Identifying the types of bacteria on raw meat

[deleted-297831]

In my science experiment I plan on testing the types of bacteria that grow on raw meat after it has been left out at room temperature for 30+ minutes. From my research the types of bacteria that can grow will vary. I want to identify if the bacteria is the same for all three types of meat that I plan on using which is chicken, pork, and beef. The problem I am now encountering is after I incubate my petri dishes and identify the results, how will I know which bacteria is which? For example how will I know if it is E. coli vs salmonella? From their structures they look the same but I'm not quite sure if the colonies can be easily identified.

[deleted-224136]

Hello,
So there are multiple ways that you can compare bacteria, but it will be difficult to say with complete certainty the species of bacteria that you have. You can have data/observations that support a specific type of bacteria, though, or at least narrow down your list.
With each type of meat you'll likely get several types of bacteria on each plate. Each isolated colony is a single type of bacteria, but on the plate you may have multiple colonies of the same bacteria, though you won't know for certain. For testing, look at each colony and note the colony color and shape (http://microbeonline.com/colony-morphol ... -colonies/).
If you have access to a microscope you can observe the specific bacterial cells and describe their morphology (rod shaped, circular, etc). You'll also be able to do a Gram Stain, which will break the bacteria into categories of "gram positive" or "gram negative" based on their cell wall.

Hope this helps.

[deleted-297831]

I really appreciate the link on how to identify and describe the bacteria, it will be very helpful when I get to that step of my experiment! As of right now I feel as if I'm doing an experiment of something that is already known so basically I'm just confirming these results. I would like to make this project as innovative as I can and I'm not quite sure how.
Also when I plan on testing the bacteria of each meat, should I consider the type of agar that I will use to grow them on?

Another thing that I ran into was if after I bought the meat and tested a piece of it almost right away, the rest of the meat I would put back into the freezer until I get my results from the previous one. When I plan on doing another set of tests, will the fact that I froze the meat affect its results? Now I would have to consider how I thaw the meat. Should I leave it at room temperature? Microwave? Is that something that I should compare in my data? For example, what if the first batch of tests resulted in not as much bacteria, but the second batch that was thawed before testing came out with much more colonies should that be something that I would use in my hypothesis?

[SciB]

Hi,

You have asked several questions and not gotten a reply so let me try to answer them for you.

Your first question is really the main one--what is your hypothesis? As you said simply growing the bacteria from three kinds of meat tells you what? Nothing. You have to vary some part of your procedure like the length of time that you leave the meat out or whether or not it has been frozen.

Do you have an idea about the question you want to ask? Are you interested in knowing how many different kinds of bacteria are on meat that you buy at the grocery store? In that case I would think you would simply take the meat from the package and immediately swab a piece of it on the agar. Some meat handling facilities are cleaner than others so the plates from their meats should show fewer bacterial colonies. You could buy beef, pork or chicken from several stores and do a comparison of bacterial counts.

Are you concerned about leaving meat at room temperature before cooking it? If that is the case then I would do a time course using one piece of meat with samples taken at time 0, right after you open the package, then at T = 1, 2, 4, 8, 12, 24 hours or something like that. This will give you a clear idea how exposure to room temperature affects the bacterial count on meat. Be sure to record the temperature each time you work with the meat.

The freezing question is an interesting one. I know under some conditions some types of bacteria will survive and remain viable after freezing, but I have no idea how freezing would affect the types and numbers of bacteria on meat. This would make a simple and relevant project. I buy chicken in 10 lb bags and freeze most of it. I know the bacteria can't grow at 0 degrees F but does this temperature also kill some of them? If you do this experiment you should thaw the meat in the refrigerator at 34-38F. Microwaving will kill bacteria on the surface of the meat and thawing at room temperature may allow them to grow.

You also asked about the type of agar to use. I think nutrient agar, the kind you can buy from Carolina Bio is suitable for your experiment. There are a bunch of specialty agars for different organisms but you want to see all the bacteria so stick with the one that people use most.

