Note We posted this blog post to enlighten operators who handle LN2 to improve their skills for safely LN2 handling. This information is just reference, operators (users) are solely responsible for how to handle LN2. We do not take any responsibility for the direct or indirect loss, damage and disadvantage that caused by referring to this blog page.
LN2 is condensed nitrogen. The boiling point of LN2 is around -195.8C (-320F; 77K), so LN2 must be stored in special containers. Nitrogen is colorless, odorless and transparent, and its specific gravity is close to that of the air. Therefore, there is the risk of hypoxia in the case a room is filled with vaporized nitrogen. Additionally, since there is also the risk of frostbite, it must be handled with care.
Nature of LN2
When LN2 is vaporized, the volume of it will become 650-800 times as big as liquid state.
Nitrogen could reduce oxygen concetration in a room even if a small amount of LN2 is spilled out.
When using LN2 in a room, turn on a ventilation fan, open windows and doors for sufficient ventilation.
Never place LN2 containers close to your face when handling LN2.
If you became hypoxic, the following symptoms are shown; headache, nausea, drunkenness, weakness, increased breathing and pulse rate. If you have symptoms above, escape from the place immediately to well ventilated place. Besides, make sure no one gets closer to the place.
Use personal protective equipment
Glove selection (When handling LN2 directly) When handling LN2, wear special and dry gloves. However, do not use fabric gloves. Especially, never use wet work gloves. Because they are frozen during work if LN2 adheres to them. It may cause long-time cold exposures and lead to frostbite.
Glove size Do not use gloves that fit your hands perfectly. Use the ones which are a little bigger so that they can be removed quickly in case of emergency.
Shoes You have to be careful about shoes because your feet might be exposed to LN2 when it spills out. LN2 could touch your feet directly if you wear slippers. If LN2 goes into your socks, it might cause a long-time cold exposure. It is recommended to use waterproof shoes.
Face guard or goggle Use a face guard or goggles just in case LN2 getting into your eyes.There is a risk of blindness in the case LN2 gets in eyes.
In the case you are exposed to LN2 directly
In the case clothes get wet with LN2, take off the clothes immediately to keep LN2 away from skin. In the case clothes stick to the skin, never try to remove them forcibly. Instead, warm them with running water and slowly remove them. In the case LN2 gets in your eyes, go to see a doctor immediately.
Frostbite
In the case of exposure to LN2, it might cause frostbite. When handling LN2, make sure to use personal protective gear such as gloves mentioned above. If you have frostbite or if you think you have it, go to see a doctor immediately. If you cannot go to see a doctor right away, perform the following first aid.
First aid for frostbite
If you have frostbite, the degree of severity depends on the color of affected area, and the severity gets higher in order of the color red < violet < black. Basically, go to see a doctor as soon as possible. If you cannot go to see a doctor right away, perform the following emergency treatment.
[How to perform emergency treatment]
Warm the affected area with water at around 40C.
If there is no warm water, warm the affected area by covering with clothes or being touched by other skin area.
If frostbite is severe, warm the affected area with cold water first.
If you get strong pain due to warming, take painkiller. You could feel strong pain as the feeling returns.
If there are blisters on the affected area, protect them with cotton cloths.
Never do the following things
Warming the affected area with extremely hot things like boiling water or fire might result in burns. Be careful with water temeprature when warming the affected area because you do not feel the heat when you lose your feeling.
Make sure not to massage or rub the affected area, it may make the symptom worse.
In the biological-related field, LN2 is mainly used to preserve biological samples such as sperm, blood and cells at ultra-low temperature (-150 to -196C). At these temperature, biological samples can be stored stably for a long time because all metabolic activities and chemical reactions stop.
Liquid phase preservation and gas phase preservation
There are 2 methods to store biological samples: liquid phase and gas phase. In Japan, storing in gas phase is popular, but gas phase storage is not always superior to liquid phase storage because the storage method differes depending on the purpose. Select a proper storage method based on the advantage and disadvantage of each method, the characteristics of the samples to be stored and sample storage containers.
Features of liquid phase preservation
There is a risk of contamination by microorganisms in LN2. When using cryotubes, LN2 might enter the tube in the case the cap is loose. In that case, the tube might blow up after it is taken out of the container. It can minimize temperature change in the container when samples are put in and taken out.
