Frozen Life Pankaj Talwar
Absorbed dose 162
Actual cooling curve 125
Air space inside straws 122
Alcohol bath 126
Assisted reproductive technologies
freezing 198, 216
embryo selection 198
general guidelines 198
media preparation 198
method 200
ampule loading 200
freezing program 200
media handling 200
straw loading 200
thawing 200
Auto-fill system 131, 132
Automatic seeding 128
Biocontainment 134
Blastocyst freezing 514
blastocyst freezing media 515
blastocyst freezing simplified 515
granding of embryos 514
why freeze embryos at the
blastocyst stage 514
Blastocyst thawing 521
blastocyst thawing simplified 522
evaluation of thawing 521
injuries during blastocyst
thawing 521
transfer of frozen-thawed
embryo 521
Blastocyst vitrification 180
artificial shrinkage 183
clinical results 184
artificial shrinkage
procedures 185
perinatal outcome 186
vitrified blastocysts transfer 184
grading of blastocysts 182
patients, embryo culture 182
troubleshooting 181
warming of blastocysts 181
Catastrophic failure 131
CBS high security straws 103
CBS high security vitrification kit 112
Choline substitution 39
Closed-pulled straw 273
Controlled rate freezing 94
Conventional freezing of embryos 296
cryoprotectant additives 297
cryoprotective agents 297
detrimental effect 300
embryo cryopreservation protocols 301
blastocyst freezing method 301
freezing of pre-implantation stage embryo 301
ethical considerations 302
evaluation of embryo survival 300
freezing at different developmental stages 300
principle of cryopreservation 297
safety of the procedure 302
steps involved 297
cooling rate 299
embryo loading 298
embryo thawing 299
equilibration 298
seeding 298
storage 299
washing and dilution of cryoprotectant 300
Conventional slow cooling 139
Cooling 124
rates 94
Cooling and warming 91
rates 95
Cord blood banking 402, 418
advantages 402
collection of blood 418
components 403
accreditation agencies 414
accreditation of cellular therapy 414
American Association of Blood Bank 414
biosafety cabinet 405
CD34 and its relevance 412
cell based assays 409
controlled rate freezer and storage 406
cord blood collection process 404
counseling and informed consenting 403
cryopreservation 408
donor screening 404
effects of CB collection on the neonate 404
engraftment 412
equipments required 405
existing limitations of usage of UCB 413
factors that could affect UCB engraftment 412
forming cell (CFU) assays 409
graft assessment by colony 409
graft versus host disease 411
HLA antigens 409
HLA compatibility 411
HLA match search process 411
human leukocyte antigen tests 409
infectious diseases testing 408
infrastructure 405
labeling 408
liquid nitrogen storage system 406
manpower 407
methods used for DNA-based typing 410
microbial cultures 408
platelet engraftment 412
post-thaw removal 411
post-thaw washing 411
processing 407
regulatory perspectives 413
role of HLA matching 411
setting up of collection centers 403
success cord blood transplant 413
testing of cord blood units 408
transportation to transplant centers 411
cryopreservation 420
automated devices for processing 422
containers for storage 422
controlled rate freezing 421
cryoprotectants 420
freezing rate 421
principle 420
release cord blood 422
testing 422
thawing 422
washing step 423
donor recruitment 418
future trends 423
processing 419
setting up of a cord blood bank 403
types of banking
autologous banking 402
family banking 402
types of transplantation
related UCB
transplantation 403
unrelated UCB transplantation 403
Cryobanking 88, 132
brief history 88
overview 88
Cryobiology 90, 139, 453
conventional freezing methods 141
CPA-free sperm
cryopreservation 142
macromolecules 140
sugars 140
cryoperservation of human ovarian
tissue 143
cytoskeletal stabilizer 142
dehydration of embryo and
blastocyst 142
immature human oocyte
cryopreservation 143
principles 139
conventional slow cooling 139
vitrification 140
recent advances 142
Cryocans 5
Cryogenics 453
Cryoinjury 140
Cryoloop 273
Cryonics 453
Cryopreservation 32, 453
cryoprotectants 32, 34
mechanism of action 33
nonpermeating 35
permeating 34
early clinical experience 37
disruption of the meiotic
spindle 37
zona pellucida hardening 38
methods of freezing
rapid freeze (vitrification) 36
slow freeze method 36
outcomes 40
recent developments 38
choline substitution 39
slow freeze method 38
trehalose 39
vitrification 39
stages 33
Cryopreservation of human
spermatozoa 217
Cryopreservation of semen
contaminated with hepatitis C
virus 284
background 284
materials and methods 285
design of the study 285
detection of HVC RNA 285
human protamine-2 gene
amplification 285
influence of the
cryoprotectant 285
seminal samples 285
results 285
analysis of the rinsing
water 285
influence of the cryoprotectant
on 285
material controls 286
Cryoprotectants 32, 34, 118, 194, 455
Cryoprotective agents 93, 94, 327
Cryosurvival 129
Cryotanks 132, 136
Cryotip 273
Damage caused by freezing 91
Dehydration threshold 90
Devitrification of embryo 510
quick warming 510
warming using cryoloop technique 512
Disinfection 123
Divided storage 133
Dose equivalent 163
Egg freezing 253
Electron microscopic grids 272, 508
Embryo freezing 192, 252, 477
disadvantages 193
embryo characteristics 195
embryo freezing simplified 478
cryoscan TM 486
fertisafe 485
particle counting 485
pH meter 484
thermodisk 486
VF hygiene monitoring 485
volatile organic compound monitoring 484
equipments for freezing 195
ethical presentation 202
historical background 192
limitations 194
materials required 478
need for cryopreservation 193
principles 194
protocols and methods 196
blastocyst freezing 198
cryopreservation of
prezygotes 198
principles of cryofreezing of human embryos 477
selection of embryos 478
Embryo freezing laboratory 49
Embryo thawing
cryoprotectants used 488
principles 487
thawing simplified 488
time of thawing and endometrial
preparation 488
Embryo vitrification 176, 502
alternative unique vitrification
device 508
principles of vitrification 502
protocols and clinical results 176
vitrification using crytop 178
vitrification using conventional cryostraws 176
vitrification using cryoloops 177, 505
troubleshooting 179
conventional vitrification 179
