Procedure: Basic Lab Skills
Micropipettes
The unit of measurement is the microliter (µl).
The capacity of each micropipette is indicated by
a number usually on the top, expressed as a
min/max or just a max.
Note:
Each micropipette is engineered to measure
within a particular range of volumes. The range
is usually given on the top of the pipet. For
example 2 – 20 designates a functional range of 2
– 20 µl. Sometimes only the maximum range is
given, i.e. P200 indicates that maximum range is
200 µl. In this case, the minimum range can often
be inferred from the accompanying pipets in the
set. If a P20 is being used, one would choose to
NOTES:
use the P20 instead of the P200 to measure
accurately below 20 µl. One must be familiar
with the pipets used in the particular lab to
determine the appropriate pipet to use.
CAUTION: DO NOT SET THE
MICROPIPETTES FOR VOLUMES OUTSIDE
THEIR RANGES. THIS WILL CAUSE
MISCALIBRATION AND INACCURATE
MEASUREMENTS.
INSERT PICTURES
Setting the micropipette:
Again, the technique for setting the micropipette
varies with the manufacturer. Often there is a dial
or in the housing of the micropipette and a
window with three digits. Turning the dial rotates
the digits through cycles of ten to turn the next
unit, much like an odometer. The digits represent
different units by orders of ten depending on the
range of the micropipette. For example, on a P20
the top digit represents the 10’s, middle digit 1’s
and bottom digit 0.1’s. On the P200, the digit
values are 100’s, 10’s, and 1’s respectively.
Operation:
1. Always use a clean, disposable tip.
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2.
3.
4.
5.
6.
7.
Depress the plunger to the first position.
Insert the tip in the liquid.
Slowly release the plunger.
Withdraw the pipette from the liquid.
Insert the pipette tip in the new tube.
Slowly depress the plunger to the second
position.
8. With the plunger depressed, remove the
pipette tip from the liquid.
9. Depress the eject button to remove the
disposable tip when finished.
Activity: Use the “Micropipette Challenge” to
practice your pipette skills.
Electronic Balances
Operation:
1. Check that the balance is plugged in and
turned on.
2. Position the balance away from drafts or
vibrations.
3. Press the tare (zero) button. The display
should read 0.00.
4. Place the object on the balance.
5. Read the mass in grams on the display.
NOTES:
If a container such as a weigh boat or beaker is used
to contain a substance during measurement, be sure to
tare the empty container before adding the substance.
Activity: Measure 2 g of NaCl in a test tube. Save the
NaCl for the next activity.
Serological Pipettes
The unit of measurement for serological pipettes is
milliliters. Most serological pipettes require a pump.
Operation:
NOTES:
1. Insert the top of the pipette firmly into the
base of the pump.
2. Check the pump plunger is in the fully
depressed position.
3. Insert the tip of the pipet into a liquid.
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4. Turn the wheel of the pump clockwise to draw
the liquid up.
5. Dispense the liquid by turning the wheel
counterclockwise.
Activity: Add 10mL water to the container
containing 2 g of NaCl.
Vortex
The vortex is used to mix solutions.
Operation:
1. Set the toggle switch to touch.
2. Adjust the speed control to 5.
3. Hold the upper third of the test tube firmly
between your index finger and thumb.
4. Place the bottom of the test tube on the rubber
platform and push the test tube down firmly.
5. Hold for 5 – 10 seconds.
NOTES:
Activity: Vortex the salt solution.
Calculate the molarity of this solution?
(Molecular weight of NaCl= 58.5 grams/mole)
Molarity= Moles of solute / Liters of solution
Glassware washing
1.
2.
3.
4.
Pour the salt solution into a sink.
Scrub the test tube with a test tube brush.
Rinse the test tube 10 times with tap water.
Rinse the test tube 3 times with distilled
water.
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NOTES:
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Centrifuge
The centrifuge is used to spin samples at high speeds
resulting in a separation of components by mass.
Activity: Obtain a microcentrifuge tube of E.coli.
Centrifuge the E.coli solution to create a bacterial
pellet.
