MICRB 202: Introductory Microbiology Lab
Unit III: Physical Controls on Growth
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Unit III: Physical Controls on Growth
Activities:
5.1 Temperature Effects (Ex 9)
5.2 UV Light: Lethal Effects (Ex 10)
Appendix 3.1: Spectrophotometer Operation
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Introductions:
A growing bacterium is tolerant of the physical and chemical conditions in its environment.
Physical and chemical factors that can control the rate, and even stop, bacterial growth include
temperature, barometric pressure, quality and intensity of light, pH, osmotic pressure, and oxygen
supply. Bacteria may grow within a range of values for any specific factor, called the tolerance range.
Any physical or chemical factor above or below the tolerance range for a bacterium will stop growth,
regardless of the availability of nutrients. This concept is known as Shelford’s Law of Tolerance.
In this laboratory unit, both temperature and ultraviolet light will be explored as physical
controls of bacterial growth. We will also demonstrate that not all bacteria respond similarly to
changes in environmental conditions.
5.1 Temperature Effects: (Ex 9)
Temperature is an excellent example of how an environmental factor can control, and even
prevent, growth. Temperature measures how rapidly molecules vibrate and collide into each other.
Temperature controls growth by directly affecting the activity of enzymes. As temperature increases
from some minimum value, an enzyme collides more frequently with its substrate and the reaction rate
increases until some optimum temperature is reached; the reaction rate is at its maximum. Increasing
temperature above the optimum value results in the weakening of bonds within the enzyme, causing
structural distortion and a steady reduction in activity. At some maximum temperature, the enzyme is
denatured to the extent it looses all function; it becomes inactivated. A bacterium has no means of
controlling cell temperature; thereby every enzyme, and hence the bacterium as a whole, is subject to
the temperature in the environment. We can define, for any bacterium, a minimum growth
temperature (lowest value with any growth), an optimal growth temperature (value for greatest
growth), and a maximum growth temperature (highest value with any growth) (Figure 3.1)
The temperature tolerance range and optimum temperature may differ dramatically for any two
species of bacteria. We can categorize bacteria into one of 5 groupings based on their growth response
to temperature (Figure 3.2). Species adapted to life in very cold environments, and have optimum
growth at ≤ 15 ºC, are called psychrophiles. Other bacteria that can tolerate cold temperatures but
have optimum growth temperatures of 20 to 30 ºC are called psychrotrophs. Most bacteria living on
and in our bodies are mesophiles, and have optimum growth temperatures of 30 to 40 ºC.
MICRB 202: Introductory Microbiology Lab
Unit III: Physical Controls on Growth
Thermophiles, have even higher optimum growth temperatures of 50 to 65 ºC. Amazingly, some
prokaryotes can grow at over 100ºC. These hyperthermophiles have optimum growth temperatures
around 90ºC. The later organisms have become the source of extreme heat tolerant enzymes for
various biotechnology, domestic, and industrial applications.
Figure 3.1. Response of bacterial growth rates to increasing temperature.
Figure 3.2. Categories of bacteria based on their growth response to temperature.
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MICRB 202: Introductory Microbiology Lab
Unit III: Physical Controls on Growth
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Exercise 12 investigates the effect of temperature on growth for a variety of bacteria (Part A),
as well as on the activity of a specific enzyme (Part B). In Part A, each student will culture their
bacterium at 5 different temperatures ranging from 5º C to 55º C. Growth will be measured based on
culture turbidity using a spectrophotometer. Students will then compare growth results and conclude
on differences in optimum temperature for all bacteria studied by the class.
In Part B, the goal is to determine the effect of temperature on activity of an enzyme called
prodigiosin. Prodigiosin catalyzes the production of a red pigment in the bacterium, Serratia
marcescens. Each student will grow S. marcescens on agar slants at different temperatures and assess
activity differences based on colony color development. How do you think the temperature for
maximum prodigiosin activity will compare to the optimum growth temperature for S. marcescens?
EXERCISE 9:
Part A: Bacterial Growth Response to Temperature:
Materials:
Nutrient broth cultures:
S. marcescens
Bacillus stearothermophilus
B. megaterium
Escherichia coli
Micrococcus luteus
Staphylococcus epidermidis
5x tubes nutrient broth
Procedure:
1. Label the five nutrient broth tubes with your bacterium initials and one of the following
temperatures each: 5º C, 25º C, 37º C, 42º C, and 55º C.
