What type of bacteria should I test on?

[figureskatingactress]

Thank you so much for your wonderful help! Yes, I am writing a background paper right now, these links are exactly what I need. This is a much more advanced project than anything I have ever done, and I wanted a challenge so I have gotten it! Thanks again!

[figureskatingactress]

So everything has been going smoothly, the bump in the road is I cannot find how the antibacterial compnenets in the plants inhibit bacterial growth! Could you help me out? Thanks:)

[donnahardy2]

Hi,

I think you are discovering just how complex your project really is. Here is an example of how to find the information you need:

First do a Google search on "chemical composition of your sample." In this case here is an article that includes the chemical composition of cloves:

http://www.ncsu.edu/bioresources/BioRes ... urkish.pdf

The primary chemicals in cloves are eugenol, eugenyl acetate, and b caryophyllene. Since eugenol is more than 80%, I next did a search for "antibacterial mechanism of eugenol." and found this article:

http://www.ajol.info/index.php/ajb/arti ... 0106/48360

Eugenol works against bacteria by disrupting the cell membrane, causing the the contents of the cell to leak out. Great way to kill bacteria.

Why don't you try a similar search for the garlic and thyme Let me know if you need more help.

Donna Hardy

[figureskatingactress]

Yes, I found the chemical compositions of all the herbs I am studying. Thank you for the information on eugenol, it seems to be about trying to find the right search query, I spent hours browsing websites with little luck, however I am doing okay now. MY background paper is finished and I am working on step-by-step method to prepare for when the samples I have ordered arrive. I did buy the Bacillus brevis from Carolina Bio, and read the care sheet. Am i correct in saying it arrives in broth and i will need to transfer it from the broth in the tube to a petri dish with nutrient agar using a sterilized loop?

[donnahardy2]

Hi,

Great. I’m glad you were able to find information on the chemical composition of your samples. I know it was a lot of hard work.

Yes. If your culture arrives in broth, you will need to transfer it to agar and confirm that you have pure colonies. Here is the information from the science buddies website on preparing agar and growing and storing bacteria strains. You will want to keep a stock culture on an agar slant if you can.

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

https://www.sciencebuddies.org/science- ... hnique.pdf

Read everything carefully. Let me know if you have more questions.

Donna Hardy

[figureskatingactress]

Is there a common household alternative to aa bunsen burner, or should I buy disposable inoculating loops?

[donnahardy2]

Hi,

The disposable inoculating loops are expensive but they would work. You will be using an inoculating loop to streak plates to verify that your culture is pure, so you won't need that many inoculating loops. You can use sterile swabs to spread the lawn of bacteria on the prepared plate and you could use the stick end of the swab as an improvised inoculating loop. If you have a regular inoculating loop available, you could wrap it in foil and bake it for an hour at 200 degrees Centigrade or soak it in 70% isopropanol for an hour. If you use the isopropanol, you will have to make sure the loop is air dried before you use is.

A propane torch can be used as an alternative to a Bunsen burner if you have a safe place to use it.

http://www.ehrs.upenn.edu/media_files/d ... ralgas.pdf

I hope that helps.

Donna Hardy

[superluis2451]

[figureskatingactress]

Hello, I have a bunch of questions today!
:)I am wondering- is there a way to get just a specific, consistent amount of bacteria from a slant to a petri dish using an inoculating loop? And also I was planning on graphing the bottom of the dishes to measure the growth- do you think that or counting colonies would be more easy & accurate? Finally, I am planning on sealing the dishes with somethiing I am pretty sure is called parafilm, it is a waxy sort of tape that seals petri dishes tightly, would this be a good idea or would it deprive the culture of oxygen? Thanks again!

[donnahardy2]

Hi,

When transferring from a slant, you won't know how many live bacteria you will be transferring. The technique for streaking on a plate includes streaking with a small visible amount of the bacteria, sterilizing the loop, and then streaking again over the original transfer and covering a new area of the plate. This technique is repeated two more times, and generally, there will be individual colonies in the third or fourth sections of streaking. I think you will have to try this couple of times to develop your technique, but I promise it will work well. Go ahead and try it!

