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Preparation of Source Material
BASIC MICROBIOLOGY

    The content of biochemistry is mostly grounded in studies of micro-organisms, which are the simplest packages of almost pure metabolism. Thus beside occasionally using plants and animal tissues for our source materials, we shall also use yeast and bacteria. Pure cultures of yeast can be bought in the supermarket, but bacteria cannot. So we must grow our own.

    This week and next we shall make use of some tricks in microbiology that are a hundred years old, are the basis of sanitary assays for environmental studies, used in hospital clinical labs, AND will produce just the right enzyme that we want to study next week.

P R O T O C O L

  1. (Re-)Familiarizing yourself

    1. The lac-operon (which produces lactase, aka β-galactosidase)
      1. review your biology notes
      2. review your genetics notes
      3. read through the "lecture" part of

  2. Planning which available E.coli strains you will use

  3. Planning your petri plates and contents

    1. Give four reasons why you want to use MacConkey Agar
      1. E.coli will grow on it (why?)
      2. Its lactose will induce the lac-operon to produce the enzyme you want
      3. Its dyes will tell you that the gene is turned "on" and lactose is being digested (how is this?)
      4. The dyes prevent most airborne contaminants from growing.

    2. Give reasons for why you also want to use nutrient agar plates
      1. The red and purple dyes are absent and won't interfer with the subsequent analyses of the enzyme in which yellow dyes are used.
      2. You can have some plates with lactose and others without lactose (what's important about this?)

  4. Producing your sterile plates, ready for inoculation

    1. Making MacConkey Agar Plates
    2. Making Nutrient Agar plates with and without 0.5% added lactose
      INDEPENDENCE! You are provided with bottles of lactose and nutrient agar powder. Hop to it!
    3. Sterilizing your empty glass petri plates and your contents
      1. Using the autoclave in room 201 (takes about 2 hrs)
      2. Using a home pressure cooker right in the lab (takes about 45 min).
    4. Labelling the plates
    5. Pouring the plates
    6. Inoculating* the plates
    7. Incubating the plates
    8. Storing the plates

  5. See you next week!

* Since our laboratory will institute a novel penalty program for those you misspell certain words such as inoculate and complementary, or unduly break glassware, it might help to know what the penalties are. The main word is "donuts" (aka doughnuts). These are in the shape of "O". The "felon" in the class must bring to lab the next time either some donuts or donut-holes for everyone to eat, and if someone already is providing such and there is a second felon, drinks are in order, right?

Now, about the word inoculate: if you are in wonderment as to its spelling, just write down I-N- and at this point you don't know if there is another N or it goes to O. Think of the penalty, which looks like an O, and write O, and continue on. By the way, inoculate and innocuous have two different roots.