PREPARATION OF MEDIA

Transport saline  |  Oocyte Collection Medium  | Oocyte Maturation Medium
TL solutions  |  TALP media    |  10X SP-TL  | 90% Percoll   |  KSOM  |  CR1aa  |  SOF

Transport Saline (0.9%)
Prepare 0.9% saline  (9 g NaCl per liter) in double distilled water and autoclave.  Label as "Sterile 0.9% saline, date made" and store indefinitely at 4°C.

Oocyte Collection Medium (OCM)
1. Dissolve TCM-199 powder (without phenol red, and without glutamine) (Hyclone) for 10 L and 3.50 g NaHCO₃in 9 L ddH₂O. Adjust pH to 7.2-7.4 and bring volume to 10 L.  Sterile-filter 400 ml medium into 500 ml bottles and keep indefinitely at 4°C.  Labels should read "Oocyte Collection Medium, - Supplements and date made".

2. On night before use, add the following: 1 aliquot of stock 4: BSS+Hep, 1 aliquot of stock 11: glutamine (4 ml), and 1 aliquot of stock 15: Pen/Strep (4 ml).  Change label to "+supplements", change date and use within a week.

Oocyte Maturation Medium
1.  Prepare 87 ml aliquots of TCM-199. and store at 4°C until use.

2.  On night before use, add the following to an aliquot of TCM-199:
    1 aliquot stock 3: BSS
    1 aliquot stock 8: gentamicin
    125 ml stock 6: Folltropin
    200 ml stock 5: estradiol
    1 ml stock 2: Na Pyruvate
    1 ml stock 10: Glutamine

Change label to read "Oocyte maturation medium" and use within 1 week.

TL Solutions - For Making TALPs
Note that these media can also be purchased from Caisson,  Millipore or Lonza (formerly Biowhittaker).

1.  To prepare media, mix the ingredients as described in Table 1 (all volumes are in milliliters), adjust the pH, check osmolarity (if osmometer is available) and sterile-filter the solution.

2. Write expiration date on the label (use within one week) and store at 4°C.

Table 1.  Recipes for  preparation of TL solutions

TALP (Tyrode’s Albumin Lactate Pyruvate) Media

Recipe 1: using media from Caisson.  Click for the formulation.

1.  To prepare media, mix the ingredients as described in Table 2 and sterile-filter the solution.

2. Write expiration date on the label (use within one week) and store at 4°C.

Table 2. Recipes for TALP media using Caisson media.

Recipe 2:  using Millipore (Specialty Media) media

1.  To prepare media, mix the ingredients as described in Table 3 and sterile-filter the solution.

2. Write expiration date on the label (use within one week) and store at 4°C.

Table 3.  Recipes for TALP Media using Specialty Media media.

* BSA, EFAF=essentially fatty-acid free Fraction V
** Save the 12 ml remaining after preparation of medium since it can be used for Percoll preparation.

*** The optimal heparin concentration will vary between bulls - if one bull is used frequently, test various concentrations of heparin in a preliminary experiment.

10X SP-TL
1.  Prepare 10x SP-TL stock solution by dissolving the following in 100 ml water:
         NaCl -  4.675 g
         KCl  -  0.23 g
         NaH₂PO₄+H₂O -  0.40 g
         HEPES   2.38 g

2.  Adjust pH to ~7.3, sterile filter and store indefinitely at 4°C.

90 % Percoll
1.  Place 4 ml of 10X SP-TL in a small beaker and add 0.084 g sodium bicarbonate and 90 ml Na lactate (Stock 1).
2. Stir until bicarbonate dissolves.
3. Add 36 ml Percoll.
4. Add 158 ml MgCl₂ (Stock 12) and 78 ml CaCl₂ (Stock 13).
5. While stirring, adjust pH to 7.3-7.45 and filter with a 0.45 mm filter (50 ml filter tube or similar bottle-top filter).  If a precipitate forms in the Percoll solution, continue to stir.  If compounds do not re-dissolve, then start over.
It is very easy to get precipitation if acid or base is added too rapidly during the adjustment of pH.  Therefore, it is recommended that this step be done slowly.

 KSOM-BE (Potassium Simplex Optimized Medium – Bovine Embryo Modification 2)  

Recipe 1 – using KSOM+AA from Caisson (without pen/strep). Click for the formulation.

