Safe Handling and Maintenance of Liquid Nitrogen Containers (LN₂)

Safe handling and maintenance of liquid nitrogen containers refers to the set of practices and precautions required to properly use, store, and preserve cryogenic vessels (LN₂ containers) to ensure operational safety, equipment longevity, and preservation of the contents, such as frozen biological material.

Structure of Liquid Nitrogen Container

A liquid nitrogen container is a double layered vessel namely outer chamber and inner chamber. The outer chamber covers the over all parts of the container. The inner chamber is suspended into the outer chamber through the neck.

The wall of inner chamber is made of high quality insulating material and is bad conductor of heat. Vacuum is created between outer and inner chamber and the space in-between is filled with insulating material.

If the vacuum is broken, the liquid nitrogen will boil and there would be rapid loss of liquid nitrogen from the inner chamber.

The inner vessel is connected with the outer vessel with glue and it is not supported by a strong welding. Hence the neck is the weakest part of the whole refrigerator and sudden tipping on one side will cause damage to the neck.

The neck region prevents the exchange of heat from outside to inner chamber and thereby the fast evaporation of liquid nitrogen is prevented.

The addition of liquid nitrogen to the container leads to considerable increase in weight load which should be supported by the neck tube.

The sudden moves and jerks will cause the vibration of the inner chamber. The side movement of the inner chamber will leads to the damage to the neck tube and causes mechanical damage.

Liquid nitrogen refrigerators are constructed in such a way the three pathways of heat transfer, conduction, convection and radiation are minimized.

The super vacuum between the chambers prevents the transfer of heat through conduction and convection.

Cryogenic Agents

• The cost of storing material at ultra low temperatures increases as the storage temperature decreases.
• In general, the lower the storage temperature the longer the sperm motility and fertility can be maintained.
• Several cryogenic agents have been used for freezing and preservation of bovine semen viz. solid carbon di oxide (-79°C), liquid air (-183°C) and liquid nitrogen (-196°C).
• Since 1964 liquid nitrogen has almost entirely replaced solid Co₂ (dry ice) in the storage of frozen semen.
• Liquid nitrogen is the 4^th coldest substance known.
• It has a boiling point of -196°C at atmospheric pressure. Being a liquid it comes into good contact with the surface of the straws.
• Hence a constant storage temperature is maintained throughout the straw.
• Liquid nitrogen is colourless, odourless and most inert substance and hence it can safely be handled in preserving biological materials.
• It is non-toxic as such and does not produce toxic or irritating vapour.Liquid nitrogen is manufactured from atmospheric air.
• Atmospheric air is the rich source of oxygen, liquid nitrogen and contains 78% nitrogen by volume.
• In industrial manufacture of liquid oxygen, liquid nitrogen arises as by-product.
• It has been reported that greater deterioration in motility and metabolic activity of spermatozoa occurs at -79°C storage than -196°C.

Safety Aspects in Handling Cryogenic Agents

There are number of general precautions and safety practices to be observed because of two important properties of cryogenic fluid-they are extremely cold and very small of liquid is converted into very large amount of gas. When liquid nitrogen evaporates into gaseous form it occupies 700 times its volume.

Safe handling of liquid nitrogen-operator guide lines:

• Liquid nitrogen should be handled carefully as it can produce “frost bite” on the skin similar to burn, even if it is in contact for few seconds.
• The vapour of liquid nitrogen is also cold and can produce cold burns. Cold vapour can damage delicate tissues such as eyes even if exposed for short period.
• One should not look into liquid nitrogen container without eye protection
• Boiling and splashing always occur when filling up a warm container.
• Always perform these operations slowly to minimize boiling and splashing and keep your eyes away from the container.
• Always use stainless steel tongs with long handles to remove any object immersed in liquid nitrogen.
• Loose fitting cryo gloves made of woollen, asbestos or leather can be used for handling.
• Cold metal may stick to skin and tear the flesh when attempts are made to withdraw the cold object from bare hands or fingers.
• Specially made cryo gloves are also available for this purpose.
• Care should be taken to avoid spilling of liquid nitrogen into shoes.
• Use containers specifically designed to hold liquid nitrogen.
• All containers have vent or safety device to allow the escape of nitrogen vapour.
• Inadequate venting or closing tightly can result in excessive gas pressure, which can damage or burst the container.
• Use only the stopper supplied by the firm. Never plug the container tightly.
• Ventilation: Always containers in a well ventilated area to prevent excessive concentration of gas.
• Excessive amounts of nitrogen reduce the concentration of oxygen in the air.
• When the oxygen level goes below 20.5 percent can cause asphyxiation.
• A person can become unconscious without sensing any warning symptoms such as dizziness.
• Transferring of liquid nitrogen: Use a stainless steel or plastic funnel while pouring liquid nitrogen into another smaller container.
• The top of the funnel should be partly covered to reduce splashing.
• When it is not safe to tilt the container, use a discharge tube to remove liquid nitrogen.
• Specially designed transfer device is also available for safe transfer of liquid nitrogen.
• Treatment of cold burns: Remove the clothing that may constrict the circulation of the frozen area.
• Immediately place the affected part in a water bath at 42°C and apply water liberally.
• Never use dry heat which may superimpose a burn upon the frozen tissue.
• Frozen tissues are painless and appear waxy with yellowish colour. They will become painful, swollen and very prone to infection when thawed.
• Therefore do not rewarm rapidly if the accident occurs in the field.
• Morphine and tranquilizers are required to control the pain during thawing and should be done only under professional media supervision.
• The visible of frost line or water vapour at the top of the container is an indication of damaged container and rapid evaporation of the liquid nitrogen.

Handling of LN2 Container

The cryogenic containers are double walled vessels with annular space evacuated and sealed.

In addition, several types of insulation viz. vacuum alone, expanded foam, gas filled powder and fibrous material, evacuated powder or evacuated super insulation are used as thermal insulators.

The outer walls of containers are made of stainless steel, carbon steel or aluminium alloys.

Do’s

• Always fill the liquid nitrogen container slowly.
• The storage room or place should be well ventilated/aerated.
• Always keep the lid in closed manner.
• Keep the canisters in appropriate place before closing the container.
• During transport support the container with soft padding (rubber, thermocole, wooden crates etc.)
• Make regular checking of the liquid nitrogen level in the container by using a dip stick.
• A minimum of 15 cm should be maintained to keep the straws in merged position.

Don’ts

• The containers should be kept in a cold place. Exposure of the container to the direct sunlight or hot air should be avoided.
• Use rubber or jute mate to place the liquid nitrogen container. Don’t place the container on a hard/concrete floor.
• Avoid moisture floor where the container is stored.
• Avoid improper handling, drilling, puncturing and rolling of the LN₂ container.
• Don’t keep any material over the container.
• Don’t rack the container one over the other.
• Avoid abrupt contact of the container with the doors and other materials.
• The canisters are specific to the design of the container.
• If the canister’s size is not appropriate it will not fit well or will not reach the bottom of the inner chamber. So don’t inter change the canister between the containers.
• The vacuum knob should not be damaged.
• Don’t swing the LN₂ containers.
• Avoid rough handling of canisters.
• Avoid frequent cooling and warming of containers.
• Thermal stress may cause so much strain within it that the inner wall of container may crack.
• Appearance of moisture on the outer wall of container is a sign of damaged container. Do not dry the container meant for regular use.
• When the vacuum disappears, the insulating capacity is lost.
• Repairing of damaged containers has to be done by the manufacturer only.

Measuring the Level of Liquid Nitrogen

The evaporation rate of liquid nitrogen varies between the containers (even among same make and capacity), temperature of the room in which it is stored and number of times it is opened. Hence the periodic checking of the level of liquid nitrogen is essential.

The minimum level of liquid nitrogen should keep the straws completely submerged in liquid nitrogen:

• To measure the level of LN₂, the dipstick is used.
• Open the lid, pass the stick into the container till it reaches the bottom of the container.
• Keep the stick for 10 seconds.
• Remove the stick and wave in the air for few seconds which will leads to frost formation.
• Measure the level of liquid nitrogen 1 cm below the level of frost line (this allowance is given for boiling liquid nitrogen when the stick is dipped).
• A minimum of 15 cm should be maintained when the straws are stored in the container.

Liquid nitrogen has the specific gravity of 0.82 i.e. 1 litre weighs 0.82 kg (1 kg=1.22 litres). Based on the weight, the quantity of liquid nitrogen in litres could be calculated. For this purpose the weight of the empty container should also be known.