All manipulations of oocytes and em

All manipulations of oocytes and embryos should be performed using a
pulled Pasteur pipette, glass capillary or a displacement pipette. It is impor-tant to use a pipette with the appropriate size tip. For example, once the
cumulus is removed (day 1 to 3), a bore of around 175–200mM is required.
Using the appropriate size tip minimizes the volumes of culture medium
moved with each embryo, which typically should be less than a microliter.
Such volume manipulation is a pre-requisite for successful culture.
Around 4PMon the day of oocyte retrieval, label 60mm Falcon Pri-maria dishes with the patient’s name. Using a single-wrapped tip, first rinse
the tip, then place 625ml drops of G1 into the plate. Four drops should be
at the 3, 6, 9, and 12 o
0
clock positions (for embryo culture), the fifth and
sixth drops should be in the middle of the dish (wash drops). Immediately
cover drops with 9mL of tested oil (such as Ovoil, Vitrolife). Prepare no
more than 2 plates at one time. Using a new tip for each drop, first rinse the
tip and then add a further 25ml of medium to each original drop. Place
the dish in the incubator at 5% O2 and 6% CO2. Gently remove the lid
of the dish and set at an angle on the side of the plate. Dishes must gas in
the incubator for a minimum of four hours (this is the minimal measured
time for the media to reach correct pH under oil). For each patient, set up
a wash dish at the same time as the culture dishes. Place 1mL of medium G1
into the center of an organ well dish, place 2mL of medium into the outer
well, and then place the dish in the incubator. If working outside an isolette,
use a MOPS or HEPES buffered medium with amino acids. This should not
be placed in a CO2incubator, but rather warmed on a heated stage.
Following removal of the cumulus cells, embryos are transferred to the
organ well dish and washed in the center well drop of medium in the culture
dish. Washing involves picking up the embryo 2–3 times and moving it
around within the well. Embryos should then be washed in the two center
drops in the culture dish and up to 5 embryos placed in each drop of G1.
This will result in no more than 20 embryos per dish. Return the dish to
the incubator immediately. It is advisable to culture embryos in groups of
at least 2. Therefore for example, for a patient with 6 embryos, it is best
to culture in 2 groups of 3 and not 4 and 2 or 5 and 1. On day 3, embryos
can be transferred to the uterus in a hyaluronan enriched medium (221).
Day 3 Embryos to the Blastocyst Stage

Results (English) 3:

使用一个的卵母细胞和胚胎的所有操作把Pasteur pipette、玻璃毛细管或位移吸管。更重要的是要用吸管与适当大小的尖端。例如，一旦积云被删除（1天3），一个口径约175–200mm是必需的。使用适当的大小的提示，最大限度地减少了培养基的体积感动与每一个胚胎，通常应小于微升。这样的音量控制是成功的文化的先决条件。在4pmon取卵日，标签60mm猎鹰PRI玛丽亚菜与病人的名字。使用一个单一的包裹小费，第一次冲洗尖，然后将625ml滴G1到板。四滴应该是在3，6，9和12零时钟位置（胚胎培养），第五和第六滴应该在盘子的中间（洗滴）。立即盖9ml试验油滴（如ovoil，Vitrolife）。准备好没有一次有2多个板。使用一个新的提示，每一滴，首先冲洗提示然后添加介质进一步25ml每个原始滴。的地方在孵化器中的5%个氧气和6%的二氧化碳。轻轻地去除盖子盘子的，并在板的一侧的一个角度。菜必须气孵化器至少四小时（这是最小的测量值媒体的时间，以达到正确的pH值下油。对于每一个病人，设置一道洗菜的同时作为培养皿。中G1的地方1ml在一个器官好碟中心，中到外的地方2好吧，然后把盘子放在孵化器里。如果工作以外的一个小岛，用拖把或HEPES缓冲与氨基酸中。这不应该被放置在一个二氧化碳培养箱，而是温暖在加热阶段。继去除的卵母细胞，胚胎被转移到机关菜洗净在中间井培养基中的培养菜。洗涤涉及到拿起胚胎2，3次，并移动它在井内。然后，胚胎应在两个中心洗涤在培养皿中滴和多达5个胚胎放置在每一滴G1。这将导致在每盘不超过20个胚胎。将盘子归给立即孵化器。在群体中培养胚胎是明智的至少2。因此，举例来说，对于一个有6个胚胎的病人来说，这是最好的以文化在2组的3，而不是4和2或5和1。3天，胚胎可以被转移到一个透明质酸子宫富集培养基（221）。第3天胚胎到囊胚阶段