Let us know what you decide to do and we will help you with the details. Remember to read the Scibuddies guide on working with microorganisms: https://www.sciencebuddies.org/science- ... fety.shtml
Also, I just noticed a Scibuddies project on minimizing bacteria in thawing and cooking meat that you might want to read: https://www.sciencebuddies.org/science- ... p015.shtml
That project requires access to a microbiology lab so you may not be able to do it, but there may be some useful information or references.

Good luck!

Sybee

[deleted-297831]

I really appreciate your feedback! It has helped me so much and I now have so many things to consider before I begin my experiment. Figuring out how I will approach the second set of tests on the meat which require thawing it, if that'll affect my results on the experiment or not is what really has me thinking. I also plan on making my own agar instead of buying it but I want to know which food should be in the agar for the bacteria to grow. I'm focusing on E. coli, Salmonella, and Listeria. Should I have 3 type of agar plates to try and grow each bacteria? On the first set of tests would I swab the meat 3 times and each time place it on each agar plate and then just see what grows?

[SciB]

Let me see if i understand your hypothesis correctly. Are you are proposing that freezing meat will reduce the number or type of bacteria living on it? Are you going to test three types of meat as you said before? Are you also planning to leave some meat at room temperature for several hours and then test it for bacteria? It is very helpful to the experts and also for your own research that you CLEARLY state your hypothesis. That way we don't have to guess what you are planning.

As for pouring your own agar plates, that is a great idea. While you can prepare the agar from scratch, that would require buying agar, glucose, Casamino acids, sodium chloride, magnesium chloride, potassium chloride, sodium phosphate, peptone, yeast extract and some vitamins. Also, you would need an accurate digital scale, a graduated cylinder to measure the volume and a flask to put everything in. The only reason that a scientist would do this is if they wanted to adjust the formula for the agar by leaving out or adding some ingredients. I would strongly suggest just buying the powdered nutrient agar if you want to pour your own plates. Nutrient agar is not the perfect medium for growing all bacteria but it is the best for all around use.

You said you wanted to focus on E coli, Salmonella and Listeria. How do you plan to identify these bacteria? You cannot work with human pathogenic bacteria unless you have a lab that has a biosafety level 2 facility--laminar flow hood, dedicated incubator, pipettors, etc. How were you planning to do this? Do you have a microbiology lab that you can work in and someone to supervise your work?

This is a great project. We just need you to outline your experiments exactly and explain your methods better so we can help you better.

Good luck!

Sybee

[deleted-297831]

Please excuse my lack of clarity on my hypothesis it's just that it is constantly changing because my project is changing as well. At first I planned on testing three types of meat to see what type of bacteria will grow on them, and that is where I predicted that salmonella, E. coli, and listeria will grow. However I felt as if that would be something that I would be confirming and I wanted to do something different, you proposed another way to approach it and test for the meat company/factory that is less contaminated and cleaner. I now want to find about three meat factories and compare how clean and thoroughly processed their meat is. My hypothesis, once I chose my three companies, would be that the best seller would be the less clean because maybe their price would be lower than everyone else's. I feel that this would fall under quantity not quality.
Yes, I have a microbiology lab that I can work in and that someone will supervise my work. Last year I had already prepared LB agar plates and I thought it was a fun process and I wouldn't mind doing it again knowing that E. coli is best grown on LB agar plates. I also researched how salmonella grows best on Xylose Lysine Deoxycholate (XLD) agar plates, and that is the type of plate I am considering using so that way it will be easier to identify the bacteria. I'm still debating on whether or not I should buy/make the type of agar that each bacteria grows best on, or if I should just use a nutrient agar and see what grows on it.
I appreciate all your feedback and advice, it is very helpful! Thank you so much.

[SciB]

You are certainly welcome, and I do understand how a project can evolve into something different from what it started out as! That's one thing we are here to help with--developing a doable project that is new and interesting.

I sort of remember my microbiology course from college where we used different agars to identify different bacterial species but I'm no expert on it. I don't even know if Salmonella and Listeria will grow on plain nutrient agar, but you could find this online. If you have the resources to use specific agars for each species then you should do that. It will make your project more precise and the results stronger. Can you really confirm that you have E coli, Salmonella or Listeria by examining the colonies on the plates? I know there are more definitive tests like Gram stain but would you be able to get permission to work with these human pathogens directly?

