Download Aseptic Technique,_Smear Prep_Simple Stain

Wilkes Science in Motion
Revised 2014
Aseptic Technique,_Smear Prep_Simple Stain
PA Standards:
PA S. T. & E.:
3.1.B.A1. Compare and contrast the cellular structure and degrees of complexity of prokaryotic
and eukaryotic organisms.
Day 1- Aseptic Techniques
Introduction:
Whenever you work with microorganisms, you should treat them as if they were diseasecausing pathogens. In order to protect your materials from contamination and yourself from
possible infection, there are certain procedures that should always be followed. In this portion of
the investigation, you will learn and practice some of these aseptic techniques. These techniques
will be practiced without the use of microorganisms.
Objectives:
1. Students will learn how to handle bacteria safely and correctly make and stain bacterial slides.
Safety Notes:
1. Disinfect tabletops at the beginning of each lab session. Be sure to leave the table
slightly wet with the disinfectant and allow it to air dry.
2. Each student must disinfect his hands by washing with antibiotic soap for at least
20 seconds and dry with a paper towel directly from a dispenser.
3. Do not eat, drink, or store food in the laboratory.
4. Bring only necessary equipment and the laboratory manual to the work area.
Leave books and personal items in another part of the room.
5. If available, wear a protective lab coat or apron when you are working with
cultures, and roll up long sleeves before washing hands if possible.
6. Tie back long hair or pull it up.
7. Wear safety glasses when they are recommended in a laboratory procedure.
8. Carry or store cultures of microorganisms in racks or baskets. Do not leave
cultures on the table or in unmarked areas when the laboratory session is
completed.
9. Place cultures to be discarded in racks or trays designated for contaminated
material; these racks should be clearly labeled. All such materials should be
autoclaved before further handling, discarding, or washing. This will be done by Wilkes
University personnel.
10. In case of spills notify the instructor or laboratory assistant. Cover small spills
with paper towels and soak the towels well with disinfectant. Let the towels stand
for half an hour. Carefully pick them up with a broom and dustpan or with clean
towels. Place the materials immediately in a container with disinfectant or in a
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biohazard bag to be autoclaved at Wilkes.
11. Place inoculating loops or swabs in a biohazard bag which will be autoclaved.
12. Instructors should demonstrate proper laboratory techniques at the beginning of
the lab period.
13. Report any laboratory accident to the instructors so that proper procedures may be
followed. Cuts should be treated promptly. Open cuts should be protected when
working in the laboratory.
14. Individuals who have special health problems should be especially careful in
following these procedures and any special precautions recommended by their
physicians.
15. Decontaminate work surfaces after spills and at the end of each lab with
disinfectant.
16. Wash your hands before leaving the laboratory.
Materials: (per group of 2 students)
Test Tube Rack
Disinfectant
Petri dishes with agar - 2
Paper towels
Culture tubes with sterile broth-2
Sterile plastic inoculating loops- 4
Disinfectant hand soap
Distilled water- squirt bottle
Petri Plates containing live cultures- E. coli and M. luteus
Culture Tubes containing live cultures- E. coli and M. luteus
Incubator set at 37 degrees Celsius/ Secure area for Petri plates and Tubes at 25 degrees Celsius
Procedure:
1. Disinfect the lab area.
a. Place a small amount of disinfectant on the lab tabletop. Use a paper towel to spread
the disinfectant over the entire tabletop.
b. Be sure to leave the table slightly wet with the disinfectant and allow it to air dry.
2. Wash your hands.
1. Turn on the water.
2. Crank out an appropriate amount of paper towels.
3. Lather your hands thoroughly and scrub both the front and back surfaces.
4. Tear off the paper towel and dry your hands. If additional paper is needed, use
this paper towel to grasp the dispenser handle and crank out additional paper. If
you touch the handle with your bare hands you may recontaminate your hands
with microorganisms.
5. Use the towel to turn off the water.
3. Practice transferring microorganisms: (See Page 3). Each group member should do one
species of bacteria per procedure and have a chance to work with both species.
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Tube to Tube:
a. Shake the tube containing the culture from side to side to evenly distribute the
microorganisms and obtain a sterile plastic inoculating loop.
b. Hold both tubes in the left hand. Use the thumb and index finger of the right hand to
hold the loop; take care not to touch the loop or put it on the table top.
c. With the last two fingers of the right hand, remove the caps from both tubes. DO NOT
SET THE CAPS DOWN. Work quickly to avoid contamination from the air.
e. Dip the loop into the tube containing the culture then into the new tube. Shake the
loop to be sure that the microorganisms are washed off.
f. Remove the loop. Replace the cap- tighten the cap fully, then loosen by one turn and
return the tube to the rack.
g. Place the used loops into biohazard bag.
h. Incubate the tubes overnight at 37 degrees Celsius for (E. coli) or 25 degrees Celsius
(M. luteus).
i. In 24 hours you should see cloudiness in both tubes indicating bacterial growth. The
M. luteus may have a yellow tint and E.coli broth will remain the same color. It is
possible another 24-48 hours may be necessary for M. luteus growth.
