CONTAMINATION DURING CRYOPRESERVATION
Cryopreservation is the most important component in cryobiology and by using this process many biological materials such as the extinct species, the embryos, tissues, cells, organs and seeds can be preserved for future use. During the process of cryopreservation, there are some evidences showed that the major risk is caused due to contamination. This project has been done to know how the contamination occurred and the precautions that has to be taken to prevent contamination. This project is done on the basis of “simple assay system for monitoring the potential for contamination during cryopreservation (G.J.Morris).” The work reported in this study is to evaluate the contamination during cryopreservation process. Sucrose hemi-heptahydrate is a contaminant which made the study possible by its properties like being harmless to the environment. Liquid Nitrogen plays very important role in the cryopreservation due to its physical properties. Cryopreservation involves the storage of biological materials, tissues, cells and organs at Liquid Nitrogen temperature. Contamination is a major problem during cryopreservation so this study helps in estimating the level of contamination. This study was performed in Cryolab where certain conditions like room temperature 25 degrees and optimum humidity were maintained. So in this study Liquid Nitrogen is contaminated with Sucrose hemi-heptahydrate chemical which is harmless inert material. This study takes place in Cryolab where some Liquid Nitrogen is taken into the Dewar flask from storage tank. sucrose hemi-heptahydrate when added to the Liquid nitrogen Dewar flask it will contaminate the environment by its vapour phase which can be observed by placing the sucrose solution containing petriplates at various heights like high level, same level and low level and also by maintaining the petriplates with various time courses. After exposure petriplates were observed for the crystal formation which indicates that contamination was occurred. These plates were compared with the control in which pure sucrose solution is used.
CHAPTER ONE INTRODUCTION
1.1 GENERAL INTRODUCTION
The cryobiology word derived from the words of Greek "Cryo" means cold, “Bios” means life, and “Logos” means science. So, cryobiology is the study of biological materials at ultra low temperatures. The biological materials may include proteins, cells, organs, organisms and tissues. Cryobiology is important in 6 major areas. They are
1. Cold-adaptation of plants, animals and microorganisms.
2. Cryopreservation. In this cell, embryos, gametes and tissues are preserved for longer periods of time by using cryoprotectant during thawing and freezing processes.
3. Organ transplantation. In this process organs like kidneys, liver, heart are stored below hypothermic conditions.
4. Lyophilization also called freeze-drying and is used for pharmaceuticals.
5. Cryosurgery involves the destruction of damaged tissues by using cryogenic liquids and gases.
6. Physics of ice nucleation.
Cryobiology has wider applications in the field of genetic research, infertility treatments, agriculture, biomedical research and live stock breeding.
The cryobiology in genetic research involves freezing and storage of different strains of embryos in laboratory animals. This can reduce the cost of research animals.
Cryopreservation is one of the most important components in cryobiology. Cryopreservation is the process of storing the biological elements at ultra low temperatures by using liquefied gas. Cryopreservation is one of the method in which viable cells, tissues, organs and microorganisms can be stored for longer periods of time at low temperature i.e. boiling point of Liquid Nitrogen -196degrees. In 1949 Christopher Polge first cryopreserved the sperm. This process contains lot of benefits. Sperms, eggs and embryos are cryopreserved for fertility treatments. In cryopreservation process metabolic actions are reduced and in turn reduce the decaying. It is also used for preservation of plant tissues and seeds. In this process there is damage occurred due to the formation of ice crystals to prevent this damage cryoprotective agents are used. The successful cryopreservation depends on the rate of cooling and concentration of cryoprotectant.
In Agriculture field by using cryopreservation breeders can get the desirable genetic qualities. In human infertility treatments frozen embryos, eggs and sperms are stored for future fertilization treatments. First baby was born in 1984 by using frozen embryo. Cryopreservation of embryos involves water removal from the cell and replacement of organic antifreeze which will prevent the formation of crystals of ice causing the cell damage. For the cryopreservation of eggs reproductive biology associates scientists in Atlanta can use the chemical solution which is similar to the natural fluid of ovaries.
In organ preservation antifreeze proteins are used for the preservation of rat liver. These proteins can change the ice crystal structure which can reduce the damage to cells. Vitrification is used for the cooling of organs to prevent the formation of ice crystals.
Contamination is the presence of foreign particles, in any substance that causes damage to the substance. The agent or foreign particle that causes contamination is called “Contaminant”. The contaminant interferes with the chemical reaction and distorts the results in an experiment. The contaminant may be a chemical substance or a living micro organism. If the contaminant is a living micro organism then the effect of contamination will be high Cryopreservation is nothing but preserving the biological materials at lower temperatures. In the present day world cryopreservation play very important role.
If we consider the things like blood banks, gene banks, stem cell banks and tissue banks etc we can understand the importance of cryopreservation. As the cryopreservation is very sensitive technique perhaps the contamination of samples is more likely high. Cryopreserved sample must be stored at the temperature -196°C which is a boiling point of Liquid Nitrogen. The stored biological material viability is not dependent on the time of storage. The oldest cryopreserved sample is a bovine spermatozoon which does not show any reduction in viability for 50 years storage. Cryopreservation is done by the immersion of Samples in Liquid Nitrogen or storage of sample in the vapour phase of Liquid Nitrogen. Immersion of sample in liquid nitrogen causes stable storage temperature but there may be risk of potential contamination. Storage of sample in Liquid Nitrogen Vapour phase minimizes the contamination. However, in vapour phase above Liquid Nitrogen large temperature variations occur and these temperature variations because contamination in open Dewar's in which samples are transferred and also reduce the viability of cells. In vitrification process Vapour phase of Liquid Nitrogen causes contamination problems. For cryopreservation there are many cryocontainers are available. These containers are different according to whether they are sealed or suitable for liquid or vapour phase storage. Heat sealed straws and glass ampoules can cause little contamination. Conventional straws in which PVA powder is used as sealant and cryovials stored at vapour phase and cause high risk of contamination. Cryovials and conventional straws which not have seals can cause leakage during storage. Accumulation of liquid cryogen in container causes problems on thawing. When the temperature is increased to above -190°C then Liquid Nitrogen turns into gas and can cause explosion of the Cryotank. Straws are having large surface area with thin wall and small diameter and they are warmed rapidly after removal from the Liquid Nitrogen. Cryopreservation through ultra rapid freezing or vitrification needs direct contact between Liquid phase Nitrogen and freezing medium which contains embryos or oocytes. Many bacterial and viral agents can survive easily in Liquid Nitrogen and cryoprotectant.
