BIO-CHEMICAL RESEARCHES ON BITTER PIT. 11 



(6) STAINING METHODS DEPENDING ON ACIDITY. 



It was thought that it might be possible to detect different groups of cells possessing differ- 

 ent acidity to the general pulp cells around them. At first phenolphthalein was 

 employed, made just alkaline with 1, 2, 3, - - 5, etc., drops of ^ alkali (soda) 

 to 5 c.c. of the reagent (1 per cent, phenolphthalein, 5 drops added to 5 c.c. water), 

 until just no longer decolourized by the apple juice. The reagent, however, proved 

 unsatisfactory. 



Later a note in a scientific periodical called attention to a special reagent, Dinitro-hydro- 

 quinone (Henderson's Reagent), with a wide colour range. This material not being procurable on 

 the market was made in the laboratory and also used. 



The apples were sufficiently acid to give the acid colour. The cells in sections examined under 

 the microscope were yellow, and only the vascular bundles showed a difference in tint, namely, an 

 orange-red (less acid). 



Again, however, it is impossible to say that the apples used were suitable, i.e., with Pit present 

 though not yet showing the brown staining. 



A third indicator stoin, Methyl violet, has been tried, but is only suitable for very acid apples. 



As to applying these reagents to apples having Pit visibly present, to see if they are suitable 

 reagents for examining apples in which Pit is not present, there is this difficulty, that the pitted areas 

 have apparently a sponge action. The reagent is mopped up and stains deeply the tissues around 

 and beneath. Thus the pitted area appears more deeply stained to the eye. The pitted area not 

 only sponges up the reagent, but also exuded apple juice. And this brings up the greatest difficulty 

 of all in applying these methods. 



In peeling the apple the whole surface is swept by the juice escaping from the cut cells, and 

 juice from the healthy cells is carried over other cells, which for the sake of argument may be con- 

 sidered unhealthy. 



The first essential then is to carefully blot with filter paper the whole surface of the apple. 

 When the reagent is next applied it requires only a very short time for the same difficulty to arise 

 again. The surface of the apple becomes bathed in reagent containing those contents of the cells, 

 which readily diffuse out, e.g., acids, sugars, etc. 



Partly, then, from inherent practical difficulties, and possibly also from the right material not 

 being available at the time, no results of any value were obtained. 



It has been previously shown -that with the Pit incidence in apples received clean, at least 

 50 apples would have needed to be carefully mapped before it could be claimed with any certainty 

 that an apple destined to subsequently Pit had been handled. It is regretted that more apples were 

 not examined in this way, but a peeled apple is an apple lost, and the material was required for other 

 experiments. 



To press this class of experiments would require that in another season a large number, say 

 200 apples, should be completely mapped over with a suitable reagent. Should then some six or 

 eight apples show local patches, the size of Pit, staining in an anomalous fashion, then this should be 

 approximately the number of apples pitting in a control batch of 200 apples kept under observation. 

 Only then could it be legitimately assumed that the anomalous staining had shown an early con- 

 dition of Pit. 



(c) STAINING METHODS, SHOWING STARCH. 



t 



In my experience Pit has practically always been associated with starch. No desire 

 arises to contradict the statement that pitted tissue may not show starch, i.e., if 

 the disease develops before or after the starch stage. But perhaps it may be 

 said, without fear of contradiction, that in 99 cases out of 100 starch is present. 

 In other words, it would appear that the critical time for the apple is when the pulp 

 cells are loaded with starch, and before the starch has been converted into sugar. 





