Testing the Dirtiness of Coins

PROLOGUE: These are two sequential lab exercises to promote interest by giving you a goal which reqires you to take the steps necessary to arrive at that goal - investigating the validity that money is dirty. In a way, this pedagogical method is like on-the-job training, which is learning as you go. You will learn how to make the lab procedures work in your hands - and that is the main mission of this project, isn't it? Using the philosophy of "divide and conquer," the big goal is subdivided into sub-goals. And those, in turn, contain little packages of standardized procedures we will call "protocols". Each protocol has a web-address associated with it for a more generalized protocol that goes beyond this experiment dealing with coins.

THE Question: Are Coins Laden with Germs?

Introduction: Our mothers told us that coins carry germs and we should always wash our hands after handling money. But is this true? If it is, this mission ought to be simple to answer. And if mother's cautions are not true, then how can it be? One might have the suspicion that money might not be a germ laden as commonly thought because one would hope that our national health agencies would have thought of ways to minimize money as a vector of disease. So what you shall be doing is taking a look at our coinage with respect to its being a vector or microbes. (The word microbes is frequently used here to cover bacteria, fungi and viruses. The following procedures will be able to monitor bacteria and fungi, but not viruses.)

Strategies: Yes, in the plural because there are several ways to attack this problem. So if you wish to present this problem to a class of students, you might like to stear groups in different directions with the final outcome an integrated class report that shows everyone a rather comprehensive picture of techniques and strategies.

Initially you might think to look at the coins microscopically to see if there are any bacteria on them. However, distinguishing bacteria from the smaller dust granules is not easy, and you will not be able to see any difference between live and dead bacteria, and it is the live bacteria about which you are concerned. Why? (You are being encouraged to communicate with each other via these little questions interspersed throughout. Today's science is a social activity as large numbers of people work together to solve problems such as how to get to Mars and what to look for once there.)
Place some coins right out of the pocket onto the surfaces of sterilize nutrient agar in petri plates. If the coins do carry microbes, then growth should occur around the edges where the coins contact the agar - or even underneath the coins!
Coins are made out of metals. What kinds? Are these metals in ionized form likely to be toxic? Is ferric toxic? Not very. What about cupric? And so on... Perhaps the coins kill microbes that are on their surfaces. Perhaps we can sterilize some coins (just to eliminate a variable) and place them on nutrient agar surfaces upon which we have already smeared an invisible film of bacteria. Those bacteria will try to grow into a very visible film. But are there halos around the coins? If so, then the coins are toxic to the bacteria you put on the plates.
Combining the above two strategies to make a new one. Suppose we coated a bunch of coins with the bacteria used in #2. Then, at timed intervals, we'd place those on uninoculated plates as in #1. How long would it take for the coin to sterilize itself.
Do the bacteria need to be in physical contact with the coins to be killed, or do the coins exude a toxic substance? Suppose we put some sterilized coins down on the sterile surface of agar, allowed them to sit there overnight, and then we remove the coins. The surface is then inoculated with bacteria. If they do not grow where the coins had sat, then something was exuded from the coins.
There are other strategies also, but we have enough now to get us into the first of the two sequential lab exercises. When we finally get to the second section then other strategies will be brought forth for consideration.

The following activities that utilize the above strategies will be divided into protocols that are mostly "stand alone" standard procedures in microbiology. Each will be given its own page. After you finish this mini-course, and ou become involved in other microbiological projects, you can call upon those protocols individually to construct a route to answering your new research question. If you believe you will be involved henceforth with these sorts of micro-problems, it is suggested you consider procuring Pierce and Leboffe's manuals. Some diagrams used in this website are from those manuals, and are used with the permission of Mr. Douglas Morton, publisher.)

SUB-GOAL ONE
We need to isolate "tool bacteria" for using on some of the above strategies.

