1.1: Practicing Sterile Technique in a Vertical Laminar Flow Hood
As you proceed through this set of protocols and experiments, be sure to enter all your work into your
notebook!
Objectives
Introduction to sterile pipetting in a laminar flow hood
Improve dexterity with sterile pipetting in a laminar flow hood
Learn and adhere to safety considerations
Vocabulary
Vertical laminar flow, horizontal laminar flow, medium, media, sterile
Outline
Obtain practice solutions and vessels; transfer liquid volumes to make media without spills; transfer and
manipulate plates without spills.
Materials
sterile
0.5% Phenol red
TE, pH 8 (10mM Tris, 1mM EDTA)
175mM acetic acid (1:100 dilution from glacial acetic acid)
serological pipettes
plastic pipette tips (barrier)
50 ml conical screw cap tubes
non-sterile
37C water bath, maintained with antimicrobial additive
rack in water bath that will securely hold vials with cap above water level
rack in laminar flow hood that will securely hold cryovial
fully-stocked biosafety cabinet
kimwipes for swabbing
70% ethanol
DAY 1 (Monday)
Protocol
PREPARATION
1. Prepare the hood
a. The hood should initially be found with the sash pulled down. If it is not, close the sash.
b. If the hood is equipped with UV, you can turn on the UV with the sash closed for 30
minutes prior to initiating work in the hood.
i. UV light poses significant health risks to the investigator and cannot sterilize
crevasses or items shielded from the rays. Thus, UV sterilization should not be
used as the sole method for hood sterilization and can be completely replaced
by alternative methods.
c. Raise the sash and inspect the hood to ensure airflow is on at an acceptable flow rate.
i. Minimum flow rates to maintain laminar flow are 0.4m/s (80ft/min).
d. Clutter within the hood should be kept to a minimum and any materials should be
positioned within the hood to minimize disruption of the laminar airflow.
i. Never block the front or rear vents.
e. Using large kimwipes, swab down the work surfaces with 70% ethanol and wipe clean.
2. Collect all materials
a. Place only the required sterile items within the hood necessary for the task at hand.
b. All materials entering the hood should be sprayed and/or wiped down with 70% ethanol
to sterilize their outer surfaces.
i. Remember, your hands are on the list of items that must be sterilized every time
they exit the hood before reentry.
ii. When working with BSL 1 rated materials (such as today), gloves are not
required.
c. For today, items to be sterilized include:
i. Containers of the various stock solutions (phenol red, etc)
ii. Aspirator and corresponding pipettes
iii. Plastic pipette tip boxes and pipettors (as necessary based on your calculations)
iv. Two 10 cm plates, two 6 cm plates, a 15 ml conical screw cap tube, a 50mL
conical screw cap tube
v. Appropriate sized tube racks
LABEL THE VESSELS
3. Label all containers prior to beginning any transfers of liquids between containers.
a. After reading through the entire protocol, prelabel the plates. Include today’s date,
your initials, and your group number as well as any additional details that might be
needed in order to tell your plates apart.
b. Prelabel any sterile screw cap tubes to be used.
PREPARE THE MEDIUM
4. General guidelines:
a. Read through all steps before beginning.
b. Loosen the screw caps for the various (thawed) components, but do not remove them
from the various vessels. Be careful not to put your hands at the point where the cap
and the container meet. This is a vulnerable location for contamination.
c. Using a fresh and sterile pipette for each different item, mix each solution by gentle
swirling or by pipetting up and down a few times. Do not mix by inversion. Avoid
getting liquid up into the neck of any container.
d. Take special care to not touch the pipette tip to any unintended surfaces. Likewise, do
not touch insides of caps, edges or rims of containers with your hands.
e. If dexterity allows, do not set vessel lids down on any surface at any time. Hold the cap
until the desired volume has been removed and then immediately replace the cap on
the vessel. If you cannot manage this, place the cap open side DOWN on the sterilized
work surface of the hood. Do not rely on your partner. It is important to learn to carry
out these manipulations on your own.
f. A good rule of thumb is to pipette the largest volume first and smallest volume last.
g. After each component has been added, tighten the screw cap on the corresponding
reagent vessel.
h. After all components have been added, ensure the stock bottles are securely tightened
and then remove them from the workspace to reduce clutter.
i. Ensure the solution has been mixed to homogeneity by gently swirling the capped vessel
within the hood.