Contamination may occur in many ways like during the collection of samples in test tubes, variation in temperature, from the environment, the donor, the process of cryopreservation, the operator who is undertaking the process, transportation of samples in the Cryobanks which are contaminated before the introduction during process and contaminated while storing the samples. This contamination can be occurred by the infective agents like fungal, bacterial, or viral which can survive at low temperatures and in the presence of cryoprotectant. During cryopreservation the risk and safety identification is the most important thing to consider. The risk identification involves categories. They are unavoidable which are not under the control of operator example climatic disasters and avoidable (M.J.Tomlinson,2008) which occur during research operation without proper vigilance and care. So we must take care about the risk assessment.
The potential source of contamination is Liquid Nitrogen itself and contamination from other samples which are cryopreserved in same cryotank. Manufactured Liquid Nitrogen has small microbial count. Contamination may take place during storage and transportation. Dry shippers and Dewar's which are used for transport when they allowed to warm can accumulate condensate which are largely contaminated with fungi and bacteria. When these are refilled with Liquid Nitrogen can contaminate the sample. Straws are contaminated outside during storage seals and plugs cause leakage and particulate matter is transferred through Liquid Nitrogen in the storage tank. Sealed Cryovessels are stored in liquid phase and vapour phase without outside contaminants (Bielanski et al,2003).
Contamination of samples during cryopreservation occurs in different stages. They are
(1) Cryopreserved samples contamination occurs during long term storage and by the process of vitrification and controlled rate freezing.
(2) Contamination occurs from the other cryopreserved samples.
(3) Contamination occurs through Liquid Nitrogen itself.
(4) Contamination through integrity of different cryovials, straws during the storage in Liquid Nitrogen.
Sample may be contaminated during the cryopreservation. Sample loss may be occurring due to mislabeling, contamination and misidentification and these are caused by insufficient storage. For example sample loss in the vessel occurs because it is not effectively secured on cryocane. The transport of cryotanks may cause minor changes in the positive temperature and results in the damage of sample. For long storage of cryovessel results in the repeated cycles of re warming and cooling. So sentinel cryovials are used for testing the stability of cryo vessels as well as biological stability and physical security for long time. Cryovials are prepared for the purpose of tolerating at ultra low temperatures and prevent the passage of Liquid Nitrogen when it leaks into the vial may lead to the cross contamination. The penetrated Liquid Nitrogen may produce pressure in the Cryovial when vapour is formed. It may lead to explosion on re warming. The exploded contents of Cryovial produce aerosols which cause pathogen transmission or microbial contamination. The liquid phase of Liquid Nitrogen is causes more contamination than vapour phase of Liquid Nitrogen.
1.2 LIQUID NITROGEN CONTAMINATION:
Liquid Nitrogen is a liquefied gas and it causes many damages. The direct one include cold injury, frostbite and asphyxiation and indirect one include mechanical injury caused during the storage of Liquid Nitrogen container and pathological and toxicological injury occur in Cryovial by the release of pathogen, virus or organisms into them. Liquid Nitrogen causes cold burns and its vapour can produce eye damage and frostbite. The storage vessels must be resistant to Liquid Nitrogen vapour and liquids because cryogenic equipment has different tolerances for different states of Liquid Nitrogen. Dewar's can be explosive by ice plugs which produce pressure in them and leakage of Liquid Nitrogen in Cryovial container because expanded vapour on re warming can cause explosion. Liquid Nitrogen poses lethal problems for personnel.
During long term storage of organs in Liquid Nitrogen tank causes accumulation of ice crystals in the tank and is the source of contamination (M.Piasecka-Serafin,1972). Liquid Nitrogen contamination can occur by the entrapment of air born microbes in ice crystals which form the air above the Liquid Nitrogen (G.J.Morris,2005).When lid of the tank is opened then it will generate the turbulence which causes the mixing of Nitrogen gas with outside air and causes the Liquid Nitrogen contamination. In mammals, cross contamination is observed during the storage of biological sample in Liquid Nitrogen (R.S.Tedder et al,1995.,D.Fountain et al,1997.,A.Bielanski et al,2003). Aspergillus contamination is observed in hematopoietic stem cell storage in Liquid Nitrogen tank (R.S.Tedder et al,1995).In bone marrow preservation contamination of virus Hepatitis .B is observed by nucleotide sequence analysis in Liquid Nitrogen tank. Survival of viruses like stomatitis virus is observed on direct exposure to Liquid Nitrogen (Schafer et al., 1976), Adenovirus and Herpes simplex virus (Jones and Darville, 1989).
Sperm banks are important in the artificial insemination. Semen preservation not involved sterile technique. Semen is collected by dip and wipes method and overfilled straws are cracked during freezing and contaminate the Liquid Nitrogen. Straws which are not properly sealed would absorb the Liquid Nitrogen and causes the cross contamination while using the thawed semen for clinical use. The containers which are made up of polyvinyl alcohol sealing powder have the potential risk of contamination because this powder would aggregate the microbes. Semen storage in cryovials has direct contact with Liquid Nitrogen.
