4.7 Storing Biohazardous Materials

4.7.1 Storing/ Holding Cultures on the Bench, in Refrigerators and 4˚C Freezers

All cultured biological material must be stored in closed containers (e.g., lidded plates, capped tubes, etc.); lids/caps can be loosened enough to allow for maintenance of aerobic conditions as needed within the culture vessel, but must not be so loosely placed that they can fall off or be easily dislodged from vessel openings. Culture plates/tubes and their lids may be further secured with a parafilm seal, as needed.

All stored tubes (e.g., screw-cap tubes, snap-cap plastic tubes, eppendorf tubes, conical screw-cap tubes, etc.) containing culture material must be secured in tube racks or boxes. Racks/boxes of culture tubes must not be placed precariously on the bench or refrigerator/freezer shelves; make sure they are solidly placed.

Stacks of stored culture plates must be secured in plate carriers, pans, or plastic sleeves with taped closures to prevent spillage.

All stored culture vessels must be legibly labeled with contents and date. If vessels are too small for full labeling, use a coding system and a log or record that coordinates each culture code with: 1) complete identity information for contents, 2) date, and 3) rack or box location of the culture, 4) name of researcher.

All refrigerators, freezers and dewars used for biohazard storage must be labeled with the biohazard symbol.

4.7.2 - 80˚C Freezer Storage

This type of freezer storage is essential for preserving valuable sample inventories in many labs. Most freezer models are equipped with alarms to warn of systems failure and warming interior temperatures. If inventory maintenance is critical, the PI or lab manager needs to implement some means by which personnel can be immediately notified of alarm conditions.   

Keeping a daily temperature log for any -80 freezer is highly recommended for quality assurance purposes, i.e., to monitor the range of daily fluctuations, for malfunctions/ error messages, and for trends of deteriorating performance.  

Hazards: The operating temperature of - 80˚C (-112˚F) is capable of inflicting frostbite very quickly; skin must be protected from direct contact with surfaces at this temperature, especially metal surfaces, by using insulated gloves.  

Chronic mishandling of these freezers can shorten their useful life and/or result in sudden operational failure, risking loss of irreplaceable samples.   

The following guidelines are provided to avoid this outcome:   

• - 80 freezers do not automatically defrost; in fact, ice builds up in them at a rate that requires defrosting with some regularity to avoid accumulations that will interfere with door closure. DO NOT FORCE DOOR CLOSURE OVER ICE BUILDUP; THIS CAN DEFORM THE DOOR AND HANDLE, MAKING THE UNIT INOPERABLE. In such a situation, immediate defrosting is called for rather than forcing closure.
• Ice buildup also can make sample retrieval challenging; be sure your samples are well organized and in well-labeled racks or boxes that are intended for -80 freezer storage.
• Keep a diagram of exactly where you store materials in the freezer so you can go right to what you need, vs. searching randomly with the freezer door open and contents warming.
• OPEN FREEZER DOORS FOR THE MINIMUM TIME POSSIBLE, AND FOR THE MINIMUM NUMBER OF TIMES OF DAY POSSIBLE. Coordinate with coworkers to access freezer contents in one or two trips to avoid multiple freezer openings throughout the day. Each opening challenges the cooling capacity of a -80 freezer to recover its set temperature; the longer and more frequently freezers are open, the more challenge is imposed, which can compromise long-term function.

Defrosting:

• Schedule a -80 freezer defrost procedure with plenty of lead time in which to plan where you can relocate your inventory. -80 storage space in labs is usually at a premium, so finding extra space to temporarily house your inventory may be difficult. NOTE: Holding -80 inventory in -20˚ or -4˚ freezer space WILL NOT keep them adequately frozen. If you cannot find temporary -80 freezer space, holding inventory in loosely-lidded Styrofoam boxes with dry ice is an acceptable alternative, if you keep the dry ice well supplied.
• Expect the entire process to take two days.
• Wear appropriate PPE for this process. Remove your inventory from the freezer in an organized way so that you can return it to the -80 in good order. Take advantage of this opportunity to cull any inventory that is no longer needed.
• When your inventory is secure elsewhere, power down the freezer and place a sign on it that warns others: “Out of Service – Defrosting in Progress – Unit Will Be Decontaminated for Biohazardous Materials by: (your name) Before Return to Service.”
• Prop the door open to let thawing begin; a floor fan blowing room air into the freezer increases thawing rate. Expect complete thawing to take at least 24 hours.
• DO NOT USE SHARP OBJECTS TO CHIP ICE OUT OF THE FREEZER TO HASTEN THE PROCESS – THIS CAN DO STRUCTURAL DAMAGE TO THE FREEZER. Place pans on the shelves to catch melting ice. If the freezer stores biohazardous material, add one part bleach to 9 parts melted ice, and allow contact time before discarding melt-water down lab sink.
• Place paper towels on floor to absorb melt-water. As they get wet, discard them in solid biowaste for autoclaving and replace them with dry towels. Decontaminate damp floor area with 10% bleach or other appropriate disinfectant.
• When ice is gone, decontaminate interior and exterior surfaces of the freezer with 10% bleach or appropriate disinfectant, and allow to dry fully.
• If accessible, clean dust and dirt off the exterior coils on the back of the freezer.
• Turn power on and allow freezer to come to temperature. Leave freezer empty for at least a day to ensure that it will attain and hold temperature before returning inventory to it. It is common for freezers to fail following an out-of-service period, so monitor carefully.
• Plan the re-installation of inventory carefully so that it can be accomplished in as few sessions as possible, and as quickly and orderly as possible.
• Keep close check on the freezer after inventory has been returned to ensure that freezer returns to set temperature and holds there.

4.7.3 Liquid Nitrogen Storage

4.7.3.1 Health and Safety Hazards

Liquid nitrogen is extremely cold; it boils at -196°C. Skin can survive brief contact with – 80 surfaces, but bare skin coming into contact with liquid nitrogen (or objects cooled by it or gases evolving from it) will be severely damaged, comparable to burns caused by contact with boiling water. Insulated cryo-gloves will protect you against liquid nitrogen vapor, but will not offer complete protection against direct contact with liquid nitrogen. Skin can freeze or adhere to surfaces cooled by liquid nitrogen, causing tearing upon removal.

Nitrogen gas sublimating from liquid nitrogen can quickly displace the oxygen in poorly ventilated or closed rooms, and can cause asphyxiation. To reduce the possibility of asphyxiation, use liquid nitrogen only in well-ventilated rooms. Do not shut doors when filling containers. If you suspect or encounter a liquid nitrogen leak in your facility, leave the area immediately, alert other nearby personnel, and call for help.

NOTE: the cloudy vapor that appears when liquid nitrogen is exposed to the air is condensed water vapor, not nitrogen gas. Nitrogen gas is invisible.

Explosion Hazard: Never place liquid nitrogen (or dry ice) in a sealed container or any object that could entrap the sublimating gas.

Never mix liquid nitrogen (or dry ice) with water or water ice; never pour it down a sink drain. Ice can solidify around it, trapping sublimating gas at a high pressure and creating an explosion hazard.
 
Given the risks associated with the use of liquid nitrogen, best practice is to employ the buddy system when you have to handle this material.

4.7.3.2 Liquid Nitrogen Storage Overview

Dewar storage vessels are vacuum-jacketed tanks for maintaining low temperature storage of biological material; they are designed to safely contain liquid nitrogen as the low temperature agent. Dewars accommodate racks for small sample vials. A loose fitting cap fitting over the neck opening prevents atmospheric moisture from plugging the neck and allows sublimating nitrogen gas to escape. Thus this type of container is non-pressurized. A liquid nitrogen supply cylinder is pressurized.

