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Golden West College *

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100A

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Biology

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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

1534 1535 1536 1537 1538 1539 1540 1541 1542 1543 1544 1545 1546 1547 1548 1549 1550 1551 1552 1553 1554 1555 1556 1557 1558 1559 1560 1561 1562 1563 e e e WA B (4x) Culture Measured ODgoonm | CFU/m Dilution Ov ‘252 /:/7&/0/6 C (8x) ) D (16x) G 00 | T 3zl E (32x) 0,00 3-6353;—,\,/‘96 1. Transfer the CFU of the original culture (To) calculated in the plate count section to the 2. "CFU/ml" column Using the original culture CFU/ml, calculate the CFU/ml at a 2x dilution and put this value in the "CFU/ml" column. Repeat for the following tubes. Create a graph with absorbance on the x-axis and CFU/ml on the y-axis. You can number the y-axis indicating that the number on the axis is x 106, x 107, or x 108 as appropriate for your data. You may graph data using Excel or other graphing software or using graph paper. Follow the instruction of your instructor. Plot each data point as a dot on the graph! If you used graphing software add a trend line to your data and set the y-intercept at zero. Also make sure to have the software place the equation for the line on the graph. If you are using graph paper, use a ruler and place it along the data points making sure the ruler passes through the bottom left corner of the graph (corresponding with zero on the x-axis and the y-axis). Draw a straight line using the ruler that best matches the data points. Only draw one straight line. This line does not have to touch every data point (and it probably will not). Do not connect each data point with the ruler with multiple lines. There should only be one straight line on the graph. After creating the standard curve above you continue your research with this same bacterial species, but now instead of doing the plate counts (which took a lot of time and materials) you simply use a spectrophotometer to measure absorbance to determine CFU (cells/mL) using the standard curve. As you continue your research, you measure the bacterial samples using the spectrophotometer. Use the standard curve you created to determine the amount of CFUs in each sample in the table below based on the absorbance. a. If you used graphing software, determine CFUs using the trend line equation b. If you used graph paper, use the position of the trend line as it relates to the absorbance reading to determine CFUs 8. Important considerations Here are some important considerations to keep in mind when performing this lab: 59

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