Friday 24 February 2017

PAP Staining (Cytolog - Papanicolaou Staining)

PAP Staining


Introduction Of PAP Staining –

Cytology विज्ञान की वह शाखा है जिसमें जीव कोशिकाओं का अध्ययन किया जाता है। और Cytopathology विज्ञान की वह शाखा है जिसमें बीमार कोशिकाओं (Diseased Cells) के उपचार,लक्षण,कारण का अध्ययन किया जाता है।

 PAP Staining cytopathology Lab में सर्वाधिक प्रयोग किया जाने वाला Staining Method है। इस Staining का इजाद Dr. Papanicolaou ने किया ।
Dr. Papanicolaou ने Trichrom Stain (tri – three, chrom- color) का विकास किया जो आज सभी cytology laboratory में प्रयोग किया जाता है

Trichrom तीन प्रकार के Stains का combination होता है जिसमें

1) Heamatoxylin (Used For Nuclear Stain)
 2) OG-6 (Used For Keratin Stain)
 3) EA-36 or EA-50 (Eosin Azure-50) (Used for Cytoplasm of         Squamous Epithelial cell, Nucleoli और RBC)

उपरोक्त तीन Stains प्रयोग होते है।


Preparation of Slides – 

Cytology में कई तरह के Tumor और Cancer के सैम्पल लैबरेटरी में आते है जिसकी Slide को प्राय: पैथलाजिस्ट ही F.N.A.C.(Fine Needle Aspiration Cytology) और Bruishing  के द्वारा तैयार करते है और फिर Slide को 95% alcohol में Dip करके Smear को Fix कर लेते है।
  
PAP Staining में प्रयुक्त होने वाली सामग्री

1)      Haris Hematoxylin
2)      HCl – 0.5%
3)      OG – 6
OG 6
4)      EA – 36 or EA - 50
EA 50
5)      Ethenol (Alcohol Absolute,95%,80%,70%,60%,50%)

Alcohol
6)      Distilled Water
7)      Xylene
8)      DPX (for mounting)
9)      Cover slip
10)    Slide Stain Tub
Stain Tub



Composition और Preparation of Reagents –


1)      Harri’s Hematoxylin


a)      Hematoxylin – 5gm

b)      Ethanol – 50ml

c)       Potassium alum – 100gm

d)      Distilled water(50*c) – 1000ml

e)      Mercuric Oxide – 2-5gm

f)       Glacial acetic acid – 40ml


2)      Orange G-6


a)      Orange G(10% Aqueous) – 50ml

b)      Alcohol – 950ml

c)       Phosphotungustic acid – 0-15gm


3)      EA 50


a)      0.04 M(Molarities) light green SF – 10ml

b)      0.3 M(Molarities) eosin Y – 20ml

c)       Phosphotungustic acid – 2gm

d)      Alcohol – 750ml

e)      Methanol – 250ml

f)       Glacial acetic acide – 20ml

   
 सभी stains को प्रयोग करने से पहले Filter कर लेते हैं।


  PAP Staining की विधि –

1)      सबसे पहले 15 minute के लिए स्लाइड को 95% alcohol में Dip करते है ताकि smear fix हो जाए।


2)      अब Slide को 2 मिनट के लिए 80% alcohol में Dip करते हैं, और फिर 2 मिनट के लिए 60% alcohol में Dip करते हैं।


3)      अब Slide को दो बार पाँच पाँच मिनट के लिए Distilled Water में Dip करते है ताकि Slide से alcohol हट जाए।


4)      अब Slide को 3-4 मिनट के लिए Hematoxylin stain में Dip करते है।
Hematoxylin Stain


5)      अब Slide को 3 मिनट के लिए Distilled Water  में Dip करते है।
6)   अब Slide को 0.5% HCl में 10 सेकन्ड के लिए Dip करते हैं।

7) अब Slide को 3 मिनट के लिए Distilled Water  में Dip करते है।

8)  For Dehydration, Slide को दो-दो मिनट के लिए 60% फिर 80% और फिर 95% concentration के alcohol मे Dip करते है।


9)  अब Slide को 3 मिनट के लिए First Counter stain OG-6(Orange Green-6) में Dip करते है। 

OG-6 Stain

10)  अब Slide को 2 बार(2 time) 2 मिनट के लिए 95% concentration के alcohol मे Dip करते है।


11)   अब Slide को 3 मिनट के लिए Second Counter stain EA- 36 or EA- 50(Eosin Azure Stain- 36 or Eosin Azure Stain- 50) में Dip करते है।
EA-50 Stain


12)   अब Slide को 2 बार(2 time) 2 मिनट के लिए 95% concentration के alcohol मे Dip करते है।


13)   अब Slide को 2 बार(2 time) 2 मिनट के लिए Absolute Alcohol (100% alcohol) मे Dip करते है।


14)   अब Slide को 3 बार(3 time) 2 मिनट के लिए Xylene में Dip करते है ताकि smear से alcohol निकल जाए ।


15)   अब smear को Dry होने देते है, जिसके बाद Smear  पर एक drop DPX  डालते है और smear पर cover slip रखके smear को permanent बना लेते है।


16)   अब हमारा Slide microscopic Examination के लिए तैयार है।   
(This is regressive nuclear staining technique of PAP Staining)


धन्यवाद



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