Guidance for Preparing Standard Operating Procedures (SOPs)

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Selanastrum B23.0 Rev. #: 1 Date: 12/99 Page 4 of 8 10. Centrifuge 11. Centrifuge bottles, 40 ml and 250 ml 12. Tygon of silicone tubing (for aeration) 13. Syringe needle on tip of sterile glass pipette 14. Light meter 15. Sterilized inoculation loop 16. Ceriodaphnia food preparation log book 17. Hydrochloric acid 18. Acetone 19. Purified water, Super-Q 20. Chemicals for MBL medium (see section 5.2) 21. Stock culture of Selenastrum capricornutum. 5. Procedure 5.1 Cleaning of Glassware a. Glassware is cleaned according to SOP L0118.1, SOP for Cleaning Laboratory Glassware, except that the final rinse is with Super-Q water. b. Glassware should always be oven dried to obtain partial sterilization. c. Glassware used for algal culturing should be kept separate from general laboratory glassware and should be used only for cultivating algae. 5.2 Preparation of Stock Solutions a. Prepare micronutrient stock solutions by adding the chemical(s) to Super-Q water to make 1L in a volumetric flask. For stock solution #3, add chemical in the order shown. 1. Stock Solution #1: 4.36 g Na 2 EDTA H 2 O 2. Stock Solution #2: 3.15 g FeCl 3 .6H 2 O 3. Stock Solution #3: 0.01 g CuSo 4 .5H 2 O * 0.01 g CuSO 4 .6H 2 O * 0.022 g ZnSO 4 .7H 2 O ** 0.18 g MnCl 2 .4H 2 O 0.006 g Na2MoO 4 .2H2O *** 1.0 g H 3 BO 3 * 0.01 g Na 2 SeO 3 QA/G-6 D-4 March 2001
Selanastrum B23.0 Rev. #: 1 Date: 12/99 Page 5 of 8 * weigh out 100.0 mg and dilute to 100 ml. Add 10.0 ml of this solution to Stock #3. ** weigh out 220.0 mg and dilute to 100 ml. Add 10.0 ml of this solution to Stock #3. *** weigh out 60 mg and dilute to 100ml. Add 10.0 ml of this solution to Stock #3. b. Prepare macronutrient stock solutions by adding the chemical to 1L Super-Q water in a volumetric flask. 1. Stock Solution #4: 36.76 g CaCl 2 .2H 2 O 2. Stock Solution #5: 36.97 g MgSO 4 .7H 2 O 3. Stock Solution #6: 12.60 g NaHCO 3 4. Stock Solution #7: 8.71 g K 2 HPO 4 5. Stock Solution #8: 85.01 g NaNO 3 c. Add 1 ml of each stock solution per liter of medium, except add 2 ml of stock solution #2. Use a sterile pipette for each stock solution and dispose of after use. d. Keep all stock solutions, except #3, in the dark at room temperature. Keep stock solution #3 at 4 o C. Retain stock solutions for no longer than 24 months. 5.3 Starting an Algal Culture a. Use a 2 L Erlenmeyer flask to prepare a 1.5 L batch of MBL medium. Add 1500 ml of Super-Q water to the cool oven-dried flask. Add 1.5 ml of each stock except #2, using a sterile 2.0 ml pipette for each solution. Add 3 ml of stock solution #2. b. Inoculate the MBL medium with a slightly visible amount of inoculum from an agar slant using a sterile inoculating loop and aseptic technique. An alternative inoculum can be used by transferring 1-2 ml of a previous culture maintained at 4 o C to the newly prepared mMBL medium. c. Place a sterile pipette in a flask with a beveled syringe needle fixed to the pipette tip. Attach to a filtered air source. This is done to prevent the algae from settling and to provide adequate gas exchange. Stopper the culturing flask with a gauze/cotton plug. d. Cultures are kept at 25 + 2 o C (or at ambient temperature) at a light intensity of approximately 400 ft-c. Lighting is continuous, i.e., 24 hours per day. QA/G-6 D-5 March 2001
Page 1: United States Office of Environment
Page 4 and 5: QA/G-6 ii March 2001
Page 6 and 7: QA/G-6 iv March 2001
Page 8 and 9: SOPs are needed even when published
Page 10 and 11: eviewing historical data and is cri
Page 12 and 13: data processing and evaluation (inc
Page 14 and 15: 5. Reference Section - Documents or
Page 16 and 17: U.S. Environmental Protection Agenc
Page 18 and 19: 4.0 Interferences This section shou
Page 20 and 21: 17.0 Tables, Diagrams, Flowcharts a
Page 22 and 23: SW Sampling Rev. 1.0 Date: 1/01 Pag
Page 30 and 31: C-47 Color Rev. #: 2 Date: July 200
Page 32 and 33: 5.3 Absorption of ammonia by the st
Page 34 and 35: C-47 Color Rev. #: 2 Date: July 200
Page 36 and 37: Selanastrum B23.0 Rev. #: 1 Date: 1
Page 44 and 45: Data ML005 Rev. # 0.0 Date: 11/00 P
Page 58: Data ML005 Rev. # 0.0 Date: 11/00 P

Guidance for Preparing Standard Operating Procedures (SOPs)

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