FLORENCE'S REACTION 



127 



FLUORESCENCE MICROSCOPY 



They are floated out by the use of hyper- 

 tonic salt and other solutions, some- 

 times with the aid of centrifugal force 

 (E. C. Faust, in Simmons and Gentz- 

 kow, p. 684). 



Florence's Reaction. The standard test for 

 choline in seminal stains. As described 

 by Pollak, O. J. Arch. Path., 1943, 35, 

 140-196: Place one drop semen, or of 

 aqueous extract of seminal stain, on 

 slide. Add drop of reagent (Pot. 

 iodide, 1.65 gm.; iodine, 2.54 gm.; aq. 

 dest., 30 cc), cover and examine micro- 

 scopically. Dark brown, rhombic crys- 

 tals appear, about 25m long and Sm wide 

 with bifurcated ends resembling swal- 

 low tails and Teichmann's hemin crys- 

 tals. In polarized light these show 

 double contours. 



Fluids. Samples of body fluids are often 

 presented for microscopic examination. 

 In a human being containing, say, 100 

 lbs. of water the}^ are naturally of great 

 variety even under normal conditions. 

 Abnormal fluids are usually described 

 as transudates or exudates. The for- 

 mer compared with the latter are 

 mainly filtrates, are more watery, have 

 lower specific gravity, less albumin, no 

 bacteria and are the result of mechani- 

 cal forces rather than inflammation. 

 See: 



Aqueous humor Intracellular phase 



Cerebrospinal Pericardial 



Duodenal Peritoneal 



Endolymph Pleural 



Extracellular phase Sjiiovial 



Tissue 



Fluoran Derivatives. As explained by Conn 

 (p. 144) fluoran is not a dye but a prod- 

 uct of phthalic anhydride containing a 

 xanthene ring and a lactone ring with 

 introduced hydroxyl groups and halogen 

 atoms in particular positions. Ex- 

 amples : eosin B and Y, erythrosin 

 bluish and yellowish, ethyl eosin, 

 fluorescein, mercurochrome 220, methyl 

 eosin, phloxine, phloxine B, rose bengal. 



Fluorescein (CI, 766) is simplest fluoran 

 dye. It stains very poorly but is highly 

 fluorescent. Its sodium salt is called 

 uranin. 



Fluorescence Microscopy. Details pro- 

 vided by Dr. W. L. Simpson of The 

 Barnard Free Skin and Cancer Hospital. 

 Supplemented by Dr. T. B. Rosenthal, 

 Dept. of Anatomy, Washington Univer- 

 sity, St. Louis. June 6, 1951. 



Fluorescence is the property, pos- 

 sessed by many substances, of convert- 

 ing short wavelengths of light into 

 longer wavelengths. In the field of 

 microscopy those structures and sub- 

 stances are of most interest that convert 

 ultraviolet light into light of the visible 

 spectrum, since it is only these sub- 



stances that can be observed directly. 

 Though fluorescence microscopes de- 

 signed for this type of observation have 

 been available commercially for many 

 years, their use has been limited until 

 recently by their relatively high cost 

 and by the apparent failure of biolo- 

 gists to appreciate the possibilities of 

 this type of observation. Recent tech- 

 nological developments in the glass 

 and electric lamp industries now make 

 it possible to assemble an apparatus 

 for fluorescence microscopy at a cost 

 well within the budget of most labora- 

 tories. Evidence of heightened interest 

 in this field is found in the numerous 

 papers concerning fluorescence micros- 

 copy within the past 10 years. Al- 

 though several reviews of the subject 

 already exist (Haitinger, M., Flu- 

 orescenz-Mikroscopie, Akademische 

 Verlagsgesellschift, Leipzig, 1938; Ham- 

 perl, H., Virchows Arch. f. path. Anat., 

 1934, 292, 1-51; Sutro, C. J., Arch. 

 Path., 1936, 22, 109-112; and McClung's 

 Handbook of Microscopical Technique, 

 New York, Paul B. Hoeber Inc., 1937), 

 the technique will be described as it 

 can be used with an assembly of low 

 cost apparatus available in the United 

 States at the present time. 

 Apparatus required: 



1. An intense source of ultraviolet 

 light that is rich especially in the region 

 from 300 to 400 millimicrons. Certain 

 electric arcs using electrodes of special 

 metal alloys (the Haitinger Arc, C. 

 Reichert — Vienna) have been developed 

 for this purpose. More easily avail- 

 able, low in cost, and having an intense 

 output in the desired region, are the 

 medium pressure mercury vapor arcs in 

 capillary quartz tubes (the A H 4 lamp 

 of the General Electric Company or 

 Westinghouse Electric Co. and lamps 

 made by Hanovia Chemical Co., etc.). 



2. Filters that remove all or nearly 

 all of the visible light. A considerable 

 selection of glass and liquid filters may 

 be used for this purpose. Since most 

 of the so-ca,lled ultraviolet filters pass 

 also a certain amount of red light, 

 supplemental blue filters must be used 

 with them. A solution of copper sulfate 

 in a cell or tube of quartz, or of ultra- 

 violet transmitting glass, is satisfactory 

 and readily available. A combination 

 of Shott glass filters U G 2 and B G 14 

 are recommended by Jenkins (R., Stain 

 Techn., 1937, 12, 167-173). Corning 

 Filters ^5840, 5860, or 5874 used with a 

 copper sulfate solution are satisfactory 

 in our experience. An entirely liquid 

 filter, using solutions of cobalt sulfate 

 and nickel sulfate, is described by 

 Backstrom (H. L. J., Arkiv. for Kemi. 



