Counting bacteria colonies (microbiology)

[lbritsch]

Regarding an experiment growing bacteria colonies in a salted broth, varying the concentration of salt.
1) will a low salt vegetable broth work as a substitute for a low salt chicken broth?
2) is there a way to quantitatively compare the bacteria colonies at home, rather than in a science lab?

[sunmoonstars]

Hi,

Growing bacteria can be a very rewarding project because they are easy to grow and pretty easy to work with. I tried finding a protocol using chicken broth to grow bacteria but I did not find one. Can you tell me a little more about the proect you are helping with?

Are you interested in growing colonies such as in this experiment, or liquid cultures in something like an LB broth in test tubes? Where will you get the bacteria from?

http://www.sciencebuddies.com/science-f ... z&from=TSW

[lbritsch]

Sunmoonstars,
Thanks for the quick reply.
It is one of the Science Fair Project Ideas here on Science Buddies, How Do Food Preservatives Affect the Growth of Microorganisms?

https://www.sciencebuddies.org/science- ... 1&from=TSW

The bacterial colonies are supposed to grow on the (purchased) LB agar plates, streaked with the broth (hopefully vegetable bouillon cubes will work as well as chicken bouillon cubes) which are to be observed on days 1, 3, 5 and 7. I am guessing regular chicken bouillon cubes are quite salty and probably are in a high enough concentration to inhibit bacterial growth, thus the instructions to use low salt chicken bouillon cubes. My daughter is a vegetarian and objects to the CHICKEN bouillon cubes.

Is the salt content of the vegetable bouillon cubes I have low enough? I can't seem to find "low salt" chicken cubes to compare to. I have found "sodium free" and "lower salt" but not "low salt" chicken cubes.
From the Internet,
regular chicken bouillon cubes have 1100 mg salt.
Herb-Ox brand sodium free chicken bouillon cubes have 5 mg salt
Wyler's brand Lower Sodium Chicken bouillon cubes have 620 mg salt
Low Salt Vegetarian bouillon cubes from Whole Foods (that we have purchased with the intent of using for this experiment): 130 mg.

My electronic scale measures down to 2 grams, but not 1 gram, so I am going to increase all volumes/weights by a factor of 10. For the experiment, the lowest concentration of ADDED salt we will have is 12 g (which would be 12000 mg) of salt in about 5000 ml (on the order of 1.3 gallons) of broth. To create the broth, we will add 20 bouillon cubes (containing 2600 mg salt) to 5000 ml of hot water. So the added salt is about an order of magnitude greater than the salt contained in the bouillon cubes. As I said, I don't know what is meant quantitatively by the phrase "low salt chicken bouillon cubes." If the bouillon cubes had so much salt to begin with such that bacterial growth was inhibited, it wouldn't be much of an experiment. It may be possible to get Vegetable bouillon cubes with almost no salt, but I would need to look again.

Then I would like to know how to quantify the results. I am expecting that higher concentrations of salt will have fewer bacterial colonies.

[sunmoonstars]

That s a very interesting project! Thanks for the link.

I did similar concentrations and agree that the vegtable cubes have a much lower salt content than you will be adding, so it should work fine. You just want to lowest salt you can find (or no salt), for the reason you stated.

As a side note - that's a good idea to scale up the volumes so your scale can be used. I am not sure you need to scal up 10x; I think I would have opted for 3-4x. Are you going to test more than one preservative (or salt)?

Yes, we expect higher salt concentrations to give lower bacterial colony counts, indicating that using a high concentration of salt is most affective at inhibiting bacterial growth. This has interesting applications in the food industry, where many packaged products contain high concentrations of salt. To quantify this, counting and recording the data for each plate should be done. Count all colonies on each plate. In order to present your data, you may decide to average your replicates (the two jars with the same test condition/concentration), and then draw a bar graph, using a different bar for each average.

Awesome project - your daughter will enjoy this one!

[lbritsch]

I did similar concentrations and agree that the vegtable cubes have a much lower salt content than you will be adding, so it should work fine. You just want to lowest salt you can find (or no salt), for the reason you stated.

As a side note - that's a good idea to scale up the volumes so your scale can be used. I am not sure you need to scal up 10x; I think I would have opted for 3-4x. Are you going to test more than one preservative (or salt)?

