University of Pittsburgh at Bradford
Science In Motion
Biology Lab 017
Introduction to Microbiology
Safety Notes:
1. Students should were safety glasses at all times.
2. Students must wash hand before and after completion of lab.
Introduction:
Whenever, you work with microorganisms you should treat them as if they were disease causing
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.
Materials:
Disinfectant
Paper towels
Hand soap
Petri dishes (2)
Culture tubes with caps (4)
Distilled water
Wire loop
Alcohol burner
Glow Germ Kit (optional)
Procedure:
1. Disinfect the lab area.
a. Place a small amount of disinfectant on the lab table top. Use a paper towel to spread the
disinfectant over the entire table top.
b. Be sure to leave the table slightly wet and allow it to air dry.
2. Wash your hands.
a.
b.
c.
d.
Turn on water.
Crank out an appropriated amount of paper towel.
Use soap and lather your hands. Scrub both the front and back surfaces.
Tear off the paper towels and dry your hands. If you need more paper towels, use the towel
you already have to cranks out more. DO NOT TOUCH THE HANDLES WITH YOUR
BARE HANDS. Use the towel to turn off the water.
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3. Practice transferring the microorganisms.
a. Tube to Tube
□ Gently shake the tube containing the culture from side to side to evenly distribute
the microorganisms.
□ Hold both tubes in the left hand. Use the thumb and index finger of the right hand
to hold the wire loop.
□ With the last two fingers of the right hand, remove the caps from both tubes. DO
NOT SET THE CAPS DOWN.
□ Use an alcohol burner to flame the wire loop and the open the end of both tubes.
□ Dip the wire loop into the tube containing the culture then into the new tube. Shake
the wire loop to be sure that the microorganisms are washed off.
□ Flame the open ends of both tubes and replace the caps. Place the tubes into the
test tube rack. Flame the loop and set it aside.
b. Tube to Petri Dish
□ Shake the tube containing the culture from side to side to evenly disperse the
culture.
□ Hold the culture tube in the left hand and the wire loop in the right hand.
□ Remove the cap with the last two fingers of the right hand. DO NOT SET THE
CAP DOWN.
□ Flame the wire loop and the open end of the tube.
□ Dip the wire loop into the tube and gently remove it.
□ Flame the open end of the tube and put the cap back on. Set the tube into the rack.
□ Tilt the lid of the Petri dish to open it just enough to insert the wire loop.
□ Rube the wire loop back and forth over the surface of the agar. Be careful not to
dig holes into the agar.
□ Close the Petri dish.
□ Flame the loop and set it aside.
c. Petri Dish to Tube
□ Flame the wire loop. Open the Petri dish just enough to insert the wire loop.
□ Select a colony on the surface of the agar and use the wire loop to scrape up a small
amount from the colony.
□ Close the Petri dish and set is aside. DO NOT SET THE LOOP DOWN.
□ Pick up the tube with your left hand. Remove the cap with the last two fingers of
your right hand. Flame the open end of the tube.
□ Insert the loop into the tube and shake the loop to wash off the microorganisms.
□ Remove the loop and flame the open end of the tube. Replace the cap and set the
tube into the test tube rack.
□ Flame the wire loop and set it aside.
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d. Petri Dish to Petri Dish
□ Flame the loop. Open the Petri dish and select a colony. Use the loop to scrape up a
colony.
□ Open the new dish just enough to insert the wire loop and rub the wire loop back
and forth over the surface of the agar. Be careful not to dig holes into the agar.
Close the dish.
□ Flame the loop and set it aside.
4. Disinfect the lab area.
5. 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 and then passed through a flame. The flame kills the bacteria and fixes 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
Microscope slides
Alcohol burner
Matches
Wire loop
Sharpie
Lens paper
Test tube rack
Forceps
Bacterial cultures (may vary)
Pseudomonas aeruginosa
Micrococcus luteus
Escherichia coli
Paper towels
Hand soap
Culture tube
Distilled water bottle
Procedure:
1. Thoroughly clean a slide with soap and warm water. FROM THIS POINT ON HANDLE THE
SLIDE ONLY BY ITS EDGES.
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2. Use the marker to MARK THE INITIALS of the bacteria you are using on the left end of the
slide.
3. Transfer bacteria to slides
a. Bacteria from agar on a Petri dish or slant
□ Place a small drop of distilled water onto the center of the slide.
□ Flame the wire needle and use it to pick up a small amount of bacteria.
□ Completely mix the bacteria into the drop of water and spread the mixture out over the
center of the slide making a circle approximately ½ inch in diameter.
□ Flame the wire loop and set it aside.
b. Bacteria from a broth culture
□ Gently agitate the culture to mix the bacteria throughout the broth.
□ Flame a wire loop and use it to pick up some of the broth; place a small drop of the
culture in the center of the slide.
□ Flame the loop a second time and transfer a second drop to the same spot on the slide.
□ Use the loop to spread the broth out over the center of the slide making a circle about ½
inch in diameter.
□ Flame the loop and set aside.
□ Allow the slide to air dry completely.
□ When you are sure the slides is completely dry, hold the end of the slide with a pair of
forceps and pass the slide over a alcohol burner flame to kill the bacteria. This will
also fix the bacteria to the slide so that they will not wash off.
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. Stain adds 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 (100x) 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.
Objectives:
1. Students will stain the bacteria so that they may be seen with a light microscope.
2. Students will observe bacterial cells by using the oil immersion lens on their microscopes.
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Materials:
Bibulous paper
Wooden Sticks
Staining Trays
Distilled water
Compound microscope
Methylene blue stain
Your bacteria smear slide (day 2)
Immersion oil
Lens paper
Procedure:
1. Place the bacterial slide on a wooden stick bridge over the staining tray.
2. Cover the smear with methylene blue for ONE MINUTE THEN GENTLY RINSE the slide
with distilled water.
3. Use bibulous paper to CAREFULLY BLOT THE WATER OFF THE SLIDE; DO NOT
WIPE THE SLIDE.
4. Using LOW POWER, focus on the initials you placed on the slide. Then switch to high power
and refocus using the fine adjustment.
5. Reposition the slide so that the smear is in the center of the field of view.
6. 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.
7. 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.
8. USE THE FINE ADUJUSTMENT ONLY to get the bacteria into sharp focus and observe as
much detail as you can.
9. 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 OF THE OIL OFF OF THE
IMMERSION LENS.
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Name_____________________________
Date______________________________
Introduction to Microbiology
Student Evaluation:
Observations:
1. Make a drawing that shows what your bacterial cells looked like when magnified.
Pseudomonas aeruginosa
Micrococcus luteus
Escherichia coli
Analysis/Conclusion:
1. List several techniques that allow you to be able to handle bacteria safely.
2. Why must the neck of the culture tube be flamed when you are transferring microorganisms?
3. Give two reason for heating the slide after the smear is air dried.
4. Why do bacteria need to be stained?
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