ACTIVITIES (point and click):
Make Slants (DO FIRST!)
How to handle commercially procured bacterial cultures
Pouring Petri Plates
Plating Lac-Operon Mutants
Testing for ß-galactosidase
Testing for Antibiotic Resistance
Microscopy

Making Slants for Storing Cultures

THIS IS THE FIRST THING YOU SHOULD DO! So many recipients of cultures act as if they have not received anything living. THESE ARE LIVING, AND THESE THINGS MULTIPLY - F A S T ! So take advantage of it!

Using a 500 ml flask, make up about 100 ml of nutrient agar according to the recipe written on the side of the container. Don't make a liter of the expensive and only use 100 ml. Make 100 mls ONLY. And use a 500 ml flask or larger!

1. Weigh the beige-colored powder directly into the flask. Use that tool called a funnel to improve your aim! And remember that all these media powders are very hygroscopic (dictionary anyone?), so show your students that you have taken physical ed and that you can get the cover back onto the bottle and screw it tightly closed! Otherwise, the next time you want to use your $40 bottle of media, it will require dynamite! (Yes, I am being facetious so that you will get angry at me for treating you this way! But you will remember to do the little tasks along the way.) Oh, yah, clean up any spilled powder from the counter top and balance, unless you want a sticky mess the next day, and maybe ants also!

2. Add 100 ml of cold tap water. (What happens when you make gravy using hot water? Lumpy! Well, agar is a carbohydrate also - like starch or flour.) Swirl to mix the contents of the flask. Notice that there is a grittiness to the "solution." That grit is the agar component: agar will not dissolve until the liquid is about 97c.

3. Heat the liquid with continuous stirring or swirling - you don't want to burn your "gravy", do you? (This author prefers to swirl the large flask over a low burner until it just begins to boil. (BUT, watch out, agar solutions foam profusely when they boil!)
   Helpful hint: if you drop the flask with the molten agar, don't run for the sponge! Allow it to solidify on the desk or floor, and then scoop it up. Just worry about not getting the hot liquid on yourself!

4. Set your open screw-topped test-tubes in a rack - only in the holes along the edges of the rack.

5. Pour about 50 ml of the molten agar into a 100 ml GLASS beaker, and use the beaker to pour agar into the tubes - filling them to about 30% full. (Note that this size beaker can be held by its flange with bare fingers - good control during pouring!) Of course refill the beaker when needed.

6. Before you do anything else: rinse out the flask and beaker with hot water. It's easier to do it when the agar film is molten than after it has solidified!

7. Now screw the tops onto the tubes. (You'll be happier later if you put them on tightly - no the tubes won't explode!)

8. Place them vertically into the pressure cooker with the amount of water prescribed in the directions for the pressure cooker. And then run the cooker so that it is under "canning" pressure for 20 minutes. Remove from heat and allow to decompress on its own - DON'T suddenly let the steam out by removing the weight!

9. As soon as the pressure is down, take out the tubes with their molten contents, and lean them far over so that the meniscus in the tubes extends to about 60% the way up the tube. Allow the agar to solidify. Voila! Slants!!!

Now when you receive cultures by mail from your supplier, you should immediately transfer some of each using a flamed loop into pairs of properly marked slants. But there are tricks to this also. Have someone stand about 5 feet from you and read these to you as you do what they say. You should be able to complete all of following steps in a matter of about 20 seconds!

1. Light your burner or plumber's torch

2. Pick up the wire loop as if it were a pencil - but at the end of the handle, and then put the wire part into the flame and get all of it to glowing red, and then waft the lower part of the handle through the flame to incinerate any dust on it.

3. Pick up your supplier's culture tube by the bottom end.

4. Grab the cap of that tube with the little finger of your loop hand, and then unscrew the cap of the tube. Keep the tube close to horizontal. Keep the cap encircled by your little finger. Do not put your little finger inside of the cap unless you have flamed your little finger first!

5. For slightly less than a second hold the open end of the horizontal tube in the flame to dry it and burn off any dust (spores).

6. Insert the wire loop. (If you miss the hole and hit the outside of the tube, reflame your loop!)

7. Insert the wire under some of the agar in the tube to cool it.

8. Lightly touch where you see some of the bacterial growth. Please don't be greedy and scoop up big gobs of the bacteria! You only need a few hundred thousand (barely visible amount; not zillions!).

