Numbers refer to steps in the student procedure) 2. Yeast cells usually come from the supplier growing on agar slants. To keep contamination at a minimum, it is usually best to limit the use of the original stock culture and have students work from subculture plates. For this experiment, one subculture plate will supply enough yeast to make suspensions for over one hundred plates. Store the original stock in a refrigerator to keep viable for up to nine months.
^L^ It is best to have relatively fresh yeast cultures for your experiments. You may wish to standardize the subculturing procedure for your classroom and then use the same procedure each time you subculture yeast cells.
It takes one milliliter of suspension to make one plate. Figure the number of plates you wish to make and then use the appropriate amount of water. It is possible to store the yeast suspension in the refrigerator and use it during several class periods within a day or two. A "just turbid" suspension has approximately one million cells per milliliter.
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Making the UV exposure plates:
5. If the agar plates are fresh, the water will take
considerably longer than ten minutes to soak into the agar. It
is best if the plates are poured and then allowed to set out at
room temperature for a week before using them for this
experiment. You may also make a more concentrated suspension,
put less than one milliliter (one tenth mL) on each plate and
then use sterile paper clip spreaders to spread the suspension
over the surface of fresh plates.
9. On cloudy days or in the winter, it is possible to use a 300
watt quartz-halogen security light (with the glass cover off) as
a source of artificial sunlight. (See "A Closer Look...
Modeling UV Effects.")
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Questions and Answers:
1.How does the plate look different the day after it was exposed
to the sun (3rd day)? The lawn of yeast should be just becoming
visible and some growth difference between the covered and
uncovered portions should be visible. (If the plate is incubated
at room temperature, the growth may not be visible until 4th
day.)
2.Describe the plate on 4th day (5th day, if incubated at room
temperature). Note the amount of growth on the plate: 1) Where
the paper or card shaded the cells, 2)where cells were exposed to
direct sunlight through the lid, and 3)where anything was placed
to test its ability to protect the cells. There should be little
or no growth where the cells were fully exposed. Where they were
fully shaded, there should be a heavy layer of yeast covering the
agar. The experimental portion(#3) can be compared with these
two extremes.
3.How can you tell if you gave the exposed half of the plate a
lethal dose? Did all of the cells die? The cells on the
covered part will grow normally. If however, the exposure is
great enough, all the exposed cells will be killed. If the dose
is not enough to kill all of the cells, the growth may still be
reduced.
4.Why did you cover a portion of the plate with a piece of opaque
material? The material will shade the cells protecting them
from the sun. This is an untreated control for the experiment.
It simply tells you what the cells are like when they are not
being stressed by environmental conditions.
5.Why is it important to hold the plate perpendicular to the
sun's rays? The surface of the plate should be perpendicular to
the rays coming from the sun to expose the cells to the maximum
dose of UV.
6.How can you tell if you have the plate perpendicular to the
sun's rays? Put a card or similar object that will show a shadow
behind the plate. The shadow will be smallest when the surface
of the plate is perpendicular to the sun's rays and the radiation
is most concentrated on the surface.
7. How does the angle of the sun affect your chance of getting
asunburn? When the angle of the sun is small, your shadow is
shorter than your height and your risk of getting sunburned from
prolonged exposure is greatest. If the sun's angle is great
enough to make your shadow longer than your height, the risk of
getting sunburned is less. .
8. How does the angle of the sun affect the amount of UV that
falls on the cells in a given amount of time? If the sun were
directly overhead (zenith angle = 0o), which only happens in the
tropics, its radiation would travel the shortest possible path
through the atmosphere. When the sun's zenith angle is greater
than 0o, it must pass through a greater amount of atmosphere
including the ozone layer. You will then need a longer exposure
to see the effect on cells or to get a sunburn. (The technical
term for this relative thickness of the atmosphere is the "air
mass".)
9. How does the time of day and time of year affect the sun's
angle and the time required for the cells to receive a lethal
exposure? Each day, the sun is highest at noon. Throughout the
year, the sun is highest at the summer solstice (approximately
June 22 in the northern hemisphere) and lowest at the winter
solstice (approximately December 22 in the northern hemisphere).
Therefore, at any particular location, assuming the sky is clear,
the time to receive a lethal exposure will be shortest at noon on
June 22 .
10. How does the geographic location affect the time required
for the cells to receive a lethal exposure at noon on June 22,
when the sky is clear? As you travel north of the tropic of
cancer (23.5o north latitude) the zenith angle of the sun
increases. Therefore, you need a longer exposure time.
Objectives and Applications:
The video segment Serial Dilution & Viable Cell Count on video
tape III illustrates this procedure. You may wish to pause the
tape after each step is illustrated to allow the students to
complete each step before viewing the next step in the procedure.
Preparing a "just turbid" suspension:
(See Laboratory Methods: Estimating the Number of Yeast Cells in
a Culture)
Teacher tip - 2
See Laboratory Methods: Dilution Plating Procedure
You may wish to review exponential notation with your students.
106 = 1,000,000
Teacher tip - 3
Numbers refer to steps in the student procedure:
2. Three drops from a bulbed pipet equals one tenth milliliter.
If one fills the pipet to the one milliliter mark with water and
then drops out three drops there is nine tenths milliliter left
in the pipet which can then be transferred to a sterile tube.
3. As you transfer one tenth milliliter to each tube be sure to
return any unused suspension to the previous tube. If you want
to conserve pipets it is possible to use one pipet for the entire
procedure. When using one pipet thoroughly rinse the pipet in
each cell suspension as the suspension is made. The pipet may
also be returned to its sterile plastic sleeve when it is not in
use.
