TISSUE CULTURE 347 



It is not possible within the scope of a single chapter to describe 

 more than the essential features of the technique and to emphasise 

 the more common errors and pitfalls. A list of text-books for 

 further study is recommended at the end of the chapter. 



779. Preparation of Glassware and Instruments. All glass- 

 ware coming in contact with the media and the tissues should be 

 of Pyrex or Jena, non-alkali glass. Cleaning, prior to sterilisation, 

 requires special attention. First boil in water with a refined soap, 

 or if the glass is new, in 5 per cent. H2SO4. The remains of clotted 

 plasma in pipettes or on coverslips may be removed in a boiling 

 bichromate-sulphuric acid mixture such as : — 



Pot. bichromate .... 4-6 grm. 



Cone. H2SO4 ..... 6 c.c. 



Water ...... 100 c.c. 



But great care must be taken to remove the acid subsequently by 

 ammonia and then by prolonged washing. Wash finally in several 

 changes of distilled water (free from heavy metals) and drain. 

 Immersion in re-distilled 95 per cent, alcohol may be preferred 

 before drying. Coverslips should be washed individually with 

 great care and stored in absolute alcohol. After polishing with 

 linen or silk, examine for traces of fluff and remove it by flaming, 

 then store the coverslips in a Petri dish on a piece of greaseproof 

 paper. Wrap the glassware in thin smooth paper before sterilising. 

 Pipettes may be stored in large boiling tubes fitted with corks. 

 It is convenient to have glassware sufficient for several weeks' 

 work sterilised at the same time and stored in a clean cupboard. 

 Rubber stoppers and teats, when new, often require boiling in 

 dilute alkali to remove traces of acid. Always sterilise rubber by 

 placing it in a glass vessel in a little water in the autoclave. 



Glassware to contain blood, freshly prepared plasma (up to 

 forty-eight hours old) and saline solutions must be coated internally 

 with refined paraffin wax. Fill the sterile vessels or tubes with 

 melted wax which has been sterilised by heating. While they 

 are still quite hot, spread a sterile towel on a table, empty each 

 vessel of its wax and rest its mouth downwards on the towel to 

 drain. Place a sterile cork or rubber stopper in each after the 

 wax has set evenly on the interior. Cover the corks and stoppers 

 with sterile paper caps and a rubber band if they are to be stored 

 for any length of time. 



780. Saline Solutions. The popular conception that living 

 tissue can be placed without harm in any sterile physiological salt 

 solution containing the approximate proportions of salts obtaining 

 in plasma is quite erroneous. The saline solutions which come in 

 contact with the living tissues to be explanted and which form 

 part of the media, must be prepared with extreme care and 

 accuracy. A short period in an inaccurately prepared saline of 

 unsuitable pH is sufficient to hinder or entirely suppress the 



