SODIS project

[cjp]

Hello. I'm hoping that you can give me some advice so that i can help assist my daughter in her science fair project. She is using Learn How to Disinfect Contaminated Water from your Microbiology section as a model for her project. I had thought that the control group would be from the untreated creek water, but today, her teacher said that she should have positive and negative controls. These terms weren't really familiar to me; I did read some of your comments about them, but I'm still not entirely sure. I can't ask my daughter as she has just left for a weekend band competition, and I would like to be up to speed when she gets back.

The teacher suggested that the positive control would be water from a freshly opened bottle. She also said that the negative control could be animal fecal matter mixed in with some water. So does this sound correct? (my daughter was probably distracted with thoughts of the band trip when talking to her teacher) If so, how should we make our negative control? We have two dogs so no problem there, but should we combine it with the creek water which we are going to use ? And sorry to be graphic, but should we just let some fecal matter sit in the water for awhile and remove the rest, or try to mash/stir it up into the water? Also, when writing out the experiment, would the creek water that we take directly from the creek and leave untreated still be considered a "regular" control along with the positive and negative controls?

Thanks so much for any help you can give. We are both interested in the idea of SODIS treatment and are looking forward to her performing the experiment.

[deleted-71827]

Hi,
I'm not sure in what context your daughter's teacher was referring to two controls for the project. I think that the control for the project should be taking creek water and simply letting it sit there so that you compare the effectiveness of the SODIS treatment on the water. The purpose of the control in a project is to have a baseline standard to which you can compare the results. In this project, the point is to see if SODIS really causes the samples to have decreased amounts of bacteria, which is accomplished by comparing the SODIS samples to the non-SODIS control of plain creek water. One control seems to be sufficient for this project, because having the control of animal fecal matter + water does not help you analyze your results. If you treat the animal fecal matter + water, it may or may not work depending on how strong the SODIS treatment will work, so it is not a reliable sample to compare your results to. Hope this helps!

[cjp]

*deleted what I originally posted here*... please see below. And staryl13, I was thinking what you had posted.. that the untreated water would be the control, but I guess my daughter's teacher is looking for something additional.

[cjp]

Okay, still doing some reading and I came across a simple definition of positive and negative controls. Positive - something you know will produce a certain result and Negative - something you know should not produce a certain result. Okay then, when looked at in terms of the SODIS process, which is about achieving the inactivation and killing of bacteria, the positive control would be using water from a freshly opened bottle because we know consumer ready products wouldn't have bacteria in them (and this is what SODIS aims for). And a negative control would be adding the animal fecal matter to the creek water because that will just add lots of bacteria to the water (and it's negative because we don't want more bacteria when using SODIS) . Am I finally on the right track? If so, would the untreated creek water (with no fecal matter added) be considered a "regular" control? Thanks. I appreciate all you guys do; you really do provide such valuable help!

[deleted-71536]

My understanding is that a positive control is something that is known to produce the desired effect with treatment, while a negative control is known not to respond to the treatment. Using those definitions, I guess your bottled water could serve as a positive control, because you expect that it is fairly pure and will thus come out "purified" with treatment.

Perhaps the water with the fecal matter should remain untreated, and thus serve as your negative control. You know it's dirty, so it will should measure as unclean.

The untreated creek water would serve as a "regular" control, as you put it.

You could run the SODIS treatment with samples from all three types of water (bottled, creek water, and fecal matter added), with the expectation that the creek water should fall out somewhere in the middle. Or, if the SODIS treatment is quite effective, the treated creek water might look like the treated bottled water. I think the idea is that the other two types of water give you a measure of whether the SODIS treatment worked, by providing two extremes.

Does this make sense? Please post back if you have more questions.

Heather

[cjp]

Hi heather,

Thanks.. yeah, i see what you are talking about. I do have a couple more questions if anyone could help. My daughter and I have been practicing preparing the agar plate with the creek water just to practice our technique and.. yikes. We both had the bacteria growing together resulting in groups, "clusters" rather than easy to count single units. I do know that there have been questions about inoculation techniques in the "Ask an Expert" section before and I have read them and plan to try some later today during our second "practice" session. But do you have any tips for me on getting the best result for counting? Should I use a glass stirring rod (sterilized of course), sterile swabs, sterile spreaders.. ?? any suggestions?? and as for technique...."zigzags" with a little space between? a straight line down the middle with perpendicular lines meeting the center line? Any ideas and suggestions would be Greatly appreciated. Also, at the very last, when she has the data do we call the the count colonies or colony forming units... or are the two the same?

Thanks for helping an old mom out

[deleted-71536]

There are several ways you can "count" your bacteria on plates, even if you cannot distinguish individual colonies. One way is to estimate the percentage of the plate that is covered in bacteria. This technique does not require you to count individual colonies, but instead just looks at the surface area covered by bacteria.

Another option is to dilute each sample the same amount with sterile, distilled water, to reduce the amount of bacteria on each plate; or, you could plate a much smaller amount of each sample. In both cases, I would spread the water samples evenly (definitely with sterile spreading implements), and see if the dilutions allow you to count individual colonies.

Does this help?

Heather

[cjp]

Hey.. Hope everyone here is having a great holiday season!