Transfer vessels are designed specifically for containing and transporting liquid nitrogen; i.e., they provide carrying handles, pressure relief valves, and venting lids. Only use such a transfer vessel designed for transporting liquid nitrogen to supply a dewar. After filling, a transfer vessel may be carried between two people with its handles, or placed on a cart to transport. If a cart is used, secure the vessel to the cart so it will not tip over when the cart is conveyed over a threshold, etc.

Always label tubes/vials well for liquid nitrogen storage, and record their placement and removal on a dewar inventory log; include tube/vial location within the storage box/can, as well as the designation of the storage box/can. This is best practice because: 1) samples can be efficiently located prior to retrieval, which keeps the time that the dewar has to be open to a minimum; 2) it prevents sample mix-ups, losses, etc.

Liquid nitrogen must be maintained at a certain volume within the dewar to keep samples at the appropriate low temperature. Levels should never go below 2 inches. Dewars can be outfitted with monitoring alarms which will alert users if the LN2 level drops to a critical point, but these alarms may not be accurate for displaying actual fill levels. The level also can be monitored by immersing a stick reserved for this purpose, to see where the liquid level is detected on the stick. Dewar liquid nitrogen levels should be checked regularly and refilled as indicated. Extra care must be taken and arrangements must be made for holidays/ semester breaks, etc., to prevent depletion when the lab is closed.

Vapor phase storage is strongly recommended 1) because contamination can be transmitted to submerged vials by liquid nitrogen, and 2) because storing in the liquid phase heightens the potential for explosion of improperly sealed vials when you retrieve the vials to use them.

4.7.3.3 Equipment and Supplies

DO NOT TRANSPORT OR HOLD LIQUID NITROGEN IN OPEN CONTAINERS, OR IN OTHER CONTAINERS THAT ARE NOT DESIGNED FOR USE WITH LIQUID NITROGEN.

Storage dewars on wheels must not be rolled on lengthy routes for relocation, or for filling at the liquid nitrogen source. Moving/ jostling the dewar contents could irreparably damage it by cracking the inner metal wall. Moving a partially-to-fully filled dewar also creates a liquid nitrogen spill hazard. Storage dewars must be filled by bringing a supply of liquid nitrogen to them.

Always use tubes/vials that are recommended for cryostorage; even these products will not withstand prolonged submersion in liquid nitrogen, so be sure to store tubes/vials in the interphase space in the dewar (i.e., in the vapor layer, not the liquid nitrogen layer).

Always place tubes/vials in cans, canes or boxes that are recommended for dewar storage, or are part of the dewar storage system, before placing them within the dewar.

4.7.3.4 Using a Liquid Nitrogen Supply Cylinder to Fill a Transfer Vessel

1. Always wear eye/face protection, buttoned lab coat, long pants, closed toe shoes, and insulated gloves when dispensing liquid nitrogen into a transfer vessel.
2. Hold the filling hose with a secure grip while turning on the tank valve to avoid unpredictable nozzle motion and spillage when flow begins.
3. To prevent splashing, place the filling hose at or below the mouth of the receiving vessel.
4. Slowly turn on the tank valve to begin flow of liquid nitrogen.
5. If flow seems to be mostly vapor and squeals loudly, the tank is almost empty or completely empty.
6. Determining when your transfer vessel is full can be challenging. DO NOT FILL TRANSFER VESSELS UNTIL IT IS OVERFLOWING AS A METHOD OF OBTAINING A FULL TRANSFER VESSEL. Instead, turn off the storage tank valve, remove the hose and check in the vessel periodically to see how quickly it is filling. You will have to wait for the white vapor to clear to see the fluid level. Flow rates for LN2 may not fill transfer vessels quickly, so be patient. Do not fill to very top of vessel.
7. Place lid on the transport vessel before moving. NEVER use a tight-fitting lid on a vessel containing liquid nitrogen.

