CLARK AND LUBS BUFFERS 



68 



CLEARING 



more: Williams & Wilkins, 1928, 717 

 pp.). Prepare: (1) A solution contain- 

 ing M/5 boric acid and M/5 potassium 

 chloride made by dissolving 12.368 gms. 

 of H3BO3 and 14.912 gms. of KCl in aq. 

 dest., and diluting to 1 liter. (2) A 

 M/5 sodium hydroxide (carbonate free) 

 solution by dissolving 50 gms. of NaOH 

 in 50 ml. (cc). aq. dest. in a Pyrex 

 flask. Let stand overnight to allow the 

 sodium carbonate to settle, or filter 

 through a Gooch or sintered glass cruci- 

 ble. (Exclude air to prevent formation 

 of more carbonate by atmospheric CO2.) 

 Keep the strong alkaline solution in a 

 paraffin-lined glass bottle. Dilute with 

 aq. dest. which has been boiled to re- 

 move the excess CO2 so that the solution 

 is about 1 N. Then make an approxi- 

 mately M/5 solution of the alkali which 

 can be accurately standardized against 

 potassium acid phthalate. 



To make buffer of the desired pH add 

 to 50 cc. of (1 ) M/5 H3BO3-KCI the desig- 

 nated amount of (2) M/5 NaOH and 

 dilute to 200 cc. with aq. dest. Or 

 combine the two in similar proportions 

 but in larger amounts to minimize error 

 in measurement. 



Cleaning Glassware. Pulverize 20 gms. 

 potassium bichromate. Dissolve this in 

 200 cc. aq. dest. with aid of a little heat. 

 Add slowly 20 cc. sulphuric acid C.P. 

 Before treating beakers, graduates, 

 bottles, etc. with this acid cleaning solu- 

 tion first wash them in soap and hot 

 water. Rinse in water to remove the 

 soap. Leave in cleaning solution 2 hrs. 

 or more. Rinse in running tap water and 

 di-y with opening downward on drying 

 racks as in biochemical laboratories if 

 possible in a dust free cupboard. For 

 neiv slides and cover glasses wash in the 

 same way and after final rinsing in tap 

 water store in fresh 95% alcohol in 

 covered dishes until they are required 

 for use when they should be wiped with 

 gauze. For old slides and cover glasses 

 soak in xylol to permit separation and 

 removal of most of balsam. Then leave 

 in waste alcohol several daj^s. Soak for 

 a day or more in strong soap solution. 



Wash in running water. Clean in clean- 

 ing solution. Wash in water and store 

 in 95% alcohol. Unless strict economy 

 is necessary it is hardly worthwiiile to 

 use slides and covers twice especially 

 when the former have been marked with 

 diamond pencils. 

 Clearing is a process in microscopic tech- 

 nique which is required in three different 

 situations. 



1. As the step following dehydration 

 in paraffin imbedding. The tissue be- 

 comes translucent but this is not the 

 essential feature of the process. What 

 is necessary is for the alcohol, which is 

 not a paraffin solvent, to be removed by 

 the clearing agent before the tissue is 

 infiltrated with paraffin. Consequently 

 the agents must mix freely with alcohol 

 on the one hand and with paraffin on the 

 other. Of them xylol is by far the most 

 widely used and rightly so. Two 

 changes of half absolute alcohol and xylol 

 within 1 hr. and 2 changes of xylol within 

 the next 3-4 hrs. are usually sufficient 

 for slices of tissue 4-6 mm. thick, but the 

 time should not be extended beyond 

 that needed to attain translucency be- 

 cause so doing causes a hardening and a 

 shrinkage of the tissue. 



Several other substances can be used 

 in place of xylol. Cedar wood oil is ac- 

 cording to Lee (p. 80) the very best 

 clearing agent for paraffin imbedding. 

 It penetrates rapidly, does not make the 

 tissues brittle, and, when not entirely 

 displaced by paraffin, does not seriously 

 interfere with sectioning. First treat 

 the tissue with ^ absolute and xylol for 

 about 2 hrs. The time required in the 

 oil of cedarwood is however a little longer 

 than in the case of xylol used alone, say 

 12 hrs. Some recommend 2 changes of 

 xylol (about 30 min.) after the oil of 

 cedarwood before entering | paraffin and 

 cedarwood oil. 



Methyl benzoate is now quite popular. 

 Pass the tissue from absolute alcohol 

 through 2 changes of pure methyl ben- 

 zoate within 12-24 hrs. When it has 

 been definitely cleared remove benzoate 

 by 2 changes of benzol (^1 hr.) before 

 passing into paraffin, or half benzol and 

 paraffin. 



Chloroform penetrates poorly and 

 should not be employed unless called for. 

 It has the further disadvantage that 

 unless completely removed in the paraf- 

 fin bath, it will make the final paraffin 

 block soft and unfit for cutting. The 

 usual practice is to clear very sm.all 

 pieces for about 12 hrs. in 2 changes, or 

 as long as may be necessary to make 

 them transparent, and in the imbedding 

 to use 4 changes of paraffin. 



A more rapid method is to pass di- 