Tube to Petri Dish:
a. Obtain a sterile plastic inoculating loop. Shake the tube containing the culture from
side to side to evenly disperse the culture.
b. Hold the culture tube in the left hand and the sterile loop in the right hand.
c. Remove the cap with the last two fingers of the right hand. Do not set the cap down.
e. Dip the loop into the tube and gently remove it.
f. Quickly put the cap back on the tube. Set the tube into the rack.
g. Tilt the lid of the Petri dish to open it just enough to insert the loop.
Diagram Shows Steps h to k below
h. Streak the loop containing the bacteria at the top end of the agar plate moving in a zigzag horizontal pattern until 1/3 of the plate is covered. (A)Be careful not to dig the
loop into the agar.
i. Rotate the plate about 60 degrees and spread the bacteria from the end of the first
streak into a second area using the same motion in step. (B)
j. Rotate the plate about 60 degrees and spread the bacteria from the end of the second
streak into a new area in the same pattern. (C)
k. Finally, draw a quick, squiggly streak in last quadrant. (D)
l. Place used loop in biohazard bag.
m. Replace the lid and invert the plate. Incubate the plate overnight at 37 degrees Celsius
for (E. coli) or 25 degrees Celsius (M. luteus).
n. In 24 hours you should see bacterial cells growing in streaks and in isolated areas. The
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E. Coli should appear as a translucent cream color, the M. luteus should appear bright
yellow. It is possible another 24-48 hours may be necessary for M. luteus growth.
Petri Dish to Tube:
a. Obtain a sterile plastic loop. Open the Petri dish just enough to insert the loop.
b. Select a colony or small area on the surface of the agar and use the loop to scrape a
small amount of bacteria into the loop; take care not to dig holes in the surface of
the agar.
c. Close the Petri dish and set it aside. DO NOT SET THE LOOP DOWN.
d. Pick up the tube with your left hand. Remove the cap with the last two fingers of your
right hand.
e. Insert the loop into the tube and shake the loop to wash off the microorganisms.
f. Remove the loop. Replace the cap- tighten the cap fully, then loosen by one turn and
return the tube to the rack.
g. Incubate the tube overnight at 37 degrees Celsius for (E. coli) or 25 degrees Celsius
(M. luteus).
h. Place the used loop into the biohazard bag.
i. In 24 hours you should see cloudiness in both tubes indicating bacterial growth. The
M. luteus may have a yellow tint and E.coli broth will remain the same color. It is
possible another 24-48 hours may be necessary for M. luteus growth.
Petri Dish to Petri Dish:
a. Obtain a sterile plastic loop. Open the Petri dish and select a colony. Use the loop to
scrape a small amount from one colony.
b. Open the new dish just enough to insert the loop and rub it back and forth over the
surface of the agar following the same steps as h to n under Tube to Petri Dish.
4. Disinfect the lab area, and wash your hands.
Day 2 - Smear Preparation Introduction
The successful staining of bacteria depends upon the preparation of a smear of the bacteria on a
clean, dry microscope slide. As the name implies, a small amount of bacteria are smeared onto a
slide, allowed to air dry. A warming tray is then used to fix them to the slide. If the smear is done
properly, the bacteria will remain on the slide through the application and washing off of a
staining agent or agents.
Objectives:
1. The students will safely transfer microorganisms from a test tube to a glass slide, fix those
bacteria to that slide, and kill the bacteria.
Materials:
Disinfectant
Wax pencil or permanent marker Bacterial cultures: (Tube or plate)
Microscope slides
Test tube rack
E. coli, M. luteus
Slide warmer
Paper towels
Sterile plastic loop
Hand soap
Distilled water bottle
Forceps or clothes pin
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Procedure:
1. Use a marker to mark the initials of the bacteria you are using on the left end of the slide in
the frosted glass area.
2. Practice transferring microorganisms.
Bacteria from agar on a petri dish:
1. Place a small drop or distilled water onto the center of the slide.
2. Use a sterile loop to pick up a small amount of bacteria.
3. Completely mix the bacteria into the drop of water and spread the mixture over the center of
the slide making a circle approximately 1 cm. in diameter.
4. Place the used loop in the biohazard bag.
5. Allow the slide to air dry completely.
6. When you are sure the slides is completely dry, hold the end of the slide with a pair of
forceps or clothes pin and allow the slide to sit on a heated warming tray for 20 seconds to
kill the bacteria. This will also fix the bacteria to the slide so that they will not wash away
when stained, rinsed, or oil is applied.
Bacteria from a broth culture:
1. Gently agitate the culture to mix the bacteria throughout the broth.
2. Use a sterile loop to obtain some of the broth; place a small drop of the culture in the
center of the slide.
