Embryo medium

Embryo medium/1% agarose dishes with furrows to hold embryos in place. 

Embryo medium (E3) 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaClj, 0.33 mM MgSO, and 0.1% Methylene Blue (standard medium with a fungicide suitable for most embryo work). 

Wash the eggs out of the trap with system water, rinse them well, and transfer them to Petri dish(es) containing embryo medium or conditioned water. 

The eggs/embryos can then be washed out of the breeding boxes, rinsed, and transferred to Petri dish(es) with embryo medium. In a healthy, productive colony, most clutches from pair crosses will comprise around 100-300 eggs, with the majority having been fertilized. 

Strain embryos/eggs in a soft mesh sieve, such as a tea strainer, to remove feces and scales, and rinse well with additional system (or tank) water. Transfer embryos/eggs to a dish of clean embryo medium. 

Transfer each selected embryo to a single well of a multiwell tissue culmre plate (see Note 8) filled with embryo medium containing the substance to be tested (see Note 9). In most cases, the test substance will be evaluated at several concentrations along with a vehicle and/or untreated control, as appropriate. If possible, just prior to transferring embryos pre-fill the multiwell plates with the appropriate medium and test solution... 

Place embryos of the appropriate stage (1-4 cells) in embryo medium in 1% agarose dishes with grooves. 

Arrange the embryos for ease of injection. Removing most of the embryo medium from the plate will provide surface tension on the chorion and will prevent sticking of the needle in the embryo following injection. 

There is evidence that the chorion may reduce exposure of zebrafish embryos to some compounds (31), and the environmental conditions in which embryos are grown may affect chorion permeability (32). However, the degree to which the chorion serves as a barrier to chemical exposure is controversial and poorly understood. Some have concluded that the effects are too small to justify the efforts of dechorionation (5). Dechorionation removes the possibility of decreased exposure to chemicals in the embryo medium, but damage to the embryos can sometimes result. It is, therefore, important to ensure that removal is done with as much care as possible. 

Embryo medium is highly aqueous, and the potential for developmental toxicity increases when significant concentrations of solvents are added. Consequently, one of the greatest limitations of any zebrafish embryo assay is the need for adequate aqueous solubility of the test substance. Solubility in embryo medium with the intended vehicle should be tested over the desired concentration range prior to initiation of an embryo assay. An assay plate can be filled with embryo medium with the test substance concentrations to be evaluated, heated to 28.5°C, and viewed with a stereomicroscope in order to detect precipitation. 

Assessing Oculotoxicity in Zebrafish Using the VMR The VMR assesses the locomotor movements of zebrafish larvae in response to lights being turned on and off (Emran et al., 2008 Yin et al., 2012). In brief, individual larvae (5 dpf that have been previously treated for 48 h with either vehicle or selected compounds) are placed in embryo medium in a well of a 96-well clear microtiter plate (12 fish/treatment group). The plate is then placed inside a recording chamber... 

Embryo medium This is 10% (v/v) Hank s with magnesium and calcium at full strength. 

Raising of zebrafish embryos has traditionally been done in a defined salt solution or embryo medium. We find that this is not necessary as long as an antifungal agent,... 

For embryos with their chorions, an injection chamber is made by placing a glass microscope slide into an 85-mm diameter disposable plastic petti dish. Flood the injection chamber with embryo medium (6), and allow a thin film of liquid to form between the slide and the dish. Remove the excess liquid using a glass pipet. 

Following injection, tilt the injection chamber, and add embryo medium to the embryos. Transfer the embryos into a clean dish, and incubate them in a humidified incubator at 28.5°C. Later, remove all dead or uncleaved embryos. 

Semliki Forest arborvirus was grown in chick embryo tissue culture. The infectious tissue culture liquid was decanted and diluted with medium 199 to give a preparation containing between 10 and 10 mouse IDso virus/ml. 

Representative nickel-sensitive aquatic species show sublethal effects at 11.7 to 125 pg Ni/L. These effects include altered immunoregulatory mechanisms in tissues of the rainbow trout at 11.7 pg/L (Bowser etal. 1994), inhibited reproduction of daphnids at 30 pg/L, growth inhibition of freshwater and marine algae at 30 to 125 pg/L, reduced growth of rainbow trout at 35 pg/L, accumulation from the medium by mussels at 56 pg/L, and abnormal development of sea urchin embryos at 58 pg/L (NRCC 1981 WHO 1991 Outridge and Scheuhammer 1993 Table 6.7). 

Incubation time and hatching itself generally depend on a multitude of factors, such as temperature, photoperiod, oxygen availability, and the presence of hormones, both natural and synthetic, in the medium. These factors may stimulate or inhibit the synthesis by the embryo of the hatching enzyme which is finally responsible for the emergence of the organism from the egg [97]. 

LeBoeuf, R.A., and Kerckaert, G., Enhanced morphological transformation of early passage Syrian hamster embryo cells cultured in medium with a reduced bicarbonate concentration and pH, Carcinogenesis, 8, 680, 1987. 

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