PREPARATION OF AMPHIBIAN PRIMARY CULTURES FROM
SOLID TISSUE (EXPLANT METHOD)
Contributed by San Diego Zoo's Institute for Conservation Research, Genetics Division
Materials and Reagents Needed
• sterile glass plate or petri dish
• sterile forceps and scalpel
 70% alcohol
• T25 culture flasks
• T75 culture flasks
• sterile pipets (5- and 10cc), pipettors or pipet bulbs
• growth medium (with 10% FBS, 1% glutamine, 1% pen/strep/fungizone)
• Hank's balanced salt solution (HBSS)
• trypsin-EDTA solution
• inverted microscope
• 6% CO2 incubator at 27° C
Protocol (Aseptic technique must be strictly followed)
1. On a sterile glass plate (or petri dish) mince tissue biopsy into tiny fragments with
sterile forceps and scalpel using clear cuts. Do not shred or tear the tissue.
Do not let fragments dry out; keep moistened with media.
2. Carefully place pieces of tissue onto the bottom of a T25 culture flask. Space the
fragments so that there is sufficient room for outgrowth.
3. Leave the flask at room temperature until the fragments have adhered but do not
let the pieces dry out!
4. Add 5 cc complete medium per flask. Incubate in a 6% CO2 incubator.
5. After 24 hours check cultures. If there are many fragments floating in the
medium, replant these pieces into another flask.
6. Check and gently feed the cultures twice a week.
7. When outgrowth is sufficient the cells must be "passed" to a larger flask
(secondary culture). Remove the medium. Rinse with 2 cc Hank's balanced salt
solution (HBSS). Remove HBSS. Add 1 cc Trypsin-EDTA or TryplE and swirl
around flask to coat. Place flask on warming tray set at 37° C until cells have
detached.
8. Add 10 cc growth medium to the flask and swirl around to get all the cells into the
medium. Transfer to a T75 culture flask. Refeed the 1° cultures. Incubate both
1° and 2° cultures. Secondary cultures should be checked every other day and
fed accordingly.
9. Cultures may be passed indefinitely, frozen when sufficient numbers of 2° flasks
have reached confluency, or harvested when active growth is most evident.