1.3 CRYOGENIC EQUIPMENT:
Cryogenic vessels are made for the transportation and storage of liquid gases at ultra low temperature. Special techniques are used for the manufacturing of cryotanks. Cryogenic tank is double walled and cylindrical in shape. For the prevention of evaporating gases, insular material is filled in the annular space between outer and inner vessel and high vacuum pump is attached. Cryogenic vessel design is compact, sturdy and its operation is easy. Top end filling of liquid reduces the inside pressure of tank and bottom filling increases the inside pressure. So, stable pressure is maintained during constant liquid delivery to vaporizer by regulating the both top and bottom filling valves opening. Capacity of cryotank storage temperatures (-196°C) ranging from 1000 Liters to 20000 Liters. Their installation and maintenance is easy. Inner wall is made up of stainless-steel and outer vessel is made up of carbon steel. Indicator is fitted with this tank to show the liquid level and safety valves, vacuum sensor, pressure gauge and pressure buildup valves. This is used for the transportation of liquid and vapour gases. Thermo siphon system is used for the purpose of cylinder filling to curb losses. All safeties and valves have good standard codes, approved and granted by CCE. Cryogenic vessel is entirely painted with epoxy white.
1.4 MICROBIAL CONTAMINATION VIA CRYOGENIC EQUIPMENT
There are three types of cryogenic equipment which involved in the microbial contamination. They are storage cryotanks, controlled rate freezers which are programmable and dry shippers. (B.W.W.Grout, G.J.Morris 2008) Were tested these apparatus for microbial contamination by using fungal pathogen i.e. Sclerotina species. It was used to cool a programmable Liquid Nitrogen freezer and after one run cooling it gives the positive results. In dry shippers analysis of microbial growth of vapour phase gives the positive results for the presence of pathogens. These experimental results confirm that pathogens will survive in Liquid Nitrogen and contaminated the cryogenic equipment. Bielanksi experiment(A.Bielanski 2005a.,2005b,) involves the contamination of bovine semen and embryos samples and also 2 different dry shippers (MVE SC2.VI holding 1.5 L of Liquid Nitrogen and an MVE Mini-Mover holding 2.9L of Liquid Nitrogen) are tested with bacteria. The contamination is tested for transmission from the contaminated dry shipper to germplasm, contaminated and non contaminated cryopreserved germplasm and a stock culture of pathogenic agents and germplasm. The embryos and semen which are stored in vapour phase of Liquid Nitrogen is tested between contaminated and no contaminated samples for 7 days in open containers. There is no cross contamination is observed in dry shippers. From this he can conclude that dry shippers can provide protection on long term storage of germ plasm in vapour phase of Liquid Nitrogen. There is some contamination occurring but it can be prevented by taking some precautions like disinfecting the dry shippers. If one sample which has contamination is placed along with other samples in cryotank for preservation then it will lead to the contamination of entire contents in the cryotank. So before preserving samples must be screened for any infections.
1.5 VIRAL TRANSMISSION:
The study (G.N.Clarke,1999) reported the viral transmission during the cryopreservation of human tissues. The study of (Bielanski et al.,2000) used ultra rapid freezing or vitrification which requires the direct contact of samples with LN. Bovine embryos are used for finding the viral contamination. In this test infected embryos are cryopreserved via vitrification which contains DMSO, sucrose and ethylene glycol. The infected and controls were taken in different containers with different sealants and cryopreserved for 3-5 weeks in same Cryotank or Dewar in Liquid Nitrogen of liquid phase. The sealed ones are non infective with virus when compared to non sealed ones. To prevent the contamination while embryo preservation use sealed container or double bagging, avoidance of direct contact between freezing medium and Liquid Nitrogen and using separate storage tanks for infected and non infected germplasm. The vapour phase of Liquid Nitrogen is safe for short term storage of samples.
The study(Mazzilli et al.,2006) identified the survival of pathogens in Liquid Nitrogen have both positive and negative results. In human sperm cryopreservation the concentration of Candida spp., reduction is observed. Neisseria gonorrhea will survived for 18 months in Liquid Nitrogen. During cryopreservation bacterial contaminants can be detected easily because bacteria can attach to the polymer surface of Cryovial. The process may cause the collection of bacteria which leads to reduction of CFUs. Long term storage leads to the accumulation of microbes which are entrapped in ice during the process in the cryovessel. The contamination occurs at different times in long storage. The Dewar with short operational time had high microbial count.
Kyuwa et al. (2003) used mouse embryos for testing the cross contamination. These are tested after 6-12 months storage in liquid phase of Liquid Nitrogen in cryotank. There is no contamination occurs in progeny mice which are derived from the cryopreserved embryos. Freezing and thawing cycles will increase the risk of contamination because these processes increase the permeability of the membrane to virus. In plant germplasm cryopreservation, cryoinjury leads to damage to cell structures which give entry for virus.
1.6 MINIMIZATION OF CONTAMINATION:
Cryovials are put into the plastic cryosleeve before immersing into the Liquid Nitrogen which prevents the technical problems while handling.
Manufacturers can produce many types of Cryovial with different features according to the customer's choice of storage system. Internally-threaded vials incorporating a silicone ‘O' ring gasket and externally-threaded cryovials are used for storage, in mechanical freezers and vapour phase vessels; Polyethylene membrane sealants for cryovials (H-I.Chen et al,2006a). They provide security for cryoconservationists used for fully immersed liquid phase Liquid Nitrogen storage (TMNunc,2005, TMNunc,2008).The study (Chen et al. 2006a) produce partial membrane sealant which prevents the passage of Liquid Nitrogen in the submerged storage tank. To prevent the risk of contamination of samples in cryotanks involves the precautions like double-containment (in this sample outer surface is covered with sterile encasement),preventing the direct exposure of samples to Liquid Nitrogen and storing the samples in Liquid Nitrogen vapor phase causes the deposition of contaminants like microbial flora are concentrated. It is very difficult to get sterile Liquid Nitrogen in Cryobanks. During long storage of samples, they must be separated from Liquid Nitrogen by putting the double bagging around sample container. This double bagging must be sterile on its outer and inner surface. The prevention of viral contamination involves cryopreserved samples are screened for blood borne viral infections and using the double sealed cryobag. This prevents the exchange of virus from sample to Liquid Nitrogen. To prevent the contamination while embryo preservation use sealed container or double bagging, avoidance of direct contact between freezing medium and Liquid Nitrogen and using separate storage tanks for infected and non infected germplasm. The vapour phase of Liquid Nitrogen is safe for short term storage of samples. The risk of contamination during long term storage can be reduced by placing the cryovessel in clean rooms with clean air, using containment or double bagging of Cryovial and stored in vapour phase of Liquid Nitrogen. Semen storage in cryovials has direct contact with Liquid Nitrogen so cryoflex must be used for sealing to prevent contamination. Cryovials which are sealed with o ring absorb Liquid Nitrogen and showed no evidence of condensed Liquid Nitrogen inside Cryovial when stored in vapour phase of Liquid Nitrogen. Through IMV technology new straw system (Cryo bio system) is developed. It will reduce the risk of contamination because it is made up of shatter-proof ionomeric resin. It prevents the contact between sample and straw. Cryovials does not maintain its seal when immersed in Liquid Nitrogen, so straws should be stored inside cryoflux. Human semen can be stored for 1year without loss of motility and capacity of fertilization at a temperature of–79°C.While using the Cryoflux in liquid phase of Liquid Nitrogen will have some inconvenience, to overcome this single step liquid nitrogen vapour cooling system is used. While using dry shippers for storage a little contamination occurs and this can be prevented by using disinfectant.
Now with improved technology in cryopreservation gene banks are effective for cryopreservation of samples upto10-15 years without contamination via cryogenic equipment. The pathogen contamination in cryopreservation of plant germplasm can be prevented by phytosanitary control, includes safe transfer and storage in gene banks, quarantine, detection of pathogen, diagnosis and evaluation tests will results in free pathogen plants.
The collection of cryopreserved samples must have three responsibilities; they are purity, identity and stability. These all are supported by good quality assurance practices. Quality Assurance (QA) and cryoprotective treatments are used in constructing the Cryobanks. The cryoprotective treatment involves exposure times, clean culture rooms and subculture intervals. Quality control includes the total process which must be reliable and should be maintained to fit for purpose. Accreditation maintains the procedures and formal standards. Changing of cryobiological research into cryobanking practice involves complexity and it not only requires diligent technical knowledge but also ethics, risk assessment, regulatory affairs and biosafety. These all influence the efficiency of technological transfer and validation of storage of cryogenic methods.
The future success of cryopreservation will depend on the safety, regulatory and technical terms. The development of cryobanking will depend on many factors like needs and expectations of stakeholders, users and beneficiaries.
1.8 AIM AND OBJECTIVE
To find out how the contamination occurs during cryopreservation by using Sucrose hemi-heptahydrate as a contaminant.
Contamination can be identified by using experimental methods but they require modern methods and these are very time consuming. So by using sucrose particles in laboratory it is easy to identify the contamination in different stages during cryopreservation.
CHAPTER TWO MATERIALS AND METHODS
Sucrose is the single most pure organic chemical, complex carbohydrate and disaccharide. It is made up of two sugars which forms bond between hydroxyl groups of one sugar with carbon of another sugar. It is commonly called as table sugar. It is produced from sugarcane (20%/w) and from sugar beets (15%/w).On acid hydrolysis sucrose produces equal amounts of glucose and fructose. Unlike other disaccharides it does not exhibit mutarotation because it is not reducing sugar. Sucrose is naturally obtained from every vegetable and fruit. Naturally sugar is produced from plants by photosynthesis. It is white crystalline powder or monoclinic spheroidal crystals, it has characteristic caramel odor. 1gm of sucrose is soluble in 0.5ml of water. Sucrose solutions are neutral to litmus and density is 1.59.Melting point is 160-186degrees (320-367F).It is stable under normal conditions.
18.104.22.168 Sucrose hemi-heptahydrate
The chemical which is used in this study was harmless and was provided by Dr.John Morris (Asymptote Ltd., Cambridge). It is a phase 2 Sucrose hydrate. Sucrose crystallizes in anhydrous form and the hemi-heptahydrate is formed due to the crystallization of sucrose at low temperature (-34°C). It has a eutectic temperature of -9.5°C and melting temperature of +27.8°C. These crystals are useful for measuring the optical properties in aqueous solutions. These crystals are orthorhombic and are in the form of needles and blades when crystal growth was rapid and are prismatic rods when growth was slow. Optical properties like refractive index (+0.3) and optical axial angles are 2E=84°, 2V=52.5° which are calculated from β and 2E, 2V=55° which is calculated from α, β and γ. Dispersion is weak.
Molecular formula : C12H22O11.3.5H2O
22.214.171.124 Liquid Nitrogen
It is colorless, odorless and inert gas with non corrosive and cold in nature. Nitrogen at very low temperatures in liquid state is called Liquid Nitrogen. Industrially it is prepared by the fractional distillation of liquefied air. It is colorless, odourless and tasteless liquid. It is cryogenic fluid which causes frostbite when contact with biological tissue. It can be stored and transported in vacuum flask. Because of its low temperature it is widely useful in every aspect. It is used to store cells, blood, tissues and other biological materials. It is used as coolant, used in cryo therapy and also used in promession process. Liquid nitrogen expansion ratio from liquid to gas is 1:694. When Liquid Nitrogen is vaporized then large amount of force is released. Careless handling of Liquid Nitrogen will result in cold burns because of its extreme low temperature. It also acts as an asphyxiant because when nitrogen evaporates it will results in the reduction of oxygen. In 2 storage tank is used for the storage and transportation of Liquid Nitrogen at low temperature. It is double walled and insular material is present in between two walls and is cylindrical in shape.
