10/07/15
Name_______________________________
Section___________________________
Introductory Microbiology Pre-lab Assignment
Isolation of an Antibiotic Producer from Soil
1. (1pt) Name four genera of microbes and their microbial type that produces a vast
number of antibiotics.
2. (1pt) Name two ways industrial microbiologists may chemically modify antibiotics.
3. (1pt) What are the advantages of modifying antibiotics?
4. (1pt) True or False. Actinomycetes are categorized as fungus.
5. (1pt) Define antibiosis.
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Lab Exercise
ISOLATION OF AN ANTIBIOTIC PRODUCER FROM SOIL
Soil is the major repository of microorganisms that produce antibiotics
capable of inhibiting the growth of other microorganisms. Clinically useful antibiotics
have been isolated from several groups of soil microorganisms, including bacteria
(Streptomyces sp., Bacillus sp.) and fungi (Penicillium sp. Cephalosporium sp.).
Although many organisms in soil produce antibiotics, only a small portion of new
antibiotics is suitable for medical use. Also, industrial microbiology is directing its
energies toward chemical modification of existing antibiotic substances. This is
accomplished by adding or replacing chemical side chains, by reorganizing
intramolecular bonding, or by producing mutant microbial strains capable of excreting a
more potent form of the antibiotic. These chemically modified drugs have been
responsible for slowing antibiotic resistance, minimizing adverse side effects in the host,
and increasing the effective spectrum of a given antibiotic.
In this experiment, an attempt will be made to isolate antibiotic-producing
Actinomycetes from soil. These spore forming bacteria are abundant in most soils and
account for much of the breakdown of organic matter that occurs there. In fact, the odor
of freshly turned soil comes from the volatile compounds these bacteria produce.
However, the greatest impact Actinomycetes have comes from the ability of some of
them to produce antibiotics. Most antibiotics in current use are produced by species of
Streptomyces. On an agar plate inoculated with soil, numerous Streptomyces colonies
develop. The colonies are easy to recognize by their pastel colors, characteristic texture
(a hard mass that extends into the agar), and soil-like odor. Some of the filaments grow
into the agar. The rest extend up from the agar and form chains of spores at the tips. The
spores give the colony its characteristic pastel color.
Although their general growth pattern is fungus-like, Actinomycetes are distinctly
prokaryotic in size and structure. In cross section an actinomycete mycelium looks like a
typical Gram positive bacterial cell.
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Day 1 – Primary Isolation
Unless the organisms in a soil sample are thinned out sufficiently, the isolation of
potential antibiotic producers is nearly impossible. As indicated in the diagram, it will be
necessary to use a series of six dilution tubes to produce a final soil dilution of 10-6.
Procedure Overview
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Day 1 - continue
Materials per group of 2 students:
soil sample (will share with bench)
6 test tubes
0.9% sodium chloride (physiological saline) in flask
3 glycerol yeast extract agar plates
6 - 1ml pipets & pump
1 - 10ml pipet & pump
bactispreader
95% ethyl alcohol in screw top jar
Bunsen burner
striker
wax pencil
spatula
Materials for entire class:
scale
weigh boats
vortex
scissors
parafilm
Procedure:
1.
2.
3.
4.
5.
6.
Label six test tubes #1 through #6 with a wax pencil. Using a 10ml pipet,
dispense 9ml of saline into each tube.
Weigh out 1g of soil and deposit it into tube 1. Cover with parafilm.
Vortex mix tube 1 until all soil is well dispersed throughout the tube. Mix for at
least 1 minute.
Make a tenfold dilution from tube 1 through tube 6 by transferring 1ml from tube
to tube. Use a fresh pipet for each transfer and be sure to pipet mix thoroughly
before each transfer.
Label three glycerol yeast extract agar plates with your initials, lab section, and
the following dilutions: 1:10,000, 1:100,000, and 1:1,000,000.
Transfer 1ml from the soil mixture in tubes 4, 5, and 6 to the appropriate plate.
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7.
Spread the organisms over the agar using the bactispreader: Procedure for alcohol
sterilization of glass bactispreaders with alcohol sterilization:
a. Dip the glass bactispreader into the ethanol (in beaker). DO NOT ALLOW
THE ETHANOL TO DRIP DOWN THE HANDLE OF THE
BACTISPREADER!!!
b. BRIEFLY pass the bactispreader through the flame.
c. Immediately move the flaming bactispreader away from the flame and the
ethanol.
d. Allow the flame to burn out.
e. Rub the bactispreader on an area of the agar away from the bacteria to cool.
f. Spread the cells over the agar of plate A.
g. Flame the bactispreader again.
8.
Incubate the plates at 28-300C for 5 – 7 days.
Day 2 – Colony Selection and Inoculation
The objective in this laboratory period will be to select Actinomycete colonies
that may be antibiotic producers. The organisms will be streaked on TSA plates that have
been seeded with Staphylococcus epidermidis. After incubation, students will look for
evidence of antibiosis. Antibiosis is an association between two or more organisms that is
detrimental to at least one of them. In this experiment, if the soil organism is an antibiotic
producer, the growth of S. epidermidis may be inhibited.
Materials per group of 2 students:
primary isolation plates from previous lab session
4 TSA deeps
4 sterile petri plates
TSB culture of Staphylococcus epidermidis
1 – 1ml pipet
pipet pump
Bunsen burner
striker
inoculating needle
positive control- culture plate of soil antibiotic producer (see step 8 below)
Materials for entire class:
hot plate with 500ml beaker half filled with water
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Procedure
1.
Place 4 TSA deeps in boiling water until the agar melts (about 5 minutes). While
the agar is melting, go to step 2.
2.
Label Petri plates #1 through #4 and with your initials and lab section. Plate 4 will
be the positive control.
3.
Remove the deeps from the water and allow them to cool until you can
comfortably hold the tubes in your hand (about 8-10 minutes).
4.
After TSA deeps have cooled, inoculate each one with 1ml of S. epidermidis
5.
Pour the contents of each inoculated tube into separate Petri plates. Allow the agar
to solidify (about 10 minutes).
6.
Examine the three primary isolation plates for the presence of actinomycete
colonies. They have a dusty appearance due to the presence of spores. They may
be white or colored.Typically are small, opaque, compact, frequently pigmented (brown,
yellow, pink, etc.), often leathery, and appear dry and dull looking. Typically, a depression in
the agar surface may be observed around the colony. Avoid molds. They usually form much
softer, fuzzy colonies if present. Select three different suspect colonies
7.
8.
9.
Using a sterile inoculating needle, scrape spores from actinomycete colonies
on the primary isolation plates to inoculate the seeded TSA plates labeled #1
through #3 (see diagram on previous page). Use inoculum from a different colony
for each of the three plates.You are not streaking to isolate colonies so you do not
need to perform the quadrant streak.
Repeat Step 7 using the positive control culture for plate #4.
Incubate the plates at 300C until the next laboratory session.
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Day 3 – Evidence of Antibiosis
Students will examine the four plates from the previous laboratory session for
evidence of antibiosis (inhibition of S. epidermidis growth) and confirm results.
Materials per 2 students:
plates from previous laboratory session
Procedure:
1.
2.
3.
Observe plate for presence of antibiosis (clear zone around actinomycete colony).
Record your results in the lab report.
Make a smear and simple stain of your soil isolate(s), control, and Streptomyces
species.
Smear preparation procedure from solid media:
1. *Flame the loop the length of the wire only until it glows red hot. Cool ~15 seconds. *DO NOT
SET THE LOOP DOWN OR IT WILL HAVE TO BE FLAMED AGAIN!
2. Place one loopful of distilled water in the center of the slide. *Flame and cool the loop again.
3. Remove the lid of the petri plate. *DO NOT SET THE LID DOWN!
4. Gently touch the center of one of the bacterial colonies with the loop. ONLY A VERY SMALL
AMOUNT OF BACTERIA IS NEEDED. Replace the lid on the petri plate.
5. Disperse the bacteria on the loop in the water so that you have a dime-sized specimen in the center
of the slide. *Flame the loop again.
6. Allow the smear to dry COMPLETELY at room temperature.
7. Attach one end of the slide to the clothespin, and pass the slide through the flame several times to
heat-kill and fix the organisms to the slide.
8. You may now stain the specimen.
Simple staining with methylene blue
When only one stain is used, the procedure is called simple staining. Most stains are cationic
(positively charged). This allows the stain to adhere to the cells because the cells are slightly negatively
charged (anionic).
Procedure:
1. Cover the smear with methylene blue for one minute over the sink or appropriate container.
2. Wash the stain briefly (2 seconds) with the distilled water in the wash bottle. Do not squirt the water
directly on the specimen! Let the water flow over the smear.
3. Blot the stain carefully with KimWipes. Be gentle. Dry the bottom of the slide.
4. Put a coverslip over the specimen on the slide.
5. View the specimen under the microscope.
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Name____________________________
Section_________________
Introductory Microbiology Lab Report
Isolation of an Antibiotic Producer from Soil
1. (1pt) How are Streptomyces colonies recognized on agar plates?__________________
________________________________________________________________________
________________________________________________________________________
2. (1pt) Evidence of antibiosis would be observed if Staphylococcus epidermidis growth
is ________________________________________________.
3. (1pt) Describe the procedure used to detect evidence of antibiosis.
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
4. (1pt) Results: Evidence of Antibiosis
Plate #
Antibiosis (+ or -)
1
2
3
4
5. (1pt) Microscopic observations under 100X magnification after smear and simple stain.
Soil isolate
Choose a soil
isolate
sample that
was positive
for antibiosis.
Control
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Streptomyces sp.
Student Microscope Checklist
When finished using the microscope, complete the following.
Check off each step when completed.
Name:
Date:
Lab Section:
Microscope Number: HCC
Bring the stage to its lowest level.
Click the 4X objective into place.
Remove the slide and dispose of appropriately. Wipe prepared slides until ALL oil/dirt
is removed (top and bottom).
Clean objective lenses with swabs and liquid lens cleaner. Dry each objective after using
the liquid.
Clean eyepieces with dry swabs.
Clean condenser lens with dry swabs.
If necessary, clean the stage with a damp Kimwipe.
Turn the light switch OFF. Do not move the microscope for a few minutes
before putting it away.
Fold the cord and place it in the appropriate drawer.
Replace the dust cover.
Return the microscope to the appropriate location in the cabinet.
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