2. Inoculate each tube with broth culture of your assigned bacterium. Use sterile technique.
3. Place your inoculated and well labeled tube into the test tube rack for the appropriate
temperature.
4. Test tube racks will be place for incubation at each temperature. After 24 hours of incubation,
all culture growth will be suspended by storage at 4º C.
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5. Resuspend any settled growth in the tubes by pipetting the culture up and down to mix.
6. Using the spectrophotometer, read the turbidity of each tube as absorbance at 640 nm. (see
Appendix 3.1 for instructions on operating the spectrophotometers)
7. Write your data on the chalk board with the rest of the class data. Record all class data.
8. Enter data into an Excel spreadsheet and plot absorbance versus temperature for each bacterium
on a single graph. Attach the plot into your lab book (e.g. using a glue stick).
9. Discuss the optimum growth temperature for each bacterium, and argue which temperature
category each bacterium falls into (see Figure 3.2).
MICRB 202: Introductory Microbiology Lab
Unit III: Physical Controls on Growth
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Part B: Temperature Effect on an Enzyme:
Materials:
Nutrient broth culture S. marcescens
2x Nutrient agar slants
Procedure:
1. Label each slant with your initials and either 25º C or 37º C.
2. Inoculate each slant with S. marcescens (recall to drag your loop over the agar surface from the
slant bottom to the top.
3. Incubate each slant at the appropriate temperature for 48 h.
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4. Include your result in the tally for the class.
5. Discuss how the optimum temperature for growth of S. marcescens compares to the
temperature of highest prodigiosin activity. Was this what you had expected? Explain.
5.2 UV Light: Lethal Effects: (Ex 10)
Living organisms are exposed to a broad spectrum of electromagnetic radiation of varied
intensities (Figure 3.3), some of which is damaging, some harmless, and some beneficial, depending
on the organism. Ionizing radiation (gamma- and X- rays) are extremely damaging and used by man
for sterilizing materials sensitive to traditional heat sterilization; i.e. autoclaving. However, natural
ionizing radiation of cosmic origin is nearly completely absorbed or reflected in Earth’s upper
atmosphere. The visible and infrared wavelengths of light that penetrate to Earth’s surface are
largely harmless. Of course, visible and some invisible light are required for photosynthetic
organisms. The short wavelength invisible solar radiation that we call ultraviolet (UV) light is
injurious to most life at intense enough doses (e.g., think of your summer sunburn!). Nonphotosynthetic bacteria are particularly sensitive to UV light damage; although, tolerance range of UV
dosage does vary between bacteria.
Ultraviolet light damage to cells is largely due to creation of lethal mutations in the DNA due
to a specific narrow region of the UV spectra centered on 265 nm. One common DNA mutation
caused by UV light is the thymine dimer (Figure 3.4). Two adjacent thymine bases on a DNA strand
will covalently bind together as a result of excitation by 265 nm light. If this damage is not repaired
prior to DNA replication a lethal mutation may be passed on to a daughter cell, resulting in death.
Bacteria do have a variety of enzyme systems for the repair of thymine dimers and other forms of
DNA damage. However, high intensity UV and/or long exposure times will result in a lethal dose; the
repair systems simply can’t keep up with the new damage. It should come as no surprise that UV light
is commonly used to sterilize surfaces and equipment in hospitals, laboratories, and even barbershops.
UV light is also employed in the final stages of drinking water purifications at some water treatment
plants, so to kill any remaining pathogenic microbes prior to distribution. Germicidal UV lights used
in the above applications are equipped with low pressure mercury vapor lamps that mostly (90%)
generate 245 nm light, well within the lethal range.
MICRB 202: Introductory Microbiology Lab
Unit III: Physical Controls on Growth
Figure 3.3. The wavelength spectrum of electromagnetic radiation, with details of visible light
wavelengths and adjacent ultraviolet and infrared wavelengths.
Figure 3.4. The thymine dimer within a strand of DNA. The pentose sugars (yellow) represent the
deoxyribose – phosphate diester “backbone”. Adjacent pyrimidine bases of thymine (blue) are
covalently bound together (dark blue lines) due to UV light exposure.
