Valerie, The technique you are using is the re-hydration technique of air-dried slides of Ng et al (Acta Cytolog 38(1):56-64, 1994)- 30 sec in saline followed by fixation in 95% ethanol (at this stage, slides can remain in ethanol before staining). Please note that it is a re-hydration, not a de-hydration step. I have found that slides that have been air-dried for longer than two days do not rehydrate well. Cells appear pink with large air-dried, pale staining nuclei - lacking chromatin details. Unfortunately there is little you can do in these cases.
Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC) Laboratory Manager & Senior Scientist Tel: 612 9845 3306 Fax: 612 9845 3318 the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -----Original Message----- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Valerie R. Sent: Saturday, 1 May 2010 10:49 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Re: How to troubleshoot a cytology slide withdehydration problems I forgot to include the complete protocol that my lab uses so you can help me better: 1.After dehydrating in sodium chloride for 25 seconds you have to dip the slides in alcohol 95. 2. 10 dips in water then 1.30 minutes in hematoxylin, it used to be 1 min, then rinse in water. 3. 1 min in rinse solution 4.10 dips in water. 5. 1 min in bluing solution 6. 10 dips in water. 7. 10 dips in alcohol. 8. 10 dips in alcohol. 9. 1 min in orange solution 10. 15 dips in 95 alcohol. 11. 15 dips in 95 alcohol. 12. 3.15 mins in EA solution 13. The slides are then dipped in 3 containers of 95 alcohol (20 dips each) 14. Slides are dipped in 3 containers of 99 alcohol (15 dips in each) 15. Then they are dipped in Isoxylene which is a combination of xylene and 99 alcohol (15 dips) 16. And lastly Xylene 10 dips When the slides do not absorb the h20 saline seems that the slides absorb more EA than hematoxylin. I don't know why. On Fri, Apr 30, 2010 at 5:34 PM, Valerie R. <vrodrigue...@gmail.com> wrote: > I am an HTL but I have to stain pap smears. In the Papanicalou > procedure for fine needle aspirations I have to stain the slides in > sodium chloride first for 25 seconds in total (15 seconds in one > container and 10 in another container). This for me is the trickiest > part when staining cytology because if slides are not well dehydrated > in saline then the cells are not going to absorb the stains well, and > the result will be a pinkish slide, when it is suppose to look > blue-greenish. > > I had this problem today where I stained an entire rack of slides, and > all the slides turned blue (which means they stained correctly) except > 4 slides which turned out pinkish (which is a sign that they did not > dehydrated properly in sodium chloride (h20 saline). > > Is there a solution to this issue. Can these slides be re-stained? > > I know this newsgroup is for histology only but I would like to know > if histotech and cytotechs have encountered this issue in their labs > and how they ended up solving the problem. > > Thanks in advance > > > _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ********************************************************************************* This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient, please delete it and notify the sender. Views expressed in this message and any attachments are those of the individual sender, and are not necessarily the views of The Children's Hospital at Westmead This note also confirms that this email message has been virus scanned and although no computer viruses were detected, The Childrens Hospital at Westmead accepts no liability for any consequential damage resulting from email containing computer viruses. ********************************************************************************* _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet