Name:
Section:
Introductory Microbiology Prelab Assignment
Pure Cultures and Aseptic Technique
1. (1pt.) List two aseptic techniques that will be performed in this lab.
2. (0.5pt.) How is a pure culture different than a mixed culture?
3. (1pt.) List the three bacterial species that will be used in this lab and the color of their colonies.
ALWAYS correctly write scientific names.
4. (1pt.) Define subculturing.
5. (1pt.) What does aseptic technique prevent?
6. (0.5pt.) The nutrient agar deep should not be melted before pouring into the petri plate during the pour plate
method.
a. True
b. False
NOTE: BRING YOUR MICROSCOPY LAB EXERCISE AND THE WIDESPREAD DISTRIBUTION
EXERCISE WITH YOU TO THIS LAB. YOU WILL NEED THESE FOR MICROSCOPE
INSTRUCTIONS AND INSTRUCTIONS FOR STAINING THE BACTERIA ON DAY 3 OF THIS
LAB.
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Lab Exercise
Pure Cultures and Aseptic Technique
Feb. 6, 2015
Skills in aseptic technique will, in part, determine the successful completion of the laboratory portion of this course.
Those entering health related fields undoubtedly were introduced previously to aseptic technique. Aseptic technique includes
all procedures that prevent contamination. In this exercise, students will practice these skills. In addition, pure cultures (one
species present) will be prepared from a mixed culture (contains more than one species) using the process of subculturing
(transferring bacteria from one culture to fresh media). Three techniques will be performed: the pour plate, the spread plate,
and the quadrant streak. These techniques allow single colonies to be separated in or on the agar.
Procedure: Pour Plate Method
The cultures must be diluted (decreasing the number of cells/ml) before pouring the plates. This allows isolated colonies on
the agar to be obtained.
Day 1
Materials:
Group of 4 students (1/table): 1 tube of mixed culture
Group of 2 students:
test tube rack
2 TSA tube deeps
2 blank tubes containing 9.9ml of 0.9% sterile NaCl (saline)
bunsen burner and striker
1 500 ml beaker half-filled with water
test tube tong
safety glasses
1 pipet pump
3 1.0 ml pipets
2 empty, sterile petri plates
hot plate
tape
1. Place the agar tubes in the beaker. Heat until the agar is completely melted (it will appear transparent), then cool until the
tubes can be held comfortably. Complete steps 2-6 while the tubes are heating and cooling.
*Do not remove the lids of the petri plates until required.
2. Label one sterile petri plate "0.001 pour plate". This plate will contain a culture diluted 1000X. There will be 1/1000 (0.001
or 10-3) the number of cells per ml on this plate as are in the original mixed culture. Also be sure to label all plates with your
initials and lab section.
3. Label the other sterile petri plate "0.00001 pour plate". This plate will contain a culture diluted 100,000X. There will be
1/
100,000
(0.00001 or 10-5) the number of cells per ml on this plate as are in the original mixed culture.
4. To disperse the bacterial cells uniformly, vortex or swirl the tube containing the mixed culture. Remove the
cap (*do not set the cap down), and *flame the neck of the tube.
*aseptic technique
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Figure 1. Dilution and Plating Procedure: Pour Plates
Transfer 0.1
ml of mixed
culture into
saline tube
#1.
Transfer 0.1 ml from
saline tube #1 into
saline tube #2.
#1
9.9 ml saline
Pipet 0.1 ml
into center
of 0.001
plate.
#2
9.9 ml saline
Pipet 0.1 ml into
center of 0.00001
plate.
0.001 pour plate
0.00001 pour plate
5. (Fig. 1) Pipet 0.1ml of the mixed culture into a tube containing 9.9ml of saline. *Flame the neck of the saline
tube after removing the cap and before replacing the cap. *Do not set the saline tube cap down. *Flame the neck
of the mixed culture tube. Replace the cap. Label this saline tube “#1”.
Note: The pipet will remove 0.1ml of liquid when the liquid has reached the 0.9ml demarcation on the pipet, not
the 0.1ml demarcation!!
