Bacteria on Ice
If cryogenics and liquid
gases could be used
more widely in medical
and other applications,
the environment and the
lives of many people
The subject of this project is cryogenics, the science of producing and maintaining low
environments. The experiment was to see whether or not and how cryogenic freezing usi
nitrogen affects the growth of bacteria.
The bacteria is frozen for varying lengths of time using liquid nitrogen, then streaked onto an agar
plate to grow. The growth is measured every 24 hours for 3 days and the results are charted and
aphed. The incubation temperature and humidity must be controlled, the storage container must
not allow much heat transfer, and the bacteria must be pure and alive.
What was discovered through the study was that the unfrozen bacteria grew the best, b
samples that were frozen for a long time grew better than the frozen samples frozen for a short time.
The bacteria can survive being frozen at cryogenic temperatures and still remain viable.
The most profitable explanation for these results
is that when the bacteria is frozen many cells
die, but some form spores to resist the extreme temperatures. These cells multiply and replace the
dead cells, causing the bacteria to have more viable cells when it is inoculated onto the agar plates.
he bacteria is only frozen for a short time, the cells do not have as much time to reproduce
and there are less living cells that will grow on the agar plates.
It would be interesting to do a follow
up project to see if that statement is true. How wo
uld you go
about your experiment? How would you test this hypothesis? It would make a great experiment.
The subject of this project is cryogenics. Cryogenics is the study of producing and maintaining low
temperature environments. Cr
yogenics has become more and more important in the last few years
with applications in the medical field, the food industry, and in the transportation of gases. With more
knowledge, gases liquefied using cryogenic procedures could become common place as a
emissions fuel to replace fossil fuels.
This project focuses on whether or not living material can be cryogenically frozen, thawed, and
then regain all its normal functions. If this could be done, masses of cells like organs could be frozen
then thawed as needed.
If bacteria is frozen using cryogenic procedures, then thawed, will it retain all its normal
1. Will the material survive being frozen?
2. What cryogen must be used i
n the freezing process and what temperature
will have the best results?
3. What processes must be taken to ensure proper freezing?
4. Does the length of time the material is frozen affect the chances of survival?
amount of time a bacteria is frozen is inversely proportional to its growth rate and chances for
This researcher believes that material frozen for short periods of time will have a greater chance
for survival than those frozen for long per
iods of time because the longer it is frozen, the more
destructive events can take place. Also, the cells may not survive due to separation from their
The subject of this project is cryogenics. The general problem is
whether or not cryogenic freezing
affects the growth of bacteria on agar plates. The material will be frozen cryogenically for different
lengths of time to see which group has the best growth rate.
1. Temperature of Cryogen Used in
the Freezing Process
This is important to the freezing process because the colder the cryogen, the faster the
freezing. With a different rate of freezing, more or less destructive events within the cells
The cryogen used fo
r freezing in this experiment will be liquid nitrogen. It will be at 77K and
will be used in the same amount for each test.
2. Type of Bacteria to be Frozen
This could greatly affect the outcome of the experiment because if the samples are different,
growth rate will be different, and the shape/ color/ type of growth will be different. Also, if
the samples are contaminated the results will not be accurate.
All the bacteria will be the same age and come from the same source. The bac
teria to be
used will be Bacillus Cereus. They will all be pure samples bought from a science supply
3. Amount of Bacteria
This is very important because bacteria will grow faster on the agar plates if there is more
bacteria to multiply. Also, if
there are different amounts of bacteria frozen the results could
Exactly 1 ml of bacteria will be frozen in each vial. A sterile swab will be placed into the
bacteria after it is thawed and swabbed on the dish in a zig
4. Temperature the Bacteria Will Be Kept at Until It Is Frozen
This could greatly affect the outcome of the experiment because, if the samples are not kept
in optimum conditions until freezing, they could die.
All the bacteria
will be kept at a constant temperature. They will all be kept together in the
5. Container the Bacteria Will Be Stored In While Frozen
This is very important because, if the containers lose differing amounts of heat, the results
quite inaccurate. The bacteria could become thawed before they are intended to
and might be harmed.
All samples will be stored in an insulated thermos filled with liquid nitrogen. It will be
insulated with an ice chest filled with Styr
6. Temperature of the Area Where the Bacteria Will Be Stored
This is very important because if samples are placed in differing environments, some
containers could lose more heat than others.
All the containers will be st
ored at room temperature in a place exposed to no sun.