Features of liquid phase preservation
Storage temperature is higher than that of liquid phase (approx. -160C). Gas phase containers are easier to handle than liquid phase containers. In general, gas phase container chamber is vulnerable to temperature changes when the lid is opened and closed, or samples are placed in or teken out. LN2 does not enter sample tubes because they are not immersed in LN2. The risk of contamination is low.
In this topic, we classify LN2 preservation containers into the following 7 types a) to g).
a) Small/medium cryopreservation containers (kane/straw type)
175L or less volume of cryo-containers belong to this type. In this type, almost all containers are liquid phases type, and samples can be stored at -180C or lower. The difference between the small and the medium are the number of samples that can be stored in and the size of the opening of the container. Cryokane and straw are stored inside the storage tool called canister. This a) type is more popular than b) box type below.
b) Small/medium cryopreservation containers (box type)
It can store freeze boxes in a dedicated canister. The storage capacity is lower than a) type containers, but it is easier to manage samples. In general, 750-6000 of tubes can be stored in b) type containers.
c) Large cryopreservation containers
The containers of this type are used when operators want to supply LN2 automatically and store 3000 or more cryotubes. Both liquid phase and gas phase are available for this type. Customers can select the vessels according to their requirements for sample types and storage method. This type is mainly used for storage of freeze boxes due to the wide opening of the container. It can accomodate 3200-35200 cryotubes. It equipes an automatic LN2 supply device called level master, and it is composed of controller, liquid surface sensor and temperature sensor. The characteristics of large containers are LN2 automatic supply, liquid and temperature monitoring, alarm function.
d) Dryshipper
The container is used when frozen samples are transported at -150C or below. When it is used, let LN2 be absorbed in advance, so sample tubes can be transported in the gas phase. When transporting it by courier, use special containers with keys. By using containers of this type LN2 never spills out from the cases in the case the containers fall down during transportation. Some containers can maintain the temperature at certain degree for up to 20 days but basically it is recommended to use it for the period of 1-2 days less than the specified in the catalog.
The structure of cryotransportation container (Dryshipper)
Please refer to the left image for the structure of cryotransportation containers (Dryshipper)
1. Chamber 2. Neck tube 3. Exterior 4. Insulation material 5. Handle 6. Cap plug 7. Canister 8. LN2 absorbent
LN2 absorbent is very important for Dryshipper. It allows an operator to transport samples safely because
the absorbent absorbs LN2 and prevent LN2 from spilling out in the case the container falls down. A user should select a proper container according to the types of container, the number of samples and the purpose of use and so on. When it comes to Dryshipper, you should also be aware of holding period. At first, select the products according to the purpose, preservation method, the type of container and the number of samples. Secondly, select the best container according to evaporative loss, re-filling period, price and so on.
Containers of this type have casters and can transport approx. 42L to 200L of LN2 (filling volume is 80% of container volume). They are closed system, nitrogen gas is released into the air through the safely valve when the internal pressure increases. By connecting to a large cryopreservation container through a flexible hose or piping, LN2 can be supplied automatically.
f) Dewer vessel
They are open-system container that can store small amount of LN2 and is also used for physicochemical experiments and simple transportation. As well as cryopreservation containers, Dewer vessels have vacuum double-layer structure, which prevents LN2 from vaporizing.
It is the storage container mainly used for sperm storage. It is generally stored in the liquid phase.
b) Cryotubes (vials)
Cryotube is used to store samples such as cells. The screw cap type is mainly used. However, since LN2 has a chance to enter the tubes when being stored in the LN2, there is a risk of contamination and expansion damage to tube during thawing. Hence, cryotube is not suitable for liquid phase storage.
c) Glass vial
Glass vials are made of glass and have excellent sealing properties because the opening is melted and welded. Since it is made of glass, operators should be careful not to break it.
d) Cryobag
Cryobags are used for storing blood samples like cord blood, peripheral blood and so on. Cryopreservation bag can be stored in the LN2.
