Team:BYU Provo/Notebook/Phage Purification/Winterexp/Period1/Exp/4.8BacteriaDeterminingTiter

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Phage Purification March - April Notebook: Experiments

4.5 Phage Spot Test

I) Purpose

Choose a bacterium to grow phage in.

II) Expected Outcome

Larger plaques in the bacteria where the phage were able to travel and infect the best.

III) Reagants Used

BL21

W3110

LB

1 X LB Top Agar (TA)

Phage:

1L

10L

40T4

T4DOS

T4 Infected

IV) Actual Procedure

We placed 100 µL of LB broth into 6 tubes, labeled 0 to -5. We repeated this for all 5 strains of phage. Next, 10 µL phage was transferred to the tube labeled '0'. The tube was vortexed and 10µL was transferred to the next tube. this was repeated to the concentration of -5.

While performing this procedure, we had innoculated .5 mL LB broth with W3110, and another .5 mL culture with BL21. This was done six times for each bacterium, so each phage type would have it's own plate split into 6 concentrations of phage.

Next we plated the bacteria using 4.5 mL TA and .5 Bacterial cultures. The plates were segmented, and we spotted 5µL of the different concentrations of phage onto the various segments of the plates.

To finish, the plates were incubated overnight at 37°C.

V) Results

Phage 10L w/ w3110 had large scale infection every concentration

Phage 10L w/ BL21 had infection in very large concentrations

Phage 1L w/ w3110 had infection in very large concentrations

Phage 1L w/ BL21 had infection in very large concentrations

Phage T4 DOS w/ w3110 had infection in very large concentrations

Phage T4 DOS w/ BL21 had infection in very large concentrations

Phage 40 T4 w/ w311o had no phage infection

Phage 40 T4 w/ BL21 had large scale infection at every concentration

Phage T4 inf w/ w311o had large scale infection at every concentration

Phage T4 inf w/ BL21 had large scale infection at every concentration

We had a significant amount of running that occurred on almost every plate, so the results were a little difficult to read. We concluded that the phage is a high enough concentration that we would have to do another phage titer to a smaller dilution in order to determine actual concentration. The controls were all a lawn of bacteria.

@@ Line 54: / Line 54: @@
 '''IV) Actual Procedure'''
-:: We placed 100 µL of LB broth into 6 tubes, labeled 0 to -5. We repeated this for all 5 strains of phage. Next, 10 µL phage was transferred to the tube labeled '0'. The tube was vortexed and 10µL was transferred to the next tube. this was repeated to the concentration of -5.
+:We placed 100 µL of LB broth into 6 tubes, labeled 0 to -5. We repeated this for all 5 strains of phage. Next, 10 µL phage was transferred to the tube labeled '0'. The tube was vortexed and 10µL was transferred to the next tube. this was repeated to the concentration of -5.
-: :While performing this procedure, we had innoculated .5 mL LB broth with W3110, and another .5 mL culture with BL21. This was done six times for each bacterium, so each phage type would have it's own plate split into 6 concentrations of phage.
+:While performing this procedure, we had innoculated .5 mL LB broth with W3110, and another .5 mL culture with BL21. This was done six times for each bacterium, so each phage type would have it's own plate split into 6 concentrations of phage.
-: :Next we plated the bacteria using 4.5 mL TA and .5 Bacterial cultures. The plates were segmented, and we spotted 5µL of the different concentrations of phage onto the various segments of the plates.
+:Next we plated the bacteria using 4.5 mL TA and .5 Bacterial cultures. The plates were segmented, and we spotted 5µL of the different concentrations of phage onto the various segments of the plates.
-: :To finish, the plates were incubated overnight at 37°C.
+:To finish, the plates were incubated overnight at 37°C.
 '''V) Results'''
@@ Line 71: / Line 71: @@
 :Phage T4 inf w/ BL21 had large scale infection at every concentration
-:: We had a significant amount of running that occurred on almost every plate, so the results were a little difficult to read. We concluded that the phage is a high enough concentration that we would have to do another phage titer to a smaller dilution in order to determine actual concentration. The controls were all a lawn of bacteria.
+:We had a significant amount of running that occurred on almost every plate, so the results were a little difficult to read. We concluded that the phage is a high enough concentration that we would have to do another phage titer to a smaller dilution in order to determine actual concentration. The controls were all a lawn of bacteria.
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