My daughter has been working on her project (Learn How To Disinfect Contaminated Water from your microbiology section).. with me trying to give a little assistance... and we are nearing the end of the experiment. We have a couple of questions about the results and hope that someone can give us a little help or direct us where to go to find some answers. First of all, her teacher had her expose water to UV-A radiation for 8, 12, 48, and 72 hours... a little different than what the experiment calls for. Now:

1. We have noticed that water under going this SODIS process for 48 and 72 hours have few colonies growing on agar at the 24 hour observation time; then, in most of the trials, there was a rather big jump in the count at 48 and 72 hour observation times. We aren't sure why. Does the UV-A radiation that the water was exposed to just slow reproduction of the bacteria down, but it recovers after a couple of days growth? Also, the information that we have read about the SODIS process says that environmental bacteria or algae (both harmless) could grow inside a bottle undergoing the SODIS process. So, could the high counts of bacteria in the 48 and 72 hour SODIS plates could just be this environmental bacteria growing?

2. We have noticed that in the water that has undergone 8 and 12 hour SODIS treatment we see larger colonies of bacteria growing on the agar plates than on the plates inoculated with water that has undergone 48 and 72 hour SODIS treatment. Almost all the bacteria on these agar plates first appear as small, white, round, and smooth, but then usually by the 48 hour observation mark, some colonies on the 8 and 12 hour plates will have grown larger and spread out, losing their perfect circular shape. However, this happens to fewer colonies in the 48 and 72 hour plates, where only 2-3 colonies might have grown noticeably larger. Any explantions/guidance would be much appreciated.

Happy New Year to all staff/experts! Thanks for all the good work you do here.

[deleted-71536]

Hello and happy holidays!

You have some great questions, and I will do my best to answer them.

1. We have noticed that water under going this SODIS process for 48 and 72 hours have few colonies growing on agar at the 24 hour observation time; then, in most of the trials, there was a rather big jump in the count at 48 and 72 hour observation times. We aren't sure why. Does the UV-A radiation that the water was exposed to just slow reproduction of the bacteria down, but it recovers after a couple of days growth? Also, the information that we have read about the SODIS process says that environmental bacteria or algae (both harmless) could grow inside a bottle undergoing the SODIS process. So, could the high counts of bacteria in the 48 and 72 hour SODIS plates could just be this environmental bacteria growing?

One idea I have for the growth patterns you observed is that the jump in growth you see comes from the colonies you first observed. The colonies you did see must have survived the treatment process. Because there are fewer colonies on the agar plate, there is more room for those few colonies to expand. Keep in mind that bacteria grow exponentially (when they have space and enough resources). So you are probably observing further growth of your "resistant" bacteria. When you continue to incubate bacterial colonies, you often see "satellite" colonies, originating from the initial colonies.

2. We have noticed that in the water that has undergone 8 and 12 hour SODIS treatment we see larger colonies of bacteria growing on the agar plates than on the plates inoculated with water that has undergone 48 and 72 hour SODIS treatment. Almost all the bacteria on these agar plates first appear as small, white, round, and smooth, but then usually by the 48 hour observation mark, some colonies on the 8 and 12 hour plates will have grown larger and spread out, losing their perfect circular shape. However, this happens to fewer colonies in the 48 and 72 hour plates, where only 2-3 colonies might have grown noticeably larger. Any explantions/guidance would be much appreciated.

Because you have fewer colonies on the 8 and 12 hour treatment plates, there is more room for those colonies to expand and grow. On the 48 and 72 hour plates, you have more colonies using up space and nutrients. This tends to slow their growth, comparatively.

I hope this helps! Let us know if you still have questions.

Heather

[cjp]

Thanks for getting back to me so quickly, Heather. I think this is what you are telling me; please let me know if I'm totally off base. You said (in response to my question 2) that "because you have fewer colonies on the 8 and 12 hour treatment plates, there is more room for those colonies to expand and grow. On the 48 and 72 hour plates, you have more colonies using up space and nutrients. This tends to slow their growth, comparatively." Did you tell me this because at the 24 hour observation time, the plates inoculated with 8 and 12 hour SODIS water have more colonies that the plates inoculated with 48 and 72 hour SODIS water. However, by the 48 and 72 hour check points, there has been a big jump in the number of colonies growing on the 48 and 72 hour plates because the smaller number of colonies at 24 hours made exponential growth possible?? And with the larger number of colonies growing at the 24 hour check on the 8 and 12 hour SODIS plates, there isn't as much room for exponential growth, but some of the colonies in these plate do grow larger, becoming less like a circle and more like a "blob"?? Sorry, I just am not quite understanding the answer to question 2.

Thanks for your time and patience.

[deleted-71536]

I think I may have been confused, and that's why my answer was confusing to you!

For some reason I was thinking that there were fewer colonies overall on the 8 and 12 hour SODIS plates, but that's the opposite of what happened. (Your results make sense; I think my brain just did a little flip with the numbers.) I'm not sure how to explain why fewer of your colonies turned into "blobs" on the 48 and 72 hour plates than the 8 and 12 hour plates. However, I can say that seeing some colonies turning into "blobs" is completely normal. Keep in mind that each colony initially represents the growth that began from a single bacterium. However, after more than 24 hours of incubation, some of the bacteria form "satellite" colonies, and the original colonies can lose their circular shape. It's basically just the bacteria spreading out.

In general, you should expect to see the colonies that survived the SODIS treatment at your 24-hour checkpoint. You probably should not expect to see new colonies after that, unless some were introduced due to opening of the plates for observation (basically, contamination). The spreading into a "blob" still arises from the original colony that started at that location.

Does that make more sense?

Heather