ultrarapid vitrification cryotop 180
ultrarapid vitrification:
cryoloop 179
vitrification simplified 503
Embryogenesis 360, 369
Embryonic development in vitro 260
Embryonic stem cells 391
cryopreservation 393
conventional slow freezing 395
programmable slow freezing 394
vitrification 396
evidence supporting the
potential 392
surface antigen markers 392
Embryos cryopreservation 41, 194
Embryos thawing 194
Equipment quality control 54
centrifuges 54
cryopreservation equipment 55
dry shippers/transport units 55
laboratory environmental control 56
laminar flow biohazard hood 54
Eutectics 160
Fertility preservation in cancer
patients 373
cancer treatment affect fertility 373
counseling 382
effects of chemotherapy on adult men
characteristics of gonadal toxicity 374
combination regimens 376
individual drugs 375
radiation therapy 376
effects of cytotoxic agents on adult women
characteristics of gonadal 377
combination regimens 378
individual drugs 377
radiation therapy 378
effects of cytotoxic agents on children
biologic considerations in boys 378
biologic considerations in girls 379
extent of gonadal toxicity in boys 378
extent of gonadal toxicity in girls 379
genetic concerns
biologic considerations 381
pregnancy outcome 382
gonadal dysfunction after cranial irradiation 379
preservation of fertility, hormone levels and sexual function
assisted reproductive technologies 380
choice of regimens 380
gonadal shielding 380
optimization of fertility after treatment 380
pharmacologic attempts at preserving fertility 381
requirements for pregnancy 373
Fertilization rates 259
Freezing point 160
Frozen embryo transfer 359
advantages 360
clinical aspects 359
factors influencing the embryogenesis 360
freezing program 364
frozen embryo transfer 365
thawing 364
indications 359
laboratory aspects 361
optimizing embryo transfer 362
single frozen embryo transfer 363
technique of embryo transfer 362
protocol at Southened fertility 363
cryo-solutions 363
embryo selection 363
freezing 363
protocols for endometrial preparation 360
Gas micropipettes 272
Gas sensing monitor 8
Gel-loading tips 273
Glass 160
Glass transition temperature 130, 156
Gryoprotectants 31
glycerol 35
sucrose 35
Hematopoietic stem cell transplantation
cryopreservtives 424
durability 426
freezing and freezing rate 425
thawing and washing procedure 427
Hematopoitic progenitor cell 417
Hemi straw system 273
Hen’s egg yolk 120
High security straws 122
Human oocyte cryopreservation 23
effects 24
various cellular organelles
microtubules 23
nuclei and nuclear envelope 25
oocyte cytoskeleton 23
zona pellucida 25
Human sperm cryobanking 325
donor semen cryopreservation 326
fecundity of cryopreserved donor semen 326
semen autoconservation 326
historical perspective 325
issues 327
cooling procedures 334
cryoprotectants 327
packaging 329
to seed or not to seed 335
storage 335
at what temperature 335
auto-fill systems and alarms 340
cryogenic storage tanks 340
divided storage 340
liquid or vapor phase storage 338
monitoring cryogenic storage tank 340
post-thaw processing 340
reducing risk 339
risk of cross-contamination 336
sterilizing the controlled rate freezer 339
Infertility Treatment Act 1995 434
Intracytoplasmic sperm injection 313
adding sperm sample to the injection dish 317
adding sperm to injection dish 317
preparation of injection dish 317
selection of viable immotile sperm by HOS 317
cryopreservation 314
adding cryoprotectant 315
cryoprotectant 314
freezing 315
maintenance of sperm bank 316
MESA samples 315
post-thaw testing 316
preparation of sperm samples 314
TESE samples 315
disposal of frozen sperm 318
sperm preparation on the day
of ICSI 316
surgical retrieval of sperm 313
Ionising radiation 162
dose consideration in female patients 165
before treatment 167
general considerations 167
maintenance of fertility 166
precautions 167
effects of radiation 163
female fertility 164
male fertility 164
follow-up after radiation
therapy 169
absorbed dose 162
dose equivalent 163
organ dose 163
radiation exposure 162
options 168
post-chemo-radiotherapy treatment
egg storage 168
embryo storage 168
hormonal manipulation 168
hormone replacement therapy 168
ovarian cryopreservation 168
ovarian transposition 168
Isothermal vapor storage 131
IUI-ready sperm samples 119
Laparoscopy in ovarian tissues cryopreservation
effects of radiation on ovarian
function 385
ovarian tissue
cryopreservation 388
ovarian transplantation 387
surgical techniques for ovarian
resection 388
preservation of ovarian function
ovarian transposition 386
techniques for ovarian
transposition 386
Latent heat 19, 454
heat of fusion 127
plateau 147
Lethal zone 124
Liquid nitrogen 3
application 7
containers 5
first aid 9
hazards 7
identification 4
production 5
properties 4
cold boiling point 4
Leidenfrost effect 4
physical and chemical 4
smoking 4
safety measures while handling 6
Low temperature phenomena 92
Mechanical freezer 130
Meiotic spindle 37, 141
Melting point 156
Microbial and viral pathogens 289
choice of liquid nitrogen
refrigerator 292
choice of sample container 291
cryopreservation procedure 291
materials and methods 289
viability of microorganisms and viruses 289
Nucleation 156
Oocyte bank 144
Oocyte cryobiology 22
Oocyte cryopreservation 37, 40, 204
advancements in the field 205
clinical outcome and case
reports 207
historical overview 204
indication 205
limitation 207
oocyte freezing protocol 205
oocyte vitrification protocols 206
Oocyte freezing 492
current methodologies
slow freezing 492
ultra-rapid freezing (vitrification) 493
oocyte freezing simplified 493
principles of oocyte freezing 492
Oocyte thawing 499
oocyte thawing simplified 499
peculiarities of oocyte freezing and thawing 501
Oocyte vitrification 187
materials and methods 188
results 189
Open-pulled straw 272
Organ dose 163
Osmotic equilibration 196
Ovarian and testicular tissue 432
Ovarian tissue banking 207
age of the patient 208
difficulties 208
indication 208
protocols 208
risk of ovarian metastasis 208
strategies for preserving female fertility 209
types of cancer 208
Ovarian tissue cryopreservation 232
application 236
autotransblantation 236
in vitro culture 238
xenotransplantation 238
cryopreservation of intact
