1. Label 24 Falcon tubes as following: Student/ Group Name, Specimen Name, Concentration, test tube number, and the date. There should be four test tubes for each of the following four concentrations: 4.5M, 4.0M, 3.5M, 3.0M, 2.5M, and 2.0M. Each of the four test tubes for each concentration should have the following labels: blank, one, two, or three.

Falcon Tube Label Example:

Conc.	4.5M	4.0M	3.5M	3M	2.5M	2M
KCl (g)	0.20	0.18	0.16	0.13	0.11	0.09
MgSO4 (g)	2.00	1.80	1.55	1.33	1.11	0.89
NaCl (g)	25.00	22.22	19.44	16.67	13.89	11.11
Na3C6H5O7 (g) - NaCitrate -	0.30	0.27	0.23	0.20	0.17	0.13
Bacteriological Peptone (g) Brand = Oxoid #LP00034B	1.00	1.00	1.00	1.00	1.00	1.00

 

2. Pipette 5 mL of each concentration into their four respective test tubes. Be sure to use a different pipette for each morality and be sure to follow the procedure given to you for proper pipetting. If you’d like to save pipettes it is okay to use one pipette ONLY if you begin with the least concentrated substance, fill the four tubes, and move on to do the next concentrated and so on.

3. Add 100 µL of Halobacterium to every test tube of every concentration except for the test tubes labeled blank for each concentration. For pipetting, students that would like real lab experience can use bulb pipets for the 5 mL samples and automatic micropipettes for the 100 µL samples if available.

4. Add 1mL of Halobacterium to one cuvette, and 1 mL of CM to two cuvettes. Take the cell density of the Halo using the spectrophotometer and record data.

5. Incubate test tubes for 48 hours in 37 0C and at 220 revolutions per minute (rpm). Record the time and date incubation began.

6. Remove tubes from the incubator after the 48-hour incubation period and add 3 mL from each tube to an empty cuvette (the blanks will be used as the reference) The tubes may be removed from the incubator any time between 47 – 49 hours.

7. Using a spectrophotometer, measure the cell density. Use the absorbance setting at 600 nm.

8. Record and plot data. The data from each of the 3 trials should be averaged. This value should be used to create the curve.

9. Clean up your lab space and lab materials. DO NOT USE SOAP if planning on reusing materials for this lab. Any trace amount of detergent will cause the cells to lyce. Thoroughly rinse all equipment at least three times with hot tap water. Follow with deionized water to sanitize the cuvettes and glassware.

 

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