HARDWICKE'S SCIENCE-.G OSSIP. 



HOW TO DOUBLE STAIN VEGETABLE 

 TISSUES. 



HAVING exchanged a great number of slides of 

 double-stained vegetable sections through the 

 medium of the "Exchange" column in this journal, 

 and as my slides appear to give general satisfaction, 

 some of the recipients going so far as to compliment 

 me upon them and asking how they are prepared, 

 I think it will be a convenience to all parties in- 

 terested, and to your readers also, if you will give 

 me an opportunity of making known by means of 

 this paper my method of double-staining vegetable 

 tissue. 



In several papers I have seen on this subject a plan 

 of using two aniline dyes (either together or separately) 

 is given, but this method I cannot recommend, as, 

 after some little experience, I find that all aniline 

 dyes are more or less fugitive when used with vege- 

 table tissue, and that consequently double-staining by 

 this method resolves itself into using two dyes of as 

 opposite colours as possible, one to be as little fugitive 

 as can be obtained, and the other of an exactly con- 

 trary character. If any vegetable section containing 

 hard and soft tissue be dyed in such colours, either 

 together or separately, and afterwards washed to a 

 certain extent, it will be found that the more fugitive 

 dye still lingers in the harder tissue, which gives it 

 up less readily than the softer parts ; these latter still 

 retaining the permanent dye, which usually is not so 

 penetrating as the other dye, and is therefore over- 

 powered by the same in the harder tissue. Were the 

 section washed long enough, every trace of colour 

 could be removed from all parts of the same. 



To be at all successful in double-staining by this 

 method requires constant practice, so as to know 

 exactly at what stage to discontinue washing the 

 stained sections ; it is therefore not surprising if 

 amateur mounters are not invariably successful when 

 they try their hand at double-staining vegetable 

 sections. 



Such being the case has led me to think whether 

 it would not be much better to employ one permanent 

 dye and one fugitive dye, using the former first, and 

 the fugitive dye afterwards, this last being washed 

 out again to any degree required. 



I now double-stain vegetable sections by means of 

 carmine and aniline green, the former being a per- 

 fectly "fast" dye, whereas the latter is extremely 

 fugitive, more so than I wish, but is the only colour 

 I know of at present which will answer the purpose 

 required. 



Before commencing staining the sections they 

 require to be decoloured ; this is important. The 

 bleaching can be done by means of alcohol when the 

 whole of the tissue and contents will be preserved, 

 but slides prepared in this way will not be so effective 

 as if the sections had been decoloured in a solution of 

 chlorinated soda which is prepared as follows : Take 



two ounces fresh chloride of lime and dissolve same 

 in one pint of distilled or clean rain water, shake well 

 and allow it to settle, when somewhat clear add to it 

 by degrees a strong solution of common washing soda 

 until no precipitation takes place ; when the whole 

 has thoroughly settled the clear supernatant liquor 

 can be siphoned off and, if necessary, filtered. Pre- 

 serve in well-corked bottles and in the dark, otherwise 

 the solution will soon become useless. 



For the carmine dye, take — carmine 10 grains ; 

 strong ammonia 10 or 15 grains ; and dissolve in a 

 test tube with a little heat ; when the carmine is dis- 

 solved add distilled water 200 grains. Filter and 

 preserve in a well-stoppered bottle. 



For ordinary use portions of this dye will have to 

 be diluted with four or five times its bulk of distilled 

 water, but occasionally sections will be met with 

 which require stronger solutions. 



The carmine dye for vegetable sections requires a 

 "mordant," prepared as follows: (A) sulphate of 

 alumina 10 grains ; dissolved in distilled water 

 200 grains. (B) Acetate of lead (sugar of lead) 

 30 grains ; dissolved in distilled water 600 grains. 

 Add B to A till no precipitation takes place, then 

 allow the whole to settle and siphon off the clean 

 liquor which must be filtered and kept in a clean 

 stoppered bottle. When required for use, dilute a 

 portion with four or five additional equivalents of 

 distilled water and filter afresh. Stronger solutions 

 may occasionally be required, as in the case of the 

 carmine dye. 



The aniline dye is prepared by simply taking — 

 3 grains of crystals of green aniline (the brand I use 

 being "Iodine Green," prepared by Messrs. Brookes, 

 Simpson, & Spiller, of London), crushing the same 

 well in a mortar and dissolving in one ounce of abso- 

 lute alcohol ; filter the solution and preserve in a 

 stoppered bottle. 



The double staining of vegetable sections is divided 

 into three distinct stages, viz. : — 



1. Bleaching the sections ; 



2. Staining in carmine ; and 



3. Staining in green aniline and mounting. 



Any interval of time, days, weeks, or months, can 

 take place between the first and second stage or the 

 second and third. 



I will now describe these stages in succession. 



Bleaching the Sections. — An ounce or so of the 

 chlorinated soda solution having been poured into a 

 glass or wide-mouthed bottle and covered or corked, 

 the sections are placed therein, but not too many, 

 otherwise they will cling together and prevent the 

 solution acting so quickly upon them ; when the 

 sections are thoroughly bleached, which will generally 

 be in from six to twelve hours, they must be taken 

 out, lest they become disintegrated, and well-washed 

 by being soaked in several changes of boiled water 

 yet lukewarm, say, about five changes of water in the 

 twenty-four hours, at the end of which time the 