ovary 239
evaluation of cryopreserved ovarian tissues 236
in vitro culture 236
LM and TEM analyses 236
xenotransplantation 236
future 240
indications 232
autoimmune diseases 233
cancers in adults 233
cancers in children 233
methods 233
influencing factors 236
slow freezing 233
vitrification 234
procedures 233
tissue preparation 527
Ovarian tissue freezing 526
collection of ovarian tissue 527
cooling program 531
cryopreservation of the
whole ovary 526
cryoprotectant preparation 529
equilibration procedure 529
histological analysis 529
indications for ovarian
cryopreservation 527
peculiarities of ovarian tissue
freezing 531
role of vitrification in human
ovarian tissue 526
seeding protocol 530
tissue preparation 527
Ovarian tissue thawing 533
ovarian tissue thawing:
simplified 534
sites of transplant 533
Packaging devices 100
capillary-based devices 101
CBS HSV kit 102
classic straws 101
cryotubes/cryovials 100
glass ampules 100
high security straws 101
packaging devices for
vitrification 101
proprietary devices 102
risk analyses 103, 104
controlled rate freezing packaging devices 104
vitrification packaging devices 105
sanitization of packaging devices
contramination of liquid nitrogen 102
controlled rate freezer 102
cross-contamination 102
Packaging systems 121
pH meter 484
Physics of freezing 146
contamination 155
cryopreservation protocols 156
embryos 157
peripheral blood mononuclear cells 156
spermatozoa 157
effects of freezing on cells 148
procedures for ICE nucleation 152
special case of embryos and oocytes in straws 153
reproducibility of protocols 158
supercooling and cell survival 151
thawing and post thaw
handling 156
Polscope technology 242
azimuth 244
birefringence 242
retardance 243
the oosight system 244
Posthumous reproduction 431, 439
cases 442
France: parpalaix versus Cecos, 1984 443
United Kingdom Evans versus the United Kingdom (application no. 6339/05) 443
United Kingdom versus Diane blood (1997) 444
USA Davis versus Davis (1990) 443
USA Hecht versus Superior Court 1993 442
conflicts between the embryo and the mother 436
control over embryos 436
disposition of unused embryos 434
grounds for extended storage 433
Indian scenario 444
infertility treatment act 434
ownership rights for gametes and
embryos 436
posthumous use of gametes and
embryo 434
regulation of ART 438
India 439
international 438
USA 438
the status of the embryo and of
gametes 435
the theory of progressive legal
protection 435
use and control of genetic
material 433
Process mapping 76
Product family overview 106
Progress in cryobiology 252
Properties of nitrogen 3
Protocol for the using HSV kit 114
resources 117
simplified procedure chart 116
thawing 115
Protocols for using HS straws
embryos 108
semen 108
simplified procedure chart 110
embryos 111
sperm 110
Quality assurance 69
Quality control 69, 80
internal quality control and quality assurance 70
instruments and equipment 71
lab conditions and
environment 73
lab personnel 72
maintenance and quality
check 72
media and reagents 70
mouse embryo bioassay 71
sperm survival test 71
technical accuracy and error 73
third party services 71
uncertainty of measurement 74
monitoring protocols
process mapping 76
standard operating
procedures 78
user requirement
specification 77
practical implementation
frequency of assessment 74
planning strategy 74
quarterly-participation of
external quality
assessment 75
total quality management 69
cost control 70
essential principles 70
role of customer expectation 70
Quality cycle 70
Quarantine tank 133
Recrystallization 128
Regenerative medicine 391, 400
Safety from HIV under
crypreservation 319
materials and methods
experimental protocols 320
supplies and reagents 320
control straws 320
PVC straws 320
safety of cryopreservation straws
IR straws 321
PETG straws 321
Sealed opened straws 273
Seeding 126, 127, 156
Semen banking 345
acceptability of frozen donor samples
fresh vs frozen semen 349
cryobiology of sperms 346
selection of donors 347
ethical aspects of therapeutic donor
insemination 350
historical perspective 345
issues in regulation of donor insemination 350
aspects of regulation in donor
insemination 351
commissions, committees 351
current level of regulation 351
laboratory screening 352
quarantine 352
regulatory options for donor
insemination 350
semen samples 352
sperm bank 352
protocols of laboratory
techniques 348
Semen cryopreservation 46, 78
accreditation and ISO standards 86
benchmarking 85
document control 80
inventory and record
maintenance 85
legal issues 86
method 79
principle 78
quality control 79
reagents 79
risk management 83
risk management and laboratory safety 81
organizational issues 82
resource issue leading to high
risk situations 82
risk management issues 83
staffing issues leading to high
risk situations 82
safety of lab personnel 84
specimen 78
tools and solution in cryobiology 83
tools for TQM cryopreservation 80
analysis 81
root cause analysis and
troubleshooting 81
trouble shooting and root cause 81
Semen thawing 473
assessment of post-thaw
fertility 473
semen thawing simplified 474
Shipping 134
Slow freezing 124
Slush nitrogen 9, 142
Solid-surface vitrification 143
Specimen identification 97
auditing 98
inventory systems 98
labeling 97
Sperm bank 352
Sperm counting device 469
Sperm cryobiology 26
Sperm cryopreservation 211
effect of storage temperature 212
specific situations 214
absence of the partner 215
intraoperative sperm harvesting 215
non-obstructive azoospermia 214
obstructive azoospermia 214
patients with cancer 214
Sperm freezing 40
media 469
Spermatogenesis 536
Spindle apparatus 245
ensuring oocyte maturation prior to
freezing 248
gross spindle morphology 246
spindle negatives 245
spindle orientation 247
spindle retardance 246
Standard operating procedures 78
Stem cells 400
sources 401
umbilical cord blood 401
types 400
adult 401
embryonic 400, 401
Steps of cryopreservation 455
cross infection in the semen
banks 470
essentials of freeze thaw cycle 462
cooling and warming rates 463
freezing 463
packaging of semen after addition of cryopreservative 462
plastic straws 462
specimen glycerolization 462
storage 463
events during freezing 457
events during thawing 457
