Science Buddies: "Ask an Expert"

Hello, I am doing a science project where i am testing the effectiveness of natural disinfectants vs commercial disinfectants on bacteria grown from surfaces, using the disc diffusion method.

The first time I conducted a trial, i swabbed a sterile cotton swab all over a drinking fountain button, added one drop of distilled water in the centre of my agar plate and swabbed the bacteria evenly across the the agar. After letting it dry, I placed disinfectant-infused filter paper discs onto my agar and it worked! However, even though i followed the same method, i was never able to replicate my results. This is because after my first trial, i was not able to grow an even lawn of bacteria. Instead, I was only able to grow colonies. Once of the reasons why I think this may have happened is if my first trial had more moisture as compared to my other trials.

However, to make my project more reliable, I wanted to create a broth using the bacteria, to make sure that all my results will have the same variables (including the same amount of moisture). Do you have any tips on how to make this bacterial broth?

I have heard that you can pick up the colonies that have grown and you can add it to an LB broth. However, in order to pick up a colony grown from the bacteria I collect from the drinking fountain button, wouldn't I have to open the agar plate after it has been incubated? Isn't this a safety hazard?

Thank you,
Hoping to hear from you guys soon!

Hi joystick,

I hope you are having a great day! Please see my suggestions below.

I think it is a great idea to pick up some of the colonies you are seeing and inoculating them into broth. LB broth and nutrient broth are two "general purpose" mediums, meaning that a wide variety of bacteria/microorganisms will be able to grow in them. Bacteria broth comes pre-mixed and dried (as powder) and just needs to be suspended in distilled water, mixed, and autoclaved before use. Some media can also be prepared by mixing the individual components together, but I would suggest just using pre-mixed media from the company. I have included some links for reference -- LB broth is 20 g powder/1 L of distilled water (https://toptipbio.com/lb-media-recipe/), while nutrient broth is 28 g powder/1 L of distilled water (https://labmal.com/2019/08/13/nutrient- ... ent-broth/).

You could also take your colonies and streak them for isolation onto fresh plates. Again, I would recommend nutrient agar or LB agar for your purpose. Doing this streak for isolation will give you richer growth of the bacteria you are seeing so you can determine which species are growing.

Opening agar plates and picking up colonies isn't a safety hazard, but do be sure that you use aseptic techniques and sterile equipment. If possible, do all of your work under a laminar flow hood and be sure to disinfect its surface with 70% or 95% ethanol before and after each use. Additionally, if possible, use a Bunsen burner to provide a sterile environment under the hood. If you do not have access to a hood, it is incredibly important that you work with your cultures using multiple Bunsen burners and a sterile lab bench. Definitely make sure that you always open your plates under the hood (if possible) and as minimally as possible. Following these safety protocols will make sure that your plates do not get contaminated and ensure that you remain safe.

I hope I have been able to answer your questions. Good luck with your bacterial cultures! Please feel free to respond to this forum should you have any more questions/concerns or need clarification on something.

--Brandi