7. Temperature of the Area Surrounding the Agar Plates While Bacteria
This is very important because in different temperature environments the growth rate of
bacteria may be altered.
All the samples will be kept in a circulating air oven at a constant temperature of 95° F at the
same time. They will all be thawed simultaneously an swabbed onto the agar plates, then
placed in the same area, then removed and measured
at the same time the next day, and
every 24 hours for 3 days.
8. Humidity of Air Surrounding the Agar Plates
This is very important because in different humidity environments the growth rate of bacteria
may be altered.
All the samples
will be kept in a circulating air oven at 95° F. They will all be thawed
simultaneously and swabbed onto the agar plates, then placed in the same area, then
removed and measured at the same time the next day, and every 24 hours for 3 days.
Amount of Time the Material Is Frozen
This is important because, when frozen for longer or shorter periods of time, destructive
processes within the cells may differ.
There will be three groups, each frozen for a different am
ount of time. There will be four
samples in each of these groups. The first group will be frozen for one day. The second
group will be frozen for two days, and the third group will be frozen for four days. Also, there
will be a control group which is not f
rozen at all.
Bacterial Growth on Agar Plates After Freezing
This is an important factor because it shows whether the bacteria was harmed by the
freezing process or benefitted from it.
Method of Measurement:
The bacteria will be st
reaked onto an agar plate and covered. They will be left to sit for 24
hours and then the bacterial growth will be measured. The total growth in square units will
be recorded and charted.
The material to be frozen will be separated i
nto four vials.
Each group will be placed in liquid nitrogen. The group frozen for four days will be frozen
first, then two days later the next group will be frozen, then one day later the third group will
be frozen. This will allow all of the bacteria to
be grown on the agar at the same time,
keeping the temperature and humidity the same for each group.
When the time comes to inoculate the bacteria, the vials will be thawed.
An inoculating loop will be placed in the bacteria and then streaked on the aga
The bacteria will be allowed to grow for 24 hours.
Each sample will be examined and the growth will be measured.
The growth will be measured every 24 hours for three days.
The data will be recorded as described in statistical treatment.
The information will be recorded in charts and graphs. The total growth of each sample will
be recorded in square inches on a bar graph. The total growth (arithmetic mean) of each
group and the total growth of each group will also be recorded
on a bar graph. The day
day growth of each sample and the day
day growth of each group (the group growth will
be determined by an average) will be recorded on a bar graph.
Instruments and Materials:
Bacillus Cereus Culture
Agar Plates (Petri dishes and agar)
DISCUSSION OF RESULTS
Almost all bacteria samples grew on the agar plates. One sample had no growth at all, though. It
was in Group 1, the group frozen f
or one day and it only had one hundredth of an inch of bacterial
growth on it after three days. All the other samples grew well.
In most cases the growth between samples in the same group was uniform, but one group in
particular had varying results.
One plate had almost no growth, another had 1.6 square inches of
growth, and yet another had four
tenths of a square inch of growth at the end of three days.
In any case, the Control Group definitely grew the best on the first day. It had almost as m
growth as the other groups had on all three days combined. By the second day the growth had
tapered down to less than half the growth of the first day, and by the third day there was less than
fourth of the growth of the first day. All the samples
grew somewhat uniformly. Th agar in this
group started to dry up.
Group 3 came in third for total growth and it was frozen for four days. Group 3 had about one
third of the total growth of the Control Group. The growth was somewhat uniform although t
sample 3D didn't quite fit the pattern. On the first day there was some growth, but not very much.
The second day it grew really fast, but tapered down again on the third day. Some of this group's
agar also started to dry out.
The growth in Group
2 was very uniform and had a growth rate right between Group 3 and the
Control Group. It came in second for growth. Group 2 was frozen for 2 days.
JOURNALS AND LOGS
Bacillus Cerus arrived in 5ml tubes.
11:30 a.m.: Test tubes, pipett
es sterilized with rubbing alcohol and boiled 30
minutes in distilled water.
12:30 p.m.: Separated bacteria samples into four test tubes, three to be frozen,
one control (not frozen).
6:00 p.m.: Group 1 samples pla
ced in liquid nitrogen.
6:00 p.m.: Group 2 samples placed in liquid nitrogen.
6:00 p.m.: Group 3 samples placed in liquid nitrogen.
6:00 p.m.: All samples removed from liquid nitrogen and thawed. Agar
prepared and left to cool; then the plates are prepared.