Let us know when you need more help.

Good luck!

Sybee

[deleted-297831]

By examining the colonies on the plates I hope to be able to identify them with the help of the website you have requested earlier. I also purchased a microbiology textbook so I hope that also helps me on my research.

The problem that I have now encountered is that in my school laboratory I could only work with BSL 1 bacteria which is only E. coli. I wanted to test for salmonella and listeria as well but since they are BSL 2 bacteria I would not be able to do my experimentation on those other two bacteria's in class. A mentor would be really great to find but now I am questioning if I should even still test for salmonella and listeria or if I should just drop those two and focus on E. coli?

[SciB]

Hi,

Why don't you do what you proposed earlier--compare the amount and variety of bacteria on meats from several different sources to see which is cleanest right out of the package. That's something interesting and unknown, so makes a good hypothesis.

I know that schools are not able to experiment with BSL2 bacteria, but you can still grow the bacteria on agar as long as you have the lids sealed to the bottoms and don't open them. If you do as you said and use different types of agar, maybe you could show the presence of Salmonella or Listeria without having to actually work with him.

If you wanted to add to your project, you could select one piece of meat and leave it at room temperature inside a plastic container. Swab it and rub the swab on a plate when you first take it out of the package (zero time), then swab it again at 6 hours, 12 hours and 24 hours. People know that you shouldn't leave meat out of the fridge, but if you actually show them how much bacteria is growing on their food that will be an eye-opening demonstration.

Let us know if this helps in focusing your project. We're here to guide you in your experiments to do the best and most interesting possible.

Good luck!

Sybee

[deleted-297831]

I plan on testing bacteria on three types of raw meat; pork, beef, and chicken. I will be looking for three different types of bacteria as well which is Listeria Monocytogenes, Salmonella, and E. coli. My school laboratory is a BSL 1 lab and I understand that to test L. monocytogenes and salmonella it would need to be done in a BSL 2 lab. Would I still be able to conduct my experiment if I plan to do it in my school laboratory? I know that I would not be able to open the plates but would the end result still be as beneficial as if I would be doing this experiment in a BSL 2 lab?

Another question I have is if I should make the agar for these bacteria their own separate plates? Ex: E.coli will be on LB agar and salmonella will have its own plate along with L. monocytogenes having its own plate too. Or should I use the same type of agar plate for all three of the bacteria I plan to test for?

In my experiment I plan on testing the meat straight out the package at time zero, then place it in a plastic container and test it again in 1 hour, 2 hours, and 3-4 hours. I would like to see how much bacteria will grow in that time period of being left out of the freezer/fridge.

[deleted-297831]

Since I am planning on testing for three types of bacteria on three different meat, I am working with a lot of variables. Instead of just identifying the bacteria, I want an actual number of how many will be present on the plate. Should I approach this by using Polymerase Chain Reaction (PCR) for quantitation or should I use serial dilution?

[deleted-140482]

I think you'll find it much easier to do serial dilutions. Quantitative PCR is not necessarily an easy process and it requires access to a lab (probably university level) and a number of specific reagents and equipment. In contrast, for serial dilutions, all you need is access to bacterial growth plates, accurate ways of measuring/dispensing bacteria, and an incubator (which you can make at home).

I hope this helps,
JMP

[deleted-297831]

I am making a gel that will be used to track DNA from Salmonella found in meat after it has been through multiple cycles of PCR. I'm not quite sure on how much agarose that I should use for my gel. I know that the longer the molecules are in a sample, the less concentrated the gel should be and too high a concentration may impede the movement of molecules through a gel. However I am still confused on how much agarose I should measure out for my experiment and no matter the concentration, should I still dissolve it in 100 ml of buffer? Or does that volume have to be changed as well?

[deleted-71603]

Hello. Please keep all questions for your project in one thread so that our experts can best help you based on what has already been discussed. Good luck, and thanks for using Science Buddies!