=====10.SOP FOR AFB STAIN FOR LEPRAE BACILLI=====

1.**	PURPOSE OF EXAMINATION:** To lay down the procedure ZN stain for leprae bacilli in Histo-pathology section at NCHLS Surat.

2.**	PRINCIPLE AND METHOD OF PROCEDURE USED FOR EXAMINATION:** Acid fast bacilli cell wall contains waxy lipid, mycolic acid. Phenol and carbol fuscin helps dye to pass through lipid wall and attach with negatively charged ions present in bacteria. After staining with help of reducing substance cells are decolorized except acid fast bacilli.

3.**	STAFF RESPONSIBLE:** lab. technician under supervision of resident doctor/tutor.

4.**	PERFORMANCE CHARACTERISTICS:** NA

5.**	TYPE OF SAMPLE:** Histopathology section slide

6.**	PATIENT PREPARATION:**NA

7.**	TYPE OF CONTAINER AND ADDITIVES:**

8.**	POSITIVE CONTROL:** positive control for AFB.

9.**	REQUIRED EQUIPMENT AND REAGENTS:**

**EQUIPMENTS:** Coplin jars, funnel, burner, what man filter paper no. 4 

**REAGENT:** Ground nut oil, 10% sulfuric acid, Basic fuchsin, Absolute alcohol, 5% phenol, Harris hematoxylin, xylene, DPX

10.**	ENVIRONMENTAL AND SAFETY CONTROL:**

11.**	CALIBRATION PROCEDURE (METROLOGICAL TRACEBILITY):**NA

12.**	PROCEDURE(STEPS):**

**Carbol fuchsin preparation:**

•	Take Basic fuchsin 1 gm, 10 ml absolute alcohol and 5% phenol 100ml.

•	Dissolve basic fuchsin in absolute alcohol and then add phenol in it.

**1 % acid alcohol preparation:**

•	Take concentrated HCL 10 ml add this in 90%alcohol 990ml. mix well and label it

**1% methylene blue preparation:**

•	Take 1 gm methylene blue powder and mix in 100 ml distilled water. Filter in glass bottle 

**Test Procedure:**

•	Take test slide on which staining has to be performed along with positive control. 

•	Deparaffinize section with a mixture of 2 part xylene and 1 part of ground nut oil and keep it in oven for 10 minute and clear it with 2 change of xylene each for 15minute.

•	Blot with filter paper until of semidry appearance.

•	Wash section in water for 5 minute.

•	Stain with carbol fuchsin for 30 minute at room temperature.

•	Wash in tap water and blot with filter paper.

•	Decolorize with 1% acid alcohol.

•	Wash in water.

•	Counter stain with 1% methylene blue for 1 dip.

•	Dehydrate in alcohol, clear in xylene, mount with DPX

**Result**

•	M. Leprae: Red

•	Nuclei and background:  blue

13.**	QUALITY CONTROL PROCEDURE:** The newer batch of stain is tested before being put in to use by checking 
their efficiency with the batch of stain already in use.

14.**	INTERFERENCE:** Inadequate quality/improper dilution

15.**	PRINCIPLE OF PROCEDURE FOR CALCULATING RESULTS INCLUDING, WHERE RELEVENT, THE MEASUREMENT OF UNCERTAINITY OF MEASURED QUANTITY VALUES:**NA

16.**	BIOLOGICAL REFERENCE INTERVALS OR CLINICAL DECISION VALUES:**NA

17.**	 REPORTABLE INTERVAL OF EXAMINATION RESULTS:** NA

18.**	INSTRUCTION FOR DETERMINING QUANTITATIVE RESULTS WHEN RESULTS IS NOT WITHIN THE MEASUREMENT INTERVAL:** NA

19.**	ALERT /CRITICAL VALUE, WHERE APPROPRIATE:** NA

20.**	LABORATORY CLINICAL INTERPRETATION:** interpret test result after true positivity of control slide. 

21.**	POTENTIAL SOURCE OF VARIATION:** NA

22.**	REFERENCE:** Histopathological laboratory techniques by Culling’s.