Source of Safe Bacteria - Cheese
Some of you will ask: "Where do we buy the bacteria?" You won't buy it! You will isolate it from the "wild" - from the world around you. There is no shortage of bacteria in the world as there are more tons of bacteria that of all other living things put together. Remember it is the base of the ecological pyramid. And, for safety's sake, you will be isolating it from a source that you know is so safe that the bacteria are edible. Forty percent of the weight of cheese is living bacteria in a narrow group called the "dairy bacteria." (Normally one of the first steps in isolating bacteria from the wild is to spread a water extract of dirt onto an agar that is made of highly specialized ingredients. The desired organism is the only one that can grow on that agar. This is called a "selective medium." In your case you used a cow and the cheese fermentation process as your selectors.)
Method of Isolating a species of dairy bacteria from the cheese
Supplies -
- pressure cooker that consists of a pot, a cover with seal, and a pressure weight Make sure that you have big enough device so that the necessary items below can fit in it.
- scale or balance accurate to 0.1 gram
- nutrient agar powder (NA)
- "powder" funnel (one with a larger opening in its neck)
- bottles in which to mix and sterilize the NA (bottles are preferred since they have parallel sides and are easier to hold with mits when hot.
- Sterilied empty petri plates - either plastic or glass.
- Gas burner - plumber's torches are cheap and mobile.
- Inoculation loops (note: there in only one "N" in inoculate)
- Natural cheeze - a hunk and NOT a slice.(approx 1"x 1" x 4")
- Coins. Here is another opportunity for you to think collaboratively! Which denominations? Which nationalities? What compositions?

FUNDAMENTAL HINTS
1. CLEAN and STERILE have two different meanings. Examples: you can sterile dirt; you can scrub your hands clean, but there are still perhaps as many as a million microbes per square inch! Even were you to soak your hand in boiling water for five minutes, it still would not be sterile.
2. Whenever doing a step that is "aseptic" (in purple) keep your mouth closed. YOU DO NOT TALK! A bacteria laden mist comes from your mouth when you talk, and those will settle upon your work. Have a helper stand off to the side and read aloud to you the directions point by point as you do them. Don't jump too quickly: listen to the WHOLE point before beginning that point.
3. NEVER store unsterilized medium thinking that you don't have time to use it today and so will store if for tomorrow. Unless you freeze it, you will find it turbid with growth the next day - even if refrigerated. Airborne bacterial and fungal spores have wafted in and set up housekeeping.

MATERIALS AND METHODS

Preparation of Media (Agar, etc.)
(http://www.science-projects.com.MediaPrep.htm)

