The Concept Of Contamination During Cryopreservation Biology Essay

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It has been examined that the liquid nitrogen is main source to contaminate the 70% sucrose solution. The 70% sucrose solution assay plates are possible for contamination during the cryopreservation steps. The method of experiment was depends on the contaminated liquid nitrogen. The crystals of sucrose hemi-heptahydrate are used to contaminate the liquid nitrogen. These sucrose hemi-heptahydrates are stable in liquid nitrogen. In this the experiment method used is, the 70% sucrose solution assay plates exposed to contaminated liquid nitrogen vapours for about 4 hours, and these sucrose assay plates are maintained at different distances are 100cm, 200cm, and 300cm. The exposed sucrose assay plates are cryopreserved at -20oC for 5 to 15 days. It was showing that only liquid nitrogen vapours are used as control. The 70% sucrose solution plates are placed at different distances to liquid nitrogen. Expose the control samples for 4 hours and cyropreserve it for 5 to 15 days. After cryopreservation of sucrose assay plates observed and the results are noted as, in control samples there are no crystal formation seen. While those treated sucrose assay plates showing granular crystals. The treated assay plates which are closure (100cm) to the liquid nitrogen are having more crystals then the sucrose plates which are placed at a distance of 200cm and 300cm. Here least crystals are observed in assay plates which are at 300cm from the liquid nitrogen flask. The distance between the liquid nitrogen and sucrose solution plates, the exposure time period of sucrose plates to the liquid nitrogen, and the concentration of sucrose hemi heptahydrate used are conforming the aim of this experiment. According to these results the liquid nitrogen is playing major role in contamination during cryopreservation.

Cryopreservation is a process in which cells or the whole tissues containing a number of cells are preserved by cooling it to a low temperature, almost to a low temperature of 77k°C or -196°C. In this several techniques are used in which microorganisms, tissues and embryos are separated from each other. The main source of life in a living cell is water in chemical form. So to halt the metabolism in cell, the water is converted into the ice at different rates during the cooling process. During this process of slow cooling, the freezing occurs on the external parts of the cells before the intracellular parts. When the ice is formed on the external sides, the water is removed from the extracellular environment, which leads to a osmotic imbalance across the cell membrane by which the water from inside the cell is also migrated out.

So the increase in this solute concentration on both inside and outside of a cell leads to the survival of the cell. There are many compounds have tested as cryo-protective agents but glycerol and DMSO is best result in the cryopreservation. DMSO is widely used for the dehydration of cells to intracellular freezing. The cryo-protective agents are depends upon the type of the cell to be conserved glycerol usually less toxic then DMSO. In some case, during/ after the storage or cryo-preservation of cells, the contamination takes place which can of any type depending upon the nature and features of the contaminating agent.

1.6. Contamination:

Contamination in a biological science generally means an accidental or intentional introduction of foreign materials (contaminating agents) which can seriously distort the results of experiments with the stored samples. There are different types of contaminating agents which are water, lights, incubators, viruses, bacteria's, invertebrates and mycoplasma. There are different types of contaminations they are:

1.6.1. Environmental Contamination:

It has been noticed that 1-10% samples of cells and tissues gets contaminated during the clinical or laboratory manipulation and in some case also when pathogenic organisms are potentially isolated (Lieby,et al.,1997). Most the contamination cases in the laboratories have through bacteria's and fungus from different sources, for e.g. cardboard boxes used to store lab consumables. Sinks and sluices in the lab, which can cause fungal growth may also be a reason of contamination in labs, so the water baths and the cold rooms must be cleaned and sanitised regularly. The other services such as supplied distilled water used to perform various operation in the labs, can also be a case of contamination because, the contaminated distilled water sometimes contains bacteria that releases Endotoxins or other organisms such as Acholeplasma which can easily slip through the bacteria filters. Most of all the lack of training and assistance of working in the lab can also lead to the contamination. Storage quality:

The place or the medium used to store the samples can also lead to the contamination. For examples if a sample is stored in a medium, which previously contained a solution of heavy metals or organic compounds can also be a source of contamination (Fountain, et al., 1997). The quality of the storage medium can also be a reason of contamination because if the quality of medium is not good then it can lead to the leakage of the sample or the viruses which may contaminate the other samples. Contamination through incubators:

The incubators are often considered to be the major source of contamination in both biological and chemical way. The mixture of gases which are suffused through incubators can contain toxic impurities such as oils or other gases, which may be used in the same storage cylinder or tanks. Incubator problem is very rare in medical grade gases but more common in less expensive industrial grade gases. Adventitious Mediator Cell Cultures:

The purpose of in vitro cell culture for tolerant substance and the use of cell lines and their products are increasing quality of therapeutic technology. Cell lines of mycoplasma contamination will spread rapidly to other cultures if not promptly isolated and always contributes to constant infection that may change the quality of the cells. (Aula.p et al.1967). Cheap and rapid techniques are available in the cell lines for mycoplasma. (Mowles et al.1990). Cell lines and their products carry contaminants which represent a serious threat to human health such as lymphocytic choriomeningitis (LCMV) and Hantavirus. (Nicklas et al.1993). In general cell lines have to test for potential pathogens based on those likely to arise from the host species and origin of tissue that test cells derived from human sources for human pathogens (Frommer et al., 1992).

1.6.2. Microbial contamination through cryogenic equipments:

It generally means an inclusion or growth of harmful microorganisms such as clostridium botulinum. This growth can be on anything such as food or stored sample making the unsafe or unused for future purpose. Three major types of cryogenic equipment have been contaminated with nuisance micro-flora are: Controlled rate freezer, dry shippers, and storage cryo-tanks

A cryogenic application includes liquefied gases, such as liquid nitrogen and liquid helium. Liquid nitrogen is most commonly used in cryogenic process and it is also legally purchasable throughout the world. Liquid helium is also used in some case because of its nature of allowing lowest attainable temperature compare to others. Grout and Morris the two well known scientists in 2008 conducted a test in which they dosed a bulk amount of liquid nitrogen with sclerotia which is a fungal pathogen in liquid form, to cool a programmable liquid nitrogen freezer. After one cooling run, when the cryogenic instrument was tested, the whole surface showed positive which signified the presence of contamination in the instrument. The experiment conducted by Grout and Morris proved that the pathogens can survive but by contaminating the liquid nitrogen and the cryogenic device. Dry Shippers:

In 2005 another scientist Bielanski experimentally contaminated the samples of semen and embryos and also the two different models of dry shippers. 270,000embryos and 264million doses of bovine semen were cryo-preserved for the used on the year 2002. (Polge et .al 1947) human gametes are not available in the cryopreservation then infected with hepatitis viruses and HIV. Germplasm stored in the liquid nitrogen phase of cross contamination suitable to container breakage at 196 °C in liquid nitrogen. Then, he tested the pathways of bacterial contamination during the transmission from and between. It found that the dry shippers were contaminated to germplasm, some of the cryo-preserved germplasm were contaminated while some were non-contaminated and a stock culture of pathogenic agents & germplasm. After that he stored the contaminated and non contaminated samples of embryos and semen's for almost 7 days in a open container with liquid nitrogen in vapour phase. After 7 days he made a test of bacterial contamination but no evidence of cross-contamination from either contaminated to non-contaminated samples or between contaminated Dewar's to germplasm. This led to a conclusion that the vapour phase of liquid nitrogen in the dry shippers can be used for the long term storage of germplasm, even though the shippers or some of the samples are contaminated.

Cryogenic equipment manufacturer have developed new products to meet the demands of users and practitioners who requires double-skin the samples that can become either accidently or purposefully in direct contact with liquid phase liquid nitrogen. Transmission of three bacterial and two viral microorganisms was cryopreserved from embryos and semen by vapour phase of liquid nitrogen stored in the dry shipper. The absorbent forces back to the humidity absorb liquid nitrogen of vapour phase at-150 °C in the storage chamber without the liquid nitrogen release. Bacterial and viral agents was detected in all post thaw samples of embryos and semen exhibited to the agents to cryopreservation and the culture stocks used as a contaminants under the storage of vapour phase. There is no transmission of agents between contaminated and non contaminated containers stored in the vapour phase of liquid nitrogen. The concentrations of bacterial and viral cultures of the vapour shippers was exhibited possible through storage and conservation of germplasm. The microorganisms used in the two gram negative and one gram positive bacterial agent on the form single strand RNA and double standard DNA virus.(w.c.d.Hare 1985).The cryo-protective agents of germplasm infected with hepatitis C and human herpes viruses. E.g. transmissible spongiform encephalopathy's (TSE) can be harmful by LN vapours.