Cryocane is the storage tool that can hold cryotubes. Cryocane with the tubular container called goblet that holds straws is also available.
b) Cryosleeve
Cryosleeve is the tubular container that protects the cryocane. It is used to prevent cryotubes on cryocane from falling off when the cane is inserted in or taken out from a container.
c) Freez box
Freeze box is the storage container for cryotubes.
d) Canister
Canister is the tool placed at the opening of the container. Cryocanes or straws are inserted in the canister. The size of canister for cane is different from the one for straw. Use the one that suits the purpose. In addition to the tubular type, there is another type that can accomodate freeze boxes.
e) Leveler
Leveler is the tool that can measure the remaining amount of LN2 in a container. It is used by inserting it into the container through the opening.
f) LN2 level monitoring system
This is the device to prevent LN2 from running out. The amount of LN2 is calculated from the diffrence between empty and full conditions and alarm goes off when the remaining amount falls below a certain level.
Precautions when using LN2 cryopreservation containers (General precautions)
1. Don’t seal
Never seal LN2 containers. In the case the container is sealed, inner pressure could increase and it might explode due to LN2 evaporation. If there is moisture around the opening or lid, the moisture is frozen around the lid. The container might be sealed unintentionally. So don’t place containers with LN2 in the places where water splashes directly (outdoors) or in the places where humidity is high.
2. Use special vessel
Some metals become easily broken under ultra-low temperature. Glass containers must not be used. When storing and transporting, make sure to use special containers.
3. Be careful not to fall LN2 containers down
Place containers on horizontal place. In the case that LN2 containers fall over due to unstable use, it may lead to the break of container, frostbite and suffocation caused by LN2 leakage.
4. Be careful not to lose the lid
In the case container lid is lost, don’t leave the container open, and completely cover the container opening with dry cloths. If leaving the container open, it may lead to contamination with liquid oxygen has strong combustibility so it may lead to explosive reaction when liquid oxygen touchs with organic substances. If an operator uses containers which are left open for a long time, they should check the color of LN2 in advance. If LN2 appears slightly blue, LN2 could be mixed with liquid oxygen.
5. Storage for a long time
Stored for a long time or refilled many times, LN2 may increase the chance of touching liquid oxygen and this leads to the contamination. If LN2 is stored for a long time, check its color and treat it with care if it gets blueish.
LN2 cryopreservation containers (for each container type)
1. Open-type container
Open-type containers have lids that do not seal the lids completely and they have cluel structures with vacuum insulation layers. They are comperatively small and easy to transport. However, since they do not have sealed lids LN2 might spill out if the containers fall down. The vacuum insulation layer could be easily broken when the inner wall is damaged by an impact since its structure is vulnerable to lateral shaking. If the vaccum insulation layer is damaged, the thermal insulation effect gets lost, and it leads to increase the pressure inside and the amount of LN2 evaporation. Besides, condensation and frost may occur on exterior of containers.
2. Self-pressure containers
Self-pressure containers have safety valves and outlets. Make sure to regularly check if the safety valve works properly because the safety valve prevents inner pressure from increasing. If the safety valve doesn’t work properly, it might lead to explosion due to inner pressure increase. If an operator cannot take LN2 from the outlet, LN2 might create ice in the pipe and the ice might block the flow of LN2. Never look into the outlet because LN2 could suddenly blast out from the outlet. In this case, contact a person in charge of LN2 containers and take proper actions.
Precautions when transporting LN2 preservation containers
1. Transportation by hands
When transporting LN2 containers by hands, 2 or more people should carry it vertically. (except small and light weight container that 1 person can carry easily). If an operator inclines a container, there is a risk of LN2 leakage or damage to the container. Since there is a chance that LN2 may leak out, it is recommended to wear gloves during transportation in the terms of safety.
2. Transportation by trolley
When an operator transports LN2 containers places on a trolley and pass over rough roads and slopes, there is a risk of falling down. Further above, if wheels of a trolley doesn’t work smoothly, there is also a risk of falling down. To prevent LN2 containers from falling down, select appropriate road, route and trolley, and use belt or rope to fix the container. Use a trolley with wheel lock and lock it when leaving the container.
3. Transportation by elevator
In priciple, use stairs when transporting LN2 containers to upper or lower floor. It is necessary to use elevator, only the containers must be loaded in the elevator. No one must be on board together with containers. Please make sure 2 or more people should carry it in and out. Please also make sure that an operator raises the signboard that indicates LN2 container is being transported so that no one gets on the elevator. In the case an operator gets on an elevator together with LN2 containers, there is almost no risk of suffocation unless the containers fall down. Calm down and wait for power recovery or a rescue even if power failure happens.
