General Information
Gowning:
Enter the Gowning Room. Don a clean cap, making sure all hair is covered. Wash you hands and arms thoroughly to the elbow with antiseptic soap and dry with the hot air dryer. (In 104 you will use Alcare, a foamed alcohol, to sanitize your hands.) Select two shoe covers, a gown, a facemask, and a pair of sterile gloves of appropriate size. If you have a beard, a beard cover must be worn. Sit down on the bench, pull one shoe cover over your right foot, being sure your hands do not touch your shoe, and place your right foot with the covered shoe on the floor on the clean side of the bench. Repeat this with your left foot. Put your clean gown on, minimizing the touch of the outside of the gown. If your hands touch your hair or shoes, or otherwise become contaminated, rinse your hands with sterile 70% IPA. Put on your facemask, your sterile gloves and rinse your hands with sterile 70% IPA.
Environmental Tests
These tests will be performed during the lab and the colonies counted 48 hours later. Record your results.
Settling Plates:
Each location will set out two settling plates. They will be exposed for the entire duration of the lab. Please label the plates clearly with a wax pencil. Incubate the plates at 30-35 ºC and count the colonies after 48 hours of incubation.
Contact Plates: RODAC
Each student will roll the fingers of the right hand over a RODAC plate after completing their media transfers.
At the end of the lab period each location will have a surface sample done. Immediately after contact with the RODAC medium, all surfaces will be thoroughly cleaned with an alcohol saturated urethane wipe. The RODAC plates will be incubated at 30-35 C and the number of colonies counted after 48 hours of incubation. Clearly identify each plate with a wax pencil. Incubate the plates at 30-35 ºC and count the colonies after 48 hours of incubation.
Exercises
1. Student will
determine the volume of
solution held within the housing of a disposable membrane filter.
a. Withdraw slightly greater than 5 mL of solution using a 10 cc syringe and 18
g needle from an UT ampul.
b. Tap the syringe barrel with the fingers to allow air bubbles to rise to the
top, then pull the plunger back slightly to bring all the solution into the
barrel.
c. Slowly push the plunger upward to remove all the air from the needle and to
bring the solution to the exact volume desired.
d. Remove the needle from the syringe. Aseptically attach the sterile filter of
your choice and a new sterile needle to the filter outlet.
e. Carefully push the plunger upward until the first drop can be seen at the
tip of the needle. Note this volume of solution in the syringe, and subtract
the volume from the desired volume. This is the amount of solution retained in
the filter housing and the needle.
f. Inject into a LVP.
(UT NS amp, 10 cc syringe, 2-18 g needles, 0.2 micron filter disk, piggy back)
2. Hang a bottle or bag
of SWFI in the hood and spike it with an Econoset or Multiadd syringe. (Don’t forget to date, time and
initial the SWFI.) Each student to reconstitute 3- 1 g vials of ampicillin.
After reconstitution 1 g is to be injected into 50 cc NS, 2 g are to be drawn
up into 20 cc syringes and capped with syringe tips.
(1 liter SWFI - bag, Multiadd syringe, 2 vented needles, 3 - 1 g vials
ampicillin, 50 cc NS,
3 - 10 cc syringes, 3 -18 g needles)
3. Media Transfers for Aseptic Technique Validation
5 – 5 mL vials of TSB media
1 – 50 mL empty glass vial
Take 4.0 mL from each vial of media and add it to the 50 mL
vial. When adding the media to the
vial, remember to let air come back into your syringe, so you do not over
pressure the vial and cause the media to spew out. Label the 50 mL vial with your name and
date. The vial will be incubated for 7
days at 20-25 ºC, and then moved to the 30-35 ºC incubators for an additional 7
days. After the fourteen-day incubation
time the vial will be checked for growth.
Please
come and look at your vial. To
successfully complete the lab section of this class you must not have any
growth in your vial. If you do have
growth you will have a retraining session and try the Media Transfer again.
4. Simulated
cytotoxic agent at a VLF workbench.
a. Prepare the VLF workbench for use.
b. Don sterile rubber gloves.
c. Prepare the following admixture order using appropriate technique.
Investigational Drug #V 21198R, 8.0 mL
Add to 150 cc NS or D5W
Infuse over 30 minutes.
d. Reconstitute each vial of cytotoxic agent with 5.0 mL SWFI. Use a Chemo Dispensing Pin with one vial and an ordinary needle with the other, using negative pressure or a different type chemo pin.
e. Dispose of materials in specially designated plastic containers.
(prep pad, rubber gloves, 2 - cytotoxic vials, 1 chemo pin, 1 SWFI,
2 - 10 cc syringes, NS 150 mLs, 3- 18 g needles, 0.2
micron hydrophobic filter,
chemo waste containers)