X-Message-Number: 2184
Newsgroups: sci.cryonics
From:  (Christopher Michael Rasch)
Subject: C. elegans freezing protocol. Please comment
Message-ID: <>
Date: Fri, 30 Apr 93 18:50:41 GMT

Hi--

	Appended is the protocol I submitted for my Bio44Y independent 
project.  Any suggestions for improvement would be greatly appreciated.
Thanks to Nick Szabo for pointing me to the Drosophila embryo research, and
to Mike Darwin, for his advice on experimental design.  
 
Effects of cooling rate on the survival of  L4 and adult stage C. 
elegans after freezing/vitrification.

	A number of organisms can survive temperatures below 
-35 degree C, with up 65% of their total body water as ice.  
Among these animals include the woolly bear caterpillars 
(Gynaephora groenlandica), which may spend 10 months of the 
year frozen solid at temperatures below -50 degrees C, and the 
striped chorus frog (Pseudacris triseriata).  These animals 
manufacture cryoprotectants (in the case of frogs, glucose) 
which minimizes freezing damage. (Storey, 1990) 
	  This experiment is designed to see how well the 
nematode C. elegans can survive freezing/vitrification down to 
liquid nitrogen temperatures.  We will compare slow cooling 
with flash freezing in liquid nitrogn. Since Riga and Webster 
report that juvenile nematodes survive better than adult 
nematodes we will also  compare survival rates of juvenile and 
adult nematodes. (Riga, Webster 1991). 
	C.elegans, is a simple 1 mm long worm.  It has been 
extensively studied--all 302 neurons have been mapped at the 
electron microscope level. The complete cell lineages of the 
1000 somatic cells is known, as is a nearly complete genetic 
map. (Rankin, 1990, pg. 89)  C. elegans is capable of non-
associative learning; it becomes habituated to successive 
patterns of taps on its dish. (Mah and Rankin, 1992).  The 
nematode feeds on E. coli, and can be raised in a agar medium.  
Thus C. elegans is a good potential model for detailed study of 
freezing on living tissue.
	The survival of frozen cells critical depends upon the 
(1) avoidance of intracellular ice and (ii) the presence of 
intracellular molar concentrations of protective solutes. 
(Mazur, 1992)
	 Cold tolerant organisms manufacture compounds 
(often glucose) which minimize freezing damage. As cells 
cool, intracellular water flows across the cell membrane 
because of osmotic and vapor pressure gradients.  This results 
in cell shrinkage.  Glycerol, the cryoprotectant we'll be using 
in this experiment, helps minimize shrinking by reducing the 
mole fraction of other solutes remaining in the nonfrozen 
water.  Glycerol inhibits the formation of crystalline ice, and 
thus depresses the freezing point of the water.  It may also 
prevent protein denaturation by hydrogen binding with bound 
water.  Other cryoprotectants (DMSO4, ethylen glycol, etc) 
work in a similar manner.  
	As cells cool, solvent water converts to extracellular 
ice, and the increasing extracellular concentration of 
nonpermeating electrolyte or nonelectrolytes damages the cell.   
When treated with a cryoprotectant,  cells don't reach the salt 
concentrations of nontreated cells until they reach much lower 
temperatures.  Chemical reactions proceed very slowly at such 
low temperatures and consequently cellular damage is 
minimized.
(Karow,  1981)

Materials and Methods


Thermometer, range (-200 degrees C/30 degrees C)
20 glass slides w/cover slips
filter paper
50 Pasteur pipettes w/bulbs
2 M glycerol solution
2 quarts LN2 (liquid nitrogen)
20 10 mL test tubes
LN2 containers (2  1 quart thermos bottles and/or Dewar flasks)
microwave
liquid Agar medium
E. coli bacteria w/media
10 100 CC syringes
 dissecting microscope
freezer (space) (-70 degrees C)
test tube tongs
gloves

	C. Elegans hatch approximately 14 hours after 
fertilization.  C.Elegans larvae subsequently develop through 
four stages, L1, L2, L3, and L4.  We will grow a colony of 
nematodes in the liquid Agar medium; the nematodes feed 
upon E. coli bacteria.  Mazur reports that Drosophila embryos 
survive 2.1 M concentrations of cryoprotectants at room 
temperature.(Mazur, 1992).  Consequently, we will add the 
glycerol at 22 degrees C. We will transport one group of 20 L4 
nematodes to a 10 ml test tube containing 2 M glycerol; a 
second group of 20 adult nematodes will be transported to 
another test tube containing an identical solution.   
	The nematodes will bathe in the solution for 20 
minutes, to ensure that glycerol sufficiently perfuses.  After 
perfusion, the test tube will be dipped in LN2 (-196 degrees C) 
for 10 minutes or until vitrification/freezing is apparent.  
	A second group of nematodes prepared as above, will 
be placed in the freezer and cooled by ~1 degrees C/minute 
until they reach -70 degrees C.  At that point they will be 
transferred to a container of liquid nitrogen for another 10 
minutes.  
	Both groups will be warmed in a microwave until they 
reach 22 degrees  C.  At that point, the nematodes will be 
transferred to a solution containing no glycerol.  We will count 
the number of nematodes before freezing, and the number of 
surviving nematodes after freezing.  A nematode will count as 
alive if it is still capable of moving.  We will compare the 
number of surviving L4 nematodes with the number of 
surviving adults.
	The freezer, syringes, pipettes, filter paper, and glass 
slides will be used to perform preliminary  tests for freezing 
nematodes.      
	 
Expected Results 

	We expect that more L4 nematodes will survive than 
adults because in Riga's experiment   "...A significant number 
of juveniles than adults survived deep freezing." (Riga, 
Webster, 1991).  We also expect that slow freezing will survive 
better because intracellular water will have to time osmotically 
travel across the membrane, and will not become frozen as 
intracellular ice.  However, if C. Elegans prove to be chill-
sensitive, as Drosophila are, flash freezing may prove more 
beneficial.  However, we will be using  a lower molar 
concentration of cryoprotectant than Mazur used with the 
Drosophila embryos. (Mazur, 1992).

Karow, A M, Pegg, D E, Organ Preservation for 
Transplantation, 2nd ed, 1981, Marcel Dekker, Inc. pg 161.

Mah, K B, Rankin C H, "Analysis of behavioral plasticity in 
male Caenorhabditis elegans." Behavioral and Neural Biology 
58 211-221, 1991.

Mazur, P, Cole K W," Cryobiological Preservation of 
Drosophila Embryos", Science,  258:1932-1935, 1992.

Pickup, J, "Seasonal variation in the cold hardiness of three 
species of free-living Antarctic nematodes" Functional 
Ecology, 1990 4(2):257-264)

Popiel, I; Vasquez, E M, "Cryopreservation of Steinernema 
carpocapsae and Heterorhabiditis bacteriophora", Journal of 
Nematology (abstract). 1991 23(4):432-437

Riga, E, Webster, J M "Cryopreservation of the pinewood 
nematode, Bursaphelenchus", Journal of Nematology 
(abstract), 1991 23(4):438-440.

Rankin, C H, Beck, C B, and Chiba C M, "Caenorhabditis 
elegans: a new model system for the study of learning and 
memory" Behavioral Brain Research, 37 (1990) 89-92

Storey, K B, Storey J M "Frozen and Alive", Scientific 
American, December 1990, pg. 92-97.

Wood, W B ed., _The Nematode Caenorhabditis Elegans_, 
1988, Cold Spring Harbor Laboratory.

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