Substitutions for agar in bacterial cultivation

Factual Questions
1

I’m trying to show the cleaning of pond water using common materials for a Environmental Science class, and I’m wondering if I can use something besides agar as a good growth medium to show the difference between the beginning situation and the end (after bleach and filtering). I need something that will grow whatever bacteria the kids let get through their cleaning procedure.

Also, if I freeze some of the original water, wouldn’t that keep the organisms (algae, bacteria) in good condition in case I need them later?

2

Chicken or beef broth are frequently used as bacterial culture media for science fair demonstrations when agar isn’t readily available. Note: agar is readily available.

3

Some, maybe, but a lot will burst from the ice crystals. We freeze our bacteria in a glycerol solution.

4

Paging masterofnone…

In the mean time, here’s my attempt at an answer:

Agar itself is just a sort of seaweed gelatin, and alone it doesn’t have much nutritional content. Really, it’s just something to provide a solid surface for microbes to grow on. It’s actually used for food, so it should be pretty readily available. You can add agar to any sort of media (the stuff that has nutrients to feed bacteria). As mentioned, beef broth is pretty good media.

I assume you want to take a sample, and spread it on an agar plate to see how much grows? Plain old gelatin might work as a crude substitute for the agar, but it won’t be as firm and can spoil…

Here’s a nice link about agar and other sorts of gels used in cooking. That gives you some names to look for in a supermarket.

Also, for your purposes you could probably freeze samples of water. Only some fraction of the microbes would survive, but I bet there would be plenty left for your class to experiment with. You could also just refrigerate the samples for a couple of weeks.

5

Wow - I’ve been paged!

As stated above, agar is similar to gelatin, derived from seaweed, and relatively available since it is sometimes used in cooking. Since it melts at 100C and hardens at 42C, it is excellent for hardening growth media. Gelatin can be used, but will melt slightly below 37C (body temp), which is the most common incubation condition. Even if incubating below this temperature, some bacteria produce gelatinase, which will hydrolyze your gelatin, turning your media to a liquid. However, this takes days to weeks to occur. Although some species can use them, neither agar nor gelatin are particularly good nutrient or energy sources. Beef broth, chicken broth, and milk are all very rich growth media.

I assume you are just looking for relative bacterial counts before and after treatment, and you are not looking for potential pathogens? If so, the low melting point of gelatin is not so much of an issue, as you can just incubate for 48-72 hours at room temp. Much longer than this, and you will not be able to see the bacterial colonies, as molds will overgrow the plates.

Off the top of my head, I’d say do a search for nutrient gelatin to determine the gelatin concentration required for hardening (IIRC it’s 7.5%). I’d do it, but I’m home on dial-up right now, and it’s slower than death. Just add the gelatin to cold beef broth, bring to a boil, cook it in a pressure cooker for 15 minutes at 15psi, and pour the plates.

Do not freeze the water. You can get away with refrigerating it for a few days, though you will change the relative abundance of different species. Freezing without the addition of glycerol or DMSO will produce sharp ice crystals which will puncture cell membranes. Algae are more prone to ice damage, but they will not grow on your plates anyway.

Let me sleep on this and check some of my info at work tomorrow. Feel free to toss more questions my way.

6

I was wrong on the gelatin concentration - it’s 12% (12g/100 ml media).

According to the Handbook of Microbiological Media, all of the beef based media require the addition of peptone (digested animal tissue) as an amino acid source. Leaving it out will work, but you will not get as many colonies, as bacteria that cannot synthesize specific amino acids will not grow.

Here is a reference to a science fair project using beef or chicken broth media (with recipe).

Here’s a good set of recipes/info on classic media.

If this involves you purchasing anything besides ingredients, it may be more costly than it’s worth. I would suggest purchasing prepared Tryptic Soy Agar plates from Presque Isle Cultures. I buy media from them when I only need small amounts of a specific media that contain ingredients that would expire before I would use them up.

Let me know if you need help with any thing else.