EMS freeze substitution kits

Achieve excellent freeze substitution results in as little as 90 minutes with this unique kit for cells of small volume such as bacteria and tissue culture cells. Freeze Substitution is a process for low temperature dehydration and fixation of rapidly frozen cells that usually takes days to complete. For those cells of greater volume or that have significant diffusion barriers such as cuticles or thick cell walls, one can extend the time to 3 hours by simply putting a lid on the box. Kit is available with or without shaker.Kit componentsPlatform shaker, 115VHeater block for 12mm tubes (13mm block is also available)Ice chest  CryotubesTemperature probeDataloggerComponents can also be ordered separately.ProductDescriptionY3450

Achieve excellent freeze substitution results in as little as 90 minutes with this unique kit for cells of small volume such as bacteria and tissue culture cells. Freeze Substitution is a process for low temperature dehydration and fixation of rapidly frozen cells that usually takes days to complete. 

For those cells of greater volume or that have significant diffusion barriers such as cuticles or thick cell walls, one can extend the time to 3 hours by simply putting a lid on the box. Kit is available with or without shaker.

Kit components

  • Platform shaker, 115V
  • Heater block for 12mm tubes (13mm block is also available)
  • Ice chest  
  • Cryotubes
  • Temperature probe
  • Datalogger

Components can also be ordered separately.

Product Description
Y3450
EMS Freeze Substitution Kit with Shaker
EMS34500-S EMS Freeze Substitution Kit without Shaker
EMS34502 EMS 111 Platform Shaker, 115 Volt
EMS62013-10* Ice Chest
EMS34504-B Heater Block, 12mm
EMS34504-R Heater Block, 13mm
EMS34505 Temperature Probe
EMS34506 Cryo Tubes
EMS34507 Data Logger

*EMS62013-10 Ice Chest found on separate page.

Why does it work?

Freeze substitution is a process for low temperature dehydration and fixation of rapidly frozen cells that usually takes days to complete. K.L. McDonald and R.I. Webb introduced a simple and effective method for freeze substitution.  

  • In well-frozen samples water molecules do not move around very much, even as the temperatures rise to a point where  hexagonal ice formation is expected.
  • Agitation  speeds up the substitution of acetone for water molecules .
  • Water in the substitution mixture does not appear to slow down substitution, but helps improve membrane contrast

Does it work for all samples?

  • If a sample can be successfully freeze substituted by the old methods, then the quick FS method should work just as well.
  • McDonald and Webb have used this procedure with complete success for over a year and a half for all the samples they have freeze substituted.
  • If samples show evidence of ice damage then it is because they were damaged during freezing and not during freeze substitution.
Warming Curve With and Without Use of Lid

warming curve

Typical temperature curves using the EMS Freeze Substitution Kit. With the lid OFF the time to 0°C is about 2 hours. With the lid ON the time is about 3 hours. Results may vary depending on the particular setting of the shaker. For example, hood air flow can have a definite influence on the shape of the curves.

PDF Brochure

Safety Reminders

  • The equipment should be used in a fume hood in case there is a leak of osmium-acetone during a run. We suggest doing a trial run with acetone only in the cryovials to make sure that they are sealed correctly.
  • When sealing cryotubes that contain frozen fixative and sample, use a warm cap so that the O-ring is flexible and gives a good seal.
  • Take care when removing the caps after a FS run because there is some pressure built up inside the cryotubes and you can spray osmium/acetone on your hands if you are not careful. Cover the cap with a piece of lab tissue when removing and wear gloves.

C. elegans 

Cross-section through nerve cells in the head of C.elegans.

 

A BHK cell processed in 90 minutes by the quick FS method. Abbreviations: CCP = clathrin-coated pit, Cav = caveolae, EE = early endosome, IF = intermediate filaments, LE = late endosome, M = mitochondrion, MT = microtubule. Bar = 1 µm. Modified from McDonald and Webb (2011).

Acknowledgements:

  1. K.L. McDonald* and R.I. Webb**, *Electron Microsocpe Laboratory, University of California , Berkley, CA and ** Centre for Microscopy an Microanalysis, University of Queensland, Queensland, Australia
Code Title Type Pack Size Availability Price Updated: 23-04-2024
EMS34500-S EMS freeze substitution kits Kit without shaker Each See above Quote only Quote
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EMS34502 EMS freeze substitution kits Shaker, 115V Each See above Quote only Quote
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EMS34504-B EMS freeze substitution kits Heater block 12mm Each See above Quote only Quote
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EMS34504-R EMS freeze substitution kits Heater block 13mm Each See above Quote only Quote
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EMS34505 EMS freeze substitution kits Temperature probe Each See above Quote only Quote
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EMS34506 EMS freeze substitution kits Cryo tubes Pack/100 See above Quote only Quote
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EMS34507 EMS freeze substitution kits Data logger Each See above Quote only Quote
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Y3450 EMS freeze substitution kits Kit with shaker Each See above Quote only Quote
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