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BACTERIA AND TEMPERATURE In addition to being factor to consider in microbial growth, temperature is also used as a means of limiting bacterial growth or killing bacteria to pasteurize or sterilize. Chilling bacteria slows growth down significantly, and subjecting bacteria to high enough heat kills them. Clearly, the normal growth temperature of the bacteria plays a role, because pyschrophilic bacteria would prefer to be in the refrigerator and thermophilic bacteria would prefer to be at temperatures that could be used to prevent the growth of or kill other bacteria. In this experiment, we are testing the ability of some mesophilic bacteria to withstand heat treatment at different temperatures. These bacteria all have an optimal growth temperature somewhere between room temperature and 50 C, so we expect heat treatment to either temporarily stop their growth or to kill them. Also, the entire experiment takes place within 30 minutes time, so even if the bacteria could grow at the temperature used, there is little time for them to grow. High temperatures wreak havoc on large molecules such as proteins and nucleic acids. Many weak bonds, such as hydrogen bonds, begin to fall apart as the molecules jiggle rapidly from the increase in heat. When such molecules lose their three dimensional shape, they stop working, and often aggregate and tangle irreversibly. Thus the cell dies. The rate at which a population of bacteria dies depends on the bacterium, the temperature, and the time exposed to that temperature. The most useful measure of how long it will take to kill a population of bacteria is called the decimal reduction time, or sometimes just the D value. The D value is the length of time it takes to kill 90% of the population at a given temperature. Let's suppose the D value for Escherichia coli at 70 C is one minute. That means every 1 minute E. coli is exposed at that temperature, only 10% of the cells remain alive. If you graph this, you will see that this is not linear but logarithmic. If there are 1,000 cells to start with suspended in liquid medium in a tube, after 1 minute 90% will be killed leaving 10% or 100 cells. After a second minute, 10% or 10 cells will be left. After the third minute, 1 cell, and after four minutes, no cells will be left. Thus to kill all the E. coli in this tube of medium, you could heat it at 70 C for 4 minutes. Bacteria would die quicker at a higher temperature, so the D value at 100 C would be less, that is, 90% of the cells would be killed in a shorter time at 100 C than at 70 C. Note that the D value does not depend on the number of bacteria; whether there be hundreds or millions, the D value tells you how long it takes to kill 90% of the cells. That still means that the more cells there are, the longer it takes to be sure all of them are dead. When preparing media to grow bacteria or glassware to put them in, we want to be sure that these things are sterile, devoid of any life, to prevent contamination. The treatment we use, usually heat, must be sufficient to kill the most hard to kill microbes present (those that have the highest D values). These are always endospores. Endospores are resting cells with very tough walls that confer heat resistance. Thus our heat treatments must be intense enough to destroy all endospores as well as "vegetative" bacteria. Dry heat at 170 C for 2 hours (an oven) will sterilize glassware. Dry heat is not useful for liquid medium; the liquid would only heat to 100 C until all the water boiled away, then gunk left behind would heat to 170 C. For liquid-containing items we use an autoclave. Ever use a pressure cooker? The principle is identical. The material being sterilized is subjected to steam under pressure; steam can reach higher temperatures than water, and the temperature rises to 121 C. Wet heat is much more effective in killing cells and spores than dry heat, and exposures of 15-30 minutes to hot steam (under pressure) is enough to sterilize most things. Sometimes we use heat to kill some organisms without necessarily killing all of them. The most obvious example is pasteurization, in which a beverage is heated at 72 C for 15 seconds. This is long enough to kill a number of disease-causing bacteria that can be found in milk, but does not sterilize it. The remaining organisms, which are called thermoduric because they are relatively heat tolerant, can still cause a beverage such as milk to spoil, so that milk must be refrigerated. We don’t generally regard thermophiles as thermoduric since they not only withstand high temperatures but they grow at high temperatures. We think of some mesophiles as being thermoduric, able to tolerate a brief exposure to heat but unable to successfully grow at high temperatures. The Exercise In this exercise, we are going to examine the heat tolerance of three different bacteria. At least one of them will be a species of Bacillus and will thus be a sporeformer. The three bacteria will be divided up around the class, and the data from all the sections of this course will be pooled, averaged, and returned to you. You will be working in with your lab partner. Each pair will have a tube with a liquid culture of a bacterium. Your entire lab bench will be assigned a temperature, for example 50, 70, or 95 C. If you have been assigned the near boiling water (95 C), you should set up a coffee can and tripod like you did for the acid fast and spore staining techniques. Each group will have a Petri plate of growth medium. With a wax pencil or Sharpie, you should mark on the bottom of the plate so that 0' it will be divided into 5 wedge shaped areas and label them as shown below. 15 ' Using aseptic technique, obtain a loopful of bacteria 30 ' and streak it onto the section of plate you labeled 0' as shown. Be careful about inoculating the correct sections of 5' the plate. When you turned the plate upside down you 2' labeled it clockwise; viewed from the top of the right-side up plate, the labels are now counter-clockwise! Now incubate your tube (with the cap on loosely) in the water bath assigned to your bench. After 30 seconds, quickly remove the cap and take out a loopful of culture. Streak that onto the section marked 30". Continue timing the incubation, and after another 1 minute and 30 seconds for a total time of 2 minutes, remove another sample. Streak that loopful of cells on the proper section and continue until the culture has been treated for a total time of 15 minutes. Avoid removing the tube as this would allow the culture to cool and interrupt the timing. If you do need to remove the tube from the water bath, avoid holding the tube by the cap since the tube itself may slide out and break. Those doing the boiling water may need to steady the tube with a test tube holder while removing loopfuls of cells. At the end of the experiment, the plates will be incubated until the next class period. After the plates have been incubated, you will judge the amount of growth in each section using the following criteria: Score Description 5 Maximum growth, characteristic of untreated cells 4 Confluent growth, less than maximum, but more than a score of "3" 3 Small, very crowded colonies 2 More than 6 colonies (but less than a score of "3") 1 One to six colonies 0 No growth at all Since each survivor produces one colony, the amount of growth covering the plate is not as important as the number of colonies. This is important to remember, because some bacteria will produce very large colonies when there are few colonies and the cells have room to spread out. Four very large colonies may be a lot of growth, but should still only get a score of "1"! You will be asked to record your data and write it on the board or pass it into your instructor. The results from all sections of the class will be pooled, and you will receive a handout with the final results.