17 EXPERIMENT No 1 SELECTED QUESTIONS 1 You have performed t

17 EXPERIMENT No. 1 SELECTED QUESTIONS 1. You have performed the indicated series of dilutions. For each, give the titer of the original culture (show your calculations) A. Tube 10 uL 3990 LL PBS 300 LL 3700 LL PBS 200 LL 800 LL LB broth 100 LL LB agar plate Colonies on plate after 24 hr 2 Titer of original culture B. Tube 100 LL 900 LL H20 1 LL 9999 LL LB broth 1 LL 49 LL LB broth 50 uL 950 ul, LB broth 100 LLS LB agar plate Colonies on plate after 24 hr 130 Titer of original culture cfu/ml C. Tube 10 LL 3490 LL PBS 1 LL 99 LL PBS 100 LLS 1900 LL PBS 500 LL- 500 LL PBS 100 LLS LB agar plate Colonies on plate after 24 hr 353 Titer of original culture cfu/ml 2. Why do you plate on duplicate plates for each dilution (think statistics? Sticky Notes Downloads 428 Lab Manual 17

Solution

Tube A:

Dilution factor= Final volume/Sample volume

So, First Dilution: Initially 10µl in 990µl PBS= 1000/10=100.

Second Dilution: 300µl in 700µl= 1000/300=3.33

Third Dilution: 200µl in 800µl= 1000/200=5

Total dilution factor= 100 X 3.33 X 5=1665

Volume plated on agar plate is=100µl=0.1 mL

Colonies on plate=2

The colony forming units can be calculated by using following formula:

CFU/mL= (No. of colonies X Dilution factor)/ Volume of culture plate

So, CFU/mL= (2 X 1665)/0.1= 33300

Henceforth, Titer of original culture= 33300 CFU/mL

Tube B:

Dilution factor= Final volume/Sample volume

So, First Dilution: Initially 100µl in 900µl PBS= 1000/100=10.

Second Dilution: 1µl in 9999µl= 10000/1=10000

Third Dilution: 1µl in 49µl= 50/1=50

Fourth Dilution: 50µl in 950µl= 1000/50=20

Total dilution factor= 10 X 10000 X 50 X 20=100000000=10⁸

Volume plated on agar plate is=100µl=0.1 mL

Colonies on plate=130

The colony forming units can be calculated by using following formula:

CFU/mL= (No. of colonies X Dilution factor)/ Volume of culture plate

So, CFU/mL= (130 X 10⁸)/0.1= 1.3 X 10¹¹ CFU/mL

Converting CFU/ml to Log Value,

Log (1.3 X 10¹¹) =11.1139

Henceforth, Titer of original culture= 11.1139 CFU/mL

Tube C:

Dilution factor= Final volume/Sample volume

So, First Dilution: Initially 10µl in 490µl PBS= 500/10=50.

Second Dilution: 1µl in 99µl= 100/1=100

Third Dilution: 100µl in 1900µl= 2000/100=20

Fourth Dilution: 500µl in 500µl= 1000/500=2

Total dilution factor= 50 X 100 X 20 X 2=200000=2 X 10⁵

Volume plated on agar plate is=100µl=0.1 mL

Colonies on plate=353

The colony forming units can be calculated by using following formula:

CFU/mL= (No. of colonies X Dilution factor)/ Volume of culture plate

So, CFU/mL= (353 X 2 X 10⁵)/0.1= 706 X 10⁶ CFU/mL=7.06 X 10⁸

Converting CFU/ml to Log Value,

Log (7.06 X 10⁸) =8.8488

Henceforth, Titer of original culture= 8.8488 CFU/mL