Science Buddies: "Ask an Expert"

Hello,
I've been working on my CUSP project which is basically a half year high school senior project/theory, and the requirements include contacting an expert in the field of your project. My project is on the subject of microbial antagonism, and although I've been doing research and preparation for a couple months, I have just ordered my materials, including E. coli K-12 and Bacillus Subtilis. I am testing the antagonism of Bacillus Subtilis versus that of antibiotics against E. coli. I have a full procedure and lab report based on many different scientific articles and previous similar experiments, but I don't know if my way is the most effective way to test this. My entire project up to this point can be found at *1, including all of my resources so far(organized but not annotated yet). My specific grievances with my own procedure are how exactly I should be transferring my bacteria from their plate(ordered from Carolina Biological supply company at *2) to the broth, how to measure the effectiveness of each type of antagonism(current plan is by measuring the area of inhibition), and which specific agar to use(my school has many different types and I can't find much about it online). I have worked with E. coli and agar before, but I've never worked with Bacillus Subtilis. I have not yet begun the experiment - the materials are arriving next week - so any information would be very helpful to steering me in the right direction before I begin.

Thanks!

*1 https://docs.google.com/document/d/1vgs ... sp=sharing

*2 http://www.carolina.com/bacteria/escher ... stion=coli and http://www.carolina.com/bacteria/bacill ... n=subtilis

Hi Dawson,

Welcom to Science Buddies!

You have an excellent idea and project proposal for your science project. Here are some information that should help you obtain excellent results.

Even though you have some experience, please do refer to the information on microbiology techniques from this website:

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

You should use a general all purpose medium that does not contain any inhibitors and that will support the growth of both E. coli ad B subtilis. Nutrient agar would be a good choice. Look at the ingredients and choose one that contains both protein and some glucose for fastest growth. The choice of culture medium is a controlled parameter, so you should use the same medium for your entire project.

To get started once the cultures arrive, you should transfer the culture to a Petri dish and streak the plate so that individual colonies will grow. Then transfer a single colony to an agar slant to keep a stock culture. Do make a note of the colony morphology ad make sure it is typical of the organisms you are supposed to be using.

The culture that arrives from Carolina will be at least several days old, so will contain many dead and slow growing cells. You need an actively growing culture to do an inhibition experiment. So,when you are ready to set up an experiment, transfer a small amount of the culture from the stock to a broth culture and incubate it at 30 to 37 degrees C for a few hours or overnight. Then use the freshly grown culture to start your experiment. This will ensure reproducible results.

Do set up your experiment twice and measure results carefully. Make sure all parameters (medium, temperature, time, etc.) are controlled except your independant variable. Post again if you have more questions. I think this will be an excellent project.

Donna Hardy

Thanks for the reply Donna!
Another question I have would be, how exactly should I transfer the bacteria from one plate to another or from the plate to the broth because the bacteria that I ordered comes on a plate already and not in a tube. In addition do you believe that measuring the area of inhibition just with a ruler under a microscope would be a good idea?
Thanks again.

Also, can I get your email so that I can keep you updated on the project and ask more questions if I need to?

-Dawson Hettrick

Hi Dawson,

I'm sorry I was not clear. You should transfer the plate culture that you receive to a new agar plate and streak it to isolate a single colony. Refer to the resource information I posted earlier. F.irst use a sterile loop to transfer the culture to a small portion of the plate, then use a sterile loop to streak the culture to a new portion of the plate; repeat two more times and incubate the plate for 1-2 days. This should give you a single colony. If you don't have slant tubes available to store a stock culture, then use the agar plate as the source of your experimental culture, but be sure and prepare a fresh plate at least once a week to keep the culture alive.

I cannot share my personal e-mail per Science Buddies rules. This is a safety rule designed to protect you. Please reply to this post and I will respond as quickly as possible to any other inquiries you might have.

Donna

Hi Donna,
I've completed two trials of my project so far, and it has not gone very well in the area of data. In the first trial I inoculated my E. coli around a homemade antibiotic disk, and the next day there was no E. coli at all. I thought that this was because the disks had not fully dried, and the next week I tried again with new plates but the same disks, now fully dried out. This also resulted in all of the bacteria dying. I looked online at www.ijmm.org/documents/Antimicrobial.doc, and I found out that I had to dilute the antibiotics more(I bought Ampicillin in liquid form at 10 mg/ml but this site said that it needed to be at .3 micro-grams/ml). On Friday I created a new batch of disks after diluting my antibiotics to about 3 micro-grams/ml. I was just wondering your opinion on the subject, and if this solution is the correct one to fix the problems that I've been having.

Dawson