Weighing machine, Spatula,500ml volumetric flask, Magnetic stirrer, Microwave, Measuring cylinder, Distilled water, Liquid Nitrogen Dewar, Hot stirrer, Petri plates, Clingfilm, Para film, Gloves and Scissors.
Para film: It is double roll size 4in.x250Ft. It is flexible, self sealing, easily moldable, odorless, thermoplastic, moisture resistant, semitransparent and practically colorless.
Clingfilm: It is excellent for covering and wrapping all foods except pure fats and foods preserved in an oily medium. It is suitable for fridge, freezer and microwave. In microwave it is used for reheating and defrosting. Roll size is 450mmx300m. It conforms to EC directive 2002/72/EC for use with food.
Gloves: Fisher brand. Powder free nit rile gloves, 250mm length, ambidextrous, protein free, latex free, non-sterile, beaded cuff and textured fingertips.
Weighing machine: Company name—Oerting OB152. 1500g.
500ml volumetric flask: SIMAX, CZECHOSLOVAKIA.
Magnetic stirrer: Gallenhamp, property of unilever research 00217
COLWORTH Lab, section 951.C.22544.
Microwave: Prestige DS20
Measuring cylinder: Griffin GT. BRITAIN, B.S.604m (ml in 20degrees)
Dewar flask and container: DILVAC made in England.
Distilled water, spatula, scissors and freezer.
2.2 GENERAL PLAN OF THE PROJECT
Main steps involved in this study were:
Ø Preparation of 70% W/W Sucrose solution
Ø Sucrose hemi-heptahydrate was used to precheck the contamination for LN2 storage system. For this control and positive controls were performed.
Ø Petri plates were filled with Sucrose solution and placed at various heights (High, Same, Low levels) and various time periods i.e.1 to 5 hrs and 1 to 5 days respectively. Then plates were removed and incubated at a temperature of -20°C for 5 to 14 days and crystal formation was observed which indicates presence of contamination.
Ø The main objective of the study was to evaluate the contamination during cryopreservation by introducing harmless, inert material Sucrose hemi heptahydrate to Liquid Nitrogen storage system. This study was performed in Cryolab. In this study sucrose solution containing plates were placed in Cryolab where the Liquid Nitrogen Dewar flask was contaminated with Sucrose hemi-heptahydrate. These sucrose petriplates were placed up to 1 hour in Cryolab. After one hour, plates were removed from room and each plate was wrapped with Para film and incubated in the freezer at -20 degrees for 5 to14 days. After 5 days petriplates were removed from the freezer and crystal formation was observed. This is simple, direct method and requires less time.
2.3.1 PREPARATION OF 70% W/W SUCROSE SOLUTION
The Sucrose was taken and weighing machine was adjusted by placing the 500ml volumetric flask and tampered to zero. After that the volumetric flask was removed and 70% of accurately weighed Sucrose was added to it. To this volumetric flask 100ml of distilled water was added and placed in a microwave for 1 to 2 minutes. Remove the flask from the microwave and shake well for dissolving Sucrose in distilled water. Then this solution was cooled and wrapped with parafilm and stored in the refrigerator which was maintained at 4°C temp and was used whenever needed.
2.3.2 PREPARATION OF CONTROL
For this preparation 70% W/W of Sucrose solution was prepared by taking accurately weighed amount of 70 grams of Sucrose in 500ml volumetric flask and 100ml of distilled water was added and was placed in microwave for 1 to 2 minutes . Then the flask was removed from the microwave and a magnetic stirrer was placed in it and transferred to hot plate stirrer to get clear solution. After the solution was cooled and pour the Sucrose solution in each plate is covered fully. Then these petriplates were exposed in the open environment in the Cryolab without containing contaminated Liquid Nitrogen in the room. After 1 hour of time these petriplates were removed from the lab room, labeled each plate and wrapped with Para film; again the three petriplates were wrapped with cling film and incubated in the freezer at a temperature of -20°C for duration of 5 to 14 days. This control was performed to ensure the Liquid Nitrogen contamination in the lab room where this study took place.
2.3.3 PREPARATION OF POSITIVE CONTROL
Take 70 grams of Sucrose in 500ml volumetric flask and 100ml of distilled water was added and placed it in the microwave for 1 to 2 minutes. Then remove the flask from the microwave and shake it well for dissolving the Sucrose in distilled water. This flask was transferred to the magnetic stirrer until it gets the clear solution. After the solution was cooled it was poured in each petriplates; approximately to cover the entire plate and sucrose hemi-heptahydrate was added to plates and were placed in the lab room for 1 hour. After 1 hour plates were removed from the lab room and each plate was wrapped with Para film and incubated in the freezer at -20°C for 5 to 14 days. After 5 days plates were removed from the freezer and crystal formation was observed. This positive control was performed to check whether any particular kind of crystal formation was observed.
The contamination process can be observed in different ways. In this study contamination was observed by placing Sucrose solution containing plates at various heights like high level, same level and low level and various time periods like 1 to 5 hours and 1 to 5 days respectively.