3. Using a new sterile loop, transfer a second drop to the same spot on the slide.
4. Use the loop to spread the broth over the center of the slide making a circle about 1 cm.
in diameter.
5. Place the used loops in the biohazard bag.
6. Allow the slide to air dry completely.
7. When you are sure the slides is completely dry, hold the end of the slide with a pair of
forceps or clothes pin and allow the slide to sit on a heated warming tray for 20 seconds
to kill the bacteria. This will also fix the bacteria to the slide so that they will not wash
DAY 3 - Simple Stain
Introduction:
In order for bacteria to be observed, they must be stained. There are many different
staining techniques that are used to stain bacteria. Stains add color to the entire bacterial cell or
to specialized parts of the cell. In this portion of the lab activity, you will use methylene blue to
stain the bacteria selected.
Because of their size, you will not be able to see bacteria with the high power objective
on your microscope. Therefore, you will need to use the oil immersion lens (l00x) to view cells
clearly. It is a good idea to practice using this lens with a prepared slide before attempting to
view your bacterial slide. Samples have been provided.
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Bacteria have 3 basic shapes- coccus (cocci)- round, bacillus (bacilli)- rod, and spirillum
(spirilla)- corkscrew. Also, the prefix diplo means two, strepto- a chain, and staphylo- a cluster.
Objectives: Students will:
1. Observe, sketch, and color a prepared bacterial slide.
2. Stain the bacteria slide so that they may be seen with a compound light microscope,
3. Observe bacterial cells by using the oil immersion lens on their microscopes and sketch.
4. Determine the shape of the bacteria they have observed.
Materials:
Bibulous paper
Methylene blue stain
Distilled water
Clothes pin
Roasting pan
lens paper
your bacterial smear slide from day 2
Immersion oil
Compound microscope
Prepared stained slides of 3 bacterial
shapes
Procedure:
Observing prepared slide of Bacteria:
1. There are 3 different circles representing the 3 basic bacterial shapes. Using low power, focus
one circle of the bacteria slide with organisms (stains evident). Then switch to high power
and refocus using the fine adjustment.
2. Reposition the slide so that the smear is in the center of the field of view.
3. Turn the nosepiece so that the high power lens is out of the way; add a drop of immersion oil
directly onto the smear; turn the nosepiece so that the oil immersion objective lens is in
place.
4. When you look from the side of the microscope, you should notice that the oil fills the
narrow gap between the slide and the objective lens.
5. Use the fine adjustment only to get the bacteria into sharp focus and observe as much detail
as you can.
6. Sketch, label, and color what you observe on the Data sheet using colored pencils.
Staining Your Slide from Day 2:
1. Hold the bacterial slide over a roasting pan with a wooden clothes pin
2. Cover the smear with methylene blue for one minute then gently rinse the slide with distilled
water.
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3. Use bibulous paper to carefully blot the water off the slide; DO NOT WIPE THE SLIDE.
5. Using low power, focus on the initials you place on the slide. Then switch to high power and
refocus using the fine adjustment.
6. Reposition the slide so that the smear is in the center of the field of view.
7. Turn the nosepiece so that the high power lens is out of the way; add a drop of immersion oil
directly onto the smear; turn the nosepiece so that the oil immersion objective lens is in
place.
8. When you look from the side of the microscope, you should notice that the oil fills the
narrow gap between the slide and the objective' lens.
9. Use the fine adjustment only to get the bacteria into sharp focus and observe as much detail
as you can.
10. Sketch and color what you observe on the Student Data Sheet.
11. When your observations are completed, turn the nosepiece to move the lens out of the way,
remove the slide and use lens paper to clean all the oil off the oil immersion lens.
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Student Data Sheet
Name ________________________ Date ____________
Observations:
1. Make a colored drawing that shows your prepared slide of bacterial cells when magnified
1000 times. Label the shape of bacteria you observed (cocci, bacillus, or spirillum).
Shape: ___________________
2. Make a colored drawing that shows your prepared slide of bacterial cells when magnified
1000 times. Label the species and shape (cocci, bacillus, or spirillum) of bacteria you
have observed.
Species: ______________________
Shape: _______________________
3. If you have time, make a drawing that shows your partner’s prepared slide of bacterial
cells when magnified 1000 times. Label the species and shape (cocci, bacillus, or
spirillum) of bacteria you have observed.
Species: ______________________
Shape: _______________________
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Analysis/Conclusions:
1. List at least three procedures you followed that allowed you to to handle bacteria safely.
2. Why must sterile loops be used and the tops quickly replaced on tubes and Petri dishes?
3. Why did you heat the slide on the warming tray after the smear was air-dried?
4.Why do bacteria need to be stained?
5.What are important observations that can be made from your study of bacterial slides under
an oil lens at 1000 X magnification?
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