The parafilm will probably restrict the flow of oxygen, but don't worry; most bacteria have options for anaerobic fermentation and can continue to grow after the oxygen runs out. You should seal all of the plates using the same technique to ensure that this is one of your controlled parameters. It will definitely be safer for you using the parafilm-sealed plates.

Donna Hardy

[figureskatingactress]

I am wondering if using a similar method with my bacteria would be acceptable as in this experiment. I would probably use just distilled water and my different component in each dish. I think this will make measuring easier. Do you approve? http://www.all-science-fair-projects.co ... _39_1.html

[donnahardy2]

Hi,

There are some problems with this protocol, but the basic idea would work for your project. In the link you have posted, the directions are to use 3 x 100 ml samples of milk and add E. coli to two tubes and garlic extract to one of the tubes containing E. coli incubate for 2 hours. Then it says to transfer 10 ml of the milk to the surface of an agar plates. The problem here is that milk can already contain bacteria so you will have a mixture of E. coli and milk bacteria. The difference in results between tube A and tube B would help show the difference between milk bacteria and milk bacteria plus E. coli, however, this is too many variables to me. I would recommend using either milk bacteria or E. coli separately, not mixed together. And your idea of using just sterile water would work also.

Also, a 10 ml sample of water or milk transferred to the surface of an agar plate would result in excess liquid on the plate and the bacteria would be swimming in liquid rather than growing as individual colonies on the surface of the plates. The drawing shows the growth of one colony in the center, but the samples will contain possibly hundreds of colonies so the drawing really doesn’t make sense.

You could follow this general protocol with the following changes:

1. You could use just sterile distilled water if you transferred a freshly grown culture of the Bacillus brevis. If you transfer a culture that has been sitting around for a few days, then you would want to use boiled milk or beef broth so the bacteria could start growing during the 2-3 hour incubations time.
2. If you use the milk, boil it first to kill off the background bacteria, or just use milk as it is. If you use newly opened milk at least 7 days before the expiration date, the concentration of bacteria might be 100 to 1000 organisms per ml. If you use opened milk a day or two before the expiration date, it could contain up100,000 bacteria per ml.
3. Since you do want to use B. brevis, then follow the experiment, but add the B. brevis to boiled milk or boiled beef broth. The control sample should contain 0 to just a few colonies. If you use unboiled milk and B. brevis added to a sterile growth medium that would work also.
4. You should plan to set up samples in duplicate and if at all possible, do a pilot experiment with single plates and then a repeat experiment with plates in duplicate.
5. Transfer not more than 0.1 to 0.2 ml of sample to the surface of the plate. If you don’t have a sterile syringe available for each sample, use a cotton swab dipped in the sample and transfer a consistent volume to each plate; then swab it over the surface in two directions to completely coat the surface. The surface of the plate should be dry at when you start the incubation.
6. Tape the Petri dishes shut and place them in zip-lock bags and turn them upside down to incubate them. You would be counting the number of colonies on each plate after 2-3.

Let me know if you have any questions. I think you will find it helpful to write a detailed protocol of the experiment using the exact method you will use for your experiment.

Donna Hardy

[figureskatingactress]

So just to clarify- would i be able to cut the liquid (probably boiled milk) down to 2 or 3 mL, have about 1 mL of each herb, and then when I transfer will it grow in one large colony as shown? I am worried about measuring growth and this seemed like the easiest way I have found

[figureskatingactress]

also- my control would be of the milk & bacteria, not just milk. them my others would be one with bacteria, milk and thyme, one with bacteria milk and basil, and one wil bacteria, milk and cloves

[donnahardy2]

Hi,

Yes, you can reduce the volume of the liquid to 1 ml and add just a portion of the herbal extract, say 0.1 ml. It will be better to run multiple samples for each herb rather than using the entire herb supply for one sample.

If you use boiled milk, then you will have a negative control of just boiled milk to show that this sample did not contain bacteria; you would also have a positive control of milk plus B. brevis with no herb to demonstrate that your agar plates would support the growth of bacteria. You would have identical samples that each contained an herb.

Your measurement will be counting the number of colonies growing in each plate. One problem with the experimental protocol as written is that it specifies transferring a small amount of bacteria to each tube separately. It would be better to transfer a small amount of bacteria to a tube, incubate it for 3-4 hours to ensure the bacteria are actively growing and then use a constant volume of this sample to transfer to each of the tubes. That would be a much better way to control this parameter in your experiment.

