/. TECHHiaUES IN EXPERIMENTAL EMdRYOLOGy 

 Equipment and Procedures in Experimental Embryology 



GENERAL INTRODUCTION 



" Biologically Clean": These two words should be the most frequently emphasized in any 

 laboratory of experimental embryology, even as they are in any tissue culture laboratory. 

 Glassware, instruments, solutions and hands must be "biologically clean" before any ex- 

 perimental results may be considered valid. The term means that, barring any experi- 

 mental conditions imposed which might alter the situation, there is no possible contamin- 

 ation of the living material either by chemical substances, by living parasites or by 

 harmful organisms such as bacteria or viruses. The experinnental conditions should be 

 such that any embryo, introduced into that environment, would be expected to survive. 

 The following precautions, in the interest of biological cleanliness, are suggested: 



1. Glassware : Regardless of the source of the glassware used, it should be thoroughly 

 scrubbed with hot soap and water, rinsed in running tap water for at least 2 hours, 

 rinsed in distilled water and air-dried either by or under the direct supervision of 

 the person planning to use it. If the glassware is cleaned with the usual cleaning fluid 

 (potassium dichromate saturated in 10% sulfuric acid) it must be thoroughly washed 

 and rinsed for a longer period in order to remove the tenacious chemicals (Richards 

 '36). Properly cleaned glassware may be wrapped in clean paper towelling and heat 

 sterilized for 1/2 hour at 100° C. As long thereafter as the glassware remains 

 wrapped it may be considered as sterile. 



2. Hands : A surgeon usually spends as much time scrubbing his hands as in operating, 

 and such cleanliness in experimental embryology will result in more dependable and 

 reproducible results. Formaldehyde, osmic or hydrochloric acid fumes, adherent to 

 the hands, will contaminate instruments and ultimately the embryos. Thin white sur- 

 geons rubber gloves are advised in tissue culture experiments. 



3. Instruments : If the instruments have never before been used, they may be thoroughly 

 washed, rinsed, and sterilized in the autoclave at 1 5 pounds pressure for 30 minutes; 

 cooled; immersed in 95% alcohol until used. Dissecting instruments from other lab- 

 oratories should never be "trusted". Each student should provide himself with a new 

 set of stainless steel instruments (never chromium plated) and should keep them in a 

 plastic tube, or in a cotton-filled box, to be reserved exclusively for his operational 

 procedures with living embryos. 



It is often necessary to preserve eggs or embryos but the operating instruments should 

 never come into contact with any fixatives. The student may use an unsterile or even 

 contaminated set of instruments for the handling of such inaterial to be preserved. 

 Often eggs, embryos or even tissues may be introduced into a fixative with the flat 

 end of a toothpick, thus avoiding the contamination of instruments. Once an instru- 

 ment has come into contact with a fixative, it should thereafter be regarded as "con- 

 taminated" and not "biologically clean. " 



4. Embryos: Dead or dying embryos are probably the most common source of contamin- 

 ation of cultures, because they are infested with bacteria and necro-toxins. Ailing 

 embryos should be isolated, and crowding should be avoided. The culture medium 

 should, at all times, appear to be clear. Healthy embryos may be passed through 

 several changes of sterile medium in order to free them of adherent bacteria. Some 

 stages of development, particularly of aquatic forms, may tolerate brief immersion 



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