4. The most useful data will come from plating the tubes with
the fewest cells. ( 103 to 100)
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Teacher tip - 4
Questions and answers:
1. How well do your colony counts agree with the expected
dilution steps of ten?
2. If the agreement isn't perfect (and we don't expect it to be,
even with a "perfect" technician) do you think the errors could
be explained by "random chance"? Explain.
3. Is there any pattern to the errors? Do the ratios
systematically get larger or smaller as the number of colonies
increases?
4. Write down any reasons you can think of for such systematic
errors to occur in this procedure.
5. If you have a culture with 1 106 cells/mL and you wanted to
dilute it to 1 102 cells/mL, would it be better to just make a
single 10,000-fold dilution or four ten-fold dilutions? Explain
the advantages and disadvantages of each method.
Four step dilution
Objectives and Applications:
The procedure that follows assumes that
individual students or
lab groups will each do a serial dilution, plate suspensions from
several dilution tubes and expose the plates for several exposure
times. The short term goal is to determine the length of
exposure for your particular geographic location and time of year
that produces a surviving fraction of approximately 0.1 (a 0.1
surviving fraction is equivalent to 10% survival). This is
sometimes referred to as "determining the LD10." The long term
goal is to develop enough expertise so that students can
efficiently monitor UV-B over a long period of time. The amount
of UV-B reaching the surface of the earth at any particular
location varies over the course of a day and also varies by
season over the year .
Once students get skilled at these procedures
two plates should
be enough to make a UV reading. (one unirradiated control plate
and one irradiated timed exposure plate)
It's possible to envision all types of monitoring schedules. One
might take one reading each week at a particular time of day and
use the data to monitor the seasonal change of UV. One might
take one reading each hour during a day and monitor the change of
UV over a single day. Students could use this technique to
quantify the sunscreen experiments they designed in the section
Observing the Effects of Solar Ultraviolet Radiation on Cells.
The preparation procedures for this experiment
are essentially
the same as those for Serial Dilutions and Viable Cell Counts.
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Teacher tip -2
Procedure:
2. There are several video segments that illustrate this
process. They use various types of pipetting equipment and
various methods of plating cells. Figure 2 illustrates the pour
plating method in which a separate final dilution tube is
prepared for each plate and then the entire contents of the tube
are poured onto the agar surface. This method has the advantage
of not requiring the use of a flame. It has the disadvantage of
not being as reproducible.
Note that Figure 2 shows only one final tube of each type. Your
students may not choose to make all of the illustrated pouring
tubes, especially the 10,000 cells per mL. If they need several
plates of one type (for example 100 cell plates for controls)
they need to make a separate final tube to pour onto each plate.
The suspension must be allowed to soak into the agar before the
plates are exposed to the sunlight. To speed up the process make
the agar plates several days in advance and let they dry out a
bit before you pour on the yeast suspension.
Video section C: Serial Dilution & Viable Cell Counts illustrates
the pour method of plating.
When you want precise data, it is better to use an alternative
plating method. Instead of pouring the dilutions illustrated in
the second row in Figure 2 onto the plate, omit that step and
accurately pipet 0.1 mL of the dilutions shown in the first row
directly onto the agar. Use a sterile spreader to distribute the
suspension uniformly over the surface, but avoid spreading it to
the edges where it can run down between the agar and the side of
the Petri dish.
Two types of spreaders:
2. Flamed glass spreader method:
Teacher tip - 3
Name of Investigators:
Date:
Results:
Teacher tip -2
Numbers refer to steps in the student procedure
Teacher tip - 3
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CHAPTER - 2 Teacher tip - 1
This section introduces the and techniques of
quantitative
microbiology, which involves a number of valuable concepts and
skills useful for a variety of experiments, including the
following:
The concept of cell growth and colony formation.
The ability to prepare a suspension of cells
with a known cell
density by visual estimation.
The ability to plate a known number of cells
on a petri plate.
The ability to make quantitative estimates of
the number of
viable cells in a culture.
Getting Ready:
You may wish to do this step for your students. One YED
subculture plate will supply enough yeast for several classes.
To save lab time you may wish to make a subculture plate for each
lab group. Yeast usually comes from the supplier growing on agar
slants. To keep contamination at a minimum it's usually best to
limit the use of the original stock culture and have students
work from subculture plates.
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105 = 100,000
104 = 10,000
103 = 1,000
102 = 100
101 = 10
100 = 1
You may wish to save plates by not using the contents of the
other tubes. (106 to 104)
The best agreement will probably be between
the 101 and 102
plates.
See A Closer Look At... The Dilution Series and Statistics for a
discussion of possible answers.
Answers will vary.
The ratio may get systematically smaller if you use one pipet to
make the whole dilution series and do not rinse it thoroughly in
each suspension before transferring cells to the next tube.
Single step dilution
Advantage: takes fewer steps
Disadvantage: requires large quantities of sterile water and
large containers
Advantage: uses small amounts of sterile water and small containers
Disadvantage: more steps, uses more sterile pipetsChapter - 3 Teacher tip -1
1. a. Choose several exposure times that look reasonable and
will produce a surviving fraction close to 0.1 You may want to
coordinate the exposure times between the different lab groups so
that you can cover as wide a range of exposures as possible.
1. No flame paper clip method:
You can easily make an inexpensive reusable spreader by
straightening out two bends in a large paper clip, leaving a
hairpin-shaped end for spreading and a straight handle at right
angles to it. You can sterilize several spreaders together in
covered beakers or wrapped in foil or paper.
Video section D:Using Yeast to Measure Solar UV, illustrates a
flamed glass spreader method of plating.
Time:
Location:
Yeast Strain:
Growth medium:
Incubation temperature:
Diagram of Dilution and Plating Procedure: