Associated with the outer membrane, these G(-) bacteria wear three layers that are analogous to clothing. They wear their underwear, which is some form of lipopolysaccharide or LPS layer (described below). On top of that they may have outerwear called a capsule or K-layer (Kapsul, in German) There are any of the 70 or so different K-antigens in E. coli alone!, and from that might extend various of 30 or more types of E.coli frills, which are the flagella (H-antigen). Because the outerwear naughtily gapes a lot, the underwear can be seen from the outside. And, as in people, any or all of these three layers are optional to life and might be missing! Thus our bodies recognize invaders by what shows from the outside - just as we recognize other people by what they look like on the outside. (Porpoises, which use sonar, can also recognize us on the inside also. So think about this the next time you swim in the ocean!)

About six years ago, two Virginia students, an eighth grader, Eva Dickinson, and a graduating college senior, Heather Wilson, stumbled upon the fact that Gram-negative bacteria could not synthesize their lipopolysaccharide layer (LPS) at temperatures approximating those of human fever. LPS forms the outer leaf of the outer membrane of Gram(-) bacteria. (More nomenclature: LPS = endotoxin = O-antigen; cells with LPS are called "smooth" and those without are "rough.") LPS is a very complicated molecule, and can be any of more than 150 different sorts. Eva made an exciting connection: if fever prevented bacteria from making the LPS layers, and since the LPS layer protected bacteria from perforation and killing by serum complement proteins, then fever should eventually allow complement killing of the bacteria. Eva presented this work at the 1994 annual meeting of the American Society for Microbiology.

From a bacterium's viewpoint, any of the given 150 types of LPS is a complicated molecule require the convergence of many different anabolic pathways. If any little step happens to be sensitive to the increased temperature, the final LPS molecule will not be formed normally - and usually that means 'not formed.'

BUT Gram-positive bacteria do NOT have LPS; they do not even have an outer membrane. Thus looking at how fever affects them must involve something very different. What is peculiar about G(+) cells is that they have much thicker cell walls than do G(-) bacteria. Now cell walls are also complicated structures requiring lots of different converging pathways to be synthesized properly. Perhaps, Californian Jeet Minocha reasoned, fever can affect some part of that whole synthetic process resulting in thin-walled, fragile bacteria (not unlike thin-shelled eggs of birds which have grown up in a DDT-laden environment). And if it does without killing the bacterium out-right, then how can we know that fever has really done anything to the cell wall? And what sort of magnitude of difference should be looked for?

Up to this time, all reports published by professional labs around the world happily told of a "huge" thirty percent increase in killing rates. Any biochemist could tell you that such would be expected of a 5° increase considering Q₁₀ Our report on G(-) bacteria is a 3,000-fold greater rate on most of nearly a hundred strains. I think we have something akin to that with G(+) on the two strains so far tested. Get that up to 10 strains or more, then you've really got something exciting to report to the world!

(Let us pause here. 'Report to the world.' That is a very heavy statement. Up to this point in your life, you have probably never been the first person to discover something that no one has ever seen before, although you certainly have discovered many things new to yourself. This is what science is about: exploration and discovery - not the memorization of facts discovered by others last week or last year or a hundred years ago. Those facts are actually the history of science, and not science per se. So take any discoveries you make in this project VERY seriously, as you will be the first person in the history of the world to have seen this. Then you must do the other necessary part of being a scientist - publish what you saw so that others might know about it also. Do that, and you are a real scientist no matter what your age.)

This experiment on the effects of fever on G(+) bacteria is being proposed because two California high school groups led by Jeet, above, have already sent scientific "scouting parties" out and they have come back with very promising news. Now it needs to be greatly expanded to show its comprehensiveness. It looks to be a "sure thing" as positive results are extremely likely to leap into your hands.

ANALOGY: This mental image should help in designing your experiment. A G(+) cell is like a bicycle tire: an inner-tube and a restraining tire around it to keep the tube from exploding. Is it possible to have a defective tire and yet able to ride the bike? Of course. But defective tires are a safety hazard because if put under unusual stress, they will fail. Failure results in a fracture through which the innertube herniates and explodes.

Using the principle of the analogy, you might ask whether a G(+) cell that has been grown at a temperature resembling fever can be subjected to unusual stresses to see if the cell wall is defective or malformed? And what might those unusual stresses be? This would be like testing the strength of a bike tire on a rough road of sharp gravel.

Just as with the tire, the stresses on the cell can be purely physical - sudden decompressions, or bumping into sharp objects. The former can be done with the French pressure cell or with a sonic cell disruptor (both pieces of equipment I have available). And abilities to withstand the rigors of sharp objects is very simple indeed - ice crystals and how well the cells can tolerate freezing and yet survive without rupturing. Just to really give the cells a lot of stress, freeze them and thaw them through 5 or 8 cycles. What proportion of the fever-grown cells survive versus those grown at lower temperatures? ¡Mucho simplistico!

MATERIALS AND METHODS

Before you go any further, you must be skilled in the art of counting live bacteria using a method called plate counts. (Also see "Exercises for the MICROBIOLOGY LABORATORY," Burton Pierce and Michael Leboffe, Morton Press, page 111-114. There is a sneaky way to do this using far fewer plates. Go to the teacher's page and ask for help in this. It would also help if you send your fax number so that diagrams can easily be sent to you.) Once you are confident of your technique, only then continue with the following.

