Activity
1 - Instructor Teaching Strategies
TESTING PHIX174 HOST RANGE
SUGGESTED
TEACHING STRATEGIES
Level
of Difficulty: Beginner
Students
will need a printed copy of the procedure.
Inexperienced
students should first practice aseptic techniques on empty tubes before
attempting the procedure. For streaming video tips on aseptic transfers,
visit www.virology.net/Video.html, then click on "Microbiology laboratory
procedures video", and "aseptic techniques."
Make
sure students understand the solution in tube B is a1:10 dilution of the
bacteriophage stock in tube A, while tube C represents a 1:100 dilution.
For
an added challenge, students can calculate the titer of the Phi X 174 stock
solution by counting the number of plaques on a plate (ideally, they should
use the E. coli strain C plate that falls in the 30 to 300 plaques range).
The titer of the stock solution will be equal to:
PFU / ml of stock solution = Plaque Forming Units / ml of stock solution
= (the number of plaques on the plate) (fold dilution) / (0.2 ml)
For example, if a 0.2 ml of 10-fold dilution of the stock solution resulted
in 50 plaques on the plate, the stock solution titer would be equal to:
(50 plaques) (10-fold) / (0.2 ml) = 2,500 = 2.5 x 10 3 PFU/ml stock solution
TIME COMMITMENT:
Serial dilution and pour plating takes 30-45 minutes.
Counting
plaques takes 15-30 minutes.
Plates
should be taken out of the incubator after the overnight incubation. If
desired, cover the rim with a strip of parafilm. Sealed plates can be
stored at 4’C for several weeks before counting.
MATERIALS:
Phi
X 174 stock solution (should be at least 1 x 10 3 PFU/ml, but no higher
than 1.5 x 10 4 PFU/ml) labeled as "tube A" (available from
Carolina Biological Supply at www.carolina.com , catalog number 12-4425)
Healthy,
overnight cultures of four bacterial strains, including the optimal E.
coli strain C, the suboptimal E. coli strain B, and two other, non- E.
coli strains.
12 tryptone base agar plates per student group
12
x ml tryptone soft-agar tubes per student group
sterile
1 ml and 5 ml pipets
sterile,
distilled water
sterile
snap-cap culture tubes
Kimwipes or paper towels
EQUIPMENT:
pipetting
device
37’C
plate incubator
37’C
shaking incubator (for overnight cultures)
Bunsen
burner
47’C
water bath
TROUBLESHOOTING TIPS:
Using
a cell counter will make counting easier. Placing the plates onto a grid
may also help.