1.6.3. Different microbial contaminant controlling in cryo-banks/tanks:

This procedure includes objecting of risks related to the cross-contamination between samples in cryo-tanks. It largely involves the technical and management precautions which are double-contaminant, avoiding of direct contacts with liquid nitrogen, and the storage of vapour form of liquid nitrogen. There are several categories of microbial contaminations. Virus and viroids:

To know the issue of microbial contamination, the plant viruses are stored and used which thought to be useful. In the plant viruses also the DNA viruses are more stable then the RNA viruses. Many of the plant viruses can be stored in dehydrated host tissues at around 4°C for several years (Gould, 1995). During the contamination process it is advised that, the viruses when cryo-preserved should be encased in double sealed tubes. These observations on the plant viruses have put forward an important consideration in cryo-banking of plant cells which carries virus infections during the time of cryopreservation (Hatta and Francki, 1981).To prove this concept many people came up with their theories and experiments. Confirmation based risk assessment of virus transmission via cryobanks:

Before many examinations were held on evidence of know about the virus transmission using cryobanks. In 1995 Tedder came up with the first evidence for which she performed an experiment. The experimental data was consist of bone marrow and blood products from sin multi transfused patients which in stored in cryostorage for almost 25months these samples were stored in cryobanks using the cryobags. The leakage of the cryobag which is used to store the bone marrow of the first patient gets first infected with the hepatitis B virus and then it contaminates entire tank and its content so the final outcome was the patient getting infected. Due to this Tedder provided same suggestions about the steps to be followed before cryopreserving any samples.

1 Tissue of the donors must be screened to detect any blood borne viral infections.

2 Samples should be stored in double containers.

In 1997 fountain stated that both vapour and liquid phase nitrogen can also cause microbiological contamination Clarke in 1999 also came up with same similar cases of cross contamination which was caused by the storage of human tissues. But in 2000 Bielanski came up with something entirely different. This method of cryopreservation included ultra rapid or vitrification of protocols which required direct contact of freezing medium containing the tissues with liquid nitrogen. The experiment included used of bovine embryos test and a range of different animals human pathogen viruses to experimentally infect embryos. The whole thing is cryopreserved in a vitrification solution which contains DMSO, ethylene glycerol and sucrose. Both infected and uninfected samples were cryopreserved in different cryocontainers with different sealant capacities. These cryocontainers are stored in the same Dewar in the liquid phase of liquid nitrogen for around 3-5 weeks. The main finding of Bielanski concluded a threat of embryos becoming contaminated from freezing during the cryopreserved process in open containers then exposing to liquid nitrogen in liquid phase advised certain steps to be followed.

1. Make the use of container with which is hermetically sealed.

2. Avoiding the direct contact between liquid nitrogen and freezing medium

3. Always storing contaminated and non contaminated samples in different tanks.

In 2003 Bielanski expanded his study which signified no evidence of the transfer of bovine virus between contaminated and non contaminated embryos and semen's. When stored in sealed plastic straws with liquid nitrogen for a time period of 6-35years. (Berz in 2007) advised some levels of containments which are necessary for avoiding viral infection in cryopreservation of haematopoietic stem cells. To present this cross contamination he also recommend double bagging the samples in protective sleeves and infected samples which should be placed in separate cryotanks. In another incident (Husebekk et al., 2004) reported peripherals of blood progenitor cells (PBPCs) which were taken from a patient but during the process of freezing the cell suspension accidentally leaked on the surface of cryobags. Then these bags were transferred to a second protective envelop, dispensed into a steel cassette and placedin a tank which contains liquid nitrogen in vapour phase. This tank already contains other samples tested virus-free. Abioticand biotic risk assessments for virus transmission in cryobanks:

