Agar is a polymer extracted from red algae that is used to solidify culture media for growing bacteria. It is preferred over gelatin for culturing bacteria because bacteria cannot degrade agar. Agar forms a stiff gel at room temperature and remains solid up to 65°C, making it suitable for growing bacteria. Common types of agar include nutrient agar, which supports growth of many bacteria types, and LB agar, which is recommended for student use as it does not preferentially grow pathogenic bacteria. Proper preparation and handling of agar plates is required for safe experimentation.

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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

2. 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:
Supplier
Catolog
Number
Contact Info Cost
Number of
plates
Science Kit &
Boreal Laboratories
WW6564600
www.sciencekit.com
800-828-7777
see site for current prices
for pre-poured plates of
nutrient agar
10
Carolina Biological
Supplies
821045
www.carolina.com
800-334-5551
see site for current prices 20
Bio-Rad
Laboratories
166-
0600EDU
www.bio-rad.com
800-424-6723
see site for current prices
of LB Nutrient Agar
Powder
40
Sigma L7025
www.sigma-
aldrich.com
800-325-5832
see site for current per
tablet prices
500

3. Common Types of Agar
Type of Agar Brief Description
Suitable for Student
Use?
Blood Agar
Contains blood cells from an animal (e.g. a sheep); most
bacteria will grow on this medium.
No, due to potential
for contamination
from human
contact.
Chocolate
Agar
Comprised of sheep blood that provides the X and V factors
necessary for Haemophilus growth, this is a nutrient medium
which is used in culturing fastidious organisms such
as Haemophilus species and Neisseria. Chocolate agar,
however, does not reveal hemolysis data, so species
differentiation among the members of Haemophilus must be
performed in another manner.
No, due to potential
for contamination
from human
contact.
LB (Luria
Bertani)
Agar
A subtype of nutrient agar, this is the general medium for
microbiology studies and may be used for routine cultivation
of not particularly fastidious microorganisms. Also, does not
preferentially grow one kind of bacteria over another.
Yes.
MacConkey
Agar
This is an agar upon which only Gram-negative bacteria can
grow. What is more is that E.coli will grow into red colonies,
as there is a pH indicator present. It should be mentioned that
MacConkey agar powder comes in two versions: one with the
sugar lactose in it, and one without any added sugars.
Since E.coli ferments sugars to acids (thus the red color), one
can add one of the many different kinds of sugars to this
sugar-free MacConkey agar and see if red colonies develop.
If you get red colonies, you know theE.coli strain you are
using can use that sugar.
No, due to
selectivity.
Miller's LB
Agar
This common variation of LB agar appears to have the same
components as LB, just in different proportions.
Yes, but sticking
with the generic
formula is
recommended.
Neomycin
Agar
Contains the antibiotic neomycin, which found in many
medications such as creams, ointments and eyedrops.
Neomycin was discovered in 1949 by the microbiologist
Selman Waksman, and it is produced naturally by the
bacterium Streptomyces fradiae. Moreover, Neomycin has a
broad spectrum of effects, killing both gram-positive and
gram negative bacteria. It is relatively toxic to humans, and
some people have allergic reactions to it. Often, Neomycin
agar is used for culturing organisms anaerobically. Neomycin
stops the growth of gram-negative bacilli and staphylcocci,
allowing Streptococcus species to grow more abundantly.
No, due to safety
concerns.

4. Type of Agar Brief Description
Suitable for Student
Use?
Non-nutrient
Agar
Usually not suitable for growing bacteria. However, may be
used for growing other microorganisms.
No.
Nutrient
Agar
Will grow the largest number of different types of microbes -
fungi and bacteria. Yet, not all bacteria can grow on these.
Some find it too rich, and others find it deficient. The nutrient
in this is beef broth, and some extracts from yeast.
Yes, because it does
not selectively grow
pathogenic bacteria.
Sabouraud
Agar
Used for fungi and has a low pH that will kill most bacteria.
It contains gentamicin, which is a aminoglycoside antibiotic.
Gentamicin can also treat many different types of bacterial
infections, particularly Gram-negative infection.
No, due to safety
concerns.
Thayer-
Martin Agar
Chocolate agar designed to isolate Neisseria gonorrhoeae,
also known as "gonococcus," which is a species of Gram-
negative bacterium responsible for the disease gonorrhoea.
No, due to potential
for contamination
from human
contact.
Tryptic Soy
Agar
A basic medium used for culturing many kinds of
microorganisms. Tryptic soy agar is mainly used as an initial
growth medium for the purposes of: observing colony
morphology, developing a pure culture, achieving sufficient
growth for further biochemical testing, and culture storage.
No, due to
selectivity.
XLD Agar
Xylose lysine deoxycholate agar. It is used for the culture of
stool samples, and contains two indicators. It is formulated to
inhibit Gram-positive bacteria, while the growth of Gram-
negative bacilli is encouraged. The colonies of lactose
fermenters appear yellow.
No, due to
selectivity.
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.

5. 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)

6. 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, W.A.M. (2012). Dorland's Medical Dictionary. Retrieved January 17, 2013
from http://www.dorlands.com/wsearch.jsp
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. Retrieved January 17, 2013
from http://www.bd.com/ds/technicalCenter/documents.asp

7. (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
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