The fluorochromes acridine orange and rhodamine B are useful for long-term study of developmental stages. These fluorochromes are prepared in sterile distilled water at 0.1% and stored in brown glass bottles in the refrigerator. Working solutions should be prepared fresh before use. Stock solutions are diluted in membrane-filtered seawater to 0.000l% and to 0.0000l% for the two fluorochromes, respectively. Previously fertilized eggs are exposed for 15 min then washed several times by light centrifugation (500 x g) in fresh membrane filtered seawater. Other fluorochromes, acriflavine, thioflavine, auramine and phosphine 3R are useful but only for immediate observations. Vital dyes, toluidine blue, Nile blue sulfate and crystal violet are prepared identically in concentrations of 0.00001 to 0.000l%. Enhanced non-fixed preparations may be prepared at concentrations of 0.001%. Many of the same dyes can be applied to the whole cells or embryos after fixation.

Illumination

Darkfield, blue, blue violet and ultraviolet induced fluorescence can be viewed with a 85 watt mercury vapor bulb (G.E. H85A3) or equivalent. A 100 watt Halogen source can be substituted for the three former methods of observation.

Filter combinations (transmitted U.V.)

	Darkfield - Noviol O (Corning) #3060 and infrared absorbing (Corning) #4308 or Kodak #301) at light source.
	Blue green - Rhodamine B - (exciter) Either blue green (Corning,#4308) or didymium, #5120 + (barrier) red shade yellow (#3480).
	Blue violet - Acridine orange - (exciter) Violet (Corning, #5113) + infrared #4308 + (barrier) yellow (Corning, #3486) or Noviol A (Corning)
	Violet - Acriflavine, thioflavine, etc. (exciter) violet (Kodak, Wratten, #18A) + #4308 + (barrier) Noviol O (Corning, #3060)
	Ultraviolet - (exciter) Mercury line (Corning, #9863 or 18A Wratten) + barrier filters.

More efficient exciter glass or narrow band pass interference filters can be substituted for improved BV and UV illumination, e.g., for acriflavine, thioflavine (exciter) blue-violet (Schott BG 12); for acridine orange (blue exciter) a narrow band pass interference filter (450-490nm) (Zeiss 48 77 09). For UV excitation, a wide band glass exciter, UG l/UG 5 (Schott) is recommended. A green exciter interference filter (546nm) is best for rhodamine B.

Separation of inclusions by centrifugal stratification

Centrifugation of the oocyte will separate out individual groups of cytological inclusions in regular order from the centripetal to the centrifugal end (Figs. 12 and 13). Eggs can be layered over fresh sucrose (0.75 M) in plastic tubes (l/5 sucrose to 4/5 eggs and seawater). Eggs are precooled in ice and centrifuged at 0°C and 4,800 x g for one hour, or at 17,000 x g for 30 min at 4°C or at 33,000 x g for 12 min at 6°C. The centrifuged eggs are kept iced to reduce return of the stratified components. Cytological observations are made on a small drop of eggs placed in an optically polished, flat depression of a thin microculture slide. A coverslip is placed over the well so that the drop is in continuous contact with the depressed cavity and the coverslip.

Specific staining markers for cellular inclusions (pre- and post GVBD);
components stratified by centrifugation and arranged from centripetal to centrifugal poles.

Components	Vital fluorochromes/dyes*	Fixed
Fat droplets	-	Sudan black B toluidine blue Nile blue sulfate
Lipid granular cap	rhodamine B, acriflavine	Sudan black B Nile blue sulfate T.C.A./Schiffs
Germinal vesicle granules	acridine O, acriflavine	-
G.V. basophilia	acridine O	-
Nucleolus	acridine O, acriflavine toluidine blue	toluidine blue thionin
Chromosomes	acridine O	gallocyanin chrome alum
Astral granules	Nile blue sulfate	alum
Granular band I (mitochondria)	acridine O, thioflavine rhodamine B, toluidine blue	thionin Sudan black B P.A.S. rxn.
Cortical granules I	darkfield	Sudan black B
Granular band II (proteid yolk, mitochondria)	acridine O, thioflavine acriflavine, Nile blue sulfate	Nile blue sulfate toluidine blue
Centrifugal basophilia	-	gallocyanin chrome alum