indications of semen cryopreservation
biochemical and physical aspects of sperm cryopreservation 461
common indications for autologous semen banking 461
principles of cryobiology 460
biological behavior of the
phospholipids membrane of
the sperm 460
changes in valume of sperm
when exposed to
cryoprotectants 460
risk of storing biological materials
at low temperatures 458
sperm freezing medium 464
the disposables /equipment
required for cryofreezing are
enumerated as below 467
the techniques for semen
cryofreezing using liquid
nitrogen vapor cooling 464
Storage and use of ovarian and
testicular tissue 432
Storage temperatures 95
Supercooling 151, 156
Temperature measurements 92
Testicular tissue cryopreservation 536
ethical issues 539
freezing technique protocol 538
spermatogenesis and stem cells 536
testicular damage postchemo or radiotherapy 536
options 536
principles of testicular preservation
cryoprotectants 537
methods of cryopreservation 538
safety of the cryofreezing
procedures 537
Testicular tissue cryopreservation 536
ethical issues 539
freezing technique protocol 538
spermatogenesis and stem cells 536
testicular damage postchemo or radiotherapy 537
options 536
cryoprotectants 537
methods of cryopreservation 538
safety of the cryofreezing
procedures 537
Testicular/epididymal sperm
freezing 354
cryobiological principles 355
physiology and spermato-genesis 354
sperm freezing techniques 356
sperm retrieval techniques 354
Thawing kit 251
Thawing procedure 258
diluting 259
preparation of media 258
thawing 258
washing 259
Thermal shock 90, 124
Thermodynamics 453
Total quality management 69
Trehalose 39
Tri-laminate zona pellucida 248
Umbilical cord blood 417, 427
Undercooling 156
Universal contamination 133
Validation studies 106
safety 106
Validation studies 112
cooling rate study 112
embryo survival 114
Vitrification 39, 57, 94, 126, 140, 156, 194, 200, 223, 254, 266, 276, 453
alternative to slow freezing 276
aseptic vitrification 279
benefits and disadvantages 224
decreased chilling injury 228
decreased toxicity of
cryoprotectants 224
increased cooling and warming
rates 224
potential danger of disease
transmission 228
carrier system for vitrifying
procedures 201
common carrier systems 272
composition of solutions 58
non-permeating 59
permeating 58
conditions for achieving a vitrified state
first condition 277
second condition 277
factors influencing vitrification 267
buffering solutions 267
cryoprotective agents 267
disaccharides 267
macromolecules 268
future development and safety 61
high concentrations of CPs 278
history and definition 266
importance of cooling rates 201
limitation 274
method and protocol 201
procedure of ultra-rapid
cryopreservation 62
renewed interest 276
re-warming the embryo 278
slow deep-freezing and
vitrification 278
slow freezing versus
vitrification 266
technique of vitrification 268
cryo-solutions 268
vitrification 269
loading the oocytes in the
cryotip 269
oocyte vitrification 268
thawing 270
materials (expendables) 270
thawing from cryotip 270, 271
warming solutions 270
toxicity of cryoprotectants 60
types of cryoprotectants 201
vitrification of blastocysts 280
vitrification of embryos at D3 280
vitrification of human embryos 279
vitrification of zygotes 279
Vitrification devices 304
material and methods 305
results 306
deep-freezing kinetics 307
effect of the deep-freezing
medium 306
thawing kinetics 308
temperature measurements 305
deep-freezing kinetics 306
effect of the deep-freezing
medium 305
thawing kinetics 306
Vitrification of human embryos 369
Vitrification principles 23
Vitrification procedure 255
equilibration 258
handling tools 257
preparation of media 255
preparation of vitrification
container 255
storage 258
vitrification 258
Vitrification-kit 251
Warming/thawing 128
Water molecule 11
application in cryobiology 21
crystal structures of ice 13
hexagonal 14
tetrahedral 13
crystalline phases of ice 14
formation 12
H2O molecule in liquid water 16
H-bonds in liquid water 16
molecule in ice 13
properties 13
properties of liquid water 16
latent heat 19
physical 17
Chapter Notes

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1Basic Cryobiology
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Thermodynamics and Physical Properties of Liquid NitrogenChapter 1

Shehbaaz Daruwala,
Bharati Dhorepatil
The element Nitrogen (N2) was discovered in 1772 by Scott physician Daniel Rutherfield, who called it “ noxious air” or “fixed air”. It is the fifth most abundant element in the universe and makes up the major portion (78.03% by volume and 75.5% by weight) of the earths atmosphere. Nitrogen is inert and will not support combustion. It occurs in all living organisms. It is an important constituent element of amino acids which make up proteins which form nucleic acids (DNA and RNA); resides in the chemical structure of almost all neurotransmitters and is a defining component of alkaloids. The name Nitrogen is derived from Greek words “Nitron” which means ‘saltpeter’ and “genes” which means ‘forming. Gaseous Nitrogen is referred to as N2. Nitrogen is pronounced as NYE-Treh - gen.
Properties of Nitrogen
Nitrogen is a nonmetal with an electronegativity of 3.0. It has five electrons in its outer shell, therefore trivalent in most compounds. The triple bond in molecular nitrogen (N2) is one of the strongest in nature. The resulting difficulty of converting (N2) into other compounds, and the ease (and associated high energy release) of converting nitrogen compounds into elemental N2, have dominated the role of nitrogen in both nature and human economic activities.
At atmospheric pressure, molecular nitrogen condenses (liquefies) at 77 K (−195.8°C) and freezes at 63 K (−210.0°C) into the beta hexagonal close-packed crystal allotropic form. Below 35.4 K (−237.6°C), nitrogen assumes the alpha cubic crystal allotropic form. Liquid nitrogen, a fluid resembling water, but with 80.8% of the density, is a common cryogen. Under extremely high pressures (1.1 million atm) and high temperatures (2000 K), as produced under diamond anvil conditions, Nitrogen polymerizes into the single bonded diamond crystal structure, an allotrope nicknamed “Nitrogen Diamond”.
