Preparing Culture Plates Properly with E. Coli

[Essential Oil Girl]

Hi,

I am doing an experiment which involves testing the antimicrobial properties of various compounds against E. coli bacteria. Some of my preliminary experiments did not work out as I expected in that I did NOT get a lot of bacteria to grow.

Here's a summary of what I did:
Ordered E. coli strain K12 (on an agar slant) from Carolina Biological Supply Company
http://www.carolina.com/product/escheri ... estMatches
Prepared culture plates using nutient agar (in standard amounts)
Sterilized a 4-mm inoculating loop in a bunsen burner and used it to extra 1 loopful of bacteria. I then spread it over the surface of the plate, turned it 120 degrees, continued spreading, and then turned it 120 degrees again to spread some more.
Incubated the plates at 37 degrees C for 24 hours.

The results were extremely spotty and not the floor of bacteria that I expected. I thought about possible sources of error. Could others comment on the likely sources so when I get back to school, I can adjust my procedure to successfully grow E. coli?

Possible sources of error are:
1. I used E. coli straight from the test tube when I possibly should have diluted the bacteria to make some sort of bacterial broth. Is it best to use the bacteria as a broth for growing a floor of bacteria?
2. I did not let the inoculating loop cool down long enough and I may have "killed" the bacteria so it didn't grow. Do I need to let the loop cool down to room temperature before sticking it in the bacterial culture?
3. I used one loopful of bacteria for the entire petri dish, so it may not have been enough. After I performed this experiment, I found some references that recommended I should have used 3 loopfuls of bacteria (e. g. 1 loopful every 120 degrees of rotation).
4. I used an inoculating loop rather than a spreading instrument. Although I've seen some references recommend an inoculating loop to spread bacteria around the plate, I later found other references that recommend a spreading instrument to get an even floor of bacteria on the plate.
5. I used an inoculating loop instead of a sterile cotton swab. My initial references recommended using an incoculating loop. I thought that if I used a sterile swab, I wouldn't be able to ensure that I would be getting an evern amount of bacteria from plate to plate. But, does the inoculating loop provide enough coverage?
6. The incubator may not be operating properly. The incubator's temperature controls are a little touchy so it is difficult to get it to operate precisely at 37 degrees C. However, I think it does operate in the vicinity of 36 to 40 degrees C. I read that growing the E. coli at temperatures below 37 degrees C, but I don't know at what temperature is too hot to kill the bacteria in the incubator.

I appreciate any suggestions that will help me pinpoint my errors and get me to successfully grow E. coli.

Thanks!

[deleted-71670]

I think you are on the right track with your troubleshooting.

1. Making a suspension of bacteria is certainly an option.

2. Yes this is important! Just let it cool for a few seconds after you flame it. It should be back to normal color, not red-hot, when you stick it in the culture.

3. Maybe. If there aren't enough bacteria, it will simply take more time for them to grow and spread across the plate. Over 24 hours E coli make pretty small colonies, but given more time should spread.

4. A liquid culture an a spreader (just a bent glass tube or rod, sterilized) should give you a more even lawn of bacteria. The technique you used, I think from your description, is more commonly used to streak for single colonies--the exact opposite of what you want here.

5. It just depends on how much bacteria you start with. A cotton swab will spread them more, I think.

6. I think a 36-40 range is probably OK, unless you've got a particularly temperature-sensitive strain. E coli will also grow at room temperature, it just takes longer.

I think your idea of a liquid culture is best. If you innoculate a liquid and let it grow overnight with agitation, you should have a nice even suspension to plate. (You could also dilute a blob of cells in liquid media, but then the blob might not disperse completely.) You will probably want to dilute it a bit with sterile media. Then take 100 ul and spread it around on your plates with a bent glass rod. Let it sit rightside up for a few minutes so the liquid can absorb into the agar. Only then turn it upside down and put it in the incubator.

If you aren't happy with the lawn you see the next day, try letting it go another night in the incubator or starting with more bacteria.

If you have time, I think the best thing to do would be a pilot experiment just to determine which protocol gives you the best lawn. Try a few different procedures and then pick the one you like best for your "real" experiment.

