THE ROLE OF FEVER IN FIGHTING GRAM-POSITIVE INFECTION

..... INTRODUCTION. So far, a host of college and high school students and several high school teachers have worked together to show how fever fights infections caused by Gram-negative bacteria. ..It is NOT by just being too hot for the bacteria. ..Most of our intestinal bacteria can tolerate 43°C (about 115°F), and that is far higher than any human can tolerate! ..When our bodies have fever, the Gram-negative bacteria find that they cannot produce one of their outer layers (called lipopolysaccharide or "LPS"), which would act as a sort of armor that protects the bacteria from being perforated and killed by a group of proteins in our blood called serum complement.

..... But we have only a hint at how fever can be used to control infections by Gram-positive bacteria. ..Several years ago, a high schooler named Jeet in San Diego discovered that under fever conditions, the one strain of Gram-positive bacteria he used was far more sensitive to penicillin when it was grown at 41°C than when it was when grown at 25°C. Since penicillin interfers with the synthesis of Gram-positive bacterial cell walls, the hint seems to be that febrile temperatures cause the formation of weakened cell walls.

..... But how can one test for this? ..Most of us know the plight of many birds when they encounter the old pesticide DDT in their environment: ..the birds produce thin-shelled eggs that break easily. ..So Jeet subjected his two cultures (high and low temperature grown bacteria) to two types of physical stress in the hopes that the stress would more easily break open the putatively more fragile fever-grown bacteria. ..One type of stress was applied by a "sonicator" which is like putting an electronic tuning fork into the culture liquid. ..The sonic vibrations rattle the cells so intensely that weak ones break open. ..His other stress was that called "freeze-thaw." ..When a liquid culture of bacteria is frozen, tiny sharp spears of ice crystals form and can stab right through weaker cells. ..And when the thaw occurs, the punctured cells explode and die. ..Thus Jeet subjected his cultures to repeated freezings and thawings. ..After each freezing, he would take a very small sample and count the number of LIVING bacteria remaining. ..He expected the more fragile forms to die off more rapidly - and they did in his one tested species.

..... WE NEED TO TEST MORE SPECIES OF GRAM-POSITIVE BACTERIA, AND SPECIES THAT COVER A VERY WIDE RANGE.

..... METHOD. First obtain a number of small plastic tubes (please do not use glass ones unless you don't really want to do this experiment). ..These are called "microfuge tubes" in your supply catalogs. ..They have little hinged snap-on caps. ..Really very handy. ..You will need about 14 of them for each species to be tested: ..7 for aliquotes of the low-temperature-grown culture, and 7 for the higher temperature culture. ..Each set of seven tubes should be labelled zero through six. ..Since there is not much writing space on these small tubes. ..Assign each species a letter of the alphabet and write that on the caps of the 14 tubes of two sets. ..And underline one set's numbers to indicate the lower temperature, and maybe even overline the numbers in the second set for the higher temperature.

..... Now grow up your pairs of cultures in liquid medium 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":

..... 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":

..... And then do successive cycles 2 and 3:

..... And so on through 4 and here is Cycle 5:

..... After one more cycle, you will have sets of tubes in the freezer that have been frozen and thawed just as many times as the tube's number.

..... Finally your are ready for the big job of bacterial enumeration: ..how many bacteria per milliliter are in each of the tubes? ..For this you are recommended to see pages 112-114 in Pierce and Leboffe's Exercises for the Microbiology Laboratory; Morton Publishing Company (http://www.morton-pub.com). ..So while you are setting up to do this, keep all the above tubes in the freezer (and their 0's in the refrigerator cold but never frozen).

..... Finally you will need to graph your data. ..Here is how: ..The "0" for each set will be what are called "100% survivors". ..You must determine what percent survivorship each of the other tubes in that set have. ..As you know 100% = 1. ..Convert all the other percentages to fractions, and then take the log of each of those, because you are going to graph the log (fraction of survival) versus the number of freezings. ..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. ..The steeper the line drops the more fragile the cells in that culture were. ..IF the lines from the fever-grown cells are always steeper than the cooler cultures, THEN you are almost ready to PUBLISH.

..... "Almost" because it would be best to get back to the author of this procedure because you could combine your work in with the work of Jeet and the other students to make a very impressive manuscript to be submitted to a major journal for publication around the world. ..The mark of a real scientist is publication!

..... So now get on with this and let's get it done!

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