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MICRB 202: Introductory Microbiology Lab
Unit III: Physical Controls on Growth
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Exercise 13 uses a germicidal UV exposure box to test the sensitivity of two Gram positive
bacteria, Bacillus magaterium and Staphyllococcus epidermidis. Each bacterium will be exposed to
increasing doses of UV light by increasing exposure time. We will determine the minimum lethal dose
for each bacterium. Knowing that B. magaterium is spore former and S. epidermidis is not, what might
be expected as the difference in UV dose response for these two bacteria?
EXERCISE 10:
UV Dose Response:
Materials:
Saline suspension of 96 hour old plate of Bacillus magaterium
Nutrient broth cultures:
Staphylococcus epidermidis
8 x nutrient agar plates per group
Lab timer (or watch)
Ultraviolet light exposure box
Swabs for inoculations
Procedure:
1. Students will divide into two groups, one for each bacterium.
2. Label all plates with bacterium initials and exposure times indicated in Table 3.1.
3. Use the cotton tip swab with your culture to swab the entire plate as follows:
a. Dip the swab just into the surface of the culture; excess liquid needs to be removed by
pressing the swap against the inner wall of the culture tube.
b. Swab over the entire plate three times; rotate the plate about a 1/3-turn between
swabs to assure complete and homogenous inoculation.
4. Expose each plate to UV for the indicated time. But each plate in the UV exposure box with its
lid off and an index card placed over half the plate surface area (Figure 3.5) Note that one
plate is exposed for the maximum time but with its lid on, so to test the degree of UV
transparency of polystyrene plastic. We will perform exposures in batches as not all plates will
fit in the UV exposure box at one time.
5. After exposure, replace lids and incubate at 37ºC for 48 hours.
Figure 3.5. A Petri dish ready for exposure, with its lid off and ½ covered with an index card.
MICRB 202: Introductory Microbiology Lab
Unit III: Physical Controls on Growth
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6. A simple ranking scale will be used to evaluation of UV light effects. Complete growth, or
“lawn”, will be assigned a “3”; the area covered by the index card will look like this. Plates
with 5 or fewer colonies will rank “1”, and plates with moderate growth will rank “2”. Plates
with no colonies will rank “0”. Score your plates, and share the data on the chalk board.
7. Tabulate all data for both bacteria in your notebooks.
8. Discuss the minimum dose to destroy each bacterium, if any. Quantify the difference in the
level of resistance between the two bacteria. Is this result what you expected? Offer an
explanation for this result that includes molecular level mechanisms of damage and repair.
Speculate and rationalize the result you would expect for a Gram negative bacterium like
Escherichia coli. Last, what’s the deal with polystyrene plastic and UV light transparency?
Appendix 3.1: Spectrophotometer Operation:
We will periodically use a Spectronic 20 spectrophotometer in MICRB 202 labs. It is important that
you know its parts and how to properly operate the instrument to zero and make readings of unknowns.
Basic operational steps for measuring cell turbidity:
1- Turn on the machine by clockwise turning the Power switch/Zero control knob located to the left
on the front side of the instrument. Let it warm for 15 minutes before you zero and make readings.
2- After the warm up period, set the wavelength to 640 nm using the wavelength control on the top
right of the instrument.
3- Adjust the meter to 0% T with the Power switch/Zero control, located on the left side. To read the
meter properly, align the needle with its reflection in the mirror. Make sure that the sample
compartment is empty and the cover is closed.
4- Fill a clean tube with about 4 ml of sterile tryptic soy broth (TSB) making sure that there are no air
bubbles. This will be your blank. Wipe the outside of the tube very well with a tissue to remove
liquid droplets, dust, and fingerprints.
5- Place the tube in the sample compartment. Adjust the meter to 100% T with the
Transmittance/Absorbance Control knob on the front right side of the instrument.
6- Remove the tube from the sample compartment and insert an unknown sample tube into the sample
compartment. Do not forget to wipe the outside of the tube very well with a tissue to remove liquid
droplets, dust, and fingerprints.
7- Read the appropriate value (% T or A) from the meter. Your readings should be < 0.8 absorbance
(A) for accurate readings; greater values no longer represent a linear response to increasing culture
turbidity, and those samples will require dilution (see your instructor).
8- Remove the sample and repeat steps 6 through 7 for any remaining samples.
9- When all measurements are complete, leave the instrument ON if there are other students needing
to make measurements on the instrument you used. Turn off the spectrophotometer if you are the
last student to use it by turning the Power Switch/Zero Control counterclockwise until it clicks.