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An Introduction to Agar

With its distinctive smell, one can easily distinguish agar from the other materials commonly found in a laboratory. Chemically, agar is a polymer made up of subunits of the sugar galactose, and is a component of the cell walls of several species of red algae that are usually harvested in eastern Asia and California. Dissolved in boiling water and cooled, laboratory agar looks gelatinous. Although agar's chief use is as a culture medium for various microorganisms, particularly for bacteria, its other less well-known uses include serving as a thickening for soups and sauces, in jellies and ice cream, in cosmetics, for clarifying beverages, and for sizing fabrics.(1)

One might ask why agar, as opposed to regular gelatin (like that found in Jello), is used for culturing bacteria. The answer is agar, unlike gelatin, won't be degraded (eaten) by bacteria. Also, agar is firmer and stronger than gelatin. It's still possible, however, to use gelatin as a culture medium for bacteria if agar is unavailable.(2)

The Difco & BBL Manual gives more details about agar and its usage:(3)

Agar is a phycocolloid extracted from a group of red-purple marine algae (Class Rhodophyceae) including Gelidium, Pterocladia and Gracilaria. Gelidium is the preferred source for agars. Impurities, debris, minerals and pigment are reduced to specified levels during manufacture.

Agar is a gel at room temperature, remaining firm at temperature as high as 65°C. Agar melts at approximately 85°C, a different temperature from that at which it solidifies, 32-40°C. This property is known as hysteresis. Agar is generally resistant to shear forces; however, different agars may have different gel strengths or degrees of stiffness.

Agar is typically used in a final concentration of 1-2% for solidifying culture media. Smaller quantities (0.05-0.5%) are used in media for motility studies (0.5% w/v) and for growth of anaerobes (0.1%) and microaerophiles.

Specifications for bacteriological grade agar include good clarity, controlled gelation temperature, controlled melting temperature, good diffusion characteristics, absence of toxic bacterial inhibitors and relative absence of metabolically useful minerals and compounds.

The Best Agar for Student Projects

For students growing bacteria at home without the supervision of a teacher (for example, investigating bacteria growth at various places around the house), it's important to use an agar formulation that does not preferentially grow one kind of bacteria over another. The worst case would be one that preferentially grew pathogenic bacteria. Therefore, we recommend a plain nutrient agar, of which LB agar is a subtype.

There are many different suppliers for LB agar. Because some suppliers will not sell to students directly, you may have to have your teacher order for you. If you are doing a project that involves inoculation and plate streaking, we highly recommend conducting the experiment at a school lab under teacher supervision.

Some suggested suppliers are:

Common Types of Agar

Preparing Bottled Agar and Plates(5)

Pre-experiment: Keep sterile Petri dishes closed until ready to pour agar into them. Air-borne contaminants can easily invade an open Petri dish.

Although pre-poured agar plates are available, one can make agar plates from tablet, powdered, or bottled agar by following a few simple instructions. Agar kits usually come with detailed instructions on how to prepare plates, and below are sample procedures for reference. When in doubt, be sure to clearly read the instructions and ask for help if needed (either consult a teacher or call the technical help line of the agar kit supplier).

Preparing Tablet or Powdered Agar:
The formulation for LB (Luria Bertani) agar is: 9.1 g/L tryptone, 4.6 g/L yeast extract, 4.6 g/L NaCl, and 13.7 g/L agar. If using tablets, dissolve 10 tablets per 500 ml of water. For agar powders, dissolve by microwaving, 6.9 g of agar in 500 ml of water. 500 ml of agar will pour ~ 25 large Petri dishes (100 mm diameter) or 50 small Petri dishes (60 mm diameter).

Preparing Bottled Agar:

• Loosen the bottle cap, but do not remove the cap while heating.
• Warm the agar bottle in a hot water bath or in the microwave until it becomes liquid.
• After opening the cap, pass the neck of the agar bottle through a flame to sterilize it. Do not lose the cap!
• While pouring the agar, open the Petri dish lid as little as possible, hold it at an angle, and make sure the lid is kept directly over the Petri dish.
• Pour enough melted agar into each sterile plastic Petri dish to cover 1/8" of the bottom. Cover the lid of the Petri dish immediately.
• Place agar plates on a counter top to cool and set. Agar medium will set like stiff gelatin at room temperature.
• Pass the neck of the agar bottle through flame again before applying the cap.

