Background:

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. 

 Objectives:

1. You will learn the manual and mathematical skill associated with preparing Luria broth (the food-containing portion of the artificial bacteria environment).
2. You will use your artificial environment to grow and observe bacteria found in your environment

Materials: 

Tryptone Yeast Extract Sodium Chloride Agar Distilled Water 250 mL Beaker 250 mL Flask 100 mL Graduated cylinder Petri Dishes* Stir Bar Hot/Stir Plate Electronic Balance

*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.

Safety Considerations:

• When weighing out the scale, make sure the scale has the proper tare calculated before measuring out substances for the broth.
• 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.

Procedures:

1. You will be making 200 mL of Luria and Bertani (LB) broth from the following substances : tryptone, yeast extract, sodium chloride.
    
2. 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 extract)
                                                                        X  =  (1000 mL) (5 g) / 200 mL
                                                                        X  =  1 g yeast extract
    
3. Place each of the three powders in 250 mL beaker and mix with stir bar.
    
4. 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.
    
5. Add this weight of agar to 250 mL flask to make 100 mL of LB agar.
    
6. Using a graduated cylinder, separately measure out 100 mL of LB broth that you prepared and add to the agar. 
    
7. Boil agar and broth solution using hot plates or microwave
   (Suggested time for microwave: two 40 second intervals).
    
8. Pour hot agar and broth solution into Petri dishes using sterile technique*.

*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.

External Links:

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 environments.

The Biology of Bacteria

• Bacteria Museum
  An organizational website containing general information on Bacterial Biology, as well as Bacterial Evolution, Pathogenic Bacteria and Medicine
   
• Introduction to Bacteria
  A Berkeley University website that contains information on bacterial Fossils, Life History and Ecology, Systematics, and Morphology
   
• Digital 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
   
• MicrobeWorld
  An organizational website containing information about the Microbe World, Microbe Education, and activities for Solving Microbe Mysteries

Activities Using Agar Plates

• Access Excellence Activities
  The Indentification of Bacteria, Spencer Reames
  On the Microbe Trail: An Introduction to Bacteria and Aseptic Technique, Laura Ziegenhirt
  Investigation the Uses of Backyard Bacteria, Elisa Brako
  E. coli and Their Human Environment, Arthur Broga
  Selecting Soil Organisms in Compost, Barbara J. Errington
  Ecology of the Dump, Judith Brown and Randyll Warehime
  Biological Succession in Microecosystems, Sharon T. Deal
  Antibiotic Production by Soil Actinomycetes, John William Goudie
  Demonstrating an Epidemic, M. Beth Powel

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