Based on the animal medical and biotechnical literature the transmissions were needed to be clarified as the level of risk associated with cryogenic transmission of viral microorganisms. The risk Level accepted by different medicals, animals, and biotechnology communities. Although different people advised these levels of precaution that should be kept in mind before cryopreserving any samples, the potential risks for cross contamination of phytological viruses and viroids in both new and already established cryorepositories will be determined by biotic and abiotic factors. Each and every viruses or viroids have their own level of risk viruses are entirely dependent on the substance to substance transmission contamination and have complex transmission infection process which may be considered as a lower risk category with their potential of cross infection via cryogenic routes (Vander Want and Dijksta, 2006). The risk of virus transmission occurs through various cryogenic routes. These routes can be expected to be higher for those plant pathogens which are transmitted from direct contact and natural wounding, in the case for potato virus. Mycoplasma:

Mycoplasm's are the smallest free living replicating organisms which are found in mature. These organisms have a certain elementary structure containing proteins DNA RNA and enzymes. These are also classified as Mollicutes because they lack cells walls and are bounded by a triple layer unit membrane. Whereas phytoplasma is a term which is applied to mycoplasma type organisms (Namba, 2002; Lee et al., 2000). These organisms are initially designated by the taxonomic group of candidates' phytoplasm species which are found in plants such organisms can lead to serious agriculture diseases. Phytoplasmas are transmitted into the plants through vectors and can be spreaded by infecting the plants by grafting its healthy plant. Mycoplasma infections in animal and human cells cryobanks can lead to a disastrous consequence.

1.7. Prevention taken to avoid cryo-preservation risks:

1.7.1. Controlled-rate and slow freezing:

This technique was established in early 1970s. In 1984, it enabled the first human embryo frozen birth. Then all over the world these machines for freezing biological samples sample came into use for different programmable steps and controlled rates for human, animal and cell biology. In this a sample, is frozen down to better preserve it for eventual nature, before it is deeply frozen or cryo-preserved in liquid nitrogen. These machines are used for storing and freezing the samples like skin, blood products, sperm, embryos, stem cells and tissue preservation at many places such as research labs and hospitals. It has been proved in several studies that the frozen embryos stored using slow-freezing techniques are somehow better than other technique used in cryopreservation. These studies also revealed that the use of frozen embryos rather than fresh embryos reduces the risk of miscarriage and premature delivery of the tests and experiments.

1.7.2. Vitrification:

It is a technique which includes the benefits of cryopreservation without damaging the samples because of the ice crystal formation. Every cell contains water into it for living. When the tissue is cooled below its freezing point, the water molecules get together and form a grouping crystal. When the water gets totally frozen, the ice squeezes other molecules inside the tissue in a harmfully concentrated solution. First the ice is formed on the outer part of the cells and continues growing of ice over the cell causes it dehydrate and shrink. Then the cells are left damaged and squashed between ice crystals. Then the chemicals which are known as cryoprotectants are added to the water to prevent water molecules from gathering together to form ice. Instead of freezing, the molecules just move slower and slower as they are cooled and finally at a temperature below -100C molecules are turned into solid without freezing then this is said to be "vitrified". Again the cryo-protectants are added to the cells before the sample is deeply cooled. In this process there is no damage held to the cells because during the cooling no ice is formed inside the cells and finally the biological time of the cell is stopped. These samples can be stored for relatively long periods in this solid form without the concern of air contamination or water contamination.

1.8. Contamination of screening stem cells:

The materials are tested for contaminants free. By screening bacterial selection media, the most of contamination which are due to bacteria can be easily discovered .To inoculate the flask that consists of ten ml culture medium, one ampoule is used. The presence of turbidity in which white colour indicates the bacterial contamination after two weeks incubated at 37ıc.The ice sediment which is accumulate in the tanks of liquid nitrogen during storage can cause the contamination of samples(Piasecka and serafin,1972).Even though in the cryopreservation of sperms in the fish is not involve the aseptic techniques, the use of sterile techniques plays a major role for controlling the contamination(Stoss,1983:Lahnsteiner et al,2000: Chao and Liao 2001). Beilanski(1997) explained that sample of embryos that are stored in the contaminant liquid nitrogen are tested negative for detecting the presence of BVDV and BAV1 viruses. Hence for preventing contamination it is recommended that thawed straws are cut by using sterile scissors and scrapped blade. Same like the cross contamination in the long term storage of sample can be prevented by using high security straws and as well as separate liquid nitrogen. Piasecka-Serafin M, 1972, the effect of sediment accumulated in containers under experimental conditions on the infection of semen stored directly stored in liquid nitrogen, Bull Acad Pol Sci Biol, 20, 263-7.



