PAPANICOLAOU TECHNIQUES 



258 



PAPANICOLAOU TECHNIQUES 



Acid phosphotungstic 0.200 gm. 



Lithium carbonate, saturated 

 aqueous solution 1 drop 



(4) EA6S: 



Light Green SF (yellowish) — 

 0.05% in 95% alcohol 45 cc. 



Bismarck Brown Y — 0.5% in 

 95% alcohol 10 cc. 



Eosin yellomsh (water and alco- 

 hol soluble)— 0.5% in 95% al- 

 cohol 45 cc. 



Acid phosphotungstic, C.P 0.2 gm. 



Lithium carbonate, saturated 

 solution 1 drop 



National Aniline and Chemical Com- 

 pany certified stains are used for pre- 

 paring the above stains. If other dyes 

 are used, the amounts may have to be 

 adjusted. 



For the preparation of stains 0G6, 

 EA36 and EA65, it is advisable, because 

 of the low solubility in alcohol of orange 

 G, Bismarck brown and light green, to 

 make the individual alcoholic stock 

 solutions from aqueous solutions as 

 follows: 



Orange G, 0.5% in 95% alcohol— to 95 

 cc. 95% alcohol, add 5 cc. of 10% 

 aqueous solution of orange G. 

 Bismarck Brown, 0.5% in 95% alcohol— 

 to 95 cc. alcohol, add 5 cc. of 10% 

 aqueous solution of Bismarck Brown. 

 Light Green, 0.1% in 95% alcohol— to 

 95 cc. 95% alcohol, add 5 cc. of 2% 

 aqueous solution of light green. 

 Light Green, 0.05% in 95% alcohol— to 

 95 cc. 95% alcohol, add 5 cc. of 1% 

 aqueous solution of light green or 

 dilute the 0.1% alcohol solution with 

 an equal volume of 95% alcohol. 

 By this method, the alcoholic content 

 of the stock stains is less than 95% 

 but it makes no appreciable differ- 

 ence in the staining results. Some 

 of the stain may precipitate when 

 added to the alcohol, but the solubil- 

 ity may be increased by heating 

 slightly (not over an open flame). 

 The strength of the light green solu- 

 tion is less in the EA65 than in EA36 

 because smears of sputum, gastric 

 aspirates, exudates, etc. are apt to be 

 thicker than those of the female genital 

 tract and therefore require a paler 

 basophilic stain. In smears stained 

 with either EA36 or EA65, as soon as 

 the green begins to predominate at 

 the expense of the acidophilic stains, 

 the counterstain should be discarded. 

 All stains should be stored in dark 

 bottles when not in use. If used con- 

 stantly, they should be filtered daily 

 and frequently reinforced by the addi- 

 tion of fresh stain. Because of oxida- 

 tion, hematoxylin should always be 



filtered daily regardless of the volume 

 of staining. 



Vaginal, cervical and endometrial 

 smears may be stained after fixation 

 of only 15 minutes, but other smears 

 will adhere better if left in alcohol- 

 ether for an hour or more. In the 

 staining process, when slides are trans- 

 ferred from one solution to another, 

 they should be drained as much as 

 possible, particularly during alcohol 

 rinses following the counterstains, and 

 during dehydration. It is also im- 

 portant that slides be passed through 

 solutions slowly and carefully, with 

 minimal agitation of the solutions, so 

 that cells will not be washed from the 

 slides. Besides the loss of cells, there 

 is also the danger of contamination of 

 other smears with "floaters" (cells or 

 clumps of cells which have fallen into 

 one of the solutions). However, these 

 floaters can usually be recognized 

 microscopically since they are on a 

 higher focusing level than the fixed 

 smear. There is also danger of con- 

 tamination while mounting the smears 

 if the glass rod used for applying the 

 mounting medium touches the smear 

 and picks up loose cells. 



Smears which have been fixed but 

 not stained may be mailed to a labora- 

 tory for staining by the method sug- 

 gested by Ayre and Dakin. (Ayre, J. 

 E. and Dakin, E., Canad. M. A. J., 

 1946, 54, 489-91.) After the smear has 

 been fixed in alcohol -ether for one hour, 

 it is removed from the fixative and, 

 without drying it, two or three drops of 

 glycerine are placed on it. A clean 

 coverslip or slide is placed on top of 

 the smear thereby sealing it from the 

 air. When the slides are received in 

 the laboratory, thej' are placed in a 

 jar of alcohol -ether until the covering 

 slide or coverslip becomes loose and 

 slips off without disturbing the smear. 



It should be stressed that the Papa- 

 nicolaou smear technique as applied to 

 cancer diagnosis is not a substitute for 

 biopsy or other established methods of 

 diagnosis and that positive smears 

 should be corroborated whenever pos- 

 sible by biopsy or x-raj^ before an}'' 

 major surgery is performed. How- 

 ever, it can be extremely useful in the 

 discovery of earlj^ or hidden lesions. 

 This is particularly true in cervical 

 and bronchogenic carcinomas, where 

 the degree of accuracy of the test is 

 high. It has an additional advantage 

 in that it is a short and relatively simple 

 laboratory procedure and, in most 

 instances, the specimens can be ob- 

 tained with little discomfort to the 

 patient. 