5. Aliquot 39.6ml of TE, pH 8 to a 50mL conical tube.
6. Add 0.4ml of 0.5% phenol red stock and mix gently by pipetting up and down. This is your
“medium” for these exercises. It should be red.
ADD MEDIUM TO PLATES
7. Medium plates:
8.
9.
10.
11.
12.
a. Transfer 10mL of medium to each of two 10 cm plates and 5mL to the two 6 cm plates.
i. Hold the plate lid by the edges (the angle between the top and the sides) above
the plate just enough for the pipette to dispense media into the plate.
ii. Be sure to dispense the solution from the pipette gently so as not to create any
splash. Harsh pipetting can also dislodge some cell types from the bottom of the
dish.
Distribute the medium evenly by very gently rocking the plate.
a. Be sure not to swirl the plate as this tends to concentrate cells at the edges. Rock or
slide the plate from side to side a few times. Allow the medium to come to a rest, and
then rock or slide the plate from front to back. Take special care not to spill the liquid or
to get the lids of the plates wet.
b. Transfer the plate stacks to the incubator without spilling any contents or getting the
lids of the plates wet. If you spill or get any medium on any part of the lid, start over in
order to improve your technique.
Acidification of medium plates:
a. Retrieve the 10cm and 6cm plates from the incubator.
b. Add 150uL of 175mM acetic acid to one of the 10cm plates and 75uL to one of the 6cm
plates. Mix by gently rocking the plates as described above
i. Take special care not to spill the liquid or to get the lids of the plates wet.
ii. If solutions were made correctly, the solution should appear slightly orange.
This is what plates look like when cells need fresh medium (or might be
overgrown).
iii. For perspective, domestic vinegar is typically between 0.5-1M acetic acid.
c. Add an additional 150uL of 175mM acetic acid to the 10cm plate and 75uL to the 6cm
plate. Mix by gently rocking the plates.
i. The solution should appear quite yellow. These would be plates of mostly dead
cells. Lets all hope that our real plates never look like this.
Feeding your cells:
a. Use the aspirator to remove the liquid from your plates.
i. Aspirate medium at one edge of the plate, moving the tip from the surface of
the liquid down the inner wall of the plate until almost all liquid is removed.
ii. Tipping the plate slightly will facilitate aspiration of the liquid medium.
iii. Do not touch the aspirator tip to the floor of your plates. This is where your cells
would be and doing so can pull them off the plate and into the aspirator.
iv. Do not allow the “cells” on your plates to sit without medium for more than a
couple minutes.
b. Add fresh medium to your plates dribbling the medium into the plate from an edge.
i. Do not blast the media into the center of the plate as this can dislodge cells
from the plate.
Return your plates to the 37C incubator.
Tightly cap and store your remaining medium in the 50mL conical tube. Since this is not real
medium, it can be stored at room temperature.
DAY 2 (Wednesday)
Protocol
1. Remove your plates from the incubator and inspect the color of the medium in the plates.
Compare it to the medium you stored in the 50mL conical tube.
2. When finished, aspirate and properly dispose of the materials.
Questions
1. Phenol red is a pH indicator dye that is purple at alkaline pH, red near neutrality, orange at
slightly acidic pH and yellow at acidic pH.
a. Why would medium exposed to growing cells gradually turn yellow?
b. What effect would you expect from exposing your medium to a 5% CO2 environment?
2. Why is it bad to leave your cells without medium for prolonged periods of time while in the
hood?