2.1 Sucrose:

Sucrose was a compound which contains more or large number of carbons in its molecular formula. The molecular formula of sucrose is C12H22O11. Sucrose was high class of chemical compound member (Spencer, 2004). The international union of pure and applied chemistry nomenclature (IUPAC) of sucrose is β-D-fructofuranosyl-(2→1)-α-D-glucopyranoside. The common name of sucrose was table sugar, generally sugar. It is also called saccharose. The physical nature of the sucrose was crystalline sugar, white in colour, odorless. And the sucrose was sweet in taste. It generally used for human nutrition. Sucrose was disaccharide and it was obtained by the combination of two monosaccharides which are D-Glucose and D-Fructose. The bond formed between these two monosaccharides was called glycosidic linkage. The molecular formula of glucose and fructose is C6H12O6.

File:Sucrose CASCC.png

Figure: 2.1. Molecular Structure of Sucrose

The hydrolysis of sucrose occurs very slowly, in presence of catalyst or acids the sucrose break down into glucose and fructose. In this process the break-down of glycosidic linkage occurs. By the hydrolysis of disaccharide become two monosaccharides (Beevers and MeDonlads, 1952).

2.1.1. Preparation of 70% sucrose solution:

The 70grams of sucrose was weighed in weighing machine by using spatula. These sucrose crystals were taken into a conical flask or a bottle that containing with 100 ml of distilled water. Shake this conical flask and stir with stirrer. Heat the solution for about 4 to 5 minutes in microwave to dissolve the sucrose crystals in distilled water. Store this 70% of sucrose solution at 4°C for further use.

The 70% sucrose solution can also prepare as follows: 70ml of sucrose indicates 70%. So 70 grams of sucrose crystals dissolved in 100 ml of distilled water. The 49 grams of sucrose crystals are taken to dissolve in 70ml of distilled water for the preparation of 70% sucrose solution.

i.e. 70 grams sucrose -------------> 100ml

'x'grams sucrose -------------> 70ml

Therefore: this gives 49 grams.


Figure: 2.2. 70% of sucrose solution assay plate

2.2. Sucrose Hemi Heptahydrate:

An anhydrous form of sucrose crystallizes into two forms that are sucrose hemi pentahydrate and sucrose hemi heptahydrate. These crystallizes forms of sucrose occurs at very low temperature that was -34°C (Young and Jones). The molecular formula of sucrose hemi heptahydrate is C12H22O11.3.5.H.Sub.2O. This heptahydrate is phase II sucrose hydrate. This hemi heptahydrate forms at the temperature of -9.5oC. The melting temperature of sucrose hemi heptahydrate is +27.8oC. It is used to contaminate the liquid nitrogen (Young and Francis).

2.3. Liquid Nitrogen:

The liquid state of nitrogen gas occurs at very low temperature was called liquid nitrogen. It was colourless liquid. LN2 and LN are the abbreviation for the liquid nitrogen. The boiling point or the liquid nitrogen boils at the temperature of -196oC. The liquid nitrogen was cryogenic liquid, so liquid nitrogen was called as cryogenic agent that means it studies at very low temperature. Liquid nitrogen freezes rapidly, it used to freeze living tissues in fraction of time. Liquid nitrogen can easily converts into solid by placing in vacuum chamber. The freezing temperature of liquid nitrogen was -210oC. Liquid nitrogen was a source used to transport the living tissues and organs. Liquid nitrogen was also used to preserve for long period of time without any injury. Liquid nitrogen was a source of dry nitrogen gas. LN was used to cyropreserve of living tissues, animal cells, plant cells, and blood, sperms, egg and biological samples for long time. The expansion ratio of nitrogen from liquid to gas was 1:694. Generally the nitrogen was non-toxic, odourless. It was an inert gas and the nitrogen was non flammable, the liquid nitrogen was hydrophobic (Umrath 1974).

