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STUDENT HANDOUT:
STUDENT HANDOUT:
LABORATORY REPORT FORM:
LABORATORY REPORT FORM:
INTRODUCTION:
INTRODUCTION:
Genetics is a biological field of study in which scientists explore heredity (the passing of traits from parent to offspring) and the variation in those traits. The field of genetics was first established by the Czech friar Gregor Mendel who studied heredity patterns almost 200 years ago. His work involved observations, tests, hypotheses, and numerical analyses of the growth and appearance of peas. To explain his findings, he defined the unit responsible for a single trait as a gene. In addition, Mendel established that this unit of inheritance, the gene, might exist in one or more varieties called an allele.
In the early 1900's, T. H. Morgan studied the genetics of the fruit fly, Drosophila melanogaster. While observing these flies, he noted that some certain traits that were present in one generation, would disappear in the following generation, but then could come back in future generations.
Even after these significant findings, for nearly three decades little progress was made in understanding how microbial characteristics are inherited. The advent of antibiotics, and the development of resistance to them by some microorganisms, led to the discovery that bacteria exhibit much the same genetic characteristics as more advanced organisms. A series of experiments by Erwin Chargaff, Rosalind Franklin and Maurice Wilkins eventually led to James Watson and Francis Crick to build the first model of DNA and write a paper in 1953 in which they described the chemical structure of DNA and announced that genetic information resides in the nucleotide sequence of the DNA (Adenine, Thymine, Cytosine, Guanine). The genetic makeup of an organism is known as its genotype. Genotypes determine the physical characteristics, or phenotypes, of that organism.
In prokaryotes, which do not have a true nucleus, a chromosome is a threadlike molecule of DNA. The molecule is a double chain of nucleotides arranged in a helix with the nucleotide base pairs held together by hydrogen bonds. The typical prokaryotic cell contains a single circular chromosome composed of double-stranded DNA. When a prokaryotic cell reproduces by binary fission, the chromosome reproduces itself; each single strand of the DNA makes a complementary strand that is a copy of itself.
We now know that phenotypes can change. One way is by a mutation, which is a permanent change in characteristics due to changes in the sequence of DNA (Figure 1)
Figure 1. Mutation in a sequence of DNA
For example, mutations are responsible for altering the colony morphology of the fungus Saccharomyces cerevisiae (Figure 2).
Figure 2. Mutation in the DNA results in changed colony morphology of Saccharomyces cerevisiae. Image from Wikimedia Commons.
A mutagen is something that induces mutations. One well know mutagen is ultraviolet light (UV). UV induces thymine dimers which prevents DNA from replicating correctly (Figure 3).
Figure 3. UV damage to DNA. Image from Wikimedia Commons.
A second mechanism that can alter phenotypes is through environmental factors. For example, the red and blue flowers of hydrangeas is not defined by their DNA, but rather by the pH of the soil (Figure 4). Acidic soil results in blue flowers, basic soil results in red flowers.
Figure 4. Hydrangea flower color is dependent on the pH of the soil. Image from PickPik.
Taken together, both genes and the environment play a role in an organism's phenotype (Figure 5).
Figure 5. The traits of an organism (phenotype) is determined by both DNA (genes) and the environment. Image from Berkeley Evolution.
PRECAUTIONS:
PRECAUTIONS:
Handle the bacterial cultures with care. Wash your hands with antimicrobial soap before and after handling cultures, and wash work surfaces with disinfectant.
Clean up spills using disposable plastic gloves, paper towels, and disinfectant. Dispose of all cleanup materials in the biohazard bag.
Do not put fingers or any objects near eyes or mouth while working.
Do not expose skin or eyes directly to the UV lamp. Wear special protective UV goggles and plastic gloves.
EXPERIMENT #1: Environmental factors influencing gene expression
EXPERIMENT #1: Environmental factors influencing gene expression
OBJECTIVES:
OBJECTIVES:
Demonstrate that a change in characteristics is not always the result of a genetic change.
Determine if changes in characteristics are due to the environmental conditions in which the organisms are grown.
MATERIALS (Work in pairs):
MATERIALS (Work in pairs):
Nutrient broth culture of Serratia marcescens grown at 25°C for 48 hours
Nutrient agar plates (3)
Sterile cotton swabs (3) or metal inoculating loop
Permanent marker
Refrigerator
Incubator set at 37°C
Incubator set at 55°C
Masking tape
Protective gloves
Disposal beaker of disinfectant (or Bunsen Burner if using metal inoculating loops)
Biohazard bag
PROCEDURE:
PROCEDURE:
Label the bottom of each nutrient agar petri dish with your initials and the date. Also label each plate with one of the incubation temperatures:
25°C = Room temperature
37°C = Body temperature
55°C = High temperature
Aseptically place a sterile swab into the broth culture of S. marcescens. Twirl the swab to saturate the cotton and then press the swab against the side of the tube to squeeze out any excess fluid. If you are using an inoculating loop, make sure you sterilize it using the Bunsen burner.
