18 BACTERIOLOGY OF THE EYE 



Giemsa's Method of Staining. Giemsa solution for Eomanowsky stain, 1 to 

 H drops ; water containing 1 to 10 drops of TT^HJ P*- car b-> 1 c.cm. Mix fresh 

 every time, and stain from fifteen minutes to one hour or more ; wash rapidly, 

 dry, and mount. 



In a successful film the nuclei of the leucocytes are dark red, the Spirochceta 

 pallida is light red, other Spirochcetce bluish. [Griibler of Leipzig prepares and 

 sends out under the name ' Giemsalosung fur die Romanowskyfiirbung ' the eosin- 

 azur dye, the exact constituents of which can therefore be omitted.] 



Improved Giemsa Staining (Loffler). (1) 1 drop of a 0'5 per cent, solution of 

 malachite green crystal double chloride of zinc, + 3 drops of 0'5 per cent, solution 

 of natrium arsenicosum, are put on to the thinly-spread preparation, which has 

 been fixed in alcohol and ether ; (2) heat to steaming for one minute ; (3) wash 

 with water ; (4) mix 5 drops of 0'5 per cent, glycerine solution with 5 to 10 drops 

 of Giemsa's solution, heat the mixture, and pour it hot on to the preparation. In 

 five minutes wash with water. Staining is very sharp. 



Preiss's Rapid Method. Mix 20 drops of Giemsa solution with 10 c.cm. of 

 distilled water ; divide into three parts ; pour one-third over the preparation, and 

 heat it high above the flame until steam forms ; pour away the stain, and repeat 

 with each of the other thirds of the solution ; wash with water. Good preparations 

 can thus be obtained in four to five minutes. 



Schmorl (Deutsch. Med. Woch., 1907, p. 876) has lately given a method for 

 demonstrating SpirocJicetce in sections by differentiation with potash alum : (1) The 

 sections are fixed in a 4 per cent, solution of formalin for fourteen days, frozen, cut 

 without washing, soaked in distilled water or formalin, then stained with Giemsa's 

 solution for twelve to fourteen hours in very carefully cleaned dishes. (2) Transfer 

 sections to concentrated watery solution of potash alum ; after a short time remove 

 to distilled water for a few minutes ; then mount in glycerine gelatine. Or lay the 

 sections in water on the slide ; dry with filter-paper ; allow the sections to be almost 

 absolutely dry in the air ; then add xylol, and mount in cedar-wood oil. The 

 tissues shrink, and the Spirochcetes are made more obvious. Alcohol must be 

 avoided throughout. 



Silver Method of Levaditi for Sections (Ann. de VInst. Pasteur, 1906, xx. 1). 

 The tissue is cut into small pieces (the anterior part of the eye can be treated as a 

 whole), which are taken either direct into 96 per cent, alcohol, or first into 10 per 

 cent, formalin for a few days, and then, after washing with distilled water, placed 

 in alcohol for twenty-four hours. From alcohol they are placed in distilled water 

 till they sink, and then in a dark vessel in the oven in 2 per cent. arg. nit. for 

 three days (according to Gierke, better 1-5 per cent, for eight days). Wash with 

 distilled water, and put into the following developing solution for forty-eight hours : 

 Ac. pyrogall., 4; formol, 5; aq. dist., 100; embed in paraffin, and cut in series. 

 The SpirochcBta: appear black, with short and variable spirals and pointed ends ; 

 occasionally one end divides. 1 



Heim recommends Hoffmann's modification. The pieces of tissue, after fixation 

 in alcohol and formol, are suspended by linen threads in a freshly-prepared mixture 

 of 90 c.cm. 1-5 per cent. arg. nit., and 10 c.cm. purest pyridin. In this they remain 

 for three hours cold, and then three hours in the paraffin oven at 45 C. in a dark 

 glass-stoppered bottle. 



The developing solution must be freshly prepared thus : Mix 90 c.cm. 4 per cent, 

 pyrogallol with 10 c.cm. pure acetone, and add 15 c.cm. pyridin to 85 c.cm. of the 

 mixture. In this the pieces remain cold overnight. Rapid paraffin embedding. 



Contrast-staining with polychrome methylene blue is possible. All glass dishes 

 must be thoroughly cleaned with ether and alcohol, and the solutions must be fresh. 



1 Regarding the diagnostic value of this method, compare later chapter, ' Syphilis.' 



