Return to Contents

STUDENT OBJECTIVE

A natural population is simulated with beads to demonstrate the basis of the Hardy-Weinberg equilibrium. If a microcomputer with an evolution program is available, students can explore this relationship further. Mutations are examined experimentally in Serratia marscescens by exposure to ultraviolet light.

EQUIPMENT	AMOUNT
	(Class of 24 with 8 groups)
Ultraviolet light source with protective eye shield Transfer hood (see Exercise 8, NOTES) Microcomputer to run simulator (optional) LCD overhead projection screen for computer (optional) Incubator-shaker	1/lab 1/lab 1/lab 1/lab Instructor use only
MATERIALS
Three colors of beads (A. C. Supply)* 3 lb coffee can Culture, live Serratia marcescens Sterile: Petri dishes, glass or disposable, 90 mm Bottles, 200 ml Pipettes, 1 ml x 0.01 (automatic 1 ml pipette with tips) Glass rods, 12 cm hockey stick shaped Flask, 250 ml (with alcohol) Alcohol lamp or Bunsen burner Wax marking pencils Goggles, UV-absorbing Rubber gloves Population Genetics Simulation Program (Oakleaf Systems)* or Populus or BioQues Evolve Autoclave bags for waste disposal	1 can/group 1/lab 7/group 3/group 1/group 7/group 1/group 1/group 1/group 1/group pr/group 1/lab 2/lab

SOLUTIONS

0.85% saline, NaCl, sterile
King’s agar, sterile
Nutrient broth, sterile

PREPARATION

Three Weeks before Lab

1. The beads should be counted and exactly 4000 should be in each can with 1960 of one color (49%), 1680 of another color (42%), and 360 of a third color (9%). Tape cans securely closed until ready to use. (See NOTES for easy counting method.)
2. Order Serratia marcescens as a liquid culture to arrive the week prior to the lab.

One Week before Lab

1. Saline preparation:

   25.5 g NaCl/3000 ml

   Dissolve salt in water. Add 99 ml to small bottles. Screw caps on lightly and autoclave 15 minutes at 15 psi. Cool completely before tightening caps. Need three bottles per student group if each group does dilution. Instructor can do dilution for entire lab, in which case only three bottles needed.

2. Autoclave a packet of seven glass rods bent like hockey sticks and wrapped in aluminum foil.
3. King’s agar preparation:

   22.0 g peptone
   8.7 ml glycerol
   11.0 g K₂SO₄
   3.3 g MgCl₂· 6H₂O
   16.5 g agar
   1100 ml distilled water

   Dissolve the salts and agar in water in a 2-liter flask. Heat and stir until mixture is near boiling. Seal flask with aluminum foil and autoclave for 15 minutes at 15 psi. Cool to 60°C and pour on to petri dishes under a sterile hood.

   Yield: approximately 40 to 50 plates

4. Nutrient broth preparation:

24 Hours before Lab

Inoculate the flask of nutrient broth with two transfer loops of Serratia marcescens culture and place on an incubator-shaker at 25^o with a medium shaking speed. After 24 hours, the broth will take on a reddish-orange tinge due to the pigmented Serratia.

Day of Lab

1. CAUTION: The UV lamp should be enclosed so that students cannot look directly at lamp. However, adequate air circulation is needed to prevent heat buildup. Lamp intensity achieves a steady state after 30 minutes. If students are to do the UV irradiation, the person putting the plates in the UV box should wear UV-absorbing goggles, have on long sleeves, and wear rubber gloves to reduce skin exposure.
2. To prevent photorecovery, plates should be placed in a paper bag following UV irradiation. If plates will not be scored within three days, place in refrigerator immediately after experiment. Remove three days before lab and incubate.
3. The petri dishes should be incubated at 25^oC. Temperature must be under 28^oC for color to develop. Color intensity of the colonies increases with the age of the culture. To insure adequate moisture, place a large container of water at the bottom of the chamber.
4. Examine the plates periodically for pigmented colony development. After color develops, remove and count the number of white colonies per plate. These plates can be stored upside down in the refrigerator until the next lab period.

NOTES

1. Any uniform-sized small object can be used to make a simulated population. Buttons, washers, and beads are ideal objects. We use plastic beads ordered from A. C. Supply. (See List of Suppliers on page 93.)

   Counters for beads can easily be made from a 6" x 10" x 3/4" piece of Plexiglas or wood. With a drill press, drill 100 pilot holes three-fourths of depth of the board. Redrill a second time two-thirds the depth of the board using a bit with a diameter slightly larger than the diameter of the bead. In like manner, another 4" x 3/4" Plexiglas can be drilled with ten holes. These two counters should provide the multiples needed in counting the beads.

   Some practice is needed to scoop the beads into the drilled holes. Two large boxes (20" x 20" x 12") are needed. In one, pour a 2" layer of beads. Scoop up the beads and rock the board gently in a short circular motion while spilling off the excess beads. When all the holes are filled, dump the beads into the other box. Keep a tally of the number of counter boards added until desired number is obtained. Transfer beads from box to can.

2. There is variation in the intensity of ultraviolet lamps. The 18" distance from the culture should produce an ideal "kill curve" in the 80 seconds of exposure. If less than 80 seconds is needed, the intensity of the lamp can be reduced by placing metal screening between the culture and the lamp. It is a good idea to make several trial tests the week before students perform the exercise to determine specific laboratory conditions.

CLASSROOM SUGGESTIONS

1. Students are successful in scoring the Serratia colonies if their plates have 300 or less colonies per plate. If the first three plates indicate there will be greater than 300, the students can decrease the amount added or do a further dilution.
2. Students usually have not had much experience in following sterile technique procedures. A short demonstration can be helpful in reminding them where contamination can occur.
3. The experiment demonstrates the lethal and mutagenic effects of UV light. Expect to kill 80% of the cells and to induce 5—10% white colonies.
4. The program Evolve available in the BioQuest Library has a good simulator of microevolution. It is available from the Academic Software Development Group, Computer Science Center, University of Maryland, College Park, MD 20742 (phone 301-405-7600).
5. Check out the links for this lab topic at http://auth.mhhe.com/biosci/genbio/dolphin/ You will find useful materials for developing your lab introduction or summary, and in some cases, you may want to tell students to connect to a particular site for further information.

ANSWERS TO CRITICAL THINKING QUESTIONS

1. Aa = Huntington’s = 2pq
   aa = normal = q²
   AA = lethal = p²
   
   1:20,000 = .00005 = 2pq
   19,999:20,000 = .99995 = q²
   .9999749 = q; frequency = 99.99749%
2. Points to consider — Genetic drift/founder effect; sampling error.
3. Consider the frequency of alleles in the population.
   Consider whether or not the population exists in Hardy-Weinberg equilibrium.
   Consider what mechanism of microevolution is involved here. Natural selection? Non-random mating?
   Consider the effects on genetic variation in the population.

SUPPLEMENTAL MATERIALS