Science Buddies: "Ask an Expert"

Hello,

I've read that agitation vs. no agitation did have significant differences at 10 degrees Celsius, but not at 19 or 37 degrees Celsius. I also read that at higher temperatures, growth without agitation was still fine. This particular study did it at 42 degrees Celsius. If I don't have access to a shaking incubator, should I go for longer incubation, higher temperature, or higher bacteria concentration (when initially mixing with broth)?

Hi - I've asked for other microbiology experts to consider your question. From what I've read and experience, the agitation helps in aeration - providing the bacteria cells with needed oxygen for their growth and reproduction. You can do a google search to see the effects of agitation vs no agitation and how fast you would expect to get optimal growth.
Maybe without an agitator you could manually swirl the growth medium a few times a day? Gently - and at the same time for the same amount of time for each?
See what you find out with a search first.

Good luck!

Agitation is critical for the growth of E coli if you are aiming for broth cultures with a high density of bacteria. Every lab that I've ever seen that grows E coli uses a shaker--and at quite high RPM. Once when I did not have a shaker, I used an aquarium air pump to bubble air through the cultures. You have to have a special filter on the air tube to prevent contamination, though.

You don't have to use a shaking incubator, however. Most school labs have a couple of magnetic stirrer-heater combinations. Grow your E coli in a sterile flask with a magnetic stir-bar sterilized in alcohol. Just make sure you set the thermostat correctly so the temperature doesn't go too high.

Hope this helps you,

Sybee

Thanks so much!

Should the flask be open? Because it is being heated. Or will that cause the culture to be contaminated?

While I've never grown E. coli this way, they do require oxygen to grow (they are aerobic bacteria), so the flask cannot be sealed. Usually I loosely cover the top with aluminum foil or place a cap on but don't screw it tightly down, and I don't see why something like that wouldn't work fine for you too. The important thing is not to seal the flask entirely.

JMP is correct. Put a piece of aluminum foil over the top of each flask and autoclave them or bake them in an oven to sterilize them. Actually, if you are growing a broth culture then you will have to autoclave the flask AND the broth. And if you are autoclaving, then you might as well stick the stir bar in the flask at the same time so it gets sterilized too.

If you are going to use the hotplate-stirrer method, you will have to do a little experiment with a flask of water and a thermometer to make sure the setting on the hotplate gives you a temperature of about 37C. This is not going to give you nearly as constant a temperature as an incubator, but you just want to make sure the temp does not go much above 37C--maybe 39 max--because the bacterial growth may be inhibited at temperatures above 40C.

You will need to stir pretty vigorously, but be careful that the stir bar is not slung off to the side of the flask. This happens sometimes. Just turn the speed down a little. Don't fill the flask more than half full. We grow one liter broth cultures of E coli in two liter flasks, but in your case i would only put 500 mL in a 2-liter flask because the stirring will not aerate as much as vigorous shaking. If you are only growing 100 ml, use a 500 ml flask, 50 mL in a 250 mL flask, etc.

Good luck!

Sybee

The oven heating should be easy enough, but i do not have access to an autoclave. Does this present a huge problem? Could i sterilize the flask w 70% isopropyl solution?

Baking GLASS flasks at 350 for half an hour is sufficient. You can use alcohol to sterilize pipets, spreaders, small tubes, etc. Just be sure all the alcohol has evaporated before you touch the bacteria.

Is your nutrient broth sterile? That HAS to be. If you have a large pressure cooker or pressure canner, you can use that as an autoclave. Otherwise you have to boil it which is OK, but does not kill some spores.

Yes, the product information says "sterile".
Do I still need to boil or use pressure cooker?

Nope. As long as you KEEP it sterile. Don't put anything into the broth that is not sterile--except your E coli inoculum, of course.

You can cover the tops of your flasks with aluminum foil and bake them in the oven to sterilize them.

Quick question, is there a certain proportion of bacteria to broth when inoculating the broth?

I wish I could give you a quick answer, but it depends on the number of bacteria per mL in your inoculum, how old the inoculum is and on how dense you want your culture to be at some particular time. In broth at 37C, E coli can divide every half an hour--sometimes every 20 minutes if they have really good aeration, but they go through an initial lag phase before they begin exponential growth.

After several hours of log-phase growth, the rate starts to plateau and they go into what is called stationary phase. After that they begin to die off and the optical density of the culture decreases. Here's the info on bacterial growth: http://en.wikipedia.org/wiki/Bacterial_growth

If you make a 1:500 dilution of a stationary phase E coli culture into broth and incubate it at 37C on the stirrer-hotplate I would say that you would have a late log-phase culture in about 12 hours. If it is too dense, you can dilute it back with more sterile broth.

What are you planning to do with the E coli once they grow up? Most experiments are done with log-phase cultures so you need to time it so that your treatment period coincides with the exponential phase of the growth curve. It’s a little difficult to do this without some way of measuring the optical density of the culture and you need a nephelometer to do that: http://www.ncl.ac.uk/dental/oralbiol/or ... growth.htm

Let me know if you need more information.

Sybee

I was thinking, inoculate the broth first, then wait 12-15 hours, then dilute it back (1:10). It would take the broth 4-5 hours to reach log phase right? Since I'm still in school, it'd probably take me around 8-9 hours till I can get back to my experiment. Is that too late?
I'm making a bacterial lawn for disk diffusion assay. Can I just use the dense broth? Or is it imperative I dilute it?

Thanks

When we work with E coli cultures we always use a nephelometer to measure the turbidity of the culture which tells us how many colony-forming-units there are per mL. What you are aiming for on the plate is a lawn that is not too dense or too light, and it is a little difficult to guess just by looking at your inoculum if it is at the right turbidity. The culture you inoculate from should have about 10exp8 E coli per mL.

There is an old way of estimating bacterial titers by using what are called McFarland standards. These are solutions of known turbidity that the bacterial culture is compared to. Here's the link: http://en.wikipedia.org/wiki/McFarland_standards
I would use your broth culture when the turbidity reaches a Mcfarland unit of about 1.0 as shown in the photo. If you check Google images, you may be able to find some better pictures of the standards.

Pipet 50 uL of inoculum onto a 10 cm plate spread it around, wait about 5 minutes for the liquid to be absorbed then place the disks on the agar using flamed forceps. If you don't get good zones of inhibition, you may need to inoculate more or less bacteria.

Hope this helps.

Sybee

When a broth culture is too dense, i understand that I have to dilute it, but what if it is too light? Just add a bigger proportion of bacteria and 1/10 of the old broth in a fresh broth?

When the culture is not dense enough, just grow it longer at 37C until it gets denser. The problem is gauging when the density is right when you don't have an instrument to measure it. I've never used the McFarland standards but it looks like they would at least give you something to go by in estimating bacterial concentration. You do need to have the same inoculum on all your plates, otherwise the width of the zones of inhibition may vary because of the amount of bacteria in the lawn.

Were you including a positive control like 0.1% sodium benzoate? If you included one disk with this on each plate then maybe you could normalize to the width of this zone of inhibition.