Occurrence of Nitrogen
Nitrogen is the largest single constituent of the earth's atmosphere (78.082% by volume of dry air, 75.3% by weight in dry air). It is created by fusion processes in stars and is estimated to be the 7th most abundant chemical element by mass in the universe.
Nitrogen is present in all living organisms in proteins, nucleic acids and other molecules. It typically makes up around 4% of the dry weight of plant matter, and around 3% of the weight of the human body. It is a large component of animal waste usually in the form of urea, uric acid, ammonium compounds and derivatives of these nitrogenous products, which are essential nutrients for all plants that are unable to fix atmospheric nitrogen. Nitrogen occurs naturally in a number of minerals, such as saltpeter (potassium nitrate), Chile saltpeter (sodium nitrate) and salt ammoniac (ammonium chloride). Most of these are relatively uncommon, partly because of the minerals' ready solubility in water.
Liquid Nitrogen is nitrogen gas from the atmosphere in which the molecules of N2 have been pressed together using pressure.4
This is a form of chilled, condensed gaseous nitrogen. It is a colorless, odorless, nonflammable, noncorrosive and non-toxic liquid that is lighter than water.
Liquid Nitrogen is normally referred to as LN2. When nitrogen is converted to liquid form it becomes a cryogenic fluid. A cryogen is a substance used to cool other substances.
Definition: Liquid nitrogen is a cold, liquefied gas with a temperature of −321° F.
A. Has a cold boiling point…does such a thing exist?
At standard temperature and pressure, nitrogen exists in a gaseous state. At −320° F, nitrogen changes its gaseous form to a liquid state, thus making liquid nitrogen extremely cold. The same temperature at which a substance condenses is the same temperature at which it boils, so nitrogen has a cold boiling point…. It may sound weird but is true logically.
Most of us tend to think of the temperature at which something boils to be a high one… but not in the case with nitrogen, which has a unique “Cold boiling point”. It is extremely cold (LN2 boils at −3204° F or −195.8°C) hereby handling of liquid nitrogen requires the use of protective gloves and goggles.
B. Leidenfrost effect
This is the ability of liquid nitrogen to boil immediately on contact with a warmer object, enveloping the object in the insulating nitrogen gas.
The negating consequence of this Leidenfrost effect is reduction in the effective coolant property of liquid nitrogen.
C. Smoking of liquid nitrogen
When LN2 rapidly boils and vaporizes on a smooth surface with a temperature that is much higher than nitrogen's boiling point, a physical phenomenon known as the “Leidenfrost effect” is observed. The liquid nitrogen vaporizes quickly and lifts itself above the surface. It hovers, producing little or no friction on the surface. If the surface is irregular, this effect cannot occur and the vaporization is even more rapid. The nitrogen vapor spreads itself out through the air picking up water vapor along the way. LN2 smokes because of the presence of water in the air. The amount of water present in the atmosphere is dependent upon the temperature of the air. Warmer air can hold more water vapor than cooler air. LN2 cools the air to condense the water out of it. These water droplets scatter light and produce the “smoking” effect. The ability of liquid nitrogen to do this makes it very popular at parties, magic shows and in classroom demonstrations.
Physical and Chemical Properties of Liquid Nitrogen (LN2)
Physical state: Liquid
Appearance and odor: Colorless, odorless
Odor threshold (PPM): Odorless
Vapor pressure: Gas@ 70°F (21°C)
Vapor sp. gravity (air = 1): 0.967 @ 70°F (21°C)
Volatiles (% by volume): 100%
Boiling point:
-195.8°C (760 mmHg)
Freezing point:
Solubility in water (%): Slight.
Stability and Reactivity of Liquid Nitrogen (LN2)
Chemical stability: Stable
Conditions of reactivity: Heat
Hazardous polymerization: Will not occur
Incompatible substances:
Magnesium powder
Fatty substances in cryogenic grinding
Hazardous decomposition products: None
Hazard Identification of Liquid Nitrogen (LN2)
Hazard overview: Nitrogen gas is colorless, odorless and non-flammable.
The primary health hazard is asphyxiation by displacement of oxygen. Maintain oxygen levels above 19.5%. Contact with the liquid or cold gas can cause freezing of exposed tissue.
Route of entry: Inhalation, skin and eye contact
Effects of acute exposure:
Eye contact:
Can cause frostbite (liquid form).
Vapor may cause a stinging sensation
Skin contact:
Can cause frostbite (liquid form).
No adverse effects from gas
May cause dizziness.
Can cause vomiting.
May result in unconsciousness
May cause excitation, excess salivation, rapid breathing
May cause headaches and drowsiness5
May cause stinging of the nose and throat
Ingestion: Not a likely route of exposure
Effects of chronic exposure: Damage to retinal ganglion cells and central nervous system may occur due to the presence of carbon dioxide.
Production of Liquid Nitrogen
Liquid nitrogen (liquid density at the triple point is 0.707 g/ml) is the liquid produced industrially in large quantities by fractional distillation of liquid air. A common method for the production of LN2 is the liquefaction of air. Liquefaction is the phase change of a substance from the gas phase to the liquid phase. In liquid nitrogen compressors or generators air is compressed, expanded and cooled via the Joule-Thompson effect. Since nitrogen boils at a different temperature than oxygen, the nitrogen can be distilled out of the liquid air, re-compressed and then re-liquefied. Once liquid nitrogen is removed from the distillation chamber it is stored in special containers. It is then made available for commercial distribution.
Containers for Liquid Nitrogen
Typical cryogenic liquid cylinder is an insulated, vacuum-jacketed pressure vessels, which are specifically designed and made of materials that can withstand the rapid changes and extreme temperature differences encountered in working with liquid nitrogen.
Equipped with (1) safety relief valves, (2) Rupture disks, (3) Loose fitting lids to protect the cylinders from pressure build-up. These containers operate at pressures up to 350 psig and have capacities between 80 and 450 liters of liquid. Product may be withdrawn as a gas by passing liquid through an internal vaporizer or as a liquid under its own vapor pressure. Even these special containers should be filled slowly to minimize the internal stresses that occur when any material is cooled. Excessive internal stresses can damage the container. Use only containers designed for low-temperature liquids (Figs 1.1 and 1.2).