9:00 p.m.: Agar is cool and plated. All samples inoculated on agar plates.
9:30 p.m.: All samples placed in a circulating air oven at 96°F.
9:30 p.m.: All samples are measured
If cryogenics and liquid gases
could be used more widely in medical and other
applications, the environment and the lives of many people worldwide co
uld be improved.
Cyrogenics is the science of producing and maintaining very low temperatures. It is useful in many
applications, including the transportation of gases, food preservation, metal fabrication, as well as in
medicine. Cryobiology, the use of l
ow temperature environments in the study of living plants and
animals, is an important area of cryogenics. Cryogenics could quite possibly be the future of
The study of cryogenics began in 1877 when Swiss physicist Rasul Pictet liquefied oxy
gen (at 90
Kelvins under one atmosphere of pressure) for the first time. A Kelvin (K) is a degree on the Kelvin
temperature scale that uses the same proportions as a degree on the Celsius scale, but starts from
absolute zero. Simultaneously, in France, Lou
is P. Cailletet also liquefied oxygen. Both obtained
liquid oxygen through Adiabatic processes. Adiabatic compression and expansion are
thermodynamic processes in which the pressure of a gas is increased or decreased without any
exchange of heat energy wit
h the surroundings.
Soon after oxygen, nitrogen was liquified. The liquefaction of nitrogen occurs at 77K under one
atmosphere of pressure. Then, in 1898, James Dewar liquefied hydrogen. With a boiling point of just
20K, liquid hydrogen presents a se
vere storage and handling problem. For this purpose, Dewar
invented a double
walled vacuum storage vessel for the storage of liquefied gases in 1898. It is now
known as the Dewar Flask. Theories of thermodynamics say that if cooled to near absolute
tures, matter can be stored indefinitely in a container which allows no heat transfer. The
flask prevents conduction and convection heat transfer, but is impervious to radiation heat transfer.
Helium is the most difficult gas to liquefy, and was not
successfully liquefied until 1908 by a Dutch
physicist named Heike Kamerlingh Onnes. The two isotopes of liquid helium boil at 4.2 and 3.2K.
Although there were no more gases to be liquefied,, further attempts have been made at chilling
helium to near abso
lute temperatures. It is a fundamental theorem of thermodynamics that absolute
zero can never be reached, but temperatures down to 40
millionths of a Kelvin above absolute zero
have been obtained.
There are many applications of cryogenics. There are
medical applications, such as
cryopreservation and cryosurgery, and other applications in electronics, the metal industry, food
preservation, the transportation of gases, and other miscellaneous uses.
In cryopreservation, cells are frozen very quickl
y with liquid nitrogen and then stored for long
periods of time in freezers that cool to extremely low temperatures. Cryopreservation is used by
hospitals and blood banks to store large amounts of blood and tissue for use during a major
catastrophe. It is
used for breeding animals by storing sperm for artificial insemination. The
microorganisms used in cheese production can be stored and transported without the loss of lactic
acid. One of the more modern developments is the ability to store cadaver body par
ts at cryogenic
temperatures, and attach them to patients with lost fingers or toes. Although they never regain their
full function, they can feel and move.
Cryogenic surgery is another application of cryogenics in medicine. It is used to deaden or
estroy tissue with high accuracy. Tiny measured drops of liquid nitrogen are shot on the skin in the
desired areas to kill the skin instantly.
Rockets can be much lighter using liquid gases instead of gas fuel because the gases have to be
great pressure. While 100 pounds of steel must be used to contain each pound of
hydrogen gas, only three pounds of steel must be used to contain one pound of that gas in its liquid
form. This is also applied to the transportation of gases in tankers or bar
ges. It is possible to
transport more gas and spend less money on containers by using cryogenics.
The processing, handling, and preservation of food by cryogenic means has developed into a
major industry. Cryogenic food preservation provides frozen f
oods as well as freeze
Liquid oxygen, nitrogen, hydrogen and argon are all commonly
used in the metals industry. In
order to meet the growing demand for liquid oxygen economically, on
site generators have been
developed to supply the gas
from plants constructed adjacent to the user. Gas is piped directly to the
user in quantities up to 500 tons per day. Liquid cryogens are used in several different metal
fabrication processes, such as the precipitation hardening of steel at 180K with liqu
id nitrogen, the
fabrication of vessels by cold stretching with high pressure nitrogen at 140K, and in the quenching of
complex metal parts.
