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Making Petri Plates Containing Tryptone-based Agar

The plates being made here are for the growth of E. coli and related bacteria.

INTRODUCTION: While E. coli are able to grow on a single carbon source such as even formate or acetate, often one wants a bit richer medium so that the bacteria will grow with a doubling time of 30 to 40 minutes. For this, tryptone broth is commonly employed (tryptic soy broth, "TSB" is related). Growth can yet be sped up by an addition of a trace of vitamin B1 (thiamine). When one needs a solid form, agar is added. One of the values of this medium is that it is sugar-free, so that various sugars can be added in the study of their specific metabolisms. In the following, two such will be made along with a sugar-free form. If you have different needs for tryptone plates, make the necessary minor adjustments.

This procedure assumes that you know a little about aseptic technique, and understand that "clean" does not necessarily mean "sterile."

Protocol

1. Assemble the following:
   1. Tap water is always used for media because trace minerals are needed.
      
   2. Tryptone powder (very hygroscopic: keep bottles screwed tightly shut between use)
      
   3. Thiamine (vitamin B1) (don't confuse this with thymine, which is a base in DNA)
      
   4. Glucose (aka dextrose), lactose, and purified agar
      
   5. Sterile petri plates (100 ml fills 3 glass plates or 4 plastic plates that are 15mm x 100mm)
      
   6. Autoclave or pressure cooker
      
   7. A 3 inch flame such as from a Bunsen burner or a plumber's torch
      
   8. Mitt or tongs for holding hot things
       
2. Making the concoctions before sterilizing them. We shall be making 100 ml of three different media: "no sugar" ("-"), "with glucose" ("G"), "with lactose" ("L")
    
   1. Measure out 300 ml of cold tap water into a 500 ml container
       
   2. Add to the water a tiny amount of thiamine: a cube of about 1 mm in all dimensions is more than sufficient.
       
   3. To make 0.7% tryptone, you will need to add 2.1 gms of tryptone powder to the 300 ml.
       
   4. Label three 200-ml bottles or flasks with marking pen: "-", "G", and "L" for no-sugar, glucose and lactose, respectively.
       
   5. Dispense 100 ml into each of the three flasks or bottles
       
3. Add 2 grams of purified agar to each flask or bottle. (You did not add the agar to the 300 ml before dispensing because agar does not dissolve and would distribute evenly.)
    
4. Add 0.3 gm of glucose to "G", and add 0.5 gm of lactose to "L".
    
5. Cap or otherwise cover the flasks or bottles. Inverted beakers work well to cover flasks. Aluminum foil works well also. Whatever you use should extend down from the top of the container an inch or two.
    
6. Place the covered containers in your autoclave or pressure cooker in a prescribed pattern so that you will know which is which in event the wet heat removes the labels. Add a bowl of water to the autoclave, if that is what you are using. This will boil more readily than will your agar solutions during the cooling phase.
    
7. Sterilize in an autoclave or pressure cooker for 20 minutes at 15 psi. Do not release the pressure after 15 minutes, but rather allow the system to cool on its own. Notice that your flasks or bottles seemed much larger than needed. This is because you needed boiling room in them during the cooling phase.
    
8. About three minutes after opening the autoclave or pressure cooker, extract your bottles or flasks using mitts or tongs. Set them on paper towels on the countertop (some bottles break if set on a cold surface!).
    
9. One by one pick up each bottle or flask, hold it over a sink and gently swirl it. (Beware of super-heated solutions which might boil up and out the lid. That's why you do this over a sink!) You swirl the solution because the agar you added will have sunk to the bottom, and now has melted into a goo. Your swirling mixes it up into the remainder of the liquid.
    
10. Arrange 12 sterile petri plates on your cleaned countertop in three rows. Label each set of four plates on the outside of their bottoms, using a small letter near the edge: "-", "G", "L", respectively.
     
11. Distribute container "-" into four plates. You do this by lifting plate tops one by one and replacing them after you have poured that ONE plate. Do NOT take off all the lids from the plates UNLESS you are in a multimillion dollar sterile facility. Thou shalt not talk while pouring plates because a salivary mist filled with microbes comes from your mouth and settles in the open plates.
     
12. After you have poured each bottle or flask, immediately rinse it with warm water to save cleanup effort. In other words, don't let residual agar solidify in the bottle.
     
13. Repeat this process with the other two containers, "G" and "L" so that you eventually will have poured 12 plates.
     
14. Immediately, before the agar solidifies in the plates, waft a flame across the agar surfaces to burst any bubbles formed during the pouring process. Again, do this plate by plate lifting one cover at a time and replacing it.
     
15. Once the agars have solidified, proceed to the drying process.
     

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