WARNING - Unless you are really interested in this stuff, there is no need to read this page since it is very long winded.

Replacing CO2 Tanks   Lead/Lag Setup   Leak Checking

CO2 Incubators

Although not as maintenance intensive as autoclaves, incubators can be a nuisance when they act up, since their contents can be extremely valuable.

One of the major problems that we have seen over the years is the lack of understanding of the CO2 tank regulator. The purpose of the regulator is, as it's name implies, to provide a constant 'regulated' pressure to the incubator chamber.

The pressure in a full tank is approximately 900 psi. Most incubators require a maximum pressure of 15 psi. This pressure reduction is the job of the regulator. Piece of cake...right? Problems seem to occur when exhausted tanks need to be changed. Believe it or not, this simple task is responsible for the majority of incubator problems.

Replacing CO2 Tanks

1. Close the tank valve by turning handle clockwise.
2. Using an appropriate wrench unscrew the regulator from the tank. Be careful not to lose the gasket that goes between the tank and the regulator. Most tanks come with a new gasket, but if one is not furnished, the old one may be used as long as it is not damaged.
3. Place the regulator on the floor or on a suitable support. Do not let it suspend from the hose.
4. Remove the exhausted tank and replace with a new one.
5. Ensure that the tank is suitably strapped to the wall or other approved earthquake restraint.
6. Re-attach regulator with gasket to the tank.
7. Slowly open the tank valve about 1/4 turn.
8. Observe the tank pressure gauge on the regulator, it should rise to about 900 psi.
9. Congratulations!! All done.

So what's all the fuss about...? You'll notice that nowhere in these instructions does it mention anything about touching the regulator's adjustment valve. That's because there is no need to. This is where the lack of understanding occurs since some folks think this is some sort of shut off or something. Actually, I'm not sure what they think it is, but I do know that they like to play with it.
Now what happens is that when the regulator adjustment valve is screwed in (ie clockwise) the delivery pressure is increased. Depending on the range of the regulator, this pressure could be increased anywhere up to 200 psi. Now, when the new tank is opened up, this increased pressure flows through the internal 'plastic' tubing of the incubator and literally blows it apart. Not good! This will be accompanied by a loud hissing sound and ice forming on the tank. The bottom line is "DON'T TOUCH THE ADJUSTING VALVE!"

"If I can't touch it then why is it there?"
Good question grasshopper....The exception to the rule in this case is during initial setup. The regulator will typically be shipped with the adjusting valve fully backed out. Once installed on a tank and the tank valve open, the delivery gauge pressure should read 0 psi. The regulator adjusting valve can now be turned in (clockwise) slowly until the required pressure is seen on the gauge.
A really smart thing to do at this point would be to wrap tape around the regulator so that the adjuster can not be accidentally moved.

This regulator shows the 'T' handle pressure adjusting screw. Turning clockwise will increase the delivery pressure and anti-clockwise will reduce the delivery pressure to the incubator. The nut on the right side attaches to the CO2 cylinder. Don't forget the gasket that goes here. The pressure gauge on the right (nearest the CO2 tank) measures the tank pressure whilst the gauge to the left measures the delivery pressure to the incubator. The 'upside down' brass valve to the left enables the system to be isolated from the tank. It is necessary to close this valve when changing tanks if more than one tank is connected in parallel (lead/lag setup). We will discuss lead/lag setup later.

This regulator looks a little different but does exactly the same thing. In this case the center black knob controls the delivery pressure. Again, clockwise to increase and anti-clockwise to decrease the delivery pressure.

Lead/Lag Setup

How do we know when the CO2 tank is empty? That's an easy one, my young apprentice, or is it? The obvious answer is that it is empty when no more CO2 enters the incubator and all the cells die. Well, that might be a bit dramatic, since the incubator would alarm and even if it was after hours the incubator should be hooked into the remote alarm system. If it's not then your stuff in there can't be that important right!? Well not really, that's a bit harsh since there are still a lot of reliable old incubators out there that were built without remote alarm contacts.

I digressed a little so now to answer the question. Actually, the real question is "How do we know when it's almost empty?" This would be good to know so that it can be changed in a timely fashion.
The problem here is that the tank pressure does not fall linearly with the CO2 level. The gauge measures the vapor pressure exerted by the liquid CO2 present in the tank.
Here's the part that's hard to grasp....The tank gauge will read 900 psi until all the liquid CO2 has gone. As the remaining vapor is consumed, the tank gauge will now drop off, typically very rapidly. That confuses a lot of people that check the gauge before the weekend and come in on Monday morning to an empty tank.

Actually, the best way to monitor the contents of the tank is by weight. Unfortunately not many labs are equipped with scales for the tanks to sit on. There are other ways such as ultrasonically, or electronic strain gauges, but both ways are very expensive. Even with the ability to accurately measure the tank contents we would end up changing the tank before it could be completely exhausted which is wasteful.
This is where the lead/lag setup comes into play...I knew we would get there eventually!!

The lead/lag setup utilizes 2 tanks, each with its own regulator. The delivery lines are spliced together so only a single line enters the incubator, just like the single tank setup.
Initial set up:

1. Back off both regulators as far as they will go using the adjuster that I earlier said not to touch.
2. Make sure the delivery valves from the regulators are open.
3. Open both CO2 tank valves about 1/4 turn each.
4. Next, decide which tank you want as the back up. Typically this will be the most difficult one to change. Anyway, it is the lag tank that will be set up first.
5. Locate the back up tank regulator and turn in the adjuster clockwise to increase the delivery gauge pressure to 12 psi. You may be surprised, if you're not very smart that is, to see the gauge of the lead regulator also rise to 12 psi, even though you haven't touched it yet. Don't panic, think. Both regulators share a common delivery line and will therefore read the same pressure. The lag tank is set. Tape it up so nobody fools around with it.
6. Now screw in the adjuster on the lead tank until the pressure reaches 15 psi. Note, the lag pressure will also read 15 psi even though it is set at 12 psi. Tape that regulator.
7. That's all there is to it. When the incubator calls for gas, the flow will be from the lead tank since it is set at a higher pressure (15 psi) than the lag tank (12 psi)

The beauty of this is that the lead tank can exhaust all the way down to less than 12 psi before the lag tank kicks in. This minimizes waste whereby a tank may be typically changed out with 400-500 psi when used as a standalone. It also gives peace of mind knowing that there is always a full tank standing by.

Now there are other more elaborate types of lead/lag setups that I have seen on the market for you deep pocketed people. They cost a lot of money and merely do the same thing when they are not broken that is!!
Stick with the good old, time proven, lead/lag system, you'll be glad you did.

Leak Checking

All the back up tanks in the world won't help you if you have a serious leak, especially on the high side of the system. By the high side I mean between the regulator and the tank where the gasket resides.
After changing a tank follow this procedure to check for leakage:
1. Close the regulator delivery valve.
2. Open the tank valve and make a note of the tank pressure.
3. Close the tank valve and go away for 30 minutes.
4. Return and check the tank pressure. The pressure should be the same. This will confirm that there is no leakage from the tank gasket or regulator.

Internal leaks from the incubator tubing are not as damaging but should still be checked out.

1. Turn off the CO2 injection or set to zero.
2. Open the tank valve and again observe the tank pressure.
3. Close the tank valve and repeat the 30 minute test.
4. On return, if the tank pressure has not dropped, you can be confident that the internal tubing is leak free.