6. (Fig. 1) Vortex or swirl the saline tube labeled "#1". Pipet 0.1ml from tube "#1" into a second tube of saline.
*Flame the neck of all tubes after removing the caps and before replacing the caps. Label the second tube of
saline "#2".
7. (Fig. 1) After the agar has cooled sufficiently, vortex or swirl the tube labeled "#1". Pipet 0.1ml from the tube
labeled "#1" into the center of the plate labeled "0.001 pour plate". *Flame the neck of the tube after removing
and before replacing the cap. *Do not set the lid of the petri plate down.
8. (Fig. 1) Pour one tube of the agar into the plate labeled "0.001 pour plate". The agar still should be completed
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melted with no lumps. *Do not set the lid of the petri plate down.
9. Replace the petri plate lid. Gently swirl the agar while keeping the plate flat on the bench. Do not splash any
agar out of the plate. Set aside to harden (15-30 min.).
10. Gently, vortex or swirl the tube labeled "#2" to disperse the organisms.
11. Remove the cap of the tube, and *flame the neck of the tube. *Do not set the cap of the tube down.
12. Pipet 0.1ml from this tube into the center of the petri plate labeled "0.00001 pour plate". *Do not set the lid
of the plate down. *Flame the neck of the tube, and replace the cap.
13. Pour another tube of melted agar into the "0.00001 pour plate", and again swirl gently while keeping the plate
flat on the bench. Set aside until solidified.
14. After the plates have solidified, incubate inverted at 28C incubator for 48 hours.
Day 2
Compare the number of colonies found in each plate. Where are the colonies located?
You should observe 3 types of colonies. Each type has a different color and is a different species. The three
species of bacteria are:
Escherichia coli: white colonies
Serratia marcescens: red/orange colonies
Micrococcus luteus: yellow colonies
Why might you only find one or two species?
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Procedure: Spread Plate Technique (Day 1) and Quadrant Streak Plate Method (Day 2)
Day 1
Materials: group of 2 students
1 1.0ml pipet and 1 pipet pump
1 bactispreader
2 tubes of saline (use the same tubes from the pour plate experiment)
2 TSA plates
bunsen burner and striker
1. Label one agar plate "0.001 spread plate". Label the other agar plate "0.00001 spread plate".
2. Vortex or swirl the saline tube labeled "#1" to gently to disperse the organisms.
3. Remove the cap from the tube. *DO NOT SET THE CAP DOWN! *Flame the neck of the tube.
4. Using a pipet (*which has not been set down), remove 0.1ml of liquid from the "#1" tube, and place it on the
center of the agar plate labeled "0.001 spread plate". Do not gouge the agar. *DO NOT SET THE LID OF THE
PLATE DOWN! *Only take the lid off of the petri plate when required. Replace the lid as soon as possible.
5. *Flame the neck of the tube, and replace the cap.
6. A bactispreader is used during the spread plate technique. *Flame the bactispreader for a few seconds (don't
wait until it is red-hot).
*DO NOT SET THE BACTISPREADER DOWN!
7. Cool the bactispreader ~15 seconds.
8. Using the bactispreader, spread the liquid around the top of the agar gently until the top of the agar is
evenly covered.
9. Replace the lid on the petri plate. *Flame the bactispreader again.
10. Invert the plate, and incubate for 48 hours at 28C.
11. Repeat the procedure with the "#2" tube and the "0.00001 spread plate".
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Day 2: Quadrant streak
The purpose of the quadrant streak is to decrease the amount of bacteria on the agar until single, isolated
colonies can be established.
Materials: group of 2 students
3 TSA plates
1 inoculating loop
plates from Day 1
bunsen burner & striker
1. Observe the plates. Ideally there should be colonies of three different colors. Answer the questions in the lab
report concerning the spread and pour plates.
2. You will subculture each species of bacteria on a separate plate. Subculturing is the process of inoculating
fresh media with a bacterial culture from another source.
