Pour this into a sauce pan.
- Add 1 level teaspoon of agar per 2/3 cup of broth. Stir the required amount of agar into the COLD broth in the sauce pan.
- Turn on the heat, and with constant and continuous stirring and/or swirling bring just to a boil. Keep swirling, and then adding small amounts of heat, and do this until all the granules of agar are dissolved. Agar solutions burn very easily - just like making gravy (in which you have added another polysaccharide as a thickening agent). An alternative way to do it is to place the broth/agar mix into a glass bowl and place it in a microwave oven. But do this cautiously as the mix will suddenly foam up. So be ready to shut off the energy at any moment. Give the dissolving solution a swirl, and microwave a bit longer. Swirl and energize repeatedly until all the granules are dissolved.§
- Distribute the hot medium among the cups you need.
- Plate pieces of double-thick aluminum foil over the cups so that at least a half inch (1 cm) over hangs.
- To your empty, clean pressure cooker, add enough tap water so that at least a half inch of water is in the bottom. Place the device on your cooking stove.
- Arrange your cups in the bottom and add anything else you need to sterilize between and on top of the foil covered cups.
- Put the lid on the pressure cooker making sure that all the lugs are in place and the top twisted to the right (clockwise).
- Turn on the heat
- For awhile the water will boil inside the pot, but steam will not spew from the vent. Only hot air will come out.
- Finally, when steam spews from the vent, place the special weight atop the vent. Some weights have several holes in them labelled with numbers indicating the amount of pressure they will produce in the pot. Make sure that you use the hole that says "15" (which means 15 psi, which is one atmosphere above ambient air pressure).
- Soon the steam pressure inside the pot will reach 15 psi, and at that time will be able to lift the special weight, which will now jiggle and hiss as it allows excess pressure to be vented. As soon as the hissing begins, you should begin your timer.
- While the hissing and jiggling continues, you may turn down the heat a little - just so long as hissing and jibbling continues.
- After 20 minutes, turn off the heat, and go for lunch.
- Using a fork, for example, slightly lift the weight. If more steam hisses out, put the weight back down, and wait a little longer for the pot to cool.
- Finally, lifting the weight does not result in hissing. Lift off the weight and put it aside in a safe place where you won't lose it.
- Twist and remove the top from the pot. When you raise the top, lift it so that the rear edge lifts first so that the steam inside rises away from you, unless you enjoy having your face get the boiled lobster look and your hair all frizzy. Set the top aside on your lab partner's notes so that all the condensation can pour forth and ruin the notes.
- Reach in with mitts and gloves to remove the various items in there. DO NOT remove foil covers or other wrappings. Remember that the cups are half-filled with nearly boiling liquid, so lift them carefully and set on the counter top where they will cool and solidify.
- For safety, dump a pan of cold water into the pot before moving it. That will cool the hot contents so that splashes and misdirected spills will not burn anyone. Dump the pot into a sink and rinse well with warm water and set aside inverted to drain and dry.
- In an hour or so, the NA in the cups will have cooled enough to solidify.*
- Immediately before use they must be "dried".
|* ||agar is an unusual substance in that its melting and freezing points are not the same. Agar melts at about 95°c, and solidifies at about 43°c. That's why you had to boil the broth and agar in the saucepan, and later why you had to wait so long for the cups to cool.
|§ ||A totally different way, is to put a covered bottle of the cold broth/agar mix into the pressure cooker along with empty foil-covered cups. Later, after sterilization, swirl the bottle to mix the agar up from the bottom. Then arrange the cups on the countertop and one by one lift the foil and pour in agar/broth solution. Replace the foil immediately after pouring. Often some bubbles of foam form upon pouring. These should be aseptically burst by wafting a flame from the plumber's torch across the liquid surface.|
Obtaining Ready-Made Plates
Often the bacteriologist in your local hospital will give you a few petri plates that already are sterile and have the kind of agar growth medium in them that you need. Take with you a small brown paper bag (bottom = 4x4 inches) and a quart-size ziplok plastic bag for carrying the plates home. If you want nutrient agar plates, the technician may have those, or you may also use TSA (tryptic soya agar), or any general type of agar. The technician will know best. If you want MacConkey agar plates, the technician will usually have those available. Using scotch tape, put a loop of tape vertically around the short stack of plates to keep the closed and together. Slip the plates into the ziplok bag and close. Set the plates in the brown paper bag so that the agar portions of the plates are down. Why the brown paper bag? If you are riding a bus or subway, what people can't see won't get them excited. Afterall, there is nothing to get excited about as there are no bacteria in these plates.
Until you are just about to use the plates, do not play with them, do not open them, do not sniff them, and keep them away from drafts that can blow dust through the cracks and onto the agar. Generally store sterile plates in your refrigerator with the agar portions "up" (plates upside down) - so condensation droplets settle on the covers and not on the agar. Using in a refrigerator, I would suggest removing the plates from the ziplok bag. Thou shalt not freeze the plates!
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