2.4. Dewar Flask:

Dewar flasks were used to store liquids and it was used to store the liquid nitrogen in small and large quantities. Generally liquid nitrogen stores in vacuum space that present between the inner and outer shells of the Dewar flasks. Durable and aluminium (light weight) are used to make the outer shell of the Dewar flask. Insulation is used to fill the vacuum space that present between the outer and inner shell of the Dewar flask. The necktube core is also used to reduce or control the loss or deduction of liquid nitrogen. The borosilicate glass was used to maintain the liquid nitrogen service. The evaporation of liquid nitrogen was controlled or minimizes by using the polyethylene in Dewar flask.


Figure: 2.3. Dewar flask

2.5. Method of Experiment:

The following method is used to evaluation of contamination after exposing to contaminated liquid nitrogen with sucrose hemi heptahydrate for about 4 hours time period. The method was helpful to prove hypothesis and this method of experiment covers aim and objective. By this hypothesis it helps scientifically for further use in cryopreservation.

2.5.1. Preparation of Negative control:

In 100ml of distilled water add 70gm of sucrose crystals or in 70 ml of distilled water add 49gm of sucrose crystals for preparation of 70% sucrose solution. Stir the solution to dissolve sucrose or heat for about 4 to 5 minutes in microwave. Pour the 70% sucrose solution into 9 clean and fresh labelled Petri dishes. In another side transfer 1liter to 2liter of liquid nitrogen into small Dewar flask and keep 3 Petri plates near the liquid nitrogen with distance of 100cm. Use another 3 Petri dishes, keep these with distance of 200cm. And use remaining 3 Petri dishes keep them with the distance of 300cm. Expose these all 9 Petri dishes to the liquid nitrogen vapours for about 4 hours. After 4 hours close these Petri plates with Parafilm and cover with cling film later preserved these labelled Petri dishes at -20oC for about 15 days. These 9 labelled Petri dishes are uses as negative control for the experiment. Preparation of Positive control:

The positive control was used to examine the hypothesis of the experiment. The negative control was used to compare the results of the positive control. In this sucrose hemi heptahydrate was used to contaminate the liquid nitrogen. The contaminated liquid nitrogen was exposed to the 70% sucrose solution assay plates for about 4 hours at different distances of 100cm, 200cm and 300cm. After exposure of contaminated liquid nitrogen to the sucrose plates, cryopreserved them for 5 to 15 days at a temperature of -20oC. These assay plates are used as positive control for the experiment. Evaluation of contamination at different distances:

Contaminate the liquid nitrogen with the sucrose hemi heptahydrate. In this experiment method 0.5gm to 3gm concentration of sucrose hemi heptahydrate crystals is used to contaminate the liquid nitrogen. The contaminated liquid nitrogen was used to examine this experiment. Add 70% sucrose solution (that stored in 4oC) to Petri dishes for the experiment. Label all the Petri dishes. Place three of these labelled Petri dishes towards the contaminated liquid nitrogen with the distance of 100cm. Use another three Petri dishes at a distance of 200cm and similarly use another three Petri dishes at a distance of 300cm. Expose these 9 Petri dishes to liquid nitrogen vapours which is contaminated by sucrose hemi heptahydrate about 4 hours. After 4 hours exposing these 70% sucrose solution Petri dishes are closed with it lid and seal it with Parafilm. Then wrap the sealed Petri dishes with Clingfilm. And preserve these all Petri dishes at the temperature of -20oC for about 15days. After 5days observe these cryopreserved Petri dishes every day and make a note. Repeat the same experiment for about 5 to 7 times, and observe the result at all the times and make a note it. F:\Photos\x\DSC_0107.jpg

Figure: 2.4. Sucrose assay plates at different distances to liquid nitrogen

2.5.3. Data Collection:

After 5days observe these cryopreserved control Petri dishes every day, and collect the data by observing Petri dishes. Similarly collect data from the treated Petri dishes as well. Collect the data from all the Petri dishes that different distance of 100cm, 200cm and 300cm. For every repeated experiment control also needed to examine.



