Bacteria Science Fair Questions - A few basic and complex questions about bacteria (Nov/04/2005 )
It just occurred to me.... by using a spectrophotometer, wouldn't the type/concentration of the agar effect the results, depending on how dense or opaque/transparent the media is? Sure, I could compare results that use the same agar, but for my experiment there are 6 different agars. Will this effect the spectrophotometer's results at all? Dramastically? Not too much? Thanks
P.S. - I still don't know if I have one available to me.... if I don't I might have to go back to the surface area idea...
You wouldn't be using agar...
Yes, the different liquid media will block light at different rates. To correct for this, you use a blank for each type of media before you read the optical denisty of the cultures growing in that media. A blank is simply a tube with some sterile media in it, of the type your bacteria is growing in. You put this tube in the spectrophotometer, and set it to zero -- thus you've accounted for the absorbance of the media. Take the blank out, put your culture in, and the OD reading is due to your cells, since you've already subtracted the effect of the media...
You wouldn't be using agar...
Yes, the different liquid media will block light at different rates. To correct for this, you use a blank for each type of media before you read the optical denisty of the cultures growing in that media. A blank is simply a tube with some sterile media in it, of the type your bacteria is growing in. You put this tube in the spectrophotometer, and set it to zero -- thus you've accounted for the absorbance of the media. Take the blank out, put your culture in, and the OD reading is due to your cells, since you've already subtracted the effect of the media...
I have good news and bad news. The good news is that our high school does have a (maybe 2) spectrophotometers that I think I can use. The bad news is that the kit (which has already arrived at my house, and I don't think I can return it, its been opened to get the coupong for live materials to be sent in) came with agars, so maybe the spec. idea won't work after all. Theres no way to convert these agars from the kit into liquid media, is there, if not, I don't think I have enough time to make/purchase it, plus the agars/supplies I have would be a waste.
Because there isnt a lot of time left to do our experiments, and I'm a little behind because I am still slightly undecided what to do, I think I may go with the surface area idea. What are your doubts? This will still measure bacterial growth, won't it? It will still show differences between bacterial growth in more and less concentrated agars, right?
I guess my biggest concern is controlling the experiment. How will you start each culture at exactly the same width, for example, with exactly the same innoculum? The streaks will all have to be uniform both in width and amount of bacteria for you to be able to measure growth in a comparable manner.
Look at it this way: if you streak some bacteria in a line on some agar with no additive, and do exactly the same thing with the same bacteria on a second plate of the same no-additive agar, and let both plates grow overnight, there's no way the two lines will be of equal width (or area) in the morning. So you measure them and they're different -- what does that tell you? There was no agent acting upon the bacteria, so what accounts for the difference in area?
Now try and do the same with different bacteria and different media -- they're all going to be different areas, but can you derive meaninful data from the differences?
Add to this the fact that different species grow at different rates. So, one bacteria grows to cover one area, and another grows to cover a larger area. If they're different species, how can you compare one to the other by measuring area? They likely would have grown to cover different areas even if there wasn't an agent acting on them...
Also, different species grow differently on the same media, regardless if there's an additive in the agar. One species may grow vigourously of Nutrient Agar, while another may not be so happy on that media (perhaps it perfers Brain Heart Infusion or Tryptic Soy or just L agar) and will grow more slowly. These are media preference differences resulting in differences in area covered, not differences due to an agent in the media. How will you account for them?
Experimental design is key to collecting meaningful data. The kit you purchased is designed to show qualitative differences, but you want to use it to measure quantitative differences. Some of the factors I listed above can be controlled for, but as a whole, it is very difficult to think of adequate controls to insure your experiment produces meaningful data.
Look at it this way: if you streak some bacteria in a line on some agar with no additive, and do exactly the same thing with the same bacteria on a second plate of the same no-additive agar, and let both plates grow overnight, there's no way the two lines will be of equal width (or area) in the morning. So you measure them and they're different -- what does that tell you? There was no agent acting upon the bacteria, so what accounts for the difference in area?
Now try and do the same with different bacteria and different media -- they're all going to be different areas, but can you derive meaninful data from the differences?
Add to this the fact that different species grow at different rates. So, one bacteria grows to cover one area, and another grows to cover a larger area. If they're different species, how can you compare one to the other by measuring area? They likely would have grown to cover different areas even if there wasn't an agent acting on them...
Also, different species grow differently on the same media, regardless if there's an additive in the agar. One species may grow vigourously of Nutrient Agar, while another may not be so happy on that media (perhaps it perfers Brain Heart Infusion or Tryptic Soy or just L agar) and will grow more slowly. These are media preference differences resulting in differences in area covered, not differences due to an agent in the media. How will you account for them?
Experimental design is key to collecting meaningful data. The kit you purchased is designed to show qualitative differences, but you want to use it to measure quantitative differences. Some of the factors I listed above can be controlled for, but as a whole, it is very difficult to think of adequate controls to insure your experiment produces meaningful data.
Each bacteria species will be placed in the 6 agars (see them at the kit site listed a page before), 5 species, 30 plates total. I won't compare different species, but compare bacterial growth within one species, and overall show that higher concentrations of these agars reduce bacterial growth. So it doesn't matter if some of these species grow more reapidly in one agar, or if they don't "like" the agar. For measuring how much bacteria I initially spread over the plate, I guess, with the grid paper underneath, I could get pretty accurate - .2mmsquared won't matter that much; I'm just showing that overall the higher cncentrated agars won't promote as much growth. By surface area, it won't be as exact as using a spec., but the basic idea gets across and it can compare differences between species, which will show similarities including all species in one agar compared to all species in another. I'm sure that none of these bacteria are non-reactive to these agar; the Carolina company picked them out - I'm sure they know what they are doing. I know that there may be a few extraneous variables, but this experiment will still show that, due to osmotic pressure, bacterial growth will be reduced with higher contrations of sucrose-based and sodium-chloride based agars.
Okay -- good luck!
Thanks, and thanks everyone for all the help!
After some help to narrow my project to an exact problem statement, "What will be the effect on bacterial growth in different concentrations of sucrose agar, and sodium chloride agar?", I did my experiment, and my hypothesis, "Due to osmotic pressure, there will be less bacterial growth in the higher concentrated sucrose and sodium chloride agars, while there will be more bacterial growth in the more diluted sucrose and sodium chloride agars", was proved correct. In my experiment, i had 195 samples, because i divided each petri dish into four quadrants using a permanent marker on the bottom of the plates. The science fair is on wednesday, but i still dont know exactly how to pronounce the bacteria names. Does anyone know of a site that tells you how to pronounce the names of common bacteria types? Thanks
I'm not aware of such a site, but if you post the names I'm sure we can help you out with the phonetics.
_Hank