Starting with the 25°C plate, create a spread plate by lifting the petri plate slightly like a clam shell. Using the cotton swab, spread the bacteria in a straight line down the plate as shown in Figure 6. Then in a zig-zag fashion, spread out your inoculation. Rotate the plate 60° and continue swabbing the plate. Rotate the plate again 60° and continue swabbing the plate. Make sure to swab the entire plate leaving no space uninoculated. This will create a lawn of bacteria.
Figure 6. Creating a lawn of bacteria.
4. Dispose of the used swab in the beaker of disinfectant or as instructed. If using a metal inoculating loop, reflame your loop to sterilize it after use.
5. Tape the lid closed with two small pieces of tape.
6. Repeat this process for the two remaining petri plates.
7. Turn the plates upside down and incubate each one at the proper temperature.
8. Observe the amount of growth and the color produced at 24-hour intervals for 5 days.
OBSERVATIONS/CONCLUSIONS:
OBSERVATIONS/CONCLUSIONS:
Record the results in Table 1 on the Microbiology Laboratory Report Form.
At which temperature did most growth occur?
QUESTIONS:
QUESTIONS:
What alterations would you make to this procedure to prove that the results you observed is NOT due to a mutation?
EXPERIMENT #2: Mutation
EXPERIMENT #2: Mutation
OBJECTIVES:
OBJECTIVES:
Observe how ultraviolet light permanently affects microorganisms.
Determine the effectiveness of different materials in blocking ultraviolet light.
Learn the correct and safe usage of ultraviolet light in the laboratory.
MATERIALS (Work in pairs):
MATERIALS (Work in pairs):
Nutrient broth culture of Serratia marcescens grown at 25°C for 48 hours
Shortwave ultraviolet lamp ((2537 A)- Sterilamp®)
Masking tape
Nutrient agar plates (3)
Sterile swabs or metal inoculating loop
Permanent marker
UV goggles
Protective gloves
25°C incubator (optional)
Disposal beaker of disinfectant (or Bunsen Burner if using metal inoculating loops)
Biohazard bag
PROCEDURE:
PROCEDURE:
Label the bottom of each nutrient agar petri dish with your initials and the date. Also label each plate with one of the incubation conditions:
Plate 1: UV Exposure-Lid Off
Plate 2: UV Exposure-Lid on
Plate 3: Control
Aseptically place a sterile swab into the broth culture of S. marcescens. Twirl the swab to saturate the cotton and then press the swab against the side of the tube to squeeze out any excess fluid. If you are using an inoculating loop, make sure you sterilize it using the Bunsen burner.
Starting with the first plate, create a spread plate by lifting the petri plate slightly like a clam shell. Using a sterile cotton swab, spread the bacteria in a straight line down the plate as shown in Figure 6. Then in a zig-zag fashion, spread out your inoculation. Rotate the plate 60° and continue swabbing the plate. Rotate the plate again 60° and continue swabbing the plate. Make sure to swab the entire plate leaving no space uninoculated. This will create a lawn of bacteria.
Figure 6. Creating a lawn of bacteria.
4. Dispose of the used swab in the beaker of disinfectant or as instructed. If using a metal inoculating loop, reflame your loop to sterilize it after use.
5. Repeat this process for the two remaining petri plates.
6. Prewarm the short wavelength ultraviolet (UV) lamp for at least 10 minutes. Use protective gloves and UV goggles and do not look directly at the light. Place plate 1 under the lamp and remove the lid. Tilt the plate slightly from side to side to allow for uniform exposure. Expose the plate for 2 minutes. The distance from the lamp to the culture should be approximately 12 inches.
7. Repeat the UV exposure with plate 2, leaving the lid in place. Expose this plate for 2 minutes also.
8. Do not expose plate 3 to the UV light.
9. Tape all three plates together and incubate them upside down at room temperature and in a dark place for 72 hours (in a 25°C incubator if possible).
OBSERVATIONS/CONCLUSIONS:
OBSERVATIONS/CONCLUSIONS:
Record the results in Table 2 on the Microbiology Laboratory Report Form.
QUESTIONS:
QUESTIONS:
How can you prove that a mutation DID occur?
Did UV light affect the appearance of growth on the plate? Why or why not?
Can you think of a practical use of UV light as a deterrent to microbial growth?
KEY TERMS:
KEY TERMS:
allele
antibiotic
binary fission
chromosome
complementary strand
DNA
gene
genotype
hydrogen bond
lawn
mutation
nucleotide
phenotype
prokaryotes
ultraviolet light
SUGGESTED READINGS:
SUGGESTED READINGS:
During the COVID-19 pandemic, you have heard about the variants. The variants are mutations in COVID’s spike proteins. These mutations have had impacts on both testing and vaccine efficacy. Click HERE to learn more about COVID’s mutations.
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