Pre-Lab
Preparation
Preparing Potato dextrose agar plates
1. Potato dextrose agar plates or potato dextrose dehydrated
medium can be purchased from Carolina Biological Supply
Co. (http://www.carolina.com) and Ward’s Natural
Science Establishment, Inc. (http://www.wardsci.com).
If the dehydrated medium is purchased, directions for
preparation of plates will be included.
2. Potato dextrose agar plates can be prepared from potatoes,
dextrose, and agar according to the following directions:
3. Boil 200 grams of peeled and sliced potatoes in 1 liter
of water until the potatoes are soft. Strain through cheesecloth
and adjust the filtrate to 1 liter with more distilled
water.
4. Add 10 to 20 grams dextrose and 12 to 17 grams agar.
Autoclave 15 min at 121º C.
5. Pour autoclaved medium into sterile Petri plates. Makes
approximately 40 plates.
Preparing 10% (v/v) bleach
solution
- Mix one volume commercial laundry bleach, e. g. Clorox,
with nine volumes of distilled water.
Preparing fruit with brown rot for classroom use
1. For teachers who do not want to maintain or purchase
cultures, it’s easy to find this fungus just by
buying stone fruit (peaches, nectarines, plums, cherries)
and leaving them at room temperature in a plastic or
paper bag. They are often already infected, and the infection
will develop within a week, resulting in obvious brownish
spores on the fruit surface. Isolations from these fruits
may be contaminated with bacteria and other fungi, so
a more successful lab for students can be accomplished
by using fruits that have been deliberately inoculated.
2. Prepare
fruits about 1 week before they are needed. Disinfest (surface-sterilize) firm, healthy stone fruit
for 30 min. in 10% (v/v) bleach solution. Rinse with sterile,
distilled water.
3. Using a sterile dissecting needle, scrape spores from
a culture of Monilinia fructicola or a fruit with brown
rot and stab each fruit four to six times.
4.
Incubate at room temperature in a moist chamber (plastic
box lined
with paper towels moistened with sterile, distilled
water) with the lid not tightly closed. Check daily for
fungal development, which will vary with temperature in
the lab and the ripeness/susceptibility of fruit. Refrigerate
the box of infected stone fruit if necessary to preserve
good disease development for student use (i.e. don’t
let the brown rot completely destroy the fruit).
5. Although the moist chamber as described above does
not start out as a completely sterile environment, because
you do not sterilize the plastic box or the paper towels,
it does provide an environment adequate to favor the growth
of the pathogen over other organisms. A clean plastic box
and fresh paper towels usually do not introduce problems.
6.
Infected fruit can be allowed to dry at room temperature
to form
a “mummy.” It will probably be possible
to use scrapings from the mummy to begin the disease again
when needed for another class.
Immediately before these lessons:
- Purchase healthy fruit to be used to test for pathogenic
bacteria.
Activity
1. As students
enter the room, have the following question on the board
for students to respond to: “Can plants
get sick? Why or why not?”
2. Allow students a few minutes to record and share their
thoughts with the rest of the class.
3. In their laboratory groups have students observe the
diseased fruits you prepared earlier. Ask them to describe
the symptoms and signs of the diseased plums (or other
stone fruit). Have them examine the suspected pathogen
carefully both macroscopically and microscopically and
make notes about and drawings of what they see. They will
want to refer back to these recorded observations in later
steps.
4. Ask students to consider how the diseased plum looks
compared to a healthy plum. Is something growing on the
plum? Are some parts of the plum softer or firmer? What
is the color of the mycelium (hair-like, non-reproductive
fungal growth) and spores? Microscopically, what are the
characteristics of the mycelium? Is it septate, i.e. does
it have internal cross walls that divide the hyphae into
compartments? Or does the mycelium just look like long
tubes without any internal cross walls? Do you see any
spores? What are their shape, color, and size? Would you
recognize this fungus if you saw it again? (See below.)
5. Ask students how they would test to ensure that indeed
it was the fungus that was causing the disease. What variables
and controls might they need to consider? Have students
write these tentative ideas down. Then ask students to
talk within their groups about what they think might be
important factors to consider. As a class, discuss some
of the ideas that students think would be central to testing.
6. Explain to
the students that Robert Koch (1843-1910) devised a scientific
approach to confirm causation of disease
by a microbe. His criteria are referred to as Koch’s
postulates. [The students probably alluded to some of Koch’s
postulates in their ideas about how to eliminate the cause
of the fungus as the root of the disease.]