Yes, we expect higher salt concentrations to give lower bacterial colony counts, indicating that using a high concentration of salt is most affective at inhibiting bacterial growth. This has interesting applications in the food industry, where many packaged products contain high concentrations of salt. To quantify this, counting and recording the data for each plate should be done. Count all colonies on each plate. In order to present your data, you may decide to average your replicates (the two jars with the same test condition/concentration), and then draw a bar graph, using a different bar for each average.

Awesome project - your daughter will enjoy this one!
Sunmoonstars:
Thanks for your input. I will probably still stick to 10X, 3x or 4x is just not much to weigh, when it comes to the salt. Yes, now I see why prepared foods have so much salt in them! She is going to streak the agar plates, in that 4 quadrant method I have read about. But I still don't know what constitutes a "colony." Is a colony a circle? What if we see a line of 6 circles? Is that one colony or 6? How is a colony defined? Can you point me to an online reference that shows an example of counting colonies? I think I have read posts from more advanced students with access to some lab equipment indicating more sophisticated means of "counting" colonies, it sounds like under a microscope. For a first time experiment at this age I don't want to get too complicated. For that same reason, we are just going to stick to salt, no other preservatives. I'd rather do the one preservative well than several and have a lot of data to deal with. The expense of the agar plates and jars will be enough with the one preservative. Obviously there are other preservatives, but if we test others, what would we do with the data? Try to assess which one is more effective? Then I would have to get into evaluating aspects like costs and nutritional value. I like the idea of averaging the replicates, but I would appreciate an explanation of how to count colonies. We can go back to the store and see if we can get even lower salt content vegetable bouillon cubes. Thanks.

[sunmoonstars]

Hi again. you have put alot of thought into this project and it shows! I have the information on counting colonies you requested. there is alot to see in this link: http://www.sciencebuddies.com/mentoring ... ates.shtml

When using the quadrant method for streaking, the idea is to have the lines spaced differently in each quadrant. One or more of these quadrants may have the colonies grow so close together they don't appear to be individual circles anymore, but rather look like a line, or "lawn" (picture #1). These parts cannot be accurately counted. You may not see this in your earliest counts because the colonies are still small, but as they grow larger they can crowd and look like one big mass of bacteria. The idea is to streak so that each colony that grows looks like an individual circle (the bottom right of picture #3). You might consider a quick experiment before your project experiment, just to get a feel for streaking the plates and how to count the colonies.

In order for you to compare the colony counts for the multiple conditions, you will need to count all the colonies on a plate. If one is too croweded, a notation of > 165 (165 being the # of colonies you could accurately count) could work, as long as this is not a common occurance in your data.

If that is not clear, let me know.

This would be a good plate to count: http://biology.clc.uc.edu/fankhauser/la ... 201181.jpg

this would be a little "iffy" because the colonies are too big and touching each other: http://www.sciencehound.com/science_pro ... e-5333.gif

[lbritsch]

Sunmoonstars,
Thanks for the info on colonies. The last 2 links have photos with distinctive colonies, which I don't have any problem counting. It doesn't appear the 4 quadrant method of streaking was used in these last 2 links. Isn't the 4 quadrant method preferred? How do I count the colonies if it looks like the first photo in the first link, Bacillus subtilis(3)? That's the type of result I am afraid of, because I don't know how to quantify it.

[sunmoonstars]

Hi,

If your plate looks like the one in the first link, you cannot count all the colonies.

Using the four quadrant method is good for if you plan to pick colonies for further study, but not so good for counting colonies, as you have realized. The results in that picutre is just what we would expect to see using this method, as they indicate in the pdf here (see the box next to the drawing - only section four yeilds colonies):https://www.sciencebuddies.org/mentorin ... tion.shtml

I suggest streaking the entire plate like quadrant 4 - good spacing. Also, you will be counting the colonies on a few different days. The earlier days should be easy to count, with the conoies pretty spread out. When you get to the last day, you may have more closely spaced colonies. Most likely, in order to get enough bacteria to have a full, uncountable plate, you would have to let that grow for a while.

You might consider trying it once before your experiment, so you are comfortable with how the colonies wil spread. You would just make some broth and streak some right onto a plate. If you do this you will have more condifence going into your experiment with so many plates.

[lbritsch]

Sunmoonstars,
I like your idea of doing a trial run(s). That is definitely the way to go. So we will just allow enough time to do that, and have enough materials on hand to support the trials(s) as well as the final experiment. Thanks.