Have available about a dozen sterile petri plates. If plastic, they are purchased sterile in plastic sleeves. There are usually 20 plates per sleeve. If plastic and not in polyethylene sleeves: DON'T USE THEM - PERIOD! If glass, you will have to sterilize them, and that can be done when you sterilize the NA in the bottles or flasks.
Read the label on the NA bottle of powder. It will tell you how much you need to make one liter of medium. You will need about 300 ml. So how much powder to you weigh out? Hint: to minimize mess, put the bottle with the funnel in it on the scale, tare, and then spoon in the requisite amount of NA. Next add 300 ml of cool tap water, which has minerals needed for growth (this is "biology", and not chemistry). Swirl the bottle to suspend all of the powder. Agar will not dissolve until about 98°C so the swirling liquid will seem to have sand in it at first. (Also NEVER make 1 liter of NA and then distribute it to subsidiary containers! Why? The agar granules will settle as you are distributing the batches. The first batches will get little agar and will later never solidify, while the last batch will solidify like a rock!) As a teacher, you should know that agar is EXPEN$IVE!.
Sterization of Liquids and Solids
(http://www.science-projects.com.Sterilization.htm)
Loosely cap the bottles. A good way to do that is to invert a small beaker over them. Thus after the bottles are sterilized, dust won't fall into them and contaminate them.
Set them (and any glass petri plates to be sterilized (tops on the plates!)) into the pressure cooker. Often an empty test tube rack works nicely as a support to hold the plates out of the water at the bottom of the pot. Also wrap THREE sets of coins separately in several layers of paper towel (each coin separated from the other) and place them on the rack in the pot also. (Alternatively: you may sterilize solid items in an oven for 90 minutes at 450°F or 190°C. DO NOT OVEN STERILE ANYTHING PLASTIC OR WITH PLASTIC OR RUBBER ON IT! Why not? If you cannot think of why you shouldn't be sure to be prepared to explain your actions to the fire department!)
Now follow the pressure cooker's directions, such adding the correct amount of water, etc. Operate at 15 psi for 20 minutes. Allow the cooker to cool on its own until you can remove the pressure weight without its hissing out steam.
Using a damp paper towel, clean off the surface of a flat desk (not one on a tilt). Array the petri plates on the desk top.
Using a hotpad, lift out one of the bottles.Do NOT remove the lid! On this first occasion, take a look at what you are holding: you will see a molasses-like syrup at the bottom of the bottle - that is the molten agar. It must be mixed thoroughly into the remaining yellow liquid. Hold the bottle over a sink and swirl it gently. You will see the agar mix into the overlying liquid.
Pouring Agar Plates
(http://www.science-projects.com.PlatePour.htm)
Pouring the plates. Keep the lids on the plates! One by one, lift a lid (do NOT turn it over so that dust will fall into it), and pour the hot agar solution in until the plate is about a third full. Immediately replace the lid. Yes, condensation occurs on the lid, but we'll deal with that later.
Once you have emptied the bottle, immediately take it to the sink and rinse with HOT water. Imagine cleaning it out once that layer of agar has solidified. Keep the lab clean. Grubby people should be sent to the basement dungeon where lions are kept!
Now return to the plates: you will see that some have froth or bubbles on the surface of the still-molten agar. You will burst these bubbles by wafting a flame across the surface. Yes, of course you have to life off the tops - one by one as you move down the line of plates. Woe be to the person who wafts too slowly over plastic plates. The lids get welded to the bottoms!
After about 30 minutes the agar will have cooled enough to solidify. (Agar is one of the few substances that has a freezing point different from its melting point!)
On the day that you are going to use the plates, continue:
On the day you are ready to use the plates you must aseptically dry away the condensation. Here is how it is commonly done.
1. Find a corner table in the room that is draft free and has little commotion. Using a damp paper towel, wipe up the dust. Then carefully unroll some of the paper towel and spread it on the desk. (usually fairly sterile inside the roll). Then array out the plates as shown here to allow the moisture to evaporate. As you open each plate to lay them down, if you see any mold or bacerial colonies, discard that plate and its cover. Drying usually takes about 2 hrs at room temperature.
2. Once the plates are dry, reassemble the lids to the cover the bottoms and stack. Do not repeatedly open and close the plates. People with busy hands should be in mental institutions, and not doing microbiology!
Isolating Microbes from a "wild" source
(http://www.science-projects.com.IsolateWild.htm)
Now set up and light your burner again, get out the inoculating loop and the hunk of cheese. The object of the next series of maneuvers is to get some of the bacteria from the uncontaminated interior of the cheese onto the agar surfaces of several petri plates.
Take the long chunk of cheese, bend it so that it breaks in the middle exposing new, uncontaminated faces. Hold one half of the cheese new face downwards to prevent dust from getting on it.
Pass the wire part of the inculating loop into the flame so that it glows red (not white) hot. No living thing can tolerate red heat (the wire cannot long tolerate the white heat!). Carefully guide the loop's wiggly, hot wire to the fresh surface of the cheese. Stab it in to cool the wire.
Then make very small circles with the loop on the freshly exposed surface of the cheese. DO NOT allow the loop to come near the contaminated exterior surfaces of the chunk.
Set the cheese down, and open one of the plates a little. Slip the loop into the plate without touching anything but the agar surface. Make lots of wiggles with the flexible loop onto the surface of the agar. Withdraw the loop, and set the cover down.
Use the same loop, and, without going back to the cheese, wiggle the wire on the agar surface of two more plates.
Finally, heat the loop to red hot to burn off any residual bacteria. (This is a standard practice to prevent the bacteria's getting spread around in the drawers, etc. Also as some loops have sharp ends, you don't want the bacteria in you in event you prick your finger on it. While cheese bacteria are harmless in your tummy, they might not be inside your blood stream!
Label the plates 1, 2, and 3 (last), and then incubate them in a dark cabinet (not a drawer, why?) for several days (a weekend is great). Set the plates as shown in Fig. 2, above. If you place them as in Fig. 1, then any drops of condensation can drip onto the agar surface and make smears of growth.
Eat the remainder of the cheese if you wish,
Put your "toys" away, and set the plates in a dark cabinet away from fluorescent lights (ultraviolet killing).
In about three days, you should see small dots of cheese bacteria colonies growing on plates #1, and maybe the others also.
You now have your bacteria for the coin experiment.
What techniques have you learned? list them.

