Probiotics

[probiotics]

Hello!

I am in 8th grade, and I have decided to do a project on probiotics. The projects is going to be the effect of probiotics on pathogenic bacteria, such as Ecoli. I have access to a BSL2 lab. My question is, how much do I dilute the liquid Ecoli bacteria to prevent a lawn of bacteria, but have enough bacteria clusters for data?

Procedure:
Procedure 1. Gather Materials 2. Prepare the probiotic substances. 3. Prepare multiple containers to mix the probiotic substances, water, and the Ecoli bacteria. Make sure they are sterile. Put on sterile gloves 4. Mix the probiotic substances and water together, creating a liquid solution that is easier to apply on to the Petri agar dishes 5. Now, mix the Escherichia coli bacteria into a separate water container. Wait approximately three hours for the bacteria and spices to properly mix with water. Stir the solutions every 30 minutes to help the process 6 Using the plastic syringe, Transfer a small part of the Escherichia coli bacteria to each of the spice solutions. Let this rest for another 2 to 4 hours 7 Finally, dip a sterile cotton swab into each solution and then spread it onto the Petri agar dishes in a zig zagging pattern, the most effective application pattern for studying and counting bacteria. Ensure that you use separate cotton swab for every solution, and bleach and dispose of the cotton swabs as soon as the application process is completed 8, Store the Petri agar dishes in a warm and dark area, preferably at 35 37 degrees Celsius for best effects 9 Every day, take pictures of the results and log the information into your observations 10.After the final day of testing, around 4 5 days. record the final results. Then, bleach the Petri dishes, double wrap them in plastic and throw them away 11 Ensure that throughout these steps you always wear plastic or rubber gloves and ensure you wash your hand before and after the experiment to ensure you acquire the proper results. 12.Count the number of bacteria clusters of each probiotic /bacteria alternative. This was done by placing the Petri dish unrer a microscope.

[probiotics]

I also need a title

[donnahardy2]

Hum

Welcome to Science Buddies! It sound like you have picked and excellent, but challenging topic to investigate.

To make a lawn of bacteria, you could transfer 0.01 to 0.1 ml of a rapidly growing culture that is just barely visible in solution and use a sterile swab to spread it evenly over the surface of the agar plate. Most bacteria produce a visible turbidity at when the concentration reaches about 1,000,000 organisms per milliliter, so this would give you about 10,000 to 100,000 bacteria per plate.

When making a lawn of bacteria, it’s important to use an actively growing culture that has been transferred to the broth and incubated for a few hours immediately before preparing the plates.

Making the lawn of bacteria would be one of the controlled parameters of your experiment, so it’s very important to prepare every plate the same way.

I have a question about your protocol. The standard of a good procedure is that someone reading it would be able to reproduce your method and do exactly the same experiment and obtain identical results. Your protocol is missing the detail needed to meet this standard, so I can’t really tell exactly what you will be doing.

Before proceeding, I recommend that you write a list of all of the materials you will be using, and that you write a detailed protocol that includes all of the steps. Please refer to the experimental procedure and materials list in the Science Buddies project guide if you need more information:

https://www.sciencebuddies.org/science- ... ndex.shtml

For a title, I like titles that ask the question that the project experiment will answer. If you could post a little more information about why you are doing this experiment, or what your purpose is, I could probably give a more specific suggestions.

I hope this helps. Please do let me know if you have any other questions.

Donna

[probiotics]

Hello, I actually want to know how to prevent a lawn of bacteria. Could you please specify where you see a lack of detail in the procedures? Thank you for letting me know about that. Also, is it possible to culture/grow probiotics from a pill that contains bacteria in a powder form. This is the exact same experiment i am conducting except i am using probiotic substances: http://www.virtualsciencefair.org/2013/grun13l

I do not want a lawn of bacteria. I am purchasing liquid Ecoli bacteria, k12, and combining it with water and some of the probiotic substance. How much of each should I mix together to get easy to read data? And, how do you think I should measure the effectiveness of each probiotic substance's ability to prohibit the growth of Ecoli? This is a big and hard project, so thankyou for helping me. I take probiotics everyday for a reason , and I wanted to know which kind worked the best. (Yogurt, pills, fermented cabbage juice(yes it tastes like vinegar ) and others)

