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Golden West College *
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100A
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Biology
Date
Feb 20, 2024
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1486 1487 1488 1489 1490 1491 1492 1493 1494 1495 1496 1497 1498 1499 1500 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 1511 1512 1513 1514 1515 = wuon P ~Noy 0.3ml 03ml 0.3ml 0.3ml 03ml 0.3ml ... A G 2.7mI TSB: initial culture _—_— o > & Dilution factor: 1X SFFSSET S Figure 43 5. ODreading Using the spectrophotometer, record the OD of each the diluted sample (don't forget to "zero" your measures with the provided blank) . Plating and incubation Prepare and label 4 Petri dishes containing some TSA (aka TSB plus agar). The label (on the bottom of the plate) should include your name(s)/initial(s) and the dilution factor: 10°, 105, 107, and 108 Label the 4 plates accordingly, then starting from the most diluted tube, transfer 0.1m| (i.e,, 100ul) to the corresponding plate. Then, using a sterilized "spreader" (using iso- propanol and a Bunsen-burner), spread homogeneously the bacteria on the plate. Sterilize the spreader between each plate (see previous lab "spread plate") Seal the plates with tape and place them in the incubator (upside-down) for overnight incubation at 37C. The plates will be moved to the fridge (4C) after incubation. . Enumeration After incubation, count the colonies on each plate. Use a Sharpie to mark each counted colony so that you count each colony only once! The goal is to obtain between ~30 and ~300 colonies per plate! Less than 30 would be statistically problematic whereas >300 would be challenging to count! Example: If you count 57 colonies on plate "Tg" (i.e., dilution factor 1,000,000 x) the deduced number of CFU in "To" is ... [CFU]]U = HCFUTG X 10 X DF [CFU|y = 57 x 10 x 1,000,000 = 570,000,000 CFU/ml Equation 2. (x10is to account for the 0,1ml that have been plated, instead of 1mll) Measured Volume Plated #CFU counted #CFU/ml ODs0onm 0 nd nd 57
1518 1519 1520 1521 1522 1523 1524 1525 1526 1527 1528 1529 1530 1531 1532 1533 T1 (10x) T, (10,000x) Ts (100,000x) Té (1,000,000x) T7 (10,000,000x) T, (100x) ~ O OIZ T3 (1,000x) Ts (100,000,000x) - 0. 003 100ul nd [ Awn n‘/ If you cannot distinguish individual colonies (e.g., >350 colonies) write "lawn." 6. Manipulation (ii): Measure Absorbance of Diluted Samples 1. Prepare 5 tubes with 2.5ml of sterile TSB 2. LabelthetubesA,B,C,D,and E 3. Using a5 ml pipette (+ pipettor) transfer 2.5ml of To (initial tube provided to you), to tube A and homogenize Using the same pipette, transfer 2.5ml from tube A to tube B, and homogenize . Using the same pipette, transfer 2.5ml from tube C to tube D, and homogenize 4. 5. Using the same pipette, transfer 2.5ml from tube B to tube C, and homogenize 6 7 . Using the same pipette, transfer 2.5ml from tube D to tube E, and homogenize 8. Read to ODgoonm for each tube (don't forget the blank)! Tube (dilution factor) Measured ODgoonm D (16x) E (32x) - 0,066 7. Standard Curve Complete the table below: 58
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