SUB-GOAL TWO - Executing the Strategies

(Cheer up! The real experiment is much shorter. No, I don't know why preparations always take so long! Perhaps it has something to do with the second law of thermodynamics.)

You now have

three sets of sterile coins and one non-sterile set
several petri plates with cheese bacteria growing on them
several uninoculated agar plates.

A. Are the coins laden with microbes?

Take the unsterilized coins and rub them around in your hands.
Drop the coins onto the surface of one of the uninoculated plates.Just don't let the coins touch each other (possible bimetal effects). Cover the plate and label it "A"
After three or four days carefully inspect the edges of the coins. Is there sign of scuzzy growth? If there is, what does it mean?
See DISPOSAL, and clean up. The coins can be displaced easily from the agar surface by inverting the plate and rapping it on the desktop. The coins will fall into the cover of the plate.

B. Can the coins kill cheese bacteria?

Take a sterile swab and pick up some of the cheese bacteria from Plate #1. Rub the swab all over the surface of another agar plate. Rub it this way and that many, many times to well distribute the bacteria as homogeniously as possible.
Immediately set up a 250 ml beaker with ethanol in it. Set a tweezers in it.
Carefully unroll one set of sterile coins. As each one pops into view, use the tweezers to lift that coin and place it on the agar surface. (After each placement, put the tweezers back into the ethanol, then fling off excess alcohol before making the next transfer.)
Once the array is finished, label that plate "B"
Incubate your plates as you did before.
Clean up and put away your stuff.
After three or four days carefully inspect the edges of the coins. Are there halos around any of the coins? If there are, what does it mean?
See DISPOSAL, and clean up. The coins can be displaced easily from the agar surface by inverting the plate and rapping it on the desktop. The coins will fall into the cover of the plate.
Keep the coins aside because soon you will want to compare yours with those of Group D.

C. How fast is the self-sterilization of the coins?

Completely unroll the second set of sterile coins
Put on rubber gloves.
Rinse your two gloved hands in the ethanol. Fling off excess alcohol.
With one hand pick up all the coins and hold them in your greedy clutches.
With the finger tips of the other hand, pick up some of the bacteria from a plate, and then work the bacteria over and around the coins in the other hand. Be sparse about it: don't make the coins gloppy! If you can see smears of bacteria on the coins, you have put too much on!
Array the coins in a line on the sterile wrapper from which they came.
Using the repeatedly sterilized tweezers, lift the coins onto the plate's agar surface in timed intervals. It is often scientifically best to use exponential time: 0, 1 min, 2 min, 4 min, 8 min, ..30 min, 1 hr, 2 hr. and so on until your coins run out.
Incubate your plates as you did before.
Clean up and put away your stuff.
After three or four days carefully inspect the edges of the coins. Are there signs of bacterial growth around any of the coins? If there are, what does it mean?
See DISPOSAL, and clean up. The coins can be displaced easily from the agar surface by inverting the plate and rapping it on the desktop. The coins will fall into the cover of the plate.