To assess the contamination during cryopreservation, the 70% sucrose solution was prepared and treated with contaminated liquid nitrogen vapours that contaminated with sucrose hemi heptahydrate.

Results observed in negative control:

Control was used to assess or compare the results with positive control. 70% sucrose solution was exposed to liquid nitrogen that without contaminated sucrose hemi heptahydrate, these Petri dishes are used as the negative control for the treated 70% sucrose solution samples. These control and treated sample plates are cryopreserved at -20oC for about 15 days. The experiment was carried out with different distance from liquid nitrogen to 70% sucrose solution.


Figure: 3.5.1: Negative Control

The above figure contains 70% sucrose sample that has been exposed to liquid nitrogen at different distances for about 4 hours, and cryopreserved it at -20oC for 5 to 15 days.

According to Morris assay system above figure shows that after incubation for 5 days at -20ËšC. The samples containing Petri dishes that has been exposed to only liquid nitrogen at different distance (i.e. 100cm, 200cm, and 300cm) does not show any change in the sample formation and same process was prolonged for 11 days and observed still no changes has been observed, even after 15 days. This appears that contamination is not occurred by the exposure of liquid nitrogen to the sucrose solution and it can be treated as negative control sample for experiment.

Results observed in Positive control:

Different stages of contamination are showed in the following figures by taking 70% sucrose solution sample in Petri dishes maintaining at different distances to contaminated liquid nitrogen with sucrose hemi-heptahydrate.

C:\Users\krish\Desktop\100cm.jpg C:\Users\krish\Desktop\200cm.jpg

Figure: 3.5.2. a: 100 cm Figure: 3.5.2. b: 200cm


Figure: 3.5.2. c: 300cm

Figure: 3.5.2. Observation of treated sucrose plates after cryopreservation

Figure: 3.5.2. a:

According to the above figure 'a' the sucrose sample solution in Petri dishes that has been exposed with liquid nitrogen containing sucrose hemi-heptahydrate at a distance of 100cm was incubated for 5-15days at temperature -20ËšC shows more changes in the samples with their contamination by forming white granular structures. As process prolonged for 5 more days that granular structures are increased and formed like a crystal structure are observed.

Figure: 3.5.2. b:

According to figure 'b', the sucrose sample that exposed to contaminated liquid nitrogen at a distance of 200cm for 4 hours. These exposed Petri dishes showing small granular structures after cryopreservation at -20oC for 10 days. After 15 days these granules that are increased little more in number and which are diamond crystals in structure. But these numbers of crystals are bit less than other Petri dishes that are placed at 100cm distance to contaminated liquid nitrogen.

Figure: 3.5.2. c:

According to figures 'c' the sample 70% sucrose solution placed at a distance of 300cm to the contaminated liquid nitrogen with sucrose hemi heptahydrate. These Petri dishes are exposed to nitrogen vapours for 4 hours and incubated at -20oC for 15 days. After cryopreservation the crystal formation is seen in Petri dishes. Compare to other Petri dishes that are placed at 100cm and 200cm distance having more number of crystals than 300cm distance maintained with liquid nitrogen Dewar flask.

If the sucrose sample temperature is get downs from -20oC to normal room temperature then these contaminated crystal structure are stable even in dissolved condition. It can easily differentiate from the sample solution. These crystals are clearly observed under microscope.

Table: 1.

The average percentage (%) of the contamination at different distances:

Observation of contamination percentage on assay plates after cryopreservation for 15 days





First experiment




Second experiment




Third experiment




Fourth experiment




Mean of Percentage




Figure: 3.2. Percentage of contamination at different distances

According to the graph the samples that are exposed to liquid nitrogen containing sucrose hemi-heptahydrate at 100cm shows maximum crystal structure formation. That shows maximum contamination. When distance increases to 200cm from nitrogen vapours the crystal formation in the sucrose sample is decreases, if distance increased to 300cm from liquid nitrogen vapours the crystal formation is bit decreased in 70% sucrose sample. That shows sucrose sample contamination decreases as distance increases between liquid nitrogen Dewar flask and sucrose sample Petri dishes. But in control samples (that is 70% sucrose solution plates exposed to the liquid nitrogen vapours at different distances) are not showing any crystal formation in it. After cryopreservation of control plates for 15days also there are no crystal formation in it. So 0% of crystals observed in control Petridishes. According to the above result distance also plays an important role in cryopreservation for contamination with liquid nitrogen.