[probiotics]

Materials
Materials:
Sterile Cotton Swabs
Plaster syringe
Plastic gloves
Camera
Water
9 Petri agar dishes
Escherichia coli
Test Tube
Multiple glass containers (used to store different ingredients)
Plastic Bags (used for final disposal)
Bleach
Measuring tools
Paper towels
Homemade Yogurt (1/8 teaspoon)
Storebought probiotic pill (1/8 teaspoon)
Fermented Cabbage Juice (1/8 teaspoon)
Listerine (1/8 teaspoon)
Dannon Yogurt (1/8 teaspoon)
Microscope
Printer
Paper
Pencil

[probiotics]

Also, is it ok if we do not isolate the bacteria (like lactobacterius in yougurt)


Thank You So Much

[probiotics]

I'm so confused rn.

[deleted-332001]

Hi, are you looking to grow the bacteria so you can see individual colonies? If so, there are a couple of ways to streak a plate of bacteria. Here's a youtube video demonstrating one of the ways: https://www.youtube.com/watch?v=D9MqxWj6SKI

I hopes this helps!
Jen

[probiotics]

Thanks, but I want a way to count all of the bacteria colonies on a plate, not only a small section.

[probiotics]

Is there a way to do this? My science teacher recommended a graph sticker, so I can count the bacterial colonies easily. Is there a way to find out how much to dilute liquid bacteria so that you can see the colonies? I thought that if I added 1/8 tsp of the probiotic substance, 1/4 tsp of water and 1/8 tsp of liquid bacteria, and then applied it to the agar plate by dipping a qtip into the mixture and applying it onto the plate. Then, I could count the colonies.

[probiotics]

Wow!I found this on a website.

Use a clean, sterile, dry pipet to remove 0.1 ml from the bacteria sample and blow it into the 9.9 ml of dilution fluid in tube #1 and mix thoroughly by blowing lots of bubbles with the pipet for a couple seconds. Discard the pipet into the used jar for later cleaning. Notice tube #1 now contains 1/100 the concentration of bacteria in the original sample because 0.1 ml is 1/100 of 10 ml. Since nearly 0.1 ml of liquid may cling to the outside of the pipet, you must wipe the pipet with Kleenex or toliet paper before inserting the pipet into tube 1.
Using another clean, sterile, dry pipet remove 0.1 ml from Tube 1, wipe pipet, blow contents of pipet into Tube 2, continue blowing bubbles for a second or two for good mixing.
Using another clean, sterile, dry pipet remove 0.1 ml from Tube 2, wipe, blow contents of pipet into Tube 3, continue blowing bubbles for a second or two for mixing.
Plate 0.1 from Tube 3. See below for information on plating.
There are many types of pipets, we are using blow out pipets and that is indicated by a frosted ring on the pipet at the top end. Read my page on types of pipets.


How do you think I should add the probiotic substance without further diluting the Ecoli bacteria?

[probiotics]

Pls Help

[probiotics]

Pls help me! My project is due very soon.

[donnahardy2]

Hi probiotics,

I do apologize for the delay in responding to your inquiries. Here are some answers to your questions.
To prevent a lawn of bacteria, you would need to make dilutions of the original sample in sterile water until the number of bacteria transferred to the plate is between 30 and 300 colonies. This will allow a countable number of bacteria to grow on the plate.

If you don’t know how many bacteria are in the original sample, then it may be necessary to make multiple dilutions. For example, a culture that is rapidly growing and just reaching visible turbidity will have about 10,000,000 (10 to the 7th) bacteria per mL. For this sample, you would need to make a 1/10,000 and 1/100,000 (10 to the minus 5th and 6th) dilution to have countable plates.

For the detailed protocol, your directions should be specific, so that someone reading the procedure would be able to follow it and obtain the same results that you obtain. For example, you could say:

1. I used 3 samples of food that contain probiotics (Dannon and homemade), or whatever your sample is and include expiration date).