1) *Flame your loop just until it is red-hot. Cool for 15 seconds. *DO NOT SET THE LOOP
DOWN WHILE YOU ARE WAITING FOR IT TO COOL.
2) Lightly touch the center of ONE colony of a specific color with the loop. *DO NOT SET THE LID
OF THE PETRI PLATE DOWN. *ONLY OPEN THE PLATE WHEN YOU NEED TO.
3) After touching a colony with the loop, replace the lid.
4) Streak a sterile nutrient agar plate using the quadrant streak method illustrated in Figure 2.
5) *Flame your loop and cool. Touch another colony of a different color from the first. Repeat the
quadrant streak method on a new, sterile nutrient agar plate.
6) Repeat the above procedure for the last colony type.
7) Incubate all plates, inverted, at 28C for 48 hours.
Figure 2. Quadrant Streak for Isolation of Single Colonies
The loop must be flamed before each subsequent streak. DO NOT OBTAIN MORE
BACTERIA BETWEEN STREAKS! Flame the loop when finished.
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Day 3
Observe the plates. There should be one type of bacteria on each plate (pure cultures).
Observe the number of colonies in EACH streaked area (Area 1 through Area 4).
The quadrant streak also may be used after the pour plate technique to obtain pure cultures.
Simple stain (remember to smear first) your bacteria and view under the microscope.
EXAMPLES OF ASEPTIC TECHNIQUE
1. Only open plates and tubes when necessary. Replace petri plate lids and tube caps as soon as possible. It is best
only to lift part of the petri plate lid rather than removing the entire lid.
2. Never put a petri plate lid or tube cap on the lab bench. You must hold them at all times.
3. Flame the neck of the tube immediately after removing the cap and before replacing the cap.
4. Flame loops and needles until they are red hot before and after each use. Do not leave the loop in the flame after
it becomes red-hot. This will weaken the metal. Flame loops and needles up to the handle. Do not set loops and
needles down before use. You must hold them at all times.
5. Flame bactispreaders (but not until they are red hot!) before and after use. Do not set bactispreaders down
before use. You must hold them at all times.
6. Wash hands before and after the lab.
7. Disinfect lab benches before and after the lab.
8. Do not set pipets on the lab bench. Try to touch the pipet as little as possible. Dispose of them immediately
after use.
9. When subculturing colonies, only touch the center of the colony with the loop. This will decrease the possibility
of contamination from nearby colonies.
The above techniques are not always stated in the laboratory procedures of succeeding labs. However, all students
will be expected to routinely utilize aseptic technique at all times.
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Name:
Section:
Introductory Microbiology Lab Report
Pure Cultures and Aseptic Technique
Appearance of plates produced by quadrant streak method (sketch whole plate; label the four quadrants):
Escherichia coli
Serratia marcescens
Micrococcus luteus
Do your quadrant streak plates have isolated colonies in the 3 rd and 4th quadrants? If not, explain why.
Microscopic observations under 1000X magnification. (simple stain; draw and identify cell shape).
Escherichia coli
Serratia marcescens
Micrococcus luteus
1. (1pt.) Compare the number of colonies on the "0.001" and "0.00001" spread plates. Why did this occur?
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2. (1pt.) Describe the location of colonies using the pour plate method compared to the location of colonies after
performing the spread plate. Explain.
3. (1pt.) What special piece of equipment is used during the spread plate technique? How is it used? When do you
flame it?
4. (1pt.) During the quadrant streak method, how is the bacteria spread? When is the loop flamed? Compare the
number of colonies in each area (Area 1-Area 4) of the quadrant streak. Explain why there are differences.
5. (1pt) What is the purpose of the quadrant streak?
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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.
Clean objective lenses with swabs, and, if using oil, liquid lens cleaner.
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.
Rotate the head so that the eyepieces are facing forward (away from the arm).
Replace the dust cover.
Return the microscope to the appropriate location in the cabinet.
Check to be sure all waste is removed from the sink and floor.
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