You and your collaborating groups in your school AND elsewhere should have at least a dozen different species of G(+) bacteria. The more wide-ranging your collection, the greater the comprehensiveness of your final report. Suggested species can be found among the attached page on safe microbes. You might consider two or three species within the following genera: Streptococcus, Lactobacillus, Bacillus, Staphylococcus, Micrococcus.

1. ..... Grow up your pairs of cultures in liquid medium, such as nutrient broth, of the two desired growth temperatures. ..In a day or two, when the cultures have gotten very cloudy, transfer 10 to 20 microliters of each culture to new media that are already thermally equilibrated for their right growth temperatures. ..Here is a MUST: ..as soon as you can see ANY hint of turbidity (cloudiness), transfer half-milliliter amounts into the properly labeled set of microfuge tubes and set them in ice water to rapidly cool. ..Then follow this diagram labelled "START":

   

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2. To START: Leaving tube "0" in the ice-water, place all the other tubes in the freezer and allow them to freeze.
3. Leave tube 1 in the freezer and take out 2 through 6 to thaw. Immediately after thawing put them all back in the freezer. After tubes 1 through 6 have become frozen (and it shouldn't be important as to how long they remain frozen), they should be thawed in cool water. ..IMPORTANT: ..as soon as there is not more ice in the tubes, they must be immediately refrozen. ..Failure to do this will mean that the bacteria will "come back to life" and repair some of the ice damage. ..Next do what is diagrammed in "Cycle 1":
   

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4. Leaving tubes 1 and 2 in the freezer, take out 3 through 6 to thaw, and once thawed immediately put them all back in the freezer.
   

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5. Leaving tubes 1, 2 and 3 in the freezer, take out 4 through 6 to thaw....
   

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6. Keep cycling through this pattern until only tube 6 has been taken out to thaw and then returned to re-freeze.
7. Now if you take out all tubes (zero through 6) and allow them to thaw. They will have gone through just as many freeze/thaws as the number on the tube.
8. Finally you are ready to run plate counts on each of these little tubes. Matter of fact: do triplicates of the "0" tube, just in case of mishap. Your starting number is the most important number you need since against it are to be compared all the other results of that set.
9. You are now going to set up for making a "classical dose plot". In this case it will be the log of the fraction of survivors (vertical axis) versus the number of freezings (horizontal axis).
10. Record your data in the first column of a data sheet. You will eventually have three columns:
    1. #surviving bacteria per ml
    2. fraction of survivors relative to tube "0"
    3. log₁₀(fraction of survivors)
11. In your "0" tube you have by definition 100% survivors (fraction = 1.00). Determine the fraction of survivors for each of the tubes in that set that underwent various numbers of freezings and thawings.
12. Now take the log-10 of each fraction of survivorship. Of course the data from tube "0" yields a fraction of 1.0 and the log of 1.0 equals zero.
13. Take a piece of bi-linear graph paper and use the upper left-hand corner as the origin. Space the numbers zero through seven across the top of the page, and then you will have to deal with the left margin and which numbers to place there. They will be negative numbers going down to that of the value of your tube 6. ..By the way, this is really easy to graph if you use semi-log graph paper because you don't have to take the logs of the fractions of survival. ..Have your teacher demonstrate how to use that kind of graph paper, which can be bought in an office supply store.
14. Although your data points will have some scatter, they should show a distinct downward trend as more bacteria are killed by each successive freezing.
15. You should be able to determine a rate of killing, such as how many freezings are necessary to kill 50% of the cells. Usually LD₅₀ (lethal dose for 50%) is the most commonly mentioned units.
16. Do this for each different species at each different temperature. (You see why it is good to have several persons working on this - it's too much work for only one person, unless you want to flunk all your other courses!)
17. Now it might be advised to make a graph of each species showing growth temperature and number of freezings needed to kill 50%. Your fever results should, at this time, be leaping off the graph paper at you! Do you see a critical threshold temperature? How does that threshold compare with those of the other species? (The author of this protocol cannot tell you what you will see - BECAUSE - this experiment has NEVER been done before. You do it; you publish it. YOU are the discoverer!

B U T !

IF you have gotten some interesting results showing that fever-grown G(+) bacteria are indeed more susceptible to the stress of freezing and thawing, you have not shown which cellular structure is the one that was malformed and defective. Reconsider the cell's anatomy. It has two layers of packaging, just like the bicycle tire. The inner membrane and the exterior cell wall. If either one (or both!) are improperly synthesized when the cells have grown under fever temperatures, you will have gotten the results expected.

BUT now how do you tell which layer was involved?

Can you think of an antibiotic or an enzyme that targets one or the other of these two layers in G(+) bacteria? Ah, ha! Penicillin targets the process of the synthesis of bacterial cell walls. And lysozyme also targets the cell walls. Now you have a smart way to target the walls! Jeet, above, showed that his California scouting groups' two fever-grown strains were killed by about 1/70 of the amount of penicillin than was needed to kill the room-temperature grown bacteria.

Question: So-, if your fever-grown cells are indeed more sensitive to penicillin or lysozyme than those cells grown at cooler temperatures, then what does that mean? Yes! If they are, then you have a really nice added piece of information to add to your finding! Defective cells walls were made.

But what if they aren't? Well, this is a win-win situation! If the fever-grown cells were not more easily killed by these two agents, then it tells you that the cells walls were not made defectively, and that it is highly probable that the membrane was defective!

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