Science Buddies: "Ask an Expert"

Hi,

I am an eighth grader conducting a science fair project. I am testing the effectiveness of different SPFs (5, 15, 50, and no protection) of sunscreen against UV light. I have 15 petri dishes to test, should I get more? I am using E coli bacteria strain K-12 as a model because it would be unethical to use human skin cells for this project. I had my science teacher order the bacteria. I am having some trouble with my procedure in my project and I'm wondering if anybody can help me. I also have some questions about the proper way to breed and dispose of bacteria and operate a UV lamp so please tell me if something looks wrong in the procedure.

The Procedure:
1. Put agar in Petri Dish
2. Inoculate petri dish with e coli (proper way to do that?)
3. Put e. coli in incubator at (how many degrees?) for 24 hours
4. Count the colonies of remaining bacteria (proper way to do that?)
5.Cover half of the petri dish lid with foil and the other half with a certain SPF of sunscreen
6. Expose samples to UV light for (how long?)
7.Count remaining colonies after exposure.

-Will it be bad to take the Petri dish lids off to put the sunscreen on the lid?
-What are the safety precautions for using a UV lamp and what wavelength should I use?
-Where should I use the UV lamp?
-How do I dispose of bacteria?
-Any other tips/suggestions?

Hi,

That's an intriguing way of testing sunscreen. It appears to be a standard method.

I put your question into Google as "testing sunscreen with E.coli" and was surprised to find many web sites discussing exactly this project!

I found a full description of the procedure, including some similar wording to your message ("unethical to use humans") at this site -
http://www.google.com/url?sa=U&start=2& ... yTNRnoL_xA

As for details about e.coli experiments, I found the following on the ScienceBuddies web site:
https://www.sciencebuddies.org/mentorin ... Agar.shtml

Bill

[The following response was provided by Sandra Slutz.]

Hi Meaghan,

I'm one of the staff scientists at Science Buddies. Your science fair question "How effective are different strengths of sunscreen at blocking UV light?" is very interesting, and important from a health perspective. As you said, it would be difficult for you to use human skin cells to do your project. Unfortunately, looking at the survival of bacterial colonies will not work for technical reasons. But don't panic! Even with only 2 weeks left you can still answer your original question - you'll just need a different experimental plan. Read this e-mail all the way through and you'll see what I mean.

First, to explain what the technical problem is with your current project design let me start by reviewing a little bit of information about about UV light. UV light (as you probably already know from your background research) comes in a range of wavelengths. The shortest is 10nm and the longest is 400nm. The 3 types of UV light people worry about from a human health perspective are UVA (400nm - 315nm) UVB (315-280 nm) and UVC (280-100nm). Exposure to UVA and UVB can cause mutations (changes) to DNA. And both UVA and UVB manage to get through Earth's atmosphere along with the visible light in sunlight. Sunscreens are made to block UVA and UVB thus preventing the DNA in our skin cells from getting mutated (which is good because mutations could lead to skin cancer). UVC is more toxic than either UVA or UVB and will do enough damage to kill cells and is considered germicidal (meaning it can killl germs - which is why wavelengths in the 280nm-100nm range are often used to kill bacteria). But UVC is blocked by Earth's atmosphere - hardly any of it gets through. Because humans aren't exposed to UVC when they are in normal sunlight, sunscreen is not made to block UVC.

So, in your experimental setup you were planning to innoculate a petri dish of agar with bacteria, grow the colonies, count how many colonies there were, expose them to UV light (with and without sunscreen for protection) and then count how many colonies survived. But, because UVA and UVB are not germicidal (they don't kill bacteria) ALL of the colonies would survive (regardless of whether or not you used sunscreen) if you used a UV light in the 400-281nm wavelength range. On the other hand, if you used a UV light with a wavelength of 280nm or less, NONE of the bacteria would survive (regardless of whether or not you used sunscreen) because these wavelengths are germicidal and not blocked by any type of sunscreen.

But you still have a really great starting question - and we have a project on our website which only takes a few days which can help you answer that question! The project is called "Testing Sunscreen Effectiveness" and can be found at this web address: https://www.sciencebuddies.org/science- ... p015.shtml To do this experiment you'll need to purchase a UV monitor (cost is about $25) but I just checked with the vendor and they can ship it in just a few days, and once you have the monitor it'll only take you one sunny day to do the experiment.

