DARK-FIELD MICROSCOPE 



93 



DAVENPORT'S 



light will go toward the formation of 

 an image this system will require a very 

 strong light source as well as a dark- 

 adapted eye for observation. If a spe- 

 cial dark-field condenser is not avail- 

 able, one can be easily improvised by 

 adding a central opaque stop just be- 

 low the regular condenser in the fol- 

 lowing manner: 



Select an objective of not over 0.66 N. 

 A. and a condenser of at least 1.25 N. 

 A. and set up the illumination as for 

 bright field, with the slide oiled to the 

 top of the condenser. Stop down the 

 iris diaphragm until it just encircles the 

 bright central disk which is seen through 

 the tube with ocular removed. Now 

 cut from black paper or thin metal a 

 disk of diameter equal to the diameter 

 of this opening in the diaphragm and 

 cement it to a piece of glass that fits 

 the insertion slot under the condenser. 

 This central stop should block all light 

 from entering the objective if it is 

 properly centered below the iris open- 

 ing. Next open the diaphragm com- 

 pletely, thus permitting a hollow cone 

 of light to focus on the specimen but 

 not to enter the objective. If the 

 microscope is already supplied with a 

 dark-field stop it should be tested in 

 this manner. 



The great advantage of dark-field 

 microscopy is that it enables one to per- 

 ceive the presence of particles far be- 

 low the theoretical limit of resolution. 

 Even though a particle may be much 

 smaller than the wave length of light it 

 does scatter incident light laterally into 

 the objective and is imaged as a rather 

 fuzzy point of light. We have here an 

 exact analogy of the situation whereby 

 we can "see" dust particles dancing in 

 a strong beam of light when it enters a 

 darkened room. Most of these particles 

 are of a size far below the limit of visual 

 resolution. Another advantage is that 

 small, transparent objects, like chylo- 

 microns, can be seen in dark-field 

 whereas they are invisible in the glare 

 of bright-field illumination. 



For the examination of ordinary his- 

 tological sections little is to be gained 

 by dark-field observation. Neverthe- 

 less Ralph, P.H. (Stain Tech., 1942, 17, 

 7-10) recommends dark-field examina- 

 tion in the study of stained blood 

 smears. It is most helpful however in 

 the search for very small, scattered 

 bodies in a more or less empty medium, 

 such as chylomicrons or spirochetes in 

 blood plasma. Dark field examination 

 is the standard technique for the study 

 of microincineration preparations. 

 The cytological results of 20 years of 

 of microincineration have been summa- 



rized by Policard, A. (J. Roy. Micr. 

 Soc.,1942, 42, 25-35). 



The source of the light must be very, 

 very strong. To obtain the best results 

 do not rely on improvised, or home 

 made, equipment. A special condenser 

 is needed. Probably the highest ob- 

 jective that can be usefully employed 

 18 a 3 mm. oil immersion objective with 

 iris diaphragm. See Microincineration, 

 Spirochetes, Chylomicrons, etc. 

 Davenport's 2-hour method for staining 

 nerve fibers in paraffin sections with 

 protargol. 1946 modification written 

 by Dr. H. A. Davenport of original 

 (Davenport, H. A., McArthur, J., and 

 Bruesch, S. R., Stain Techn., 1939, 14, 

 21-26). Fix for 1 to 3 days in: Form- 

 amide (Eastman Kodak Co.), 10 cc; 

 paranitrophenol, 5 gm.; 95% ethyl alco- 

 hol, 45 cc; aq. dest., 45 cc. Transfer 

 thru graded alcohols to absolute, then 

 either n-butyl alcohol or xylene and 

 embed in paraffin. Sections are cut and 

 mounted in the usual manner, paraffin 

 removed and the slides run thru graded 

 alcohols to dist. water. Impregnate 

 for 1 hr. at 58-^2 °C. in a 5% aq. silver 

 nitrate. Rinse in 3 changes of aq. 

 dest. with 20-30 sec. allowed for each 

 change. The rinse water should cover 

 the slides completely, each slide sepa- 

 rate (not back to back) and the water 

 discarded with each change to prevent 

 carry-over of silver nitrate into the 

 protargol. Place the slides in 0.2% 

 protargol (Winthrop Chemical Co.) for 

 1 hour at room temperature. Rinse 

 quickly (2 sec.) in aq. dest. and reduce 

 for 1 to 2 min. in the following mixture : 

 Sodium sulfite, 5 gm. ; Kodalk (Eastman 

 K. Co.), 0.5 gm.; hydroquinone, 1 gm.; 

 aq. dest., 100 cc. Wash in running tap 

 water several minutes and rinse once in 

 dist. water. Tone in 0.1% aq. gold 

 chloride for 5 to 10 min. Wash again 

 for about 1 min. and reduce in 1% aq. 

 oxalic acid for 10-20 sec. Rinse and 

 place in hypo (10% aq. sodium thio- 

 sulfate) for about 1 min. Wash in run- 

 ning water, dehydrate and cover. 



Notes: If the stain is too dark, try 

 any or all of the following modifications : 

 rinse longer after the protargol, use 

 0.1% protargol, omit the oxalic acid re- 

 duction after gold toning. If too pale: 

 double the concentration of the pro- 

 targol, double the time of either or both 

 silver impregnations, omit rinsing after 

 protargol, double the concentration of 

 kodalk in the reducer, lengthen the 

 time of reduction in oxalic acid. The 

 technic is suitable for mammalian cen- 

 tral or peripheral nervous tissue, but 

 for sympathetic fibers in intestine and 

 uterus a moderate degree of success has 



