FAR222: STERILITY TEST

Students Info:

This is a practical to test on your ability to conform to Standard Aseptic

Technique procedures. There is NO NEED to submit a written report for this
practical class.

Discard all USED syringes/needles and Broken Ampoules into the YELOW
BIOHAZARD BINS.

Before you proceed, wipe your working area with alcohol. Make sure that the
BUNSEN BURNER IS OFF while you are swabbing the working bench with alcohol.

Each Student will be supplied with:

1 Vial
1 Ampoule
1 tube of Unknown Sample
8 Sterile test tubes
3 Plate agar

EXPERIMENT 1: Sterility Test for Vial Sample

1. For this experiment, you are given: 4 sterile test tubes, 1 plate agar and 1
syringe with needle.
2. Using a sterile pipette, aseptically dispense 5 ml. of Nutrient Broth to each of
the test tube. Label the tubes as No.1, 2, 3 and 4.
3. Using a forceps, remove the aluminium foil from the vial.
4. Swab the rubber cap of your vial with alcohol.
5. Invert the vial and using a sterile syringe/needle, draw out 5 ml. of the vials
content.
6. Transfer this drawn-out sample to test tube No 1 (as illustrated in the picture
below). Mix gently.
7. Using a sterile pipette, transfer 5 ml of the broth/sample mixture from test tube
No. 1 to test tube No. 2. Repeat the dilution for test tube No. 3 and No. 4.
8. Take an agar plate and divide the plate into 4 sectors, mark each quadrant as
1, 2, 3 and 4.
9. Using a sterile wire loop, take a loopful of the dilution and streak on each
quadrant correspondingly (i.e. Quadrant no 1 for sample from test tube 1, etc.)
 10. Incubate your sample at 37oC, overnight (this includes all the Serial Dilution
test tubes and your inoculated plate agar).

5 ml 5 ml
5 ml 5 ml

1 2 3 4

EXPERIMENT 2: Sterility Test for Ampoule Sample

1. For this experiment, you are given: 2 sterile test tubes, 1 plate agar and 1
syringe with needle.
2. Using a sterile pipette, aseptically dispense 8 ml. of Nutrient Broth to each of
the test tube. Label the tubes as No.1 and 2.
3. Before you proceed to breaking open the glass ampoule, wash your hands
thoroughly first.
4. If there is liquid at the upper tip of the ampoule (above the breakage line),
gently tap the ampoule with your finger to get all the liquid to the bottom part
of the ampoule.
5. Protect your hands from the broken glass by using paper towel or cotton wool
when opening the ampoule.
6. To open the ampoule, hold it with both hands, with one thumb against the
narrow top section. Hold the bottom of the ampoule firmly while pushing the
top section away from you with easy, even pressure (A light pressure should
cleanly snap the ampoule open, while using too much force can cause it to
shatter).
7. Using a sterile syringe/needle, transfer 2 ml of the ampoule contents into test
tube No. 1. Allow the mixture to mix well, and transfer 2 ml of the mixture from
test tube No. 1 into test tube No. 2.
8. Take an agar plate and divide the plate into 2 sectors, mark each sector as 1
and 2.
9. Using a sterile wire loop, take a loopful of the dilution and streak on each
sector correspondingly.
 10. Incubate your sample at 37oC, overnight (this includes both the Serial Dilution
test tubes and your inoculated plate agar).

EXPERIMENT 3: Sterility Test for Unknown Sample

1. For this experiment, you are given: 2 sterile test tubes and 1 plate agar.
2. Using a sterile pipette, aseptically dispense 5 ml. of Nutrient Broth to each of
the test tube. Label the tubes as No.1 and 2.
3. Using a sterile pipette, transfer 5 ml of Unknown sample to test tube No. 1.
Mix gently.
4. Subsequently, using a sterile pipette, transfer 5 ml of the broth/sample mixture
from test tube No. 1 to test tube No. 2.
5. Take an agar plate and divide the plate into 2 sectors, mark each sector as 1
and 2.
6. Using a sterile wire loop, take a loopful of the dilution and streak on each
sector correspondingly.
7. Incubate your sample at 37oC, overnight (this includes both the Serial Dilution
test tubes and your inoculated plate agar).

BEFORE LEAVING THE LAB:

Arrange your test tubes on the racks provided in this order:
4 (VIALS) 2 (AMPOULE) 2 (UNKNOWN)
3 plates, inverted

DO NOT FORGET TO WRITE YOUR NAME, AND MAKE SURE ALL THE TEST TUBES
AND PLATES ARE CORRECTLY IDENTIFIED. YOU MUST BE PUNCTUAL FOR THE LAB
SESSIONS. LATE COMERS WILL NOT BE ENTERTAINED!!!!