4. Transportation by car
It is desireble not to transport LN2 containers by car, but if it is unavoidable to load LN2 and transport it by car, be sure to fully open the window.
Note: During trasnportation, vibration may cause sudden boiling and blowing of LN2. Especially when containers are fully filled, LN2 can easily spout out. Keep your face and body away from the container opening as much as possible.
Q. What should an operator care about when filling containers with LN2?
A. In principle, LN2 should be filled to the bottom of the neck tube. If LN2 is filled to the top of the container, it may hasten the deterioration of the neck tube (it is typically made of epoxy resin) and cause breakage. In addition, when closing the lid after filling LN2, spilled LN2 may enter into the vacuum exhaust port, and it may cause container breakage. When filling a container with LN2 which is kept at room temperature, it is dangerous because a large amount of nitrogen gas is released until the container is cooled enough. Please make sure that the work place is well-ventilated or is equipped with exhausting system.
Q. Are there any difference in vaporizing amount of LN2 between liquid phase and gas phase preservation?
A. There is no big difference between them.
Q. How to dispose of LN2?
A. When diposing of LN2, evaporate LN2 completely by leaving the lid open at the well-ventilated area. Alternatively, dipose of LN2 into grassland or pebble. Do not throw it away at concrete or asphalt area and this might result in the breakage. Do not throw it away neither, by flowing it into a sewer pipe because it may cause breakage of the pipe.
Q. How to clean LN2 cryopreservation containers?
A. Spray alcohol or wipe with clean cloth dampened with alcohol after emptying LN2 container. When drying up the inside of the container, please use blower. Appliances that generates high temeprature (50C or more) like a hair dryer may give damage to the neck tube and LN2 absorbent.
Q. How to check the current state of deterioration?
A. Please check the remaining amount of LN2 periodically after filling up a LN2 container. Confirm the amount of LN2 decrease by using a leveler or a scale. If the LN2 decrease is much bigger than that of new product, the container might be deteriorated. Annual inspection is recommended by the manufacturer. To keep the long life of the container, dry up and clean it at latest 2 times a year.
Q. How to use Dryshipper?
A. The most common operation method is explained below. However, basically, please follow the product instruction manual. At first, pour LN2 to the bottom of the neck tube, and close the lid and leave it about 10-15 minutes. After that, pour LN2 to the bottom of the neck tube again. Repeat this procedure 3-4 times until LN2 does not decrease any more. Leave the container with the lid closed for 24 hours, then dispose of the excessive LN2. At this time, make sure that the weight of the container is the same as that of fully filled state (described in the catalog, etc.). Depending on how the dryshipper is used, basically, it is recommended to use a dryshipper for the period of 1 or 2 days shorter than that of the LN2 retain time described in the catalog.
Q. What should an operator care about when using dryshipper?
A. The LN2 retain time becomes shortened if water gets in a LN2 absorbent. To avoid this, it is recommended to measure the weight of vacant container right after purchasing. When measureing the weight after using and drying up, there is possibility that water is mixed in the LN2 absorbent if the measurement value is righter than that of initial value. In this case, the container might not be able to work as specifications in catalog, so dry it up completely in advance when used. Basically, dry up the container in a dry room, and then, store the container with the lid closed.
Q. Is transporting dryshipper by train legal?
A. Essentially, please use transporter when transporting a dryshipper. Please ask the train, airplain, ship and bus companies for the details. Please observe laws and regulations in each country.
Q. Are threre any tips that helps to reduce LN2 evaporation loss?
A. Since evaporation rate is proportional to the opening area, the bigger the opening area is, the bigger the evaporation amount is. Reduce the time when the lid is open, or reduce the frequency of putting in and taking out of the samples.
Q. How to retrieve samples if they are accidentally dropped in the container?
A. Waiting for a few minuites after leaving the lid open, you can see inside the container because the fog clears up as time passes. Check inside the container with light. If the sample can be seen, wear the special gloves and pick up the samples using a fire tong. If the samples cannot be seen, there is no other way to wait until LN2 evaporates completely. Never get close to a container opening when looking for samples or looking into a container because there is a risk of suffocation. Please make sure to keep your face away form the opening of LN2 containers.
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