# Timed embryo

Stephen G. Hall, Ph.D. hallsg at genmedcorp.com
Mon Jul 7 21:39:00 EST 1997

```Wild-type embryos can be exposed to different temperatures for different
periods of time. Certain morphological features of the embryos enables one
to determine age (Roberts, 1989).  Based on staging with these criteria,
the embryos' ages can be plotted as a function of temperature.  A power
equation, y= 46906.635x^-2348, is obtained mathematically where y is the
developmental age and x is the temperature in C°.  The normal
developmental temperature for Drosophila in the literature is 25°C.  Based
on a normal developmental age at 25°C (DA)25, the data at 25°C is
normalized to 1.0.  The developmental age (DA) of Drosophila at 18°C
(DA)18 is then (0.45)(DA)25, and the developmental age at 29°C [(DA)29] is
(1.4)(DA)25.  In other words, an embryo normally is staged at 25°C and
develops roughly half as fast at 18°C and twice as fast at 29°C. However,
rearing flies at greater than 25°C isn't recommended.  This data can be
compared to data presented by Powsner (1935) and summarized in Ashburner
(1989) and correlates well with these previously published reports.

Just collect virgin females and allow them to lay on agar juice plates in
the dark.  Add a dab of yeast mixed with water added to the plate helps.
It is best to take an initial 1 hour collection of embryos which females
will lay in the dark. These embryos will be older since the females will
store them.  Throw the first batch out and replace with a fresh plate.

For example, do a 1 hour collection to purge all embryos the female is
storing and throw the plate out.  Add a new plate and do a 4 hour
collection at 25°C and replace the plate.  Those embryos are 0-4 hours
old.  If you were to collect for 1 hour at 18°C, they would be 0-2 hours
old.

The collection should be done in the dark and roughly on the same
light/dark cycle that the flies are used to.  You shouldn't use females
more than say 3 weeks old since they get old and tend not to lay well.
You will notice that a week or so after they eclose they will lay embryos
the best.

The embryos at 25°C that are 0-4 hours old can be put in an incubator say
for another 4 hours.  Since there is no female present to lay new eggs,
you can be sure that all the embryos are then 4-8 hours old since they
will have aged by 4 hours.  You can also put the plate at 18°C which slows
them down.

By varying the duration of your collection time and then varying the time
you incubate the plate, you can very accurately stage embryos for any
window of time you want.

Ashburner, M.  (1989). Drosophila: A Laboratory Handbook. New York: Cold
Spring Harbor Press.

Powsner, L. (1935). The effects of temperature on the durations of the
developmental stages of Drosophila melanogaster. Physiol. Zool. 8,
474-520.

In article <Pine.A32.3.93.970703081907.119846B-100000 at tigger.cc.uic.edu>,
siau min fung <siauminf at uic.edu> wrote:

> Hi!!!!
>
> I need to collect mass amount of timed embryo for in-situ hybridization.
> But, I donot know, how you can estimate the embryo's age?? For example, if
> I want to collect embryos that is 5 hours old, how is this normally done??
> I have done some reading on how to prevent the females from retaining the
> eggs but I cannot find anything on "timed embryo collection".
>
> thank you very much
>
> siaumin
> siauminf at uic.edu

In article <Pine.A32.3.93.970703081907.119846B-100000 at tigger.cc.uic.edu>,
siau min fung <siauminf at uic.edu> wrote:

> Hi!!!!
>
> I need to collect mass amount of timed embryo for in-situ hybridization.
> But, I donot know, how you can estimate the embryo's age?? For example, if
> I want to collect embryos that is 5 hours old, how is this normally done??
> I have done some reading on how to prevent the females from retaining the
> eggs but I cannot find anything on "timed embryo collection".
>
> thank you very much
>
> siaumin
> siauminf at uic.edu

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