Alternatively, if you're interested in exploring how quickly UVC can kill bacteria check out our project called "Death Rays: What Duration of Ultraviolet Light Kills Bacteria" at this web address: https://www.sciencebuddies.org/mentorin ... p017.shtml

You may also want to look at these resources:

"Inoculation: How to Put the Bacteria You Desire on a Petri Dish" - https://www.sciencebuddies.org/science- ... tion.shtml

"Microbiology Techniques & Troubleshooting" - https://www.sciencebuddies.org/science- ... ques.shtml

The above sources may help answer most of your bacteria culturing questions.

Best of luck with your project - you really did come up with a great science fair question!

- Sandra
Science Buddies

(The following is additional information related to this science fair project and the answers provided by Sandra Slutz, Science Buddies.)

Hi Sandra,

Thank you for all of your help. I am still trying to decide exactly what to do with my project. I keep having problems with the bacteria inoculation. The first time I tried to inoculate the bacteria, I used E coli strain k-12 out of a tube and used an inoculating loop. Nothing grew. The second time I tried, I swabbed raw chicken and nothing grew. Could it be the incubator? I do not have access to an incubator so I have a styrofoam cooler with a desk lamp over it. I have a thermometer in it and I adjust the lamp until I get the right temperature. I used nutrient agar (from the same place) so could it be the agar? I am just confused with what I am doing wrong.

Thanks so much,

Meaghan

(The following is additional information related to this science fair project and the answers provided by Sandra Slutz, Science Buddies.)

Hi Meaghan,

I'll be honest that trouble shooting from a distance can be a bit hard. But I'll give it my best shot.

I'm not that surprised that nothing grew the second time when you swabbed raw chicken. It is true that chicken may have bacteria on it and one needs to be careful to wash hands and cutting surfaces carefully - but its not a guarantee that all pieces of chicken will have bacteria. And even if they do, the quantity of bacterial cells may be very small. So, we'll have to discount this trial in our troubleshooting since it is a very real possibility that you didn't transfer any bacteria to the agar plates.

OK - so lets go back to your first trial. I apologize if any of my questions seem ridiculous - but I need to make sure of some of the basics, they're the most likely places for errors to occur

1) What kind of agar are you using? Is it nutrient agar? Did you buy the plates ready-made or make them yourself? If the plates were ready-made nutrient agar from a science supply company I'd say there is a 99.9% likelihood that the plates are fine; these companies generally do a good job of producing the plates. If you made the plates yourself that is a potential source of error and you may want to double check that you didn't make any mistakes when mixing up the agar.

2) You bought the K-12 E. coli. Do you remember if it came as a liquid culture or an agar slant? If it was a liquid culture did you gently shake the tube (or mix the liquid in some way) to make sure the cells were distributed throughout the liquid rather than all clumped at the bottom of the tube? And did you use the e. coli promptly when you got it. If you didn't shake you may not have gotten any cells on your inoculating loop. And if it wasn't used promptly - the cells may have died.

If the E. coli came as an agar slant then there would have been agar in the tube, and in a "hole" in the agar you should have seen a cloudy area - that is where the E.coli would have been growing in the agar. To inoculate from that you would have needed to make sure you dipped your inoculating loop into the cloudy area and actually pulled out bacteria on the loop. You would have been able to see the bacteria as a whitish creamy substance on the loop.

3) When using the inoculating loop - did you sterilize it first by holding the loop in a hot flame before using it? If so, did you make sure the metal of the loop had cooled before you touched it to the bacteria? If not all the bacteria you scooped up on the loop were probably killed by the intense heat from the metal. Here is a link on how to streak plates that you may find useful: http://www.umsl.edu/~microbes/pdf/streakplates.pdf

4) The design for your homemade incubator sounds reasonable. The only trouble shooting question I'd have is whether the temperature (and I know you said you measured it with a thermometer) was constantly around 95 degrees F or if it might have spiked (perhaps after a couple of hours of having the lamp on) to a higher temperature which killed the bacteria. One thing which makes me a bit suspicious of this is the fact that you commented that your agar plates where drying out and shrinking quickly - this might suggest too hot an incubator. Try taking the temperature every couple of hours. Make sure to take the temperature quickly because as soon as you take off the lid you're letting in cooler room temperature air and what you really want to know is what the temperature was inside the incubator with the lid on. I'm less worried about the temperature being too low. E. coli will grow in cooler temperatures - it just takes longer, as much as 4 or 5 days to see colonies rather than the 24 hours it takes in a 95 degree incubator

I hope these questions/comments are useful.

Best of luck,

- Sandra
Science Buddies