9. Withdraw the loop from the tube; briefly flame the opening of the tube and insert its opening into the cap that has been held all the while by your "pinkie." (Don't miss the cap or you'll brand your fingers with the hot mouth of the tube!)

10. Now pick up one of the freshly made slants, and repeat all of the above in approximately reverse order! And when you run the loop over the fresh agar surface in the new tube, you don't really need to be hyper-professional with even wiggles of the loop. A simple stripe is all that is needed.

11. Usually within a day or two, you will see that your stripe has grown and become visible. YOU DID IT! (But if the tube gets moldy a few days later, well-, you didn't do it!)

12. If the inoculated slants are kept very tightly closed , and if they are stored away from UV (sunlight or fluorescent lights), they will remain viable for about a year!

If you want to store them longer: don't make slants! Make "stabs!" Don't lay the tubes down as the agar solidifies after sterilizing in the pressure cooker. Keep them vertical. Then, instead of inoculating the surfaces of the agar in the tubes, stab the loop down into the agar almost to the bottom of the tube. Again, if stored in the dark at about 15c or so, they will last for decades! This author has recovered cultures from an international archive after more than 40 years storage. Amazing critters, hey? (Some people even dip the capped ends in wax for a permanent seal (a la wine bottles in molten lead).)

Making MacConkey Agar Plates

Remember the bit about establishing an environment that inhibits contamination? Well, this is it! The reason for dwelling on plates filled with this type of agar is that this is the most forgiving type of medium and is good for primitive conditions such as the back of pickup trucks and classrooms full of novices. How so? The most common contaminants faced by microbiologists are bacteria that arise from spores that have blown into their plates. But almost all - maybe 99.999% of all wafting spores are either of fungi, which cannot take the heat, or of Gram-positive bacteria, which cannot grow on MacConkey agar, because they cannot tolerate the detergent in the agar (see the bile salts on the ingredient list?). What is more is that this sort of agar is red because it contains some dyes - pH indicators - that turn red IF the bacteria are able to convert the added sugar to acids. Very few Gram-negative bacteria can do that -, but E. coli usually can! Thus Prof. MacConkey invented this agar nearly a century ago to test for fecal pollution of surface waters. Clever! So, you and your students will usually be seeing red colonies on these plates and little else - unless you are using some mutants, which we'll get to elsewhere.

Now, how to "make" MacConkey plates:

1. Get out an appropriate number of clean, DRY petri plates - glass ones from your oven (450F for 30 min) or sterile prepackaged plastic ones, and array them with their covers ON - on a clean desk top.

2. Figure that 300 ml of agar medium will fill 10 plates.
3. Put the requisite amount of powder into a flask that is at least three-times the needed volume; add cold tap water; swirl to suspend all the powder.

4. Heat to near boiling. (Firstly, all contaminating Gram-negative bacteria are killed by this temperature; secondly, any Gram(+) bacterial spores won't be able to germinate and grow in the plates you are making.)

5. Pour the hot liquid into each plate so that they are half full.

6. Note that this type of agar is so forgiving that you can minimize the condensation that forms on the covers of the plates by replacing the covers with paper towels from the middle of a new stack (usually close to being sterile due to how they are made). The steam just goes through the paper, but dust cannot settle into the agar. (You have, of course, set aside the glass or plastic covers down on a clean desk top with their openings pointing DOWN.)

7. Waft a large, strong flame over the surfaces of the molten agar to burst any bubbles of foam that might be on the surfaces. You now have a mirror-like surface that will solidify that way!

8. Once the agar is solid replace the lids.

9. IMMEDIATELY USE THE PLATES!!!

Using the MacConkey Agar Plates

Bacteria can be applied to the surfaces of the agar in several different ways and for several different purposes. Certainly the most crude way - and this is generally all that is needed! - is by making a stripe with the inoculating loop or a sterile cotton swab. Please note the "power" of the plate: you can transfer many different bacteria to that one surface: put one here, another over there, and so on. Says Dr. Petri: "Look! Put your control here and your three experimentals over there, there and there. Wow! The whole experiment on one plate - and see how they all have the same medium! The only variable is their genes! Wow, again!" You can even be clever by painting your different strains onto the plate by using your loop to write "C", "1", "2", and "3". Great for photographs used later for posters!

Plating of the lac-operon mutants on MacConkey agar will show that some grow up pale and other dark red. Those that become dark red are making acid from the sugar in the medium, while those that remain pale, are unable to use the sugar, and so no acid is made.

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