Louisiana Administrative Code
Title 51 - PUBLIC HEALTH-SANITARY CODE
Part IX - Marine and Fresh Water Animal Food Products
Chapter 1 - Shellfish Growing Areas
Section IX-121 - Preparation for Laboratory Analysis of Shellfish Growing Waters [formerly paragraph 9:002-10]

1. Air incubators used in the preliminary incubation of tubes of test medial shall maintain a uniform and constant temperature of 35° plus/minus 0.5°C at all times.

2. Covered, circulating water baths used to incubate tubes of test media for the remaining incubation period shall maintain a uniform and constant temperature of 44.5° plus/minus 0.2°C at all times.

3. Hot air ovens used in the heat sterilization of glassware and related supplies shall be of sufficient size to prevent overcrowding, maintain uniform and adequate sterilizing temperature, and be equipped with suitable thermometers able to accurately register in the range of 160-180°C.

4. Autoclaves used in the sterilization of test media shall be sufficiently large enough to prevent interior crowding, provide uniform temperature within the chambers, including the sterilizing temperature of 121°C, and be equipped with accurate temperature and pressure recording devices. Pressure gauges and properly adjusted safety valves should be connected directly to either the saturated steam power lines or to a suitable steam generator. The autoclave should be capable of reaching the desired temperature within 30 minutes.

5. Electrometric pH meters used in the preparation of test media and reagents shall have an accuracy of plus/minus 0.1 pH unit.

6. Balances used in the preparation of test media and reagents shall provide a sensitivity of at least 0.1g at a load of 150g and be used with standardized weights. When less than 2g of materials is weighed, the analytical balance used must have a sensitivity of 1 mg under a load of 10g.

7. Water deionization units should be fitted with a 0.22 um-pore diameter filter.

1. Pipets shall be 1.0 ml serological pipets with 0.1 ml graduations and 10.0 ml pipets with 0.1 ml graduations. Pipets with damaged tips are not to be used. The error calibration shall not exceed 2.5 percent. Pipets that conform to APHA standards as given in "Standard Methods for the Examination of Dairy Products," 14th ed. 1978, American Public Health Association, 1015 18th Street, N.W. Washington, DC 20036 may also be used.

2. Dilution bottles or tubes used in the analysis of shellfish growing waters shall be of borosilicate glass or other material resistant to the solvent action of the water. The bottles shall be fitted with glass or rubber stoppers or polyethylene screw caps equipped with Teflon or equivalent liners that do not produce bacteriostatic compounds on sterilization.

3. Only satisfactorily tested laboratory pure water from stills or deionization units shall be used in the preparation of culture media and reagents and shall be tested and found free from traces of dissolved metals and bactericidal or inhibitory compounds as described in the latest edition of Standard Methods for the Examination of Water and Wastewater.

4. Butterfield's buffered phosphate diluent used in the analysis of shellfish growing waters shall be prepared as follows: Stock solution: dissolve 34.0g of potassium phosphate, monobasic, in 500 ml of laboratory pure water, adjust with 1 N NaOH to a pH of 7.2 and bring to 1000 ml volume with laboratory pure water. Dilute 1.25 ml of stock solution to 1 L with laboratory pure water and dispense into dilution bottles in amounts necessary to achieve the desired quantity within a 2 percent tolerance after sterilization. Autoclave the bottles at 121°C for 15 minutes. Store in a cold, dry place at room temperature.

5. A-1 media is to be prepared from individual components as follows: Dissolve 5g lactose, 20g tryptone, 5g NaCl, and 0.5g salicin in 1 L distilled water. Heat to dissolve ingredients, pipet in l ml Triton-X-100 and adjust pH 6.9 plus/minus.1 with 1 N NaOH solution. For 10 ml sample aliquots, prepare and use double strength medium. Single strength medium should be dispensed in 10 ml amount for 10 ml inocula. Autoclave media for 10 minutes at 121°C. Store in dark at room temperature away from possibility of excessive evaporation and contamination. Use media within seven days.

6. All laboratory glassware used in the analysis of shellfish growing waters must be thoroughly cleaned using a suitable detergent and hot water (160°F), then rinsed in hot water (180°F) to remove all traces of residual detergent, and then rinsed four times with a complete change of water, the final rinse being laboratory pure water. The effectiveness of the rinse should be established by testing the as described in the current edition of Standard Methods for the Examination of Water and Wastewater. Glassware should be autoclaved or should be sterilized for not less than 60 minutes at 170°C. If glassware is in metal containers, it must be heated to a temperature of 170°C for not less than two hours. Plasticware may be sterilized with low-temperature ethylene oxide gas. However, precautions should be taken to assure that all of the gas has been removed from containers before using.

7. Bromothymol blue (BTB) indicator solution used in the quality control of glassware shall be prepared by adding 16 ml 0.01 N NaOH to 0.1 g BTB and diluted to 250 ml with laboratory pure water to equal a 0.04 percent solution.