Dewars are designed for storing and dispensing small quantities of liquid nitrogen. Easy to operate, the snap on cap and neck-tube core assure positive closure and easy access without unnecessary exposures to the cryogen. Constructed from materials of the highest performance and quality including an aluminum exterior, these containers are not only rugged, robust, and dependable, but also have very high thermal efficiencies.
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Fig. 1.1: Dewars or cryocans
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Fig. 1.2: Liquid nitrogen cryocans of various capacities
The safe handling and use of liquid nitrogen in liquid nitrogen dewars or flasks is possible only by knowing the potential hazards. There are two important properties of liquid nitrogen that present potential hazards:
  1. It is extremely cold.
  2. At atmospheric pressure, liquid nitrogen boils at − 320° F/-196° C. Very small amounts of liquid vaporize into large amounts of gas. One liter of liquid nitrogen becomes 24.6 ft3/0.7 m3 of gas.
The safety precautions as outlined must be followed to avoid potential injury or damage, which could result from 6these two characteristics. Any attempt to handle liquid nitrogen must be done only after fully understanding the potential hazards, their consequences, and the related safety precautions.
Safety Measures while Handling Liquid Nitrogen
  1. Precautions while transferring liquid nitrogen: Use a phase separator or special filing funnel to prevent splashing and spilling when transferring liquid nitrogen into or from a dewar. The top of the funnel should be partly covered to reduce splashing. Use only small, easily handled Dewars for pouring liquid. For the larger, heavier containers, use a cryogenic liquid withdrawal device to transfer liquid from one container to another. Be sure to follow instructions supplied with the withdrawal devices.
  2. Do not overfill containers: Filling above the bottom of the neck tube (or specified maximum level) can result in overflow and spillage of liquid when the neck tube core or cover is placed in the opening. Therefore avoid overfilling the cryocans.
  3. Special kind of dipstick to be used to measure the level: When a warm tube is inserted into liquid nitrogen, liquid will spout from the bottom of the tube due to gasification and rapid expansion of liquid inside the tube. Wooden or solid metal dipsticks are recommended; avoid using plastics that may become very brittle at cryogenic temperatures, which then become prone to shatter like a fragile piece of glass. Never use hollow rods or tubes.
  4. Store and use liquid nitrogen only in a well-ventilated place: As the liquid evaporates, the resulting gas tends to displace the normal air from the area. In closed areas, excessive amounts of nitrogen gas reduce the concentration of oxygen and can result in asphyxiation. Because nitrogen gas is colorless, odorless and tasteless, it cannot be detected by the human senses and will be inhaled as if it were air. Inhaling air that contains less than 18 percent oxygen can cause dizziness and quickly result in unconsciousness and death.
  5. Disposal of liquid nitrogen: Never dispose of liquid nitrogen in confined areas or places where others may enter. It should be done outdoors in a safe place. Pour the liquid slowly on gravel or bare earth where it can evaporate without causing damage.
  6. Handling liquid nitrogen dewar: Keep the unit upright at all times. Tipping the container or laying it on its side can cause spillage of liquid nitrogen. It may also damage the container and any materials stored in it. Rough handling can cause serious damage to dewar's such as, allowing it to fall over on its side, or subjecting it to sharp impact or severe vibration can result in partial or complete loss of vacuum. To protect the vacuum insulation system, handle containers carefully.
    Do not drag or roll these units across a floor. Use a dolly or handcart when moving containers. Large units are heavy enough to cause personal injury or damage to equipment if proper lifting and handling techniques are not used.
    When transporting a liquid nitrogen dewar, maintain adequate ventilation and protect the unit from damage:
    Do not place these units in closed vehicles where the nitrogen gas that is continuously vented from unit can accumulate. Prevent spillage of liquids and damage to unit by securing it in the upright position so that it cannot be tipped over. Protect the unit from severe jolting and impact that could cause damage, especially to the vacuum seal.
  7. Keep the unit clean and dry: Do not store it in wet, dirty areas. Moisture, animal waste, chemicals, strong cleaning agents and other substances, which could promote corrosion, should be removed promptly. Use water or mild detergent for cleaning and dry the surface thoroughly. Do not use strong alkaline or acid cleaners that could damage the finish and corrode the metal shell.
  8. Protect dewar contents: Materials stored in a liquid nitrogen dewar with a wide mouth are protected by the extremely low temperature of the liquid nitrogen or the gas that issues from the evaporating liquid nitrogen. When all of the liquid nitrogen has evaporated, the temperature inside the unit will rise slowly to ambient. The rate at which the liquid nitrogen will evaporate depends upon the pattern of container use and the age and condition of the container. Evaporation increases as insulation efficiency deteriorates with age and rough handling. Opening and closing to insert and remove materials and moving the unit will also increase the evaporation rate.
    To protect valuable material stored in a liquid nitrogen cryocan, check the liquid level in unit frequently. It is of the greatest importance to check the liquid nitrogen level periodically in order to prevent critical samples from deteriorating when no liquid nitrogen is in the cryocan.
  9. When to replace existing cryocans: Condensed moisture or frost on the outer shell of a cryocan and abnormally rapid evaporation of the liquid nitrogen are indications 7of vacuum loss. If vacuum loss is evident or suspected, start thinking immediately about the procurement of a replacement dewar. It is just not cost effective to continue to use a dewar with a bad vacuum and waste valuable liquid nitrogen in the process. There is also the safety issue of excessive boil-off in an enclosed area that is not large enough to “absorb” the higher rate of nitrogen boil off.
Liquid nitrogen is valued for its coldness as well as inertness:
This combination is employed to rapidly chill and freeze food items (semen, oocytes, embryos, meat, fruit, vegetables, baked goods, dairy products). Rapid freezing results in very small ice crystals, less cellular damage, and better-quality products after thawing, i.e. the process of “Vitrification”.