D. Are the coins exuding any bacterio-toxic substances?

Set up a 250 ml beaker with ethanol in it. Set a tweezers in it.
Carefully unroll one set of sterile coins. As each one pops into view, use the tweezers to lift that coin and place it on the agar surface. (After each placement, put the tweezers back into the ethanol, then fling off excess alcohol before making the next transfer.)
Once the array is finished, label that plate "D"
Incubate your plates as you did before to give any exuding substance time to dissolve into the agar.
After 24 hours, rap your inverted plate sharply on the desk top. The coins will drop off the surface into the cover. Dump the coins onto a paper towel, and recover the plate.
Have the coins changed any? Stained? Corroded? Compare your coins with those of Group B.
Take a sterile swab and pick up some of the cheese bacteria from Plate #1. Rub the swab all over the surface of another agar plate. Rub it this way and that many, many times to well distribute the bacteria as homogeniously as possible.
Incubate your plates as you did before.
Clean up and put away your stuff.
After three or four days carefully inspect the areas where the coins had sat overnight. Are there any signs of inhibited growth? If there are, what does it mean.
See DISPOSAL, and clean up.

OBSERVATIONS

Did you see signs of microbial growth around the coins? All coins? Only some? Which ones? Metalurgy?
Did you see signs of halos around the coins? All coins? Only some? Which ones? Metalurgy? Compare your coins with those of Group D.
How long did the bacteria have to be in contact with the coin to be killed? All coins? Only some? Which ones? Metalurgy?
Were any of the prior coin positions too toxic for microbial growth? All coins? Only some? Which ones? Metalurgy? Compare your coins with those of Group B.

DISPOSAL OF BACTERIOLOGICAL MATERIALS

This is an intellectually tricky subject. Most schools and colleges treat ANY microbe as if it were an extreme pathogen because most schools and college load their safety committees with medical technicians and medical doctors, who, upon being questions, usually cannot think of any microbe that is safe. Ask almost any farmer, and, while they strain to list pathogens, they can rattle off a series of microbes that are harmless (nitrogen fixing bacteria, and the dozens of organisms in the rumens of their cows, goats, and sheep. And someone in the farmer's family will also recognize the yeast used to make bread, wine and beer. Some people will also know about the microbes that turn milk into the other dairy products - hard and soft cheeses. Still others will note that pickles and silage are fermentation products. In fact, of the zillion or microbial species, it is only a rare one that is a pathogen. Stay away from hospitals or veterinary clinics, and you tend to stay away from nosocomial diseases. (Dictionary anyone?) So you will probably be a pawn in this battle. Just follow your system's rules until they become more enlightened.

Thus, while you may throw excess cheese in the waste basket for the janitor to dispose of, the agar from your plates may have to be scooped out and placed in a biohazard bag, and the given pressure cooker treatment. You see the irony!

But perhaps your system will let you merely pour about 10 ml of household bleach in the plates to kill those safe dairy bacteria, and then you may dispose of the agar in the trash (just don't burn your skin on the bleach or get "racing stripes" on your clothing.

If as a teacher you wish to join the fray, here is a list of safe microbes that most of this nation's top microbiologists at the National Institutes of Health, the Centers for Disease Control, and elsewhere, deem totally safe and need no more special handling than were you working with a flower pot of soil.

PEDAGOGIC GOALS Upon completion of this sequence, you shall have learned...

many of the basic procedures of microbiology
main ones not included were microscopy, staining, species identification, how to grow bacteria in liquid culture, and determining how to count bacteria
the names of the microbiologist's tools and their uses
how to have fun while learning.

It is also recommended that this work can become the basis for teaching students how to search out a good projects, do it, and make the most of it by writing a scientific paper.

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