Preparing Pre-Poured Plates: If plates have been refrigerated, set them out and allow them to warm to room temperature.

Storage: Stack agar plates upside down in the refrigerator. Do Not Freeze! The purpose of placing the plates upside down is to prevent condensation from dripping down onto the agar surface which could then facilitate movement of organisms between colonies.

Please see http://www.umsl.edu/~microbes/pdf/tipsforplates.pdf for additional tips.

Additional Safety Considerations(6)

• When stirring the broth solution, one should take special note in beginning the stir scale at a low setting and adding more speed from there.
• When heating the broth, make sure to cover the flask in such a manner that will not lend itself to boiling over, but to avoid spillage.
• When pouring the broth, make sure to fill the Petri dish without burning oneself. In addition it is important in this process to make sure that the Petri dish is covered immediately to allow the substance to cool proportionately.
• Once the Petri dishes have been exposed or inoculated, students should not re-open them.

Incubation(7)

Place each Petri dish inside a zip lock bag to prevent drying out and to control odors. Turn the plates upside down and put them in a warm place. For many microorganisms, the ideal temperature for incubation is 32°C or 90°F. Bacterial growth should start to become visible in 2-3 days.

For those growing bacteria at home (for example, investigating bacteria growth at various places around the house), you may use a homemade "light bulb incubator" in place of a laboratory incubator. This page describes how to construct a "light bulb incubator:" http://www.umsl.edu/~microbes/pdf/Incubator.pdf

Disposal(8)

Once the Petri dishes have been taped shut, they should not be opened again. All microorganisms grown during the experiment should be killed before discarding. The best way to dispose of bacterial cultures is to pressure sterilize them in a heat stable biohazard bag. If autoclaves or pressure cookers are not available or large enough to make this convenient, an alternative is to bleach the plates. Saturate the plates with a 20% or "1 in 5" household bleach solution (in other words, 1 part bleach and 4 parts water). Let them sit and soak overnight in the bleach solution before disposing of them. Please note that the bleach solution is corrosive and needs to be thoroughly removed afterwards. In addition, the plates can be incinerated if access to an incinerator is available.

References

Dorland's Medical Dictionary: http://www.mercksource.com/pp/us/cns/cns_hl_dorlands.jspzQzpgzEzzSzppdocszSzuszSzcommonzSzdorlandszSzdorlandzSzdmd-a-b-000zPzhtm

University of Texas Health Science Center at Houston: http://med.uth.tmc.edu/

Endnotes

(1) "Agar plate." Wikipedia. http://en.wikipedia.org/wiki/Agar_plate, accessed January 14, 2005.

(2) "Microbiology." MadSci Network. http://www.madsci.org/posts/archives/mar98/888937612.Mi.r.html, accessed January 25, 2005.

(3) "Agars." Difco & BBL Manual. http://www.bd.com/ds/technicalCenter/inserts/Agars.pdf, accessed January 17, 2008.

(4) This is the catalog number for nutrient agar. Please follow the item description at the bottom, next to the catalog number, and not the picture caption, which says non-nutrient agar.

(5) "Agar Bottles - Preparation & Equipment Use." Science Stuff, Inc. http://www.sciencestuff.com/playground/agar_bottle.shtml, accessed January 14, 2005.

(6) Mott, et al. "Artificial Environments for Growing Bacteria." WW Bio Institute. http://www.woodrow.org, (www.woodrow.org/teachers/esi/2002/Biology/Projects/lab_skills/ls5/), accessed January 14, 2005.

(7) "Agar Bottles - Preparation & Equipment Use." Science Stuff, Inc. http://www.sciencestuff.com/playground/agar_bottle.shtml, accessed January 14, 2005.

(8) Leung, Beatrice. Science Buddies Advisor, email correspondence 1/10/05.

Credits

Shijun Liu, Science Buddies

Laurie Usinger, Bio-Rad Laboratories