[donnahardy2]

Hi Essential,

Amber has given you some good advice, and I would like to add just a couple of comments that should help. Bacteria go through 4 phases of growth when they are transferred to a new medium: lag, log, stationary, and death phase. Here is a website that gives more information on this topic: http://www.mansfield.ohio-state.edu/~sa ... lack06.htm. The culture that you received through the mail was in late stationary or death phase so did not make a good source of microorganisms to make a lawn for your experiment. Log phase bacteria are best to use for this type of testing. If you transfer a loopful of the original culture to a tube of broth and incubate it overnight, you will have a fresh, actively growing, late-log phase culture that will be perfect for making a lawn. About 0.1 ml of a barely turbid culture will contain about 100,000 actively growing bacteria, and if you spread this gently over the surface of the agar plate with a sterile cotton swab or the bent glass rod that Amber suggested, this will make a solid lawn that will be perfect for the antimicrobial testing experiment. The inoculating needle technique will also work, but takes longer. If you are going to be doing more than one experiment, it will be important to prepare your cultures the same way every time to ensure obtaining consistent results.

You should carefully check the temperature of your incubator and make sure it is not too high. E. coli will die rapidly if the temperature is over 45 degrees Centigrade. Growth will be close to optimum and best for antimicrobial testing if the temperature can be maintained at 37 degrees Centigrade, but, if necessary, you can use a slightly lower temperature if necessary, to avoid overheating the culture.

You should transfer a fresh culture to a new slant of agar to make a fresh culture to keep your culture alive until you finish your project. The science buddies website has great information on growing and storing bacteria

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

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

I hope this helps. Good luck!


Donna Hardy

[Essential Oil Girl]

Hi Amber and Donna,

Thanks for your advice on preparing culture plates of E. coli. At this point though, I'm not sure if the protocol I've used actually worked or not.

My revised protocal for preparing plates was:
1) Prepare plates with nutrient agar (I used some that I had made x weeks ago and that had been stored in the frig since they were made)
2) Used 0.5 ml of E. coli K12 in nutrient broth. Ordered this from Carolina. They did confirm that what I ordered was the appropriate bacteria for preparing a floor of bacteria and that 0.5 ml would provide enough bacteria for a nice floor.
3) Using a sterile pipette, transferred 0.5 ml of the bacteria from the test tube onto a Petri dish.
4) Sterilized a stainless steel spreader and waited a few secs for it to cool. Then, spread the E. coli as best I could around the plate.
5) Waited a few minutes for the agar to soak in the E. coli, then covered with paraffin wax and turned the plates upside down and incubated at 37 degrees C for 24 hours.
6) Finally, I took pictures of the plates (while they were upside) to test for microbial growth
7) I also repeated the procedure using 0.2 and 0.1 ml of E. coli, just in case 0.5 ml was too much.

I've attached results for the above 3 plates. At this point, I'm not sure if what I'm seeing is a floor of bacteria or just the residue of where I initially put the bacteria on the plate. Can you offer your comments based on the pictures?

At this point, I'm thinking of refining my procedure as follows. I should sterilize the spreader BEFORE I pour the E. coli on the plate. In this way, after I deposit the amount of bacteria on the plate, I can immediately begin to use the spreader to push the E. coli evenly around. With the way I did it, I think some of the E. coli started to absorb in the initial center area of the agar while I was sterilizing and cooling the spreader.

Additionally, some individual colonies of E. coli are visible in the pictures. But, I really had expected that there would be no distinct colonies, but a relatively even carpeted floor of bacteria that was observable. So, I'm really wondering if I could tell if my preliminar experimental results look like bacteria has grown.

I realize I could confirm by putting something that was known to be antimicrobial in the plate (like a Streptomycin disk that I have). But, following your advice, I do want to make sure my protocol is correct before proceeding with further experiments.

Also, I know that most sources say that the E. coli should grow in 24 hours. Perhaps I need to let it incubate for 48 hours to test the results.

I'd appreciate any follow up advice you could offer.


Thanks,
Essential

[deleted-71670]

Hi Essential,

Your .2 and .5 plates, judging from the pictures, look pretty lawnlike to me. What do you think Donna?

The single colonies you are seeing around the edges are probably because you didn't spread the bacteria all the way to the edges of the plates, but that may be ok for the experiment you are planning? If you are putting antimicrobial-soaked filter paper discs on, for example, just stick to the parts that look like an even lawn. If it makes it easier for you to spread the cells, you can add another 100 or 200 ul of sterile media to dilute the cultures, that gives you a little more to work with.

As Donna mentioned it is important to start from fresh cultures so the bacteria you begin with are in growth, not stationary phase. So your liquid culture should be fresh from overnight at 37 degrees (innoculated the night before from a single colony, and in a decent volume, around 10 mls I'd say), or the bacteria will get crowded and old and start to lyse and just not make as good a lawn in when you put them on your plate.