To use the superconductivity effect and provide adequate cooling, special electronic devices are
cryogenic temperatures. Superconductivity is an attribute obtained by certain materials
when cooled with liquid helium to just a few Kelvins above absolute zero. Superconductive materials
have zero resistance for electricity flow, which allows these comput
er chips to operate extremely
In the process of cryopumping, the residual gases left in a vacuum vessel by conventional
pumping methods are frozen out on low temperature coils. It is possible to attain an extremely high
vacuum using cryopumping
and is particularly important in devices used to simulate the vacuum of
Other uses of liquid gases are the purification of gases, protecting hazardous materials with an
inert atmosphere, the production of ammonia and fertilizers, and ox
idation processes to make
chemicals like methanol and ethylene. Cryogenics can also be used in the formulation of drugs to
release antibodies or other agents from the cells.
Special freezing and storage equipment are used in cryogenics and they all u
se liquid gases to
cool the container. The freezing device is called a controlled
rate freezer. The material to be frozen
is placed inside a well
insulated chamber of the device and liquid nitrogen is admitted in another
section of the chamber and immediat
ely vaporized, then circulated by a fan. The controller reads the
temperature inside the chamber and adjusts the rate of liquid nitrogen flow to the rate desired.
Storage equipment ranges in size from 360 1cc ampules to 90,000 1cc ampules. The smalle
containers can last 60 days without a new charge of liquid nitrogen and employ working storage for
small laboratories. For stationary storage of large amounts of frozen material, like storage of blood at
a blood bank, large freezers are used. Brief expo
sure to outside temperature does not raise the
temperature of the frozen material more than one or two degrees.
For shipping, a special container is used. The refrigerant is absorbed in disks of porous material
that can easily conform to the objects
being stored. Because the disks hold the frozen material in
place, the container can be shipped in any position.
Cryogens are liquefied gases used to maintain low
temperature environments. They must be
isolated from mixtures such as the atmosphere,
natural gas, or a chemical by
product stream to
produce a pure gas. The desired components can be separated and purified by cryogenics. There
are a few procedures used to isolate the desired cryogen. They are fractional distillation, scrubbing,
ve adsorption. Depending on the mixture, one or more of these procedures may be used.
Oxygen and nitrogen are both obtained by the fractional distillation of air. Although some
hydrogen is isolated by the electrolysis of water, which is the more expe
nsive way, most hydrogen is
isolated through the catalytic reformation of hydrocarbons. Selective adsorption is particularly
applicable for the separation and purification of the noble gases. Argon, neon and krypton are
becoming increasingly useful in indu
stry and are obtained as a mixture from air
Helium is most economically recovered by a cryogenic condensation method from several
rich (more than .3%) natural gas sources. If these sources are exhausted, helium must be
ered from the atmosphere, where its concentration is only 0.0005% by volume. Helium
recovery is expensive and extremely energy intensive. It requires the energy equivalent of « barrel of
oil to extract one cubic foot of helium at standard temperature and p
ressure. Helium can also be
obtained as a by
product of air
separation plants at lower cost and energy consumption, but the air
liquefaction industry is too small to supply more than a few percent of present requirements.
Some current uses of helium
can be met by other gases
argon in heliarc welding, for example.
Helium, however, is unique in low
temperature applications that involve superconductivity. The
discovery of high
temperature conductors offers some hope that the higher boiling cryogens ca
applied to superconductivity.
scale hydrogen liquefaction plants are in operation throughout the United States and many
helium liquefiers provide wide
distributed sources of liquid helium. Thermal insulation reduces
losses to almost negli
gible proportions so that liquid hydrogen and helium can be transported over
long distances with very small losses. The shipping containers for hydrogen and helium vary in size
up to 10,000 gallons.
expanded traffic in cryogenic liquids h
as increased the importance of accurate
metering of quantities delivered or used. Metering is complicated by losses arising from various
sources. There have been many attempts to develop mass flow meters, but the transfer of cryogens
is still usually evalu
ated by liquid
level or volume
flow measurements. Both of the common metering
methods require additional measurements of pressure and temperature to determine liquid density
Converting a stored liquid to a has produced a need for adequate p
umps and vaporizers.