7. Share with students Koch's Postulates:
I. The diseased host is observed for signs of the causal
organism and symptoms of the disease; the causal organism
is shown to be associated with all diseased individuals.
II. The causal organism is isolated into pure culture
and described.
III. This pure culture of the suspected pathogen is inoculated
into a healthy host and shown to cause the same disease
symptoms and signs as originally observed in Step 1.
IV. The same causal organism is re-isolated into pure
culture from the inoculated diseased host and shown to
be identical
to the organism described in Step 2.
8. Explain to
the students that they are going to use Koch’s postulates to determine the cause of the disease
in the stone fruit. (Step 4 can be eliminated if time is
limited). However, Koch’s postulates are not clearly
identified in the protocol. As they carry out the protocol
they should identify which one of Koch’s postulates
each step refers to.
Protocol
1. To isolate the probable pathogen on a nutrient medium,
e.g. potato dextrose agar (PDA):
a) Cut four small (2 mm x 2 mm) pieces of infected fruit
tissue.
b) Disinfest briefly by immersing the four pieces of tissue
for 15, 30, 45, or 60 sec in 10% (v/v) bleach solution.
Note: This is done to remove any surface contaminants without
killing the pathogen deeper in the tissue. Since it is
not known exactly how long this takes, several different
times are chosen to ensure a successful isolation of the
pathogen. You want to disinfest the tissue of any contaminating
organisms, but not kill the fungal pathogen.
c) Sterilize forceps by briefly passing them through a
flame and allow them to cool. Using sterile forceps, remove
the tissue from the bleach and blot dry on a paper towel.
d) Place each piece on the surface of the PDA agar in the
Petri plate. Minimize the time that the medium in the plate
is exposed to possible contamination from spores in the
air.
e) Incubate at room temperature for five to seven days.
2.
Describe the isolated pathogen in culture both macroscopically
and microscopically. Record these observations as words
and drawings. Do you think this is the same organism
that you observed on the diseased fruit in Step 1?
3. Use the isolated pathogen to inoculate healthy plums
as follows:
a) Immerse 2 healthy plums in a 10% (v/v) bleach solution
for about two minutes. This disinfests the fruit of any
surface contaminants. In the original fungal isolation
onto PDA, small pieces of cut fruit were placed in the
bleach solution for a shorter time to avoid killing the
pathogen deeper in the tissue. The 2 minute time for
whole fruits can be used because the intact skin
of the fruit
protects the inner flesh from the chlorine. Remove and
dry with paper towels. Make a V-shaped cut with a sterile
blade on the surface of the first plum. Place loosely
in a plastic bag with a moistened paper towel,
close with
a twist-tie and label. This is the control plum.
b) Repeat with the second plum with this change: inoculate
the wound with spores from your isolate using a sterile
dissecting needle.
c) Incubate for one week and record your observations
of any symptoms and signs that develop on each fruit.
4.
Koch's postulates require that the pathogen be isolated
from the inoculated
fruit-as in Step 9 (step 1 on the student protocol sheet)
to determine if it is the same organism that was originally
observed on the first diseased fruit.
5.
Once students have had time to allow the disease to grow
on the healthy
fruit and confirm the cause of the
disease, bring the class together to discuss the results
and to identify how they thought Koch’s postulates
were addressed in the protocol.
6.
Students may wish to design experiments to further investigate
factors that affect disease. For example:
1) What is the effect of temperature on infection and
disease development?
Inoculated fruit can be placed at room temperature and
in a refrigerator for a simple comparison. Why do we
refrigerate most fruits and vegetables after purchase?
2) What is the effect of wounding on infection and disease
development?
Spores can be applied to wounded and non-wounded fruit
for a simple comparison. What are some potential sources
of wounds in commercial fruit production, harvest and
shipping?
3) What is the host range of Monilinia fructicola?
Students can bring in healthy fruits and vegetables for
inoculation to determine which ones are susceptible to
brown rot. Stone fruit are the common hosts of this fungus,
but ripe apples and pears sometimes develop the disease.
Which species develop brown rot when inoculated with Monilinia
fructicola and which ones do not develop the disease?
Embedded Assessment
- Are
students able to identify variables and controls that
they should consider when attempting to identify the
cause of disease?
- Can
students identify which steps in the protocol are associated
with which of Koch’s Postulates?
- Are
students able to isolate the pathogen and if not, are
they able to identify what might have
gone amiss in
their procedures?
Homework
In
their science notebooks, have students write a reflective
conclusion.
What did they learn? What new questions do
they have? How does the lab connect to “real life?”
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