HELPFUL HINTS FOR
SUCCESSFUL IVP
Before starting or doing any part of the
procedure, here are a few things that can greatly improve the chances of
getting good results. Also, be sure to read the Guide on Use of Instruments for
Picking Up Oocytes and Embryos
Speed is Important
Results will improve as you become faster at
performing each step.
Water is Important
The choice of water depends upon local
availability of highly-purified water. We make up stock solutions using
Tissue Culture Water purchased from Sigma.
For all other media (OCM, saline, etc.), we use milliQ water. For
dishwashing, the final rinse is also with milliQ water.
Oil is Important
Often when IVP fails, the oil is the
culprit. Water-soluble contaminants in oil can kill embryos. Oil
can be cleaned by incubating it with water or medium in some sort of shaker or
mixer. We have had good luck using Sigma's
Embryo-Tested Mineral Oil.
Keep Incubators Set Properly
Check incubators regularly for accuracy of
temperature and CO2 readings and to ensure the air is humidified (i.e.
reservoir of water inside the incubator is full). Water-jacketed
incubators respond to changes in setpoint slowly so make adjustments well
before the incubator will be used.
Use Supplies for IVP Only
Set aside glassware, plasticware, and
instruments that will only be used for IVP to prevent any residue from another
application to contaminate the media and affect the oocytes/embryos. This
could be done easily by having a set of glassware that are different than the
ones regularly used in the laboratory.
Keep the Work Area Warm to Prevent Cold Shock
1. Air-conditioning vents should be covered
during IVP procedures. If the room is devoted for IVP only,
air-conditioning vents can be permanently sealed.
2. A space heater may be used to warm up the air
near the work site as well as microscope stage and bench surfaces.
Placement of a dish on a cold lab bench or microscope stage could result in a
rapid cold shock of embryos. Don’t get too close to the space heater though or you can cook
your embryos.
3. Before looking at embryos using a microscope,
make sure stage has been pre-warmed (by turning on the space heater).
Metal is an excellent conductor of cold from one material to another.
4. When washing oocytes, place beakers on
plastic mesh or Styrofoam and not on a cold surface (i.e., countertop).
5. Always place dishes (i.e. petri dishes,
X-plates) on a slide warmer set at 39°C.
Pre-Warm
Media at 38.5°C
a) Media designed for use in air should be
pre-warmed for at least 2 to 3 h in an oven, incubator or water bath set at
38.5°C. Make sure lid is on tight. Media to be pre-warmed in this
manner includes Saline, Hepes-TALP, Sp-TALP and Oocyte Collection Medium.
b) Incubator – Media designed for use in a
5% CO2environment should be pre-warmed to 38.5°C in an
incubator. In order for the pH of the medium to be equilibrated, it is
important to loosen the lid of any bottle/tube of medium placed in the
incubator. Leaving a bottle of medium buffered for a specific CO2
environment outside the incubator too long will result in a dramatic change of
pH which could severely affect embryonic viability and development. Media
that must be pre-warmed in an incubator include
Sterility and Cleanliness
1. All glassware,
plasticware and media used should be sterile.
2. Use sterile techniques when handling media.
3. When cleaning benchtops, use a commercial cleaner (we use Windex without
ammonia) to remove blood and other material from the surface and always finish
cleaning by swiping all surfaces with a rag or kimwipe soaked with 70% ethanol
(remember: ethanol is toxic to embryos
so be careful where you splash it.
4. In our experience, the citothaw used to thaw semen can be a source of many nasty microorganisms. Always rinse and dry the citothaw after each use. Periodically, it is a good idea to disinfect the inside of the citothaw with bleach.
Other Tips
1. Pipet tips
may contain toxins or other substances that might inhibit development of
embryos. As a precaution, always fill and empty pipet tip at least once before
using, especially before adding new medium or serum to a microdrop containing
embryos.
2.
When transferring oocytes or embryos from one medium to another, transfer the
oocytes/embryos in as little medium as possible.
3. Due to repeated openings of the incubator door, temperature at the front of the incubator fluctuates. Thus, place dishes at the back of the incubator to reduce exposure to changes in temperature.
this page was last
updated 05-10-05
all original material for
this website © Rocio Rivera, Peter J. Hansen et al. 2000-2002
Links to commercial sites do not constitute endorsement by the authors or the University of Florida