Developing cryogenic pumps was a difficult job, posing many low
temperature bearing, seal, and
The cost of energy needed to refrigerate and liquefy gases is the greatest concern to the
cryogenics industry. A
ctual cryogenic gas expansion refrigerators are much less efficient than the
theoretical limit. Very small refrigerators operate at 1% or less of the theoretical limit, and the largest
refrigerators operate with efficiency as high as 35%. Computer analysis
suggests that existing
refrigeration techniques can only attain 40% of Carnot's limit.
In a slow freezing process, cells collapse due to loss of water. Dehydration is very common.
When ice crystals form, they expand and can destroy cell walls. All t
he other material inside the cell
concentrates and can alter the acid
base ratio and cause salt concentrations to reach extremely high
levels. By speeding the freezing process up to the point where temperature drop is measured in
degrees per second, some o
f these destructive events can be averted. Ice crystals will form within
the cells so that less water will be lost, and the size of the crystals will be reduced tending to
decrease the destruction of cell walls. However, most of the destructive processes w
ill still take
To prevent dehydration, steps must be taken to prevent the freezing of water in the form of pure
ice. All of the cell fluids must solidify together. This will prevent concentration of the minerals that can
destroy cell walls and
prevent ice crystals from forming and rupturing cell walls.
To make all the cell walls solidify together, agents that lower the freezing point of water are used.
These include alcohols and glycols. Glycerol, a poly alcohol compatible with other bioc
materials in living cells, is frequently used in cell preservation. Besides the antifreeze additive,
refrigeration procedures are designed to control the rate of decline in temperature to the freezing
point, through the liquid
solid transition, and
below. As a result of these procedures, all the materials
in the cell and the surrounding fluid solidify together in a state resembling glass. In this form, cells
can be preserved for an indefinite time and will regain all normal functions when properly t
With preservation of a large mass of cells such as a living organ, the problems are significantly
increased. Control of the cooling rate is must more difficult, and additives may not reach cells in
sufficient amounts to depress the freezing po
int accurately. In addition, the organ may suffer from
oxygen deprivation or from interruption of its normal environment quite apart from the freezing
In the future, cryogenic engineering could be applied in several modes of transport
Superconducting magnets produced by cryogenic means are already being used, notably in Japan,
levitated trains with speeds of up to 300 mph.
The use of liquid hydrogen fuel appears promising for both jet aircraft and land tran
especially automotive. Hydrogen fuel offers the advantages of cleanliness and high efficiency,
producing no solid or carbon monoxide emissions. At 13.9K, solid hydrogen can exist together with
liquid hydrogen. This mixture, call slush hydrogen,
can flow like a liquid. Because the density of solid
hydrogen is 12% higher than liquid hydrogen, slush hydrogen can be useful as a fuel in application s
requiring smaller or lighter storage tanks. Since energy must be expended to produce hydrogen,
savings must be found before it can achieve the same value as fossil fuels. Also, procedures
for its safe handling must be established before hydrogen can become a common fuel.
Superconductivity and associated cryogenic systems can be applied to elec
tric power generation
and transmission. Superconducting magnets are being developed to provide the magnetic fields
needed for the generation of power by magneto hydrodynamics, and for energy storage in electric
power transmission systems because energy can
be stored in large magnetic fields.
Superconducting magnets are also needed for the magnetic confinement of plasmas in
thermonuclear power generation.
Some other prospective uses for cryogenics are producing fresh water from salt water and
large masses of living cells, notably organs or even bodies. The benefits are endless.
Organs from donors could be stored until needed and bodies could be frozen to prevent the spread
of disease. After a cure is developed, the frozen people could be revive
d and cured of their illnesses.
Cryogenics has endless uses and is of great importance to the world around us. Still, there are
tremendous amounts of applications for cryogenics still left unexplored. Looking into the future,
cryogenics will come to
be more widespread and will be applied in many new areas. For now
though, we have to explore medical uses, especially the preservation of large masses of cells. Is
there any living thing that can survive being cryogenically frozen? What procedures must be
What materials must be used? Hopefully these and other questions will be answered in the near
future. Cryogenics could lead us through the 21
Century, and beyond.
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2. Edeskuty, F. J., Williamson, Jr. K. D., Keller, W. E. (1993)
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pp. 555~558. New York: McGraw
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New York: McGraw
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The Software Toolworks
, Danbury, CT: Grolier Electronic Publishing, Inc.
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Cambridge: Compton's New Media, Inc.
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Freezing Point: Cold as
a Matter of Life and Death,
New York: The John Day Company