The intense cold produced by these products can also be used to make normally soft and flexible materials hard and rigid, allowing them to be ground, machined or fractured.
When liquid nitrogen is vaporized and warmed to ambient temperature, it absorbs a large quantity of heat. The combination of inertness and its intensely cold initial state makes liquid nitrogen an ideal coolant for certain applications such as freezing.
Main Uses of LN2
  1. Freezing and preservation of food: Foods can be packed, sealed and then sprayed with liquid nitrogen as it evaporates upon contact with many surfaces (including food). Thus, evaporation process allows for the absorption of heat and energy from the food as a result the molecules in the food slow down and the food freezes
  2. Cryopreservation: Liquid nitrogen is widely used in the preservation of medical specimens it is particularly useful for very long-term preservation of cells and tissues. It may be used for the rapid freezing of different tissues such as bone marrow, blood, sperms, ovarian tissue, embryos, etc. It is useful in the preservation of animal embryos, bacteria and fungi.
  3. Cryotherapy: Extremely cold temperatures can kill human tissue. Liquid nitrogen may be used in standard medical procedures such as wart removal and treatment of certain skin cancers.
  4. Cryonic preservation of humans and pets in the hope of future reanimation.
  5. Research laboratories: Many research labs around the world use liquid nitrogen to aid in research requiring cryogenic conditions (i.e. superconductivity labs).
  6. Classroom demonstrations: Liquid nitrogen is commonly used in the classroom to demonstrate its amazing chemical properties and to illustrate its cooling effects on other materials.
Rapid release of nitrogen gas into an enclosed space can displace oxygen, and therefore represents an asphyxiation hazard. This may happen with few warning symptoms, since the human carotid body is a relatively slow and a poor low oxygen (hypoxia) sensing system. When inhaled at high partial pressures (more than about 3 atmospheres, encountered at depths below about 30 m in scuba diving) nitrogen begins to act as an anesthetic agent. It can cause nitrogen narcosis, a temporary semi-anesthetized state of mental impairment similar to that caused by nitrous oxide. Nitrogen also dissolves in the bloodstream and body fats. Rapid decompression (particularly in the case of divers ascending too quickly, or astronauts decompressing too quickly from cabin pressure to spacesuit pressure) can lead to a potentially fatal condition called decompression sickness (formerly known as caisson sickness or more commonly, the “bends”), when nitrogen bubbles form in the bloodstream, nerves, joints, and other sensitive or vital areas.
  1. Cryovials stored in liquid nitrogen may “explode” when removed from the dewar. Cryo vials are not guaranteed to be leak tight if stored in the liquid nitrogen (LN2). Due to “superfluidity” of LN2 it can leak into sealed cyrovials. Although these cryovials are made of a tough plastic which prevents cracking at the liquid nitrogen temperatures of −196°C (−321°F), the vials in which the liquid nitrogen has leaked when removed from the dewar causes expansion of liquid nitrogen and vials can easily explode. To site a hypothecal situation where in simple calculation shows that 0.5 g of liquid nitrogen in a 1.5 ml cryovial will generate a pressure of 4,053 psi when it evaporates. Failure of the screw threads can turn the cap into a projectile with an initial velocity of up to 296 miles per hour (132 meters/sec), and up to 8.4% as much kinetic energy as a 22-caliber bullet! The rapidly expanding gas can be just as dangerous.
    Precautions when storing samples in LN2 wear cryogloves, face sheild, safety glasses when removing samples.8
    Various ways for liquid nitrogen to enter into a cryovial:
    1. Defective vials made of inferior quality silicone or plastic.
      Always use standard cryovials such as Nunc cryovials.
    2. The screw cap of the cryovial is overtightened or undertightened or there are droplets of water on the threads.
    3. Using expired cryovials.
      Nunc tubes must be used within three years of their date of sterilization because the silicone gasket deteriorates over time.
  2. Frostbite hazard: Contact of sufficient quality of liquid nitrogen with the body, results in a “cold burn” or a “cryogenic burn” within seconds, though not instantly on contact, depending on form of liquid nitrogen (liquid vs. mist) and surface area of the nitrogen-soaked material (soaked clothing or cotton causing more rapid damage than a spill of direct liquid to skin, which for a few seconds is protected by the Leidenfrost effect).
    Small amounts of LN2 will rapidly evaporate and will give a small sensation similar to a pin prick. The danger comes from larger quantities which do not evaporate quickly and cause more harm. Should a larger quantity come in contact with a person, the person should immediately get away.
    Prevent frostbite hazard by: Donning protective clothing to keep LN2 from contacting the body.
    Precautions when LN2 spillage occurs:
    1. If a large spill occurs, discontinue filling and leave the room until the liquid evaporates.
    2. If clothing becomes soaked, hold it away from the body until it warms, or remove the clothing.
    3. If a cold burn occurs, once warmed up, it will appear very similar to, and should be treated the same as a sunburn. Warm affected skin slowly using cold (NOT HOT) water only.
    4. Any serious cold burn should be treated by a doctor. Ample care should be taken while handling liquid nitrogen avoid spilling the liquid on clothing, since the clothes can easily become saturated with the liquid and being in contact next to the skin for a significant period of time, leading to serious burns. Thus for this reason, cloth gloves may be worse than nothing at all.
  3. Asphyxiation: Liquid nitrogen rapidly evaporates giving nitrogen gas. Just one liter of liquid produces around 700 liters of gas at atmospheric pressure, displacing significant quantities of breathable air if the gas is released in a confined space, e.g. in a laboratory, cold room, or storage area. The problem is compounded by nitrogen's tendency to accumulate at low levels where it is less easily dispersed than the ambient atmosphere. Thus, an apparently small spillage could lead to dangerously low oxygen levels, presenting a serious hazard to personnel working in the confined area. This condition is called Oxygen Deficiency Hazard (ODH). Gas sensing monitors are available to insure that this O2 level is maintained at all times is a preventive measure to avoid ODH for personnel working with liquid nitrogen (Fig. 1.3).