2.3.4 CONTAMINATION AT DIFFERENT HEIGHTS
In this process prepared 70% W/W Sucrose solution was taken in each petriplate. Some Liquid Nitrogen was taken from the storage tank to Liquid Nitrogen Dewar flask. To this Liquid Nitrogen 0.1 gram of Sucrose hemi-heptahydrate was added to study the contamination levels. The Sucrose containing plates were placed near the contaminated Liquid Nitrogen Dewar flask. Three of the petriplates were placed at the same level of contaminated Liquid Nitrogen and other three petriplates were placed below the level of contaminated Liquid Nitrogen and other remaining petriplates were placed above the level of contaminated Liquid Nitrogen. After one hour these plates were removed from the flask and each plate was wrapped with Para film. For preventing the contamination from air borne particles wrapping must be done properly. These plates were incubated in the freezer at -20°C for 5 to 14 days. After 5 days plates were removed from the freezer and crystal formation of Sucrose hemi- heptahydrate was observed.
2.3.5 CONTAMINATION AT VARIOUS TIME PERIODS (1 TO 5 HOURS)
In this study it shows how the contamination changed at different time periods. For this study 70% W/W Sucrose solution was taken and poured into each petriplates; until the entire plate was covered. These petriplates were placed near contaminated Liquid Nitrogen Dewar flask. After exposure of 1 hour; three petriplates were removed and each plate was wrapped with Para film and incubated in the freezer at -20°C for 5 to 14 days. After 2 hours of exposure time other three petriplates were removed and each plate was wrapped with Para film and incubated at -20°C in the freezer for 5 to 14 days. Similarly after three and four hours of exposure time the six (three at each hour) petriplates were removed and each plate was wrapped with Para film and incubated at -20°C in the freezer for 5 to 14 days. Similarly after five hours other three petriplates were removed and each plate was wrapped with Para film and incubated at -20°C in the freezer for 5 to 14 days. After 5 days of incubation all hourly petriplates were removed from the freezer and crystal growth of sucrose hemi-heptahydrate was observed which indicates the sign of contamination.
2.3.6 CONTAMINATION AT VARIOUS TIME PERIODS (1 TO 5 DAYS)
This study shows how the contamination spread with time. In this study 70% W/W sucrose solution was taken and poured into each petriplates; and these petriplates were placed in a room at same distance and same level where liquid Nitrogen is contaminated with sucrose hemi- heptahydrate. After one day exposure of time three petriplates were removed and labeled the time and date course on each plate with a marker and each plate was wrapped with Para film and incubated in the freezer at -20°C for 4 to 15 days. After 2 days of exposure other three petriplates were removed and was wrapped with Para film and incubated in the freezer at -20°C for 4 to 15 days. Similarly after three, four and five days of exposure nine petriplates were removed and labeled the time and date course on the plates with a marker and each plate was wrapped with Para film and incubated in the freezer at -20°C for 4 to 15 days. After 5 days of incubation all the petriplates were removed from the freezer and crystal formation of sucrose hemi-heptahydrate was observed; which indicates the sign of contamination.
CHAPTER THREE RESULTS
The results which were obtained in this study are shown below.
3.1 CONTAMINATION OBSERVED IN CONTROL AND POSITIVE CONTROL PETRI PLATES
After incubating the Control and Positive Control Petri plates at -20°C for 5 days no contamination was observed in control petriplates. This control petriplate showed clear solution whereas in positive control petriplates after incubating for 5 days at -20°C contamination was observed in the form of white crystals. There is cloudy appearance of white crystals due to long incubation time. These crystals got dissolved in 70% sucrose solution on warming at room temperature.
(a)Control petriplates with clear solution indicates no contamination,(b) Positive control petriplates with white crystals indicates contamination
3.2 CONTAMINATION OBSERVED IN PETRIPLATES AT DIFFERENT HEIGHTS
The results obtained after the contamination of the sucrose containing petriplates with contaminated liquid Nitrogen which were kept at various heights i.e. low level, same level, high level were represented in the below table 1.
In this study evaluation of contamination is done by placing the sucrose containing petriplates at various heights to the contaminated liquid Nitrogen Dewar flask and after incubating at -20°C in the freezer for 5 days crystal growth was observed. This experiment was performed four times.
The below table 1 represents the contamination at various heights i.e. low level, same level and high level heights respectively. In high level and same level crystal formation was observed in entire plate where as in low level crystal formation was observed partially. This occurs because the contaminated liquid Nitrogen did not transfer the sucrose hemi-heptahydrate through its vapour below the contaminated liquid Nitrogen Dewar flask. This study was performed four times and for every time same results were obtained.
HEIGHT OF PETRIPLATES IN DEWAR FLASK
OF CONTAMINATION (%)
Table 1: Contamination observed in Petri plates at different heights
Fig.5 represents the contamination in the form of crystals which were kept at different heights. It states that, when the sucrose plates were kept below the liquid Nitrogen Dewar flask, then the crystal formation was observed partially. When the plates were placed at same level and high level heights to the contaminated liquid Nitrogen Dewar flask crystal formation was gradually increased. When these high level and same level crystal formations was observed in three dimensional microscope then there was slight reduction of crystal growth in same level compared to high level. Thus it is confirmed that the sucrose petriplates which were kept at high level showed more contamination than same and low level height plates.
(a)Contamination in the form of crystals observed at low level, (b) Contamination in the form of crystals observed at same level and (c) Contamination in the form of crystals observed at high level heights.
In 6 low level petriplates in the first experiment showed 75% contamination and high and same level petriplates showed 100% contamination. In all the four experiments, results were same but when observed same and high level petriplates in 3 dimensional microscopes there was slight difference observed. It showed that there was less formation of crystals in same level than in high level. These height results were very promising because when petriplates kept at high level near the liquid Nitrogen Dewar flask there was more chance to get exposed to liquid Nitrogen vapour. Because vapour always travels in upward direction so, the contamination in high level is 100%. The petriplates at same level get exposed to more liquid Nitrogen vapour when compared to low level. So contamination in same level is higher than low level.
