Biologists are concerned with the living world that
surrounds them, now estimated to contain between 10 and
30 million distinct species. This diversity of life can be organized into
two meaningful groups depending on whether or not the cell(s) of which
the organism is composed contain or lack a nucleus. Most organisms that
have nucleated cells, called eukaryotes, are the multicellular plants,
fungi, and animals that you observe every day basis. Organisms lacking a
nucleus, called prokaryotes, are the unicellular bacteria. Although
bacteria are equally abundant in the environment they readily go unnoticed
and are difficult to observe. One method of observing bacteria is to grow
them in artificial food-containing environments. In this laboratory
exercise, you will learn to prepare such an artificial environment, and
will be further challenged to use your preparation in finding and
observing bacteria in your environment.
- You will learn the manual and mathematical skill
associated with preparing Luria broth (the food-containing portion of
the artificial bacteria environment).
will use your artificial environment to grow and observe bacteria found
in your environment
*The Petri dish was invented by the
German bacteriologist Julius Petri (1852-1921) as a container for growing
microorganisms. Similarly, the nutrient agar that you will be
preparing was developed by the biologists Luria and Bertani.
- When weighing out the scale, make sure the scale
has the proper tare calculated before measuring out substances for the
- 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
- 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
- You will be making 200 mL of Luria and Bertani (LB) broth from the following substances
: tryptone, yeast extract, sodium chloride.
- LB broth recipe: 1 liter (1000 mL) is made using 10 g tryptone, 5 g
yeast extract, and 5 g sodium chloride (NaCl).
Calculate the weight of tryptone, yeast extract, and sodium
chloride to make 200 mL of the solution.
Sample calculation: 200 mL (X g yeast extract) = 1000 mL (5 g yeast
X = (1000 mL) (5 g) / 200 mL
X = 1 g yeast extract
- Place each of the three powders in 250 mL beaker and mix with stir
- Calculate the weight of 1.5 % agar needed to make 100 mL.
Sample calculation: 2% agar solution is made by using 2 g of
agar in 100 mL of LB broth.
- Add this weight of agar to 250 mL flask to make 100 mL of LB agar.
- Using a graduated cylinder, separately measure out 100 mL of LB
broth that you prepared and add to the agar.
- Boil agar and broth solution using hot plates or microwave
(Suggested time for microwave: two 40 second intervals).
- Pour hot agar and broth solution into Petri dishes using sterile
*Sterile technique for pouring hot LB agar: Several inches above
the plate, gently pour approximately 20 mL of agar per plate. Hold
the cover at an angle just above the plate while pouring to protect from
materials falling onto the agar; immediately cover plate and allow 15
minutes to cool.
The following websites were selected so that those individuals
interested in creating artificial environments for growing bacteria
could find more general information on the Biology of Bacteria and
extended activities for which one could use the prepared artificial
The Biology of Bacteria
- Bacteria Museum
An organizational website containing general information on
Bacterial Biology, as well as Bacterial Evolution, Pathogenic Bacteria
Introduction to Bacteria
A Berkeley University website that contains information on bacterial
Fossils, Life History and Ecology, Systematics, and Morphology
Learning Center for Microbial Biology
A Michigan State University website containing a Microbe Zoo, Microbe
of the Month, Curious Microbes (bacteria living in extreme
environments), Microbe News, Microbe Ecology, and bacteria Scientists
- Cells Alive
A company website containing information on Cell Biology,
Microbiology, Immunology, Microscopy, Cell Models, and a cell gallery
An organizational website containing information about the Microbe
World, Microbe Education, and activities for Solving Microbe Mysteries
Activities Using Agar Plates