Papanicolaou Stain
Intended use:
It is intended to stain and check cellular morphology in clinic. During gynecological examination, Papanicolaou stain is a commonly used screening method for cervical cancer and pathological changes prior to cancer. With Papanicolaou stain, the hormonal level of woman can be observed and any infection by pathogens in genital meatus such as Candidiasis and Trichomonas can also be detected. This Papanicolaou stain kit utilizing EA36 and EA50 is a modification of EA36 and both methods are essentially the same. Generally, EA36 and EA50 are mainly intended for gynecological specimen while other modified EA stains are intended for non-gynecological specimens (i.e. exfoliative cells from chest and ascites).
Principle:
Papanicolaou stain contains Hematoxylin, Orange G, Eosin, Bismark Brown and Light Green, etc. Oxidative Hematoxylin will bind nucleic acids of karyon and appear indigo. Other stains will bind different chemical components of cytoplasm and appear in different colors. Cytoplasmic dyes are prepared with high concentration alcohol and strict measures of hydrating and dehydrating are taken during staining, hence various components of the cell will bind the dyes well. In general, the structure of nucleolus is clear, cytoplasm is bright and vivid, and the granules in cytoplasm are evident. Therefore, Papanicolaou stain is a very useful cytology staining method.
Methods:
1. Fix smears in 95% ethanol for more than 15 minutes.
2. Dip smears in 80% and 50% ethanol for 30 seconds each. Rinse with water gently.
3. Stain for 3~5 minutes with Harris.
4. Rinse with water for 1~2 minutes.
5. Differentiate in 0.5%~1% hydrochloric acid ethanol for several seconds.
6. Blue with running water for 5~10 minutes or with lithium carbonate solution, and then rinse with water for 1~2 minutes.
7. Dip smears in 50% and 80% ethanol for 30 seconds each. And then dehydrate them in 95% ethanol for 2 minutes.
8. Stain for 1~3 minutes with Orange G.
9. Wash smears in 95% ethanol twice for 10~20 seconds each.
10. Stain for 2~5 minutes with EA36.
11. Wash smears in 95% ethanol twice for 10~20 seconds each.
12. Dehydrate in 100% ethanol and then clear in xylene. Mount with neutral resin and examine microscopically.
Specifications:
|
Contents |
3Btlsx250ml |
3Btlsx1000ml |
Components |
|
Harris Hematoxylin Solution |
1x250ml |
3x1000ml |
Hematoxylin |
|
Orange G |
1x250ml |
3x1000ml |
Orange G |
|
EA36 |
1x250ml |
3x1000ml |
Light Green, Eosin, Bismark Brown |
Precaution:
1. It is normal to form a layer of oxidative film on the surface of Harris Hematoxylin and some crystal precipitates of aluminum sulphate appear at the bottom of Harris. Prior to use, the oxidative film must be removed. Filter regularly.
2. It is hard to stain with Hematoxylin in winter so the staining time should be extended properly.
3. Differentiation means to wash the extra hematoxylin, which is adsorbed by cells with hydrochloric acid to get a bright contrast between nucleus and cytoplasm. It can’t last too long, or else the nucleus stains light.
4. Orange G remained on the slide should be removed by washing in 95% ethanol. Otherwise, EA36 staining will be affected
5. Do not use the reagents beyond the stated expiration date. For kit storage, avoid exposure to extreme high or low temperature and sunlight.
Expected Results:
1. Epithelia: nuclear is indigo and nucleolus is red; keratinized cells in cytoplasm appear pink, full-keratinized cells appear orange; cells prior to keratinization are sky-blue or light green.
2. Erythrocyte: vermeil or salmon pink.
3. Leukocyte: cytoplasm is sky-blue or light green, nucleolus is indigo.
4. Mucus: sky-blue or pink.