3.3 CONTAMINATION OBSERVED IN PETRIPLATES AT DIFFERENT TIME COURSES
The contamination results observed in petriplates which were exposed at different time periods in hours near the contaminated liquid Nitrogen Dewar flask are represented in the following table 2. In this study 70% W/W sucrose solution is poured into petriplates and were exposed to different time periods like 1 to 5 hours near contaminated liquid Nitrogen Dewar flask. This experiment was performed for four times and in this study petriplates were exposed near the Dewar flask for 1,2,3,4 and 5 hours respectively. After individual exposure time each petriplate was removed and each plate was wrapped with cling film and incubated in the freezer at -20°C for 5 to 14 days.
All hourly based petriplates showed contamination when compared with control. This experiment was performed for four times. In the experiment 25% of crystal growth was observed in 1 hour plates, 50% in 2 hour plates and 75% crystal formation was observed in 3 and 4 hour petriplates and 100% crystal growth was observed in 5 hour petriplates. In the second experiment 50% crystal growth was observed in 1, 3 and 4 hour plates and 75% in 2 hour plates and 100% crystal formation was observed in 5 hour petriplates. In the third experiment 50% crystal growth was observed in 1 and 2 hour petriplates, 75% in 4 hour plates and 100% crystal formation was observed in 3 and 5 hour petriplates. In fourth experiment there was 25% crystal growth observed in 2 hour petriplates, 75% in 1 and 3 hour plates and 100% crystal formation was observed in 4 and 5 hour petriplates. On an average of four experiments 50% of crystal growth was observed in 1 and 2 hour petriplates and 75% was observed in 3 and4 hour petriplates and 100% crystal formation was observed in 5 hour petriplates from this it was concluded that as the exposure time increased contamination also increased.
TIME PERIOD EXPOSED IN HOURS
PERCENTAGE OF AVERAGE CONTAMINATION
Table 2: Contamination observed in petriplates at various time courses
Fig 7 represents the comparison of hourly based time period exposure, and it confirmed that there was same amount of contamination in first 1 and 2, and 3 and4 hours and in 5th hour exposure time course there was total crystal growth in petriplates was observed.
(a) Crystal formation observed after 1 hour exposure time, (b) Crystal formation observed after 2 hour exposure time,(c) Crystal formation observed after 3 hour exposure time, (d) Crystal formation observed after 4 hour exposure time, (e) Crystal formation observed after 5 hour exposure time.
After 5 days petriplates were removed from the freezer and crystal formation was observed. In all petriplates crystal formation was seen but when compared 5th hour plate there was less formation of crystals were observed in 1, 2 and 3 hour time plates. This study was performed for four times and the average results showed that there was no difference in 1 and 2 hour petriplates. These two have 50% of crystal growth. In 3rd and 4th hour time period petriplates; there was 75% crystal formation was observed and in 5th hour there was 100% contamination. From this study it was concluded that as the exposure time was increased then the contamination also increased.
3.4 CONTAMINATION OBSERVED IN PETRIPLATES AT DIFFERENT TIME COURSES
The contamination results observed in petriplates which were kept at different time course in days near contaminated liquid Nitrogen Dewar flask were presented in the following table3. In this study sucrose solution petriplates were exposed to contaminated liquid Nitrogen at different time course in days like 1,2,3,4 and 5 days respectively. This experiment was performed for four times. In the first study petriplates were exposed at different day periods to contaminated liquid Nitrogen, then these plates were removed according to their days and each plate was wrapped with Para film to prevent the air entrapment and incubated at -20°C in the freezer for 5 to 14 days.
In table3 all petriplates showed contamination when compared with control. This experiment was performed for four times. In the first experiment 75% contamination was observed in 1 and 3 day petriplates and 100% crystal formation was observed in 2, 4 and 5 day petriplates. In the second experiment there was 25% crystal growth observed in 1 day plates, 75% observed in 2 and 3 day plates and 100% crystal formation was observed in 4 and 5 day petriplates. In the third experiment 50% contamination was observed in 1 and 3 day petriplates where as 75% observed in 2 day plates and100% crystal formation was observed in 4 and 5 day petriplates. In fourth experiment 50% contamination observed in 1 and 2 day petriplates and 100% crystal formation was observed in 3 and 4 day petriplates. On an average 50% crystal growth was observed in 1 day petriplates, 75% observed in 2 and 3 day plates and 100% crystal growth was observed in 4 and 5 day petriplates. When these 100% petriplates observed cryo microscopy there will be less number of crystals is formed in 4 hour plates than in 5 hour petriplates.
TIME PERIOD EXPOSED IN DAYS
Table 3: Contamination observed in petriplates at various time course in days
The formation of crystals was observed in all the time period plates which were exposed to contaminated liquid Nitrogen for 1 to 5 days. But here when compared to 4th and 5th day petriplates less crystal formation was observed in 1st, 2nd and 3rd day time courses. Contamination was gradually increased as the time period was increased.
Fig 10 represents the comparisons of contamination from day to day. This showed that in first day there was 50% crystal formation was seen and in 2nd and 3rd day there was 75% of crystal formation observed and in 4th and 5th day there was 100% contamination observed. From this it is confirmed that as the exposure time of petriplate increased the contamination also increased.
Fig 10 represents the comparisons of contamination from day to day. This showed that in first day there was 50% crystal formation was seen and in 2nd and 3rd day there was 75% of crystal formation observed and in 4th and 5th day there was 100% contamination observed. From this it is confirmed that as the exposure time of petriplate increased the contamination also increased.