2. Each sample was diluted in sterile water using the following dilution: 10 to the minus 4,5, and 6 and 0.1 ml of each dilution was transferred to a sterile nutrient agar plate and applied with a sterile cotton swab.

3. Etc. If you write down exactly what you are going to do, this will help you plant the experiment and also avoid unexpected events.

You need to describe how you make the homemade yogurt and fermented cabbage juice.

Here is an example of a standard protocol for a bacterial plate count for reference:. You should write down exactly what you are going to do.

http://www.fda.gov/Food/FoodScienceRese ... 063346.htm

Your material list is good, but you should list the measuring tools you are using and define the type of agar used in your Petri dishes.

You do not need to isolate the bacteria in your samples; it sounds like you will be counting the bacteria in each sample.
I recommend reviewing the information on microbiology techniques from this website. This will help you plan your project.

https://www.sciencebuddies.org/science- ... nces.shtml\

Please let me know if you have any questions.

Donna

[probiotics]

Ok! Thank you. I know exactly hoe to dilute my e.coli bacteria, but i do not know how i should dilute/add the probitoic substances since i do not know how much bacteria is in it.

[donnahardy2]

Hi,

If you are working with freshly grown probiotics, assume that each sample has 10,000,000 (10 to the 7th) bacteria per ml. Make your dilutions to try for 100 colonies per plate. Make one 10-fold dilution below and one 10-fold dilution above your target. This usually works.

For older cultures, for example, the Dannon yogurt, assume the cultures have 1,000,000 organisms per mL omit the highest dilution and add one more lower dilution.

I hope this makes sense. Let me know if you need a more detailed description.

Donna

[probiotics]

Hi! Thanks for that info, it's very helpful. I just need to know have much of the diluted bacteria and diluted probiotic substance I should add to have good data (no lawns, or no completely empty plates)

[donnahardy2]

Hi,

I will post a detailed response on Sunday. Please let me know what volume of sterile water you will be using to dilute your samples, and what volume of pipettes you are using. I will be able to give you step-by-step instructions.

Donna

[donnahardy2]

Here are two links that should help you understand how to do serial dilutions for a plate count. The first is a detailed step-by-step protocol that uses sterile cotton swabs and would be good if you don’t have pipettes. The second is a YouTube video that shows what to do if you have sterile pipettes available.

http://www.waksman-foundation.org/labs/ ... lution.htm
https://www.youtube.com/watch?v=HZzpgjGosmg

It looks like YouTube has other selections for serial dilutions and plate counts that might be useful, but I did not review any of the other videos.

To get started in you can use a sterile swab to transfer a single colony from a plate of yogurt bacteria to a tube containing a liquid culture medium and incubate this overnight to reate of freshly growm culture. You would then plate out a 10 to the minus 4,5 and 6 dilutions to get an accurate count.

Please let me know if this still does not make any sense, or if you have any specific questions.

Donna

[probiotics]

I will be using these steps:

Starting with 5ml of liquid Ecoli:

Use clean, sterile, dry pipet to remove 0.1 ml from the bacteria sample and put it into the 9.9 ml of dilution fluid in tube #1 and mix thoroughly by using a sterile qtip.
Discard the pipet.
Using another clean, sterile, dry pipet remove 0.1 ml from Tube 1, wipe pipet, blow contents of pipet into Tube 2, continue blowing bubbles for a second or two for good mixing.
Using another clean, sterile, dry pipet remove 0.1 ml from Tube 2, wipe, blow contents of pipet into Tube 3, continue blowing bubbles for a second or two for mixing.
Plate 0.1 from Tube 3.

Now, I have a probiotic tablet, fermented cabbage juice, organic yogurt, non-organic yogurt, culturelle tablet

I will take equal amount of each and mix it with an equal amount of water.

I don't know the measurements.

Then, I will plate it on top of the Ecoli bacteria. I need enough probiotic stuff to make sure some bacteria is killed, but not too much. I need to figure out the measurements, and I need your help for that. Thank You!!



How do you think I should add the probiotic substance without further diluting the Ecoli bacteria?

[probiotics]

This is harder than I thought it would be..... Do I have to isolate the bacteria from the probiotic substances??? .

[probiotics]

Pls Help, I have to start Tommorow