1. Check that the samples to be centrifuged are
of equal size, mass, and volume.
2. Close the cover of the tubes.
3. Place the tubes in the rotor receptacles in a
balanced configuration. Maintain an equal
space between samples. Hint: Align the tube
hinges facing out. The pellet should form on
the same side as the hinges.
4. Close the cover of the centrifuge.
5. Adjust the timer to the desired time, usually in
minutes.
Note: Setting the timer to pulse will allow you to
manually control the time of centrifugation. The
rotor will only spin while the start button is
depressed.
6. A red light will display when the rotor is
turning.
Note: if the centrifuge vibrates excessively, it is
not balanced. Turn of the centrifuge immediately
by turning the timer to 0 in timer mode or
releasing the start button in pulse mode.
7. Wait for the red light to turn off, indicating
that the rotor is stopped.
8. Open the cover and remove the samples.
Indicate the proper
balancing of a centrifuge
when you have 3
microcentrifuge tubes
Label the supernatant and
pellet in the diagram.
NOTES
Sterile Technique
We avoid using flames with students in the lab.
Therefore our sterile technique uses disposable sterile
tips and transfer loops. A general rule of thumb for
sterile technique is to breach sterile boundaries as
briefly as possible.
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Resuspension:
1. Obtain a container labeled “Bacterial Waste.”
2. Hold the tube containing the E.coli pellet at a
45 degree angle. (A 45 degree angle
minimizes exposure to particulates in the air.)
3. Open the tube and pour the supernatant into
the container labeled “Bacterial Waste.”
4. Close the tube.
5. Use a sterile pipette tip to add 200 µl of sterile
water to the E.coli pellet. Remember to hold
the tubes at 45 degrees.
6. Resuspend the E.coli pellet by gently drawing
the pellet in and out of the pipette tip until the
solution is cloudy with no visible E.coli
chunks.
7. Cap the tube and dispose of the pipette tip in
the bacterial waste container.
NOTES:
Pouring Agar
1. Lift the lid of a Petri dish just enough to admit
the neck of the bottle. Avoid touching the
bottle to any surface.
2. Pour the enough agar to cover the bottom of
each dish.
3. Cover the dish and let it sit until the agar is
hardened. (about 30 – 45 min.)
Note: cover should not be off
NOTES:
Inoculating liquid cultures
1. Obtain an agar dish with viable bacteria.
2. Obtain 1 ml of sterile Luria Broth (with
ampicillin for this exercise).
3. Obtain a packaged sterile transfer loop.
4. Open the end of the package that does not
contain the loop.
5. Remove the transfer loop, being careful to
touch only the handle.
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6. Open the cover of the agar dish with bacteria
just enough to allow a sterile loop to touch the
surface of the agar.
7. Gently drag the transfer loop across the
surface of the agar, collecting bacterial
colonies on the loop. You may see clump of
bacteria on the end of the loop. However, it is
not necessary to see the bacteria.
8. Remove the loop and insert it into the 1 ml of
Luria Broth. Remember to hold the tube at an
angle.
9. Spin the loop in the broth to release bacteria
into the solution.
10. Remove the transfer loop, close the tube, and
dispose of the loop in the bacterial waste.
Sterile Technique Activity
Check your sterile technique by transferring 100 µl of
sterile water to a sterile agar dish.
Procedure:
1. Obtain a microcentrifuge tube of sterile water.
2. Obtain a sterile Petri dish of LB agar.
3. Label the bottom of the dish “sterile water”
along with your initials and today’s date.
4. Use a micropipette with a sterile tip to transfer
100 µl of sterile water to the agar.
5. Spread the water with a sterile transfer loop
by gently moving the sterile loop across the
agar.
Agar Slant Inoculation
a. Unwrap a sterile applicator swab.
b. Use it to collect some E. coli bacteria
from the E. coli agar plate.
c. Inoculate agar slants by gently swiping
the swab on the agar. Recap the agar
slants and incubate at room temperature
for 4 days, or in a 37ºC incubator for 2
days, until bacterial colonies are visible.
Once colonies are present, the slants can
be stored in a 4oC refrigerator.
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