A Short Course in Microbiology
for pre-college teachers
"Microbiology is a cultural course!"
WHY MICROBES ANYWAY? What good are they for teaching? Well, they are the closest living things to offering "instant gratification". And they are simple possessing just the bare essentials of life. Thus the fundamental themes of ALL living things can be quickly presented - genetics, biochemical pathways, bioremediation and decomposition, population growth - all without having feathers, fur, arms or legs, or even nuclei or mitochondria getting in the way! Later the more refined themes of other forms of life can be built upon this base.
PURPOSE of this page: Fewer than 5% of public school teachers have had any training in the handling of micro-organisms. While microbiology constitutes one of the largest scientific employers encompassing a vast realm of knowledge, there are, however, only a few simple techniques that must be mastered before success leaps before the eyes of the students and teachers! What follows are those fundamental skills. (It might be noted that this author takes pride in having run a "Laboratory of Ultra-Low Technology" for years, and has developed a number of techniques for doing sophisticated microbiology "out of the back of his pickup truck in the Amazon Delta." Thus, any classroom setting should seem a paradise where truly great things can be accomplished!)
Two guiding principles leading to the right mindset:
1. Just because it is clean doesn't mean that it is sterile!
(what you can't see CAN hurt your experiment!)
2. Establish a culture environment that inhibits contamination!
Abide by these two points and your experiments will go successfully!
"Equipment and Supplies"
|oven (kitchen)||for sterilizing dry things|
|wire inoculating* "loops"||for transferring small numbers of bacteria from place to place|
|a plumber's propane torch||to sterilize the "loops" and for clearing bubbles off the surfaces of freshly poured agar|
|petri plates||for growing microbes on a solid surface so that defined colonies can be seen|
|MacConkey Agar (with lactose)||Allows only Gram-negative bacteria to grow, plus other uses.|
|glucose||occasionally used as an additive to the MacConkey Agar.|
|hot plate||for preparation of the agar|
|tap water||microbes need minerals, too!|
|screw-topped test-tubes||for making slants for storing bacteria between semesters|
|Nutrient Agar||for use in the slants and in some plates|
|8 to 12 quart pressure cooker||to sterilize slants|
|tongs and oven mitts||use unless you want scalded hands|
|micro-organisms||read about safe microbes prior to ordering|
|a lab manual*||for troubleshooting|
(Reference manual. Of the many lab manuals for microbiology, this pair of books is recommended as they are a small compendium of highly illustrated techniques: Pierce and Leboffe: Exercises for the Microbiology Laboratory (1999) (ISBN 0-89582-462-0); Leboffe and Pierce: A Photographic Atlas for the Microbiology Laboratory (1999) (ISBN 0-89582-461-2) Morton Publishing Co. )
This author has had a longstanding rule: if any correspondent fails to spell inoculate correctly, well...! They owe me a dinner!
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
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!
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!
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:
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.