1.  Purchase KSOM from Caisson Laboratories and store frozen.  Once thawed, keep at 4°C for 2 weeks.

2.  Add 1 ml pen/strep to a new bottle of thawed KSOM.

3.  To 5 ml of KSOM stock add:

        EFAF BSA  - 15 mg  (3.00 mg/ml)

        Gentamicin stock 8A  - 2.5 ml (0.5 ml/ml)

        Nonessential amino acids, 100X -  25 ml

4. Sterile filter medium through a 0.22 mm syringe filter into a sterile 10 ml beaker.  Use immediately.

Recipe 2 – using KSOM from Millipore (contains pen/strep)

1.  Purchase KSOM MR-106-D from and store frozen.  Use with caution after the expiration date provided by the manufacturer.  Once thawed, keep at 4°C for 2 weeks.

2.  To 5 ml of KSOM stock add:
        EFAF BSA  - 15 mg  (3.00 mg/ml)
        Gentamicin stock 8A  - 2.5 ml (0.5 ml/ml)
        Nonessential amino acids, 100X -  25 ml

Note:  This formulation is different than was previously described on this Webpage. Cell and Molecular Technologies has developed a new KSOM formulation that contains essential amino acids and half the required concentration of nonessential amino acids.  

3.  Sterile filter medium through a 0.22 mm syringe filter into a sterile 10 ml beaker.  Use immediately.

CR1aa (an alternative culture medium)
Note: the patent for this medium is held by Infigen

1.  Make CR1 stock (prepare in a 100 ml volumetric flask):

    NaCl                     0.670 g
    KCl                       0.023 g
    NaHCO3              0.220 g
    Na Pyruvate        0.004 g
    Glutamine            0.015 g
    Hemi-Ca Lactate 0.055 g

Add first 5 ingredients to volumetric flask.  Add water (~90 ml).  Thoroughly dissolve constituents and then add Hemi-Ca Lactate.  Add remaining water.  Store for up to 2 days at 4°C.
Note: constituents of this medium are known to precipitate out of solution.  To minimize the chances of this occurring, make sure all constitutents are dissolved before adding hemi-Ca lactate and  use immediately after making.  If a medium appears white and cloudy, discard and start again.

2.  To prepare CR1aa, add the following to 5 ml of CR1 stock:

        EFAF BSA  - 15 mg  (3.00 mg/ml)
        Gentamicin stock 8A  - 2.5 ml (0.5 ml/ml)
        Nonessential amino acids, 100X -  50 ml
        Essential amino acids, 50X - 100 ml

Sterile filter medium through a 0.22 mm syringe filter into a sterile 10 ml beaker.  Use immediately.

 SOF (Synthetic Oviduct Fluid)

This is based on the formulation of Fischer-Brown et al. Zygote 10:341-348 (2002) except that the concentration of gentamicin is altered and the BSA is essentially fatty acid free BSA.  We purchase the medium as a custom formulation from Millipore (BSS-046-D)and supplement the  medium with 0.4 mM sodium pyruvate, 20 μl/ml MEM non-essential amino acids and 10 μl/ml BME essential amino acids, and 0.025 mg/ml gentamicin sulfate (i.e., 10x more than used for KSOM-BE2-it is not clear that this is the optimal concentration). Essentially Fatty-acid-free BSA is also added to achieve a final concentration of 8 mg/ml BSA.

To make 200X Sodium Pyruvate:

0.88 g → 100 ml H2O→ Sterile Filter + Store at 4 ْC for 1 month.

To make 50 ml (modified) SOF+ add:

0.25 ml 200X Sodium Pyruvate

1.0 ml MEM amino acids

0.5 ml BME Amino Acids

0.25 ml gentamicin sulfate

400 mg EFAF-BSA

The recipe for SOF is as follows (all concentrations are in mM unless stated)

CaCl₂ 2H₂O                                1.17

KCL                                           7.16

KH2PO4                                     1.19

MgC_l2  6H₂O                               0.49                                            

NaCl                                          107.7

NaHCO₃                                     25.07

Na lactate (60% syrup)              3.3

Na pyruvate                               0.4

BSA (mg/l)                                8000

Gentamicin sulfate (U/l)            500 000

BME essential AA  (ml/l)             10

MEM non-essential AA (ml/l)                  20

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this page was last updated  01-21-08

all original material for this website © Rocio Rivera, Peter J. Hansen  et al. 2000-2008Links to commercial sites do not constitute endorsement by the authors or the University of Florida