    The human body does not detect oxygen deficiencies very well. The feeling of being suffocated is due to excess carbon dioxide, not from a lack of oxygen, so symptoms may not be evident. The normal oxygen level in the atmosphere is 20.9%. Physical and intellectual performance may be inhibited if oxygen levels fall below 17%, and at levels less than 17%, symptoms of asphyxia, such as gasping, vomiting or collapse, will set in. Victims may well not be aware of their condition, especially if the oxygen level falls rapidly, and in the event of a major leak or spillage could fall unconscious without ever being aware of the danger. When the oxygen content of air is reduced to around 10%, unconsciousness can be immediate with virtually no warning thus creating an unsafe environment for workers. If there should be a large spill anywhere in the building, don't wait around notify everyone that they need to evacuate the area.
    Liquid nitrogen should never be carried in an elevator, because of the remote possibility that the dewar containing the liquid will go “soft” (i.e., the vacuum in the dewar flask will be lost). Never put liquid nitrogen, in styrofoam coffee cups. Although the styrofoam will keep the liquid from evaporating, the risk is too great that someone will walk by and mistake it for limca or sprite and try to take a sip.
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Fig. 1.3: Gas sensing monitor
  1. Fire and explosion hazard: Liquid nitrogen is not flammable however it is capable of condensing oxygen out of air creating oxygen rich environment. Flammable materials can ignite in the presence of condensed oxygen.
  2. Property damage: On spillage of liquid nitrogen on floor tiles it can cause cracking and damage to any rubber tubing. Cracked floors can cause a tipping hazard to personnel working in the laboratory.
First Aid for Liquid Nitrogen Exposure
If a person complains of dizziness or loses consciousness while working with liquid nitrogen, move to a well-ventilated area immediately. If breathing has stopped, apply artificial respiration, give oxygen and call a physician. Keep warm and at rest.
If skin is exposed to liquid nitrogen, restore tissue to normal body temperature 98.6° F (37°C) as rapidly as possible, followed by protection of the injured tissue from further damage and infection. Remove or loosen clothing that may constrict blood circulation to the frozen area. Call a physician. Rapid warming of the affected part is best achieved by running the exposed part under water at 108° F (42° C) for fifteen minutes. Under no circumstances should the water be over 112° F (44°C), nor should the frozen part be rubbed either before or after rewarming as it causes further tissue damage. If the finger is burned do not place it in your mouth. This could burn your mouth. The patient should neither smoke, nor drink alcohol.
  1. Wear safety goggles at all times while handling liquid nitrogen.
  2. Never place liquid nitrogen in a sealed container or any object that could cause entrapment of the gas.
  3. Never mix dry ice or liquid nitrogen with water or water ice or pour it down the sink. Ice can solidify around it, trapping the gas at a high pressure.
  4. Vials to be stored in contact with liquid phase nitrogen must always be sealed with Nunc CryoFlex, which is a type of heat-shrinkable tubing.
  5. When cooling objects using liquid nitrogen always do so slowly using tongs to prevent boiling and splashing.
  6. Use a cryoclaw to retrieve the samples that have fallen into the dewar.
  7. Liquid nitrogen should never be carried in an elevator, because of the remote possibility that the dewar containing the liquid will go “soft” (i.e. the vacuum in the dewar flask will be lost).
  8. Never put liquid nitrogen, in styrofoam coffee cups. Although the styrofoam will keep the liquid from evaporating, the risk is too great that someone will walk by and mistake it for limca or sprite and try to take a sip.
Personal Protective Equipment (PPE)
One must be thoroughly familiar with the properties and safety considerations before handling a cryogenic liquid and its associated equipment. The eyes are the most sensitive body part to the extreme cold of the liquid and vapors of liquid nitrogen.
The recommended personal protective equipment (PPE) for handling cryogens includes:
  1. Full face shield over safety glasses.
  2. Long sleeve shirts, and trousers without cuffs.
  3. Safety shoes are recommended for people involved in the handling of containers. Depending on the application, special clothing suitable for that application may be advisable.
  4. Cryogloves of thermal insulated or leather gloves which are loose fitting so they can be easily and quickly removed if cryogenic liquid is spilled on them. Insulated gloves are not made to permit the hands to be put into a cryogenic liquid. They will only provide short-term protection from accidental contact with the liquid.
Slush Nitrogen
Subcooling of the LN2 is accomplished by direct or indirect evaporative cooling which relies on the thermodynamic principle that the boiling point of a liquid of a pure substance in equilibrium with its gaseous phase is a function of pressure. Therefore, as the pressure exerted on liquid nitrogen is lowered, its boiling point decreases resulting in evaporative cooling of the liquid nitrogen. That is, the energy required for the liquid nitrogen to evaporate is supplied by the liquid nitrogen, therefore, resulting in a lowering of the liquid's temperature. For example, for LN2, at a pressure of about 1.8 pounds per square inch (psia), it will l have a temperature of about −210°C. In the receptacle of the vacuum chamber of the refrigeration system, a large volume of LN2 is subjected to a partial vacuum of from about 1.8 psia up to about 14 psia, preferably from about 1.82 to about 3 psia, such that the temperature of the LN2 subcooled refrigerant is lowered to a temperature below its normal boiling point near its triple point at which the liquid and solid nitrogen are in equilibrium and form a thickened mixture referred to as “Nitrogen slush” in which liquid and solid nitrogen coexist.10
By applying negative pressure with a vacuum, LN2 will freeze and will be transformed into a slush state with a lower internal temperature of −210°C without vaporization, thus SN2 may offer high-speed cooling rates with a possibility to increase the survival rate of mammalian oocytes as well as other characteristics after vitrifying/warming. As it is seen boiling of liquid nitrogen (LN2) occurs when a sample is immersed and results in gas bubbles around the specimen which, in turn, results in poor heat transfer.
The nitrogen slush or subcooled liquid nitrogen is commonly referred to as SN2. However, it is still controversial whether SN2 has a beneficial effect on the development of embryos after cryopreservation.
  1. National Science Foundation's Graduate Teaching Fellows in K-12 Education (GK-12) program (DGE # 0231913 and # 9979516) and Cornell University Office of environmental health and Safety University of Pennsylvania
  1. Physics of human Body with illustrative examples from Medicine and Biology (Springer, Verlag,  New York,  2000).