Science Buddies: "Ask an Expert"

I chose the How Well Do Disinfectants Work project and followed the instructions but nothing is growing on my agar plates.

I took a piece of balogna and wiped it over a cutting board and let it sit for 2 days. Then I clensed each area with a different disinfectant. Then I took a Qtip, rubben and area and then swiped it in a fresh agar dish. After doing all of them I put them in a dark warm place. It's 4 days now and I don't see anything growing in any of the plates, not even the water control plate. The instructions say 1-2 days for colony growth.

Should I have used old, moldy food? Would a different type of food be better?
Do you have any ideas about what went wrong?
Thanks!

Hi Melmic,

This is a great experiment and I agree with you that you should have had some bacteria colonies to count. Your water control was a positive control designed to ensure that the plates would support the growth of bacteria. Since you have not observed any growth at all, you can't make any conclusions about the disinfectants. You need to find out why the positive control doesn't have any colonies.

The most common cause of failure to grow is due to using agar plates that are too dry. How old were the plates and how did you store them? What type of agar were you using?

Another possibility is the temperature. Balogna/cutting board bacteria will grow well between 18 and 37 degrees Centigrade(65 to 100 degrees F). If the incubation temperature is lower than this, then keep incubating the plates for a few more days; if it was hotter, then you may have cooked the bacteria.

How about your experimental technique? Did you do the water control before using the disinfectants? Is it possible that some disinfectant was included in the water control?

If you have time to repeat the experiment, you should go ahead and set it up again. If you don't have time or agar available to repeat the experiment, then you can write up your results and discuss the possible reasons why the bacteria did not grow.

Donna Hardy

Hi Donna,
After reading your response I feel stongly that it has to do with the agar plates. I ordered them from the company suggested in the project and even though they said they can be stored in the fridge up to 2 weeks after delivery, I think we waited too long to start the project. Due to unforeseen problems, we were about 12 days into the 2 week period.

I think our technique, water control process and temperature were all ok, but I am wondering about the bologna.

Do you think that is the best thing to use?
Should it be fresh, rubbed on the board and left out for 1-2 days or should we leave the bologna out a day or so and then rub it on the board?
Also, does the material of the board matter? We used plastic.

Thank you so much for taking the time to answer our questions and helping us with the this project. My son is actually doing it with a friend and we're all excited about it. Luckily, we have until the end of the month and I have already ordered new plates. Hopefully, we'll be successful this time.

Thanks again

Hi Melmic,

I agree with you that the problem was due to dry agar plates. If the plates were 12 days old, they were close to the end of their usable life. I’m sure you will get much better growth with fresh growth medium. It’s good that your son and his friend have time to repeat the experiment.

Your question about the bologna sample is good too. Balogna has sodium nitrate and sodium nitrite added as a preservative. Have you ever noticed that bologna lasts a really long time, and doesn’t spoil readily? Since the objective of the project is to test the effectiveness of different preservatives, it would be better to use a sample that did not have any other antimicrobial compounds. If you wanted to use food on a cutting board, you could use any food that does not contain preservatives. Maybe some milk that is close to the expiration date, or some leftover freshly cooked vegetables. I would avoid using any raw meat.

Let me know if you have any other questions.

Donna Hardy

I received the new agar plates today and we're anxious to start the new project but I'm concerned about what to use to grow bacteria. You mentioned milk that is close to the expiration date or fresh vegetables but I have some questions.

If I use milk, should I make it spoil, rub it on the board and let it sit for a day or 2 or will that dry it out and kill the bacteria eventually?

Does fresh vegetables have a lot of bacteria??? ugh!

What about taking my kitchen sponge and rubbing that on the board? Would I have to wait before we clean it with disinfectants and take samples?

Thanks so much for your help. We just want to make sure it works this time.

Hi,

The kitchen sponge would be an excellent choice. Just use it to wipe the surface and let it dry out briefly and then start the experiment. Do your water control first, and then use a different section of the board for each of the disinfectants.

For a food sample, I was thinking about anything that you have in the refrigerator that has been sitting for a while. Bacteria double in number about once every 24 hours, so if you have milk, cooked vegetables, or any other leftover food that has been incubating in the refrigerator for several days, chances are the bacterial levels would have increased to significant levels even if the numbers were very low (100-1000 per gram of food) when first stored. Food will smell and taste perfectly good until the bacterial levels go up to 100,000 per gram. Milk that has a slightly off smell just before it spoils will have 10,000,000 to 100,000,000 bacteria per ml. If you do decide to use a food, just spread a small amount evenly over the surface and let it dry briefly. Remember that the source and treatment of the bacterial sample is a controlled parameter, and the only difference in the experiment will be your independent variable (the disinfectant).

Good luck!

Donna Hardy

Dear Donna,
You have been an invaluable resource for this project. We can't thank you enough.
I will let you know how it goes and yes, I do have plenty of food incubating in my refrigerator that I will look at differently from now on!
Thanks again,
Melinda

Hi Melinda,

Thanks for your reply. Please do let us know about your results, and of course, if there are any questions about writing up the project board.

Donna

Hi Donna,
Sorry I haven't posted in awhile but I've been busy growing colonies!
I'm happy to report that we were successful in growing bacteria and showing the effectiveness of disinfectants.

I ended up wiping down a new plasic cutting board with my dirty, old kitchen sponge. We used water to clean one spot and then various disinfectants on the others, like alcohol, bleach, hand sanitizer, lysol and windex antibacterial cleaner. We cultured each area and swiped the agar plates.

I placed them in a warm, dark place and put a towel around them to keep them really warm. After a day and half, still nothing and then I realized that they must need light! I moved them to a warm, dry place with lots of sunlight and within 12 hours the colonies started to grow - only in the water control dish, but they did grow and the boys had a science project they could report on.

Since I had 4 extra agar plates I thought it would be fun to see what had bacteria on it, so my son cultured some stuff I had in the fridge. We used a grape, lettuce, a tomato and frozen beef. Within a day, each plate had its own interesting colonies growing. He's bringing those dishes in also to show what the disinfectants kill when used properly.

Thanks again for all of your help. This site and your help has been invaluable to us.

Hi,

Thanks so much for letting us know that your project was successful. It sounds like it was a great project, and your sons learned a lot. Your observations on the light is very interesting. Congratulations!

Donna Hardy