You're definitely on the right track, and I am impressed at how carefully you are thinking through your protocol. Good luck!

Amber

[Essential Oil Girl]

Amber,

I'm glad you think the 0.2 and 0.5 ml plates look like I've got a lawn of bacteria. I've seen what a lawn of yeast looks like after it has been incubated, and so was expecting similar results.

When I had spoken to Carolina company, they assured me that their E. coli (in broth) was "ready to go" in that it didn't need further dilution and incubation before using. Also, when you recommend adding some sterile media (nutrients without agar), that would pose a short term problem for me since the only nutrient powder I have available at my school is the kind already mixed with agar. So, I'm afraid that if I add some additional media (of the kind I have) after I dissolve and boil the media in liquid, it will just gel and prevent a nice floor of bacteria from growing in the first place.

Based on all the above, here's what I'm going to do next time in the lab. I'll use 0.5 ml of E. coli broth and more carefully spread it around the dish. As a positive control (to see if I do have bacteria growing), I'll add some antimicrobial disks to the plate. I'll then culture the plates for 1 night, check results and let go for the second night.

Since I know the antimicrobial disks should have a zone of inhibition around them, if there is no bacteria growing, there won't be any zone of inhibition. If what I see on the plates is indeed bacteria, then the disks SHOULD have an inhibiton zone.

Finally, Carolina Company said that I could use the E. coli "straight from the test tube" for up to 2 weeks, as long as I use the liquid broth kind and it is stored at room temperature.

My next experiment should better determine whether I really need to follow the procedure you outlined above with regard to starting with more active bacteria. I'm going to keep my fingers crossed!

Thanks,
Essential

[deleted-71490]

Your photographs suggest that the surface of the agar plates was not dry whey they were inoculated. A thin film of moisture on the surface allows the introduced cells to literally spread all overthe surface of the plate.

One way to solve this problem is to leave the petri dish covers off unt the agar has set up. This must be done in a clean room or laminar flow hood. You can improvise by cutting an opening in one side of a cardboard box and doing the work inside.

Good luck.

Matthew W. Mulanax

[donnahardy2]

Hi Essential,

Thanks for sending the pictures. I agree with Amber. It looks like there are enough viable bacteria for a lawn in the 0.2 and 0.5 ml plates, and that that problem was that you just didn't spread the sample around enough to make a lawn. Matt noticed the area of very dense growth that look like areas where liquid had pooled on the plates, or maybe that was where the culture soaked into the agar before you had a chance to start spreading the lawn. You said you made the plates several weeks ago. Did you store them upside down? If so, you should be starting with a plate that does not have any free flowing liquid on it. Making a lawn of bacteria requires technique, and now that you've seen what happens, I'm sure that your results will improve next time. Do make sure the surface is "dry" before adding the antimicrobial discs and turning the plate upside down for incubation. Sterilizing the spreader before you add the culture might help, or maybe switching to sterile cotton swabs that are ready to go would help.

If you use the liquid culture from Carolina as it is, you will be using stationary phase bacteria for the lawn, and as long as you have enough sample to complete your project, you can use the culture as it is, because this would be a controlled variable. If you want to grow the E. coli in a broth, you can make homemade broth by adding 1-2 grams of sugar to 100 ml of clear chicken or beef broth and boil for several minutes or autoclave it. E. coli would grow well in any medium that has a little protein and some carbohydrate. You can transfer a small amount of a single colony to a tube of cooled, sterile broth and incubate it overnight for a fresh culture to start the experiment with. Do all of your experiments with either the culture from Carolina, or the freshly grown overnight culture.

Bacteria don't grow well on dried agar, so check your agar plates and if it looks like they have starting to dry out, you may need to make fresh plates to continue the experiment.

E. coli lawns should grow very well in 24 hours at 37 degrees Centigrade. If it takes 48 hours to grow, then something is wrong. For example, the culture may be too old or the incubation temperature is wrong.

Donna Hardy

[Essential Oil Girl]

I just wanted to thank everyone for their advice over the past few weeks. I got the E. Coli K12 in broth and used 0.5 mls of it with a bacterial spreader to create a floor of bacteria on my Petri dishes. I was definitely able to grow the bacteria in 24 hours and was also able to successfully test my samples for signs of antimicrobial inhibition.

Thank you again for your excellent advice!

Essential Oil Girl

[deleted-71670]

wonderful!

[donnahardy2]

Hi Essential,

Congratulations on getting your project done! And, thanks so much for letting us know about your successful results. We do appreciate your taking the time to let us know that our advice was helpful.

Donna Hardy