However, one problem you will encounter is that you may have too many or too few bacteria in each plate, so it would be helpful to make dilutions of each sample after it is incubated with the herb to ensure that you obtain plates that have a countable range of bacteria, which are usually 30-300 colonies.

One other consideration that I have not mentioned is dosage. This type of experiment is better if you can use more than one concentration of the herb. But this would increase the number of plates required significantly, so don’t do this unless you have lots of time and agar.

A pilot experiment using one herb, or just a negative and positive control will really help you finalize your experimental protocol and ensure good results, and I do recommend that you try tm include this extra step. When is your project due?

Donna Hardy

[figureskatingactress]

my project is due after christmas break so i really don't have time to spare- i have all my materials and have made my agar plates. I only have enough supplies to do 5 plates with thyme, 5 with basil, 5 with cloves and 5 control. I have sterile syringes to use

[figureskatingactress]

also- should i do a simple dilution or a serial dilution? And would i dilute it with moremilk?

[donnahardy2]

Hi,

You are very well prepared to complete your project on time, but you are right; you don’t have time to spare.

You should do a serial dilution 1:10 and 1:100 dilution, and maybe a 1:1000 dilution. You can do the dilutions in sterile water. If you set up a pilot experiment tomorrow with just a positive control, you can get experience in setting up the experiment and verify that you are working in the right dilution range. You will have results by Tuesday and you can set up your definitive experiment on Wednesday. That will give you a week to finish your board.

While you are waiting for your results, you can write the sections for your board and plan the layout.

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


Donna Hardy

[figureskatingactress]

My project was having some difficulty and not proceeding as hoped, and I was afraid all the bacteria on my slant would die. For this reason, I streaked a clean agar plate with a lot of it. If I am lucky and it is not dead and grows, will I be ablee to pick the colonies off the plate and use them in my experiement as planned originally?

[donnahardy2]

Hi,

Yes, it is always better to use young, actively growing bacteria for an experiment like yours. Hopefully you will have some colonies to select from on Tuesday or Wednesday. Fortunately, Bacillus brevis forms spores when the culture gets old, so I'm sure you will have viable colonies. But you will get faster results starting with young vegetative cells rather than dormant spores. . Transferring the culture to a plate will revive the culture and if you transfer a small bit of colony from the new plate to the milk, I'm sure you will get good results.


Donna Hardy

[figureskatingactress]

Hi!
So I finished my experiment and am working on graphs. I did, however, have some difficulty counting. I probably should have diluted the acteria more ( I streaked the plates with the 1:100) because counting was very difficult. On some plates there were tons and tons of tiny colonies, while one have very few HUGE colonies. Also, In the first zigzag, the colonies often merged together, making counting difficult and potentially inaccurate. I held each dish under a bright lamp and used a pin to count, but I am afraid my data was affected. Should I be worried about it, or should I include it in my conclusion that I should have diluted? Also, can the number of colonies go down? mine seem to have, and I don't know if that is normal.
Thanks again!

[donnahardy2]

Hi,

It’s not possible to accurately count more plates with more than 300 colonies; these plates are described as “TNTC” or “too numerous to count,” and all you can do is estimate the number of bacteria as greater than 300 x the dilution factor. This would definitely a topic for discussion in your conclusion section. If something does not work out right, you should always think of what you would do differently next time, and making additional dilutions would be the obviously solution. The number of colonies cannot decrease with incubation time, but if the colonies grow together, you won’t be able to tell how many you had at the beginning.

I hope this helps.

Donna Hardy

[figureskatingactress]

So, should I mark the colony count as going down on my table and graph and put it in my graph description that the plates above 300 are TNTC so are approximate and that their numbers appeared to go down because they grew together? This is the only reasonable approach I can think of.

[donnahardy2]

Hi,

I think this would be a reasonable approach. If you had a TNTC plate and the dilution was 1:100, then you could report results as >300,000. If all of your results are >300,000, then you could also make a qualitative description of the results. What were your results? Can you post the data?

Donna Hardy