Unsure about streaking my plates and bacteria culture?

[GalaxyGirl4]

I'm going to be ordering this from Carolina Labs:
http://www.carolina.com/bacteria/escher ... _id=155068

I'm slightly confused, some replies and tips have been confusing me. Can I just take my inoculating loop, dip it into this culture, and streak a fresh petri plate? Or do I have to grow the bacteria out, then take the grown bacteria and streak onto a fresh plate? Is there anything available where you can just dip into the culture and immediately streak and start the experiment?

Edit: There's an experiment family on the website, "The End Zone". It uses the same tube of E.Coli and it tells you to just streak the plates immediately after dipping a cotton swab in the culture. Can I do that, or do I have to isolate a colony to make sure it's "pure"?
https://www.sciencebuddies.org/science- ... #procedure

[deleted-143835]

Hi there,

While I'm not a microbiologist, in my experience I believe it is sufficient to dip an inoculating loop or cotton swab (as long as these are sterile before using) into the culture and then spread it onto a Petri dish. Remember not to dip it more than one time (get a new one!) and keep the Petri dish as safe from contamination as possible.

Hope this helps!

[SciB]

Hi GG,

Please keep all your posts in ONE thread so we can follow what has been discussed before. Your first set of posts about this project was in December 2013. Some scibuddy experts might not know that and their answers might be different if they knew what you were doing before. In order to help you with your project, we need ALL the information!

Your question about streaking the bacteria before you use it is a good one. People who work with bacteria such as E coli in the lab do routinely streak their cultures to isolate single bacterial colonies to insure they are working with a single population. Since you are buying your E coli from Carolina Bio, you do not have to do this because they have already done it.

There is one important point to keep in mind about the type of culture you need. E coli can grow on solid nutrient agar OR it can grow in liquid nutrient broth. The broth culture is the one you want to order. Why? In your earlier posts, you said you wanted to see if lemon grass oil kills bacteria and in order to do this, you have to spread a uniform layer of E coli on a plate. You cannot do this by streaking with a loop. Here is some good information about this problem: https://www.sciencebuddies.org/science- ... =28&t=4508

I can explain how to make the E coli 'lawn' on the plate, but first i want to tell you to call Carolina Biologicals and ask them WHICH of their catalog numbers is for a LIQUID broth culture of E coli. I clicked on your link and the description of that culture says it is grown on nutrient agar, not in broth. So, i checked the other listings for E coli and found another one http://www.carolina.com/bacteria/escher ... Cat=&sCat= that i suspect is liquid except that the description also says it is growing on agar. That is why i want you to call the company and make SURE that you order a BROTH culture of E coli. This will give you 5 milliliters of bacterial culture which is enough to make a lawn on about 30 petri dishes.

OK. So, how do you make a bacterial lawn on an agar plate? It's really simple. You do need a couple of items however--a glass or plastic dropper with a rubber bulb, a 'spreader', and some rubbing alcohol for sterilizing your spreader and dropper. Here are instructions on how to make a spreader from a large stainless steel paper clip and to use it for making a lawn of bacteria: https://www.sciencebuddies.org/science- ... #procedure

This information should be enough to get you going, but we'll be here to follow up if you have more questions.

Good luck!

Sybee

[GalaxyGirl4]

Thank you for your help! I'll post further questions on my previous post, sorry about that.
I did call Carolina and they told me that Item #155068 was a liquid broth culture (even though it only says it was grown on nutrient agar), so I have the right product chosen out. So, essentially, I have to drop a few mL of the bacteria from the tube into each petri dish on top of the pre-prepared agar, then spread it evenly across the petri dish before I put my disks in it? Am I supposed to let it dry for a few minutes after doing this, as well?

[SciB]

No not a ‘few’ mL! That’s way too much. This is a science project and for that you have to measure volumes accurately and use exactly the same amount for each plate. For standard size (100 mm diameter) plastic petri dishes, which are what I assume you are using, the usual volume is 0.1 mL or 100 microliters. If you have access to a school lab that has an adjustable micro-pipetter I would use that to measure the volume, otherwise a dropper can be used. Read the scibuddies project instructions I sent you. It explains how to make the spreader, how to sterilize it and how to use the dropper to transfer bacteria to the plate. You need to put one drop of liquid culture per plate because that is approximately equal to 100 microliters (0.10 mL). Here’s a video that shows how to do make a bacterial spread: http://www.youtube.com/watch?v=jr4-ye50Za4

Spread the liquid around evenly all over the agar surface until almost all the liquid has been absorbed, then let the plates sit for about 10 minutes. Place your test disks on the agar surface and add the test compound. Tape the lids onto the bases so that they cannot accidentally come off. E coli grow best at human body temperature, 37C. They will grow at lower temperatures but it will take longer. Try to keep them in the dark and as warm as possible and let them grow until you see a good layer of bacteria growing on the agar surface. Leave the plates upside down while you are incubating them. This is so any water that condenses on the lids does not run down onto the agar and make it wet.

Let us know how it turns out.

Sybee