4.7.3.5 Storing, Retrieving and Reviving Biological Samples in Liquid Nitrogen Dewar

Maintain a storage dewar in a safe, out-of-the-way location in the lab.

When inserting or removing racks, be careful not to come into contact with the neck of the dewar; remove or insert racks in a vertical manner to prevent scratching or otherwise damaging this vulnerable area of the vessel. If it is damaged, the vacuum jacket could rupture and ruin the dewar’s functionality.

When accessing the contents of a dewar, always provide yourself with a stable, convenient location to place the dewar top after removing it, as well as safe, stable place to put the ultra-cold storage racks or samples that you remove from the dewar (e.g., lab bench, lab cart surface, etc.).

Use tongs to remove sample ultra-cold vials from storage canes or boxes. Vials can explode when removed from the dewar, so this should be done in the BSC.

Always wear eye protection, lab coat, closed toe shoes and insulated gloves when adding or removing samples from liquid nitrogen storage.

The thawing procedure is stressful to frozen culture material (cell cultures and bacterial cultures). Using good technique and working quickly ensures that a higher proportion of cells survive thawing. Always follow instructions provided with your cells, with reagents used, or in your SOPs for best results when thawing and reviving culture material.

4.7.4 Lyophilization Storage of Biological Materials

4.7.4.1 Storage Overview

Lyophilization, also known was freeze-drying, is a process in which water is removed from a material after it is frozen and placed under a vacuum. Laboratories performing biological research sometimes 1) utilize lyophilizers for preservation and storage of bacteria, fungi, protozoa, algae, viruses, mammalian tissue cultures or plant cells, and 2) reconstitute cultures in lyophilized form to revive cultured material. Both procedures have inherent hazards. Thus, this equipment and procedures should only be used by persons who have been thoroughly trained.

Lyophilized mammalian cells, plant cells, protozoa and algae must be maintained below -150˚C in a liquid nitrogen storage system for long-term stability. Lyophilized bacterial cultures can survive storage at -60˚C for several years, and should never be stored at temperatures greater than 4˚C.

4.7.4.2 Lyophilizer Hazards

A high vacuum is generated with this equipment which can cause implosion of glass ampules. Safety glasses/goggles/ face shields and other appropriate PPE must be worn at all times when using a lyophilizer.

Lyophilizers generate low temperatures. Because ampules and other parts of the machine may cause cold burns if touched to exposed skin, protective gloves must be worn when handling these cold parts, or avoid contact.

There is a risk of electrical shock hazard if a lyophilizer is misused, or is malfunctioning. Inspect for any problems before using a lyophilizer (e.g., problems with power supply, sparks, burning smell, etc). If problems are discovered, do not use the unit and notify the individual who is responsible for it.

Never use a lyophilizer for evaporating material containing organic solvents.

4.7.4.3 Reviving Lyophilized Biological Material

The process of liberating lyophilized culture material from an ampule can create aerosols of dry culture material and present a biohazardous exposure risk. Only perform this procedure in the containment of a BSC, and ensure that work surfaces and surfaces of supplies are decontaminated following your procedure.

The glass ampules used for lyophilized cultures actually must be broken in a controlled procedure to liberate their contents. Due to the significant risk of receiving cuts from broken glass, as well as risk of exposure to biohazards, you must wear appropriate PPE (e.g., hand protection, lab coat, eye/face protection) for this procedure. Use the instructions provided with cultures you have ordered from a commercial provider. For lyophilized cultures produced in house, the following ATCC method can be used:

1. Score the ampule with a sharp file near the tip of the ampule.
2. Disinfect the ampule with alcohol-dampened gauze. (Make sure the gauze is not too wet.)
3. Wrap the gauze around the ampule, then using two hands, break the ampule at the scored area. Take care that alcohol from the gauze is not sucked into the dry culture material when the vacuum is released.
4. Rehydrate the material at once.