Appendix: Operation and Maintenance of Nuaire Incubators

June 15, 1990

Rosalie Veile

Introduction:

The Nuaire incubators provide a controlled environment for optimum growth of tissue culture cell lines. Five parameters contribute to optimum growth conditions. These are:

1. Humidity
2. Precision temperature control
3. Precise CO2 control
4. Sterility
5. Reliability

Components of the system are: The Nuaire CO2 Water-Jacketed Incubator, the Nu-1550 Automatic Tank Switch, the Trimline Regulator, and the CO2 tanks.

1. Humidity:

Humification of the incubator is achieved through water evaporation from the stainless steel water pan which is placed on the bottom of the shelf rack. Use only distilled sterile mineral-free water in the stainless steel pan. The water should be changed at least once a week. Flooding the bottm of the incubator is not recommended, since it is difficult to change the water weekly and almost necessitates the use of chemicals (copper sulfate; or other bacteria growth inhibitors) which are not recommended and may damage the stainless steel.Humidity recovery will require 15-20 minutes after door is opened for 30 seconds.

2. Precision temperature control:

37 degrees C is the set point desired, and a temperature calibration must be performed within 1 degrees C of this temperature. To obtain the desired temperature, place a glass mercury thermometer in a glass beaker filled with water. Position the beaker in the middle of the incubator for several hours. Adjust temperature reading on the display panel to coincide with temeprature of the thermometer, and program incubator to run. Temperature of the incubator should be checked daily.

3. Precise CO2 control:

Lymphoblastoid cell lines grow best using 5% CO2. It is necessary to determine the proper percentage of CO2 in the chamber with the use of a fyrite. The fyrite employs the well-known "Orsat" method of volumetric analysis involving chemical absorption of a sample gas, as CO2. The reagent used to absorb CO2 is potassium hydroxide (dyed red). Potassium hydroxide is poisonous and care should be taken to avoid accidental body contact or internal ingestion (on bodyily contact, flush area with water, then wash with vinegar). To ensure proper fyrite operation and reliable results, the fluid level should be correct as well as the fyrite fluid strength. Refer to Bacharach fyrite gas analyzer operation/maintenance manual for proper use and care. The autoflow incubator uses Matheson Instruments flowmeters. The flowmeters provide accurate and low flow measurements of CO2. To prevent dirt from the CO2 supply from interfering with the operation of the flowmeters, a 0.3 um filter (located at the autoflow inlet valve) should be used and changed monthly . Also, the 0.3 um air inlet filter should be replaced monthly. The purpose of this filter is to cleanse the room air which is drawn into the chamber. Below is a table with approximate values to achieve the desired setting in the incubator:

Adjustments in the percentage of CO2 should be made using the CO2 flowmeter only. The flowmeter ball will drift up and down and will not affect the CO2 percentage. It is essential that the percentage of CO2 in the chamber be checked every two weeks or at the same time a new tank is connected to the CO2 line. If a tank is not connected correctly or there is a leak in the line it is very important to detect the leak as soon as possible. Tubing connections should be a tight fit and brittleness in the lines should be checked monthly. The regulator's high pressure stage direct from the supply cylinder will have a range from zero to 2,000 PSI. This gauge indicates actual tank pressure. The low pressure stage should have a range from 0 to 30 PSI. This gauge will indicate the actual CO2 pressure into the incubator system. To connect the regulator, first open the CO2 cylinder slightly, to blow out dust or dirt that may have collected in the valve outlet. Keep your face away from the outlet to protect it from dust. Use some teflon tape to help seal the connection and tighten with a wrench. Be sure the disc gasket is in place before making the connection.

4. Sterility

The environment provided by the autoflow is not selective. As a result, any contamination within the chamber is subjected to the same environment as the speciments. Therefore, it is essential that all shelves and shelf brackets should be wiped off 70% alcohol, 2% alkaline gluaraldehyde or similar noncorrosive antimicrobial agent frequently and autoclaved monthly. Also, all flasks placed in the incubator must be wiped with alcohol prior to being placed into the incubator and shelves should be wiped off with alcohol every time the cultures are fed. Mild detergent should be used to clean the outside of the incubator. If any contamintated cultures are observed they should be isolated from other cultures in a different incubator and fed the proper media to rid them of contamination. The incubator in which the contaminated culture was removed from must be sterilzed.

5. Reliability

All parts of the incubator must be reliable to maintain the optimum conditions for tissue culture. The incubator requires periodic preventive maintenance to insure a reliable controlled atmosphere. The CO2 supply filter and air inlet filter should be replaced monthly. Tubing must be checked and replaced if necessary. The chamber should be cleaned and sterilized frequently. The water jacket requires no anti-bacterial agents because a copper tube producing copper sulfate which eliminates bacterial growth within the water jacket is already in place. The water jacket should be checked to insure that it is always full. Use deionized water to fill the water jacket. Flowmeters should be checked annually to insure CO2 percentage in the incubator (refer to preventive maintenance, page 29 of Nuaire manual).

References:

Nuaire Incubator, Operation and Maintenance Manual, Sept. 1988.