CHAPTER FOUR DISCUSSION
Cryopreservation is very important in the advanced technology and its increased demand and novel applications causes some disease transmission. So, prevention of the contamination and the transfer of diseases is of great concern to the authorities so the present study was conducted to evaluate potential of contamination during different stages of cryopreservation was examined. In this study Sucrose Hemi Heptahydrate was used as a potential contaminant and it indicates how the contamination was transferred to Cryopreserved samples and general laboratory room from contaminated liquid nitrogen. This study provides a well defined system for examining the basis of contamination from the contaminated liquid nitrogen. This study has the ability to monitor the contamination which formulates strategies for the control of contamination to be developed and monitored. By using this assay system some examples of applications were presented here.
The contaminants present in the Liquid Nitrogen were transmitted above vapour phase, as previously stated that the vapour phase is contaminated (Fountain et al,1997), but there are some beliefs that vapour phase safer for cryopreservation in non sealed sample. Contamination may also occur through the laboratory equipment in which liquid Nitrogen was stored. Contamination also depends on freezing time. This study has the potential for examining the contamination caused by such factors.
The height results which were discussed above chapter indicates that low level petriplates showed less contamination when compared to same and high level height. This occurred because of the vapour phase of Liquid Nitrogen. Vapour is always travels upward direction and spreads entire room so high level petriplates are exposed to Liquid Nitrogen vapour quickly than low level petriplates.
In this study non-biological material is used as a potential contaminant (G.J.Morris, 2009) so, it does not produce any contamination in itself. If needed; decontamination is done by washing surfaces with hot water or by destroying the hydrate. The height results indicates that petriplates which are placed at high level to near contaminated liquid nitrogen Dewar flask was showed with 100% contamination. This is in the form of white crystals of Sucrose Hemi-Heptahydrate because of quick exposure of high level height petriplates whereas in low level petriplates only 75% crystal formation was observed; which indicates contamination due to less exposure to contaminated Liquid Nitrogen. This contamination may occur due to transfer of sucrose hemi-heptahydrate crystals through its vapour phase of Liquid Nitrogen and there are some evidences showing this. The detailed mechanism of vapour and microbial transferred within the Dewar flask is unknown. It is assumed that there is limited or no circulation of frozen particles within the flask by the Liquid Nitrogen vapours in the absence of Liquid Nitrogen phase. This contrasts to the environment within the conventional Liquid Nitrogen Dewar's where extensive movement of both vapours and boiling Liquid Nitrogen take place during temperature changes and tank refilling. This view could be supported by report of (Fountain et al., 1977) who detected the environmental or water borne bacteria and fungi in the vapour phase above Liquid Nitrogen in the Dewar's flask. Our experimental result shows that Liquid nitrogen vapours serve as a vehicle for the transmission of contaminants.
The hourly based results in this study showed that the exposure time of petriplates to contaminate Liquid Nitrogen increased contamination is also increased. This experiment was repeated for four times. In all experiments contamination was gradually increased with exposure time. In first, second there was 50% contamination observed whereas in third and fourth there was 75% contamination observed and five hour there was 1005 contamination observed. This may be occurred due to the air borne particles in the Cryolab where the experiment was carried out.
The previous studies showed that opened and sealed straws which are stored near Liquid Nitrogen which is contaminated with sucrose hemi-heptahydrate for 6 hours and after incubation sucrose hydrate crystals are observed in open straws within 5 days after incubation at -20degrres in the freezer. Whereas heat sealed does not show any contamination. In the present study different time periods like 1-5 days petriplates showed gradual increase in the contamination in the form of white crystals this may be occurred due to the evapouration of water from the plate after incubating at -20degrees for 5days. Sucrose crystals are seen in the form of star shaped before incubating the day's petriplates. After incubation there was white crystals are formed in the petriplates. As the Cryolab is small room there may be a chance of air borne particles, so there is a possibility to get all plates contaminated. Cryoscanning electrion microscopy was essential to define the structure and shape of the crystal (G.J.Morris).
This study revealed that Liquid Nitrogen was potential source of contaminant during cryopreservation. It can transfer any biological material through its vapour phase. This study showed the evidence that possibility of contamination during long term storage of biological materials in Liquid Nitrogen. As the storage time increased contamination also increased. In this study sucrose hemi-heptahydrate was used as contaminant and it will be transferred through Liquid Nitrogen vapour and even contaminates other materials.
The contamination persists in cooling equipment and remains in vapour phase of liquid nitrogen for about 24 hours, allows the contamination of surrounding places by escaping from Dewar flask, this vapour can carry the contaminants into air and assay plates by freezing cycle. It also reveals that the risk of contamination was more when the plates kept similar height of Dewar flask, at a distance of 2.8 meters for exposing more than 1 hour. Similarly hazardous materials stored in the liquid nitrogen container can cause cross contamination of samples or other biological materials present near the storage container by liquid nitrogen vapour.
Regular cleaning of liquid nitrogen and sterilization of sample container and room sterilization can reduce the risk of contamination in cryopreservation to avoid the air born contamination.
Now a day's there is an increasing demand for cryobiology as Cryopreservation is an important aspect of cryobiology. However, increased demand for the cryopreservation technology and its applications in assisted reproductive technologies, there may be a chance of contamination while collecting the cryopreserved samples like semen and embryos. In the present study we performed various heights and time parameters. One of my friends did this project on different parameters like distance and concentration. As the concentration of Sucrose Hemi Heptahydrate increased contamination is also increased. Whereas the distance of petriplates to Dewar flask increased then contamination was decreased. Further studies must be done to get good and accurate results. In the future, height of the plates will be increased and observe the results and also time course from 1 to 10 hours, days from 1 to 15 days should be performed to get accurate results. Concentration of Sucrose Hemi Heptahydrate concentration is increased up to 10gms,then observed the results .Distance should be increased to 10mts to get accurate results.
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