This experiment has proved that the liquid nitrogen was main source of contamination during cryopreservation of 70% sucrose solution at -20oC. The distance has also played a major role in the contamination during cryopreservation of sucrose solution. Other project members has also proved that the above hypothesis, aim and objectives. That the, Ravindhar has worked with different concentration of sucrose hemi heptahydrate, which contaminates the liquid nitrogen with the results, are showing that the lower concentration of sucrose hemi heptahydrate contaminates less than the higher concentration of sucrose hemi heptahydrate in 70% sucrose samples. Another colleague Srikanth was carried out the experiment with different exposure time period to the contaminated liquid nitrogen, the results was obtained as the 70% sucrose solution is more contaminated when the exposure time was more, if the exposure time is decreases then the observation of contamination also decreased in 70% sucrose solution. This experiment has showed that the potential of contamination occurs by the liquid nitrogen vapours.

According to Morris (2005) an easy analysis was carried out the occurrence of contamination during different stages of cryopreservation at -20oC. In this assay system it was observed the single crystal in raised structure of sucrose hydrate. After 15 days cryopreservation, it was observed that a central deep depression in sucrose hydrate crystals, increased in size of crystals and fusion between the adjacent sucrose hydrate crystals. In this assay system the crystals are stable even after the sucrose solution dissolved (Morris, 2009).

The liquid nitrogen vapour phase was acted as a mediator for the transfer of contaminants from the liquid nitrogen to the source. it was mentioned that the vapour phase of liquid nitrogen may be contaminating, but the widespread was faiths that the liquid nitrogen vapour phase was 'safe' for those preserved in open or non - sealed flasks that are like cryovials are mainly used for vitrification. There was an issue rose that the outside or which are not autoclaved cryocontainers will also may inevitably making contamination during the thawing methods or procedures (Fountain and Ralston., 1997).

In the recognised recent days, the transfer of hepatitis B virus to the disease suffering patients through the transplanted bone marrow cells was stocked in contaminated nitrogen (Tedder and, 1995). Similarly same type transfer of a viral agent to patient with the liquid nitrogen also recognised in 1971 these viruses are concerns as the papovavirus (Charles and Sire. 1971). There was a chance of probable simplex virus 1 and adenovirus type 2 was transferring on cotton wool swabs into the Dewar flask and out of the Dewar flask was also recognised (Carroll, 1995).

In the suspected embryos was preserved in liquid nitrogen containing Dewar flask or tanks that has the chance to contaminate with some of the murine pathogens, if these pathogens are entered accidentally into the liquid nitrogen flasks or tanks. The previous studies explained as the evaluation of cross - contamination in between the cryotubes containing mouse 2 - cell Embryos and murine pathogens in liquid nitrogen (Shigeru 2003). If embryos are cryopreserved in liquid nitrogen and transmission of the virus has been contaminate the preserved cells or tissues. According to Bielanski the possibility of contamination is occurring in preserved experiments are relatively more (Bielanski,, 2000).

Here researchers supposed that initial sources of contamination of dry shippers are taking part, i.e. shippers itself or the liquid nitrogen and they had a chance to inadvertently contaminate with micro-organisms through the process of production methods, preservation and transportation. This contamination generally involves in non - pathogenic ubiquitous micro - organisms like which was identified in one of the dry shippers experiment (Murray 2003).



The 70% sucrose solution gets contaminating when the liquid nitrogen vapours acts as source of contamination and the sucrose hemi-heptahydrate acts as contaminating agent. While the distance between the liquid nitrogen flask and sucrose solution plates is increases then the contamination of sucrose samples are decreased. By observing these results the liquid nitrogen also plays an important role in the formation of crystals during contamination. This method of experiment proves the above hypothesis and aim.


The exposure of liquid nitrogen to the 70% sucrose solution with different time intervals as also proves the above hypothesis and aim, and different concentrations used that sucrose hemi-heptahydrate to contaminate the liquid nitrogen that is exposed to 70% of sucrose solution assay plates also proves the above hypothesis and aim.