One of the virtues of using avian embryos for experimentally analyzing early developmental events is the ease with which they can be cultured and subsequently manipulated and observed. Gastrula- and neurula-stage avian embryos can be cultured for 24–48 h on their vitelline membranes (acellular membranes enclosing the embryo and the yolk), which are stretched over a glass ring placed on an egg-agar substrate. Their development occurs similarly to that of embryos developingin ovo, yet unlikein ovo, embryos are fully accessible for experimentation. This method of cultivation on the vitelline membranes/egg-agar substrate, called New culture, was developed by Denis New in 1955 (1 ), and its usefulness has not been surpassed in the more than 40 yr since its inception (2 ). New culture provides an ideal system for experiments involving grafting of tissues from one embryo to another, microinjection of dyes or drugs, time-lapse video recording, and where superior development is required from cultured embryos during the first 2 d of incubation. Blastoderm expansion occurs essentially normally in New culture until the blastoderm comes in contact with the encircling ring. Optimal stages for starting New cultures are between the pregastrula and 7-somite stage (HH [refs.3 and 4 ] stages 1–9), with the optimum time of development in culture without degeneration of some tissues being approx 24 h or development to about 22 somites (HH stage 14), whichever comes first. Differentiation of some tissues will continue well beyond this time. Whole-egg culture plates should be used for pregastrula stage embryos (HH stages 1–2), and agar-albumen culture plates should be used for later stages (HH stages 3–9) to optimize development (see belowand Note 1 ).
