Microscopic Examination 1. PARA.LAb 3rd.

microscopic examination of fecal under microscope

1. Microscopic Examination Dr. Jawad Younas

2. Fecal Examination Principle/Objective: Microscopic techniques are more accurate than macroscopic or physical examinations.Helps in diagnosis of many intestinal and rectal parasitic diseases.Perform to check the intestinal conditions of the animals body. Microscopic technique is reliable in the most cases.Having two method. Direct method Indirect method

3. Direct Method Objective: Simplest and cheapest to facilitate the detection of intestinal parasites(helminths) that pass their infected subjects into faeces.Smallest fecal quantity is required (about 2-5 g). Contamination of stool with urine must be avoided. Material: Physiological sayline Microscopic slide Applicator sticks/Tooth Picks Cover Slips

4. Direct Method Procedure: Take a clean and dry glass slide, which is free from scratches. Take a drop of distilled water in the middle of glass slides and add small amount of feces on it. Mix it well with tooth pick/Match stick. Spread thinly and evenly and cover the cover slip. Examine smear under microscope for parasitic larvae or eggs.

5. Direct Method Precautions: A satisfactory film should slightly opaque and allow newsprint to be read through it. At least 3 smear form for negative results. A drop of iodine may be used to highlight protozoa. Remove any large parasite with forcep and the place coverslip over the smear.

6. Direct Method Hymenolepis Nana Egg showing hooklets Tinea Saginiata egg with Hooklets

7. Indirect Method It has Two Types. Floatation Sedimentation

8. Indirect Method Floatation Method Principle: Floatation based on the difference in specific gravity of parasitic eggs,larvae,cyst,debris and that of floatation solutions. Floatation solution usually have specific gravity in between 1.200-1.300. So fecal materials which has specific gravity greater than 1.200 and 1.300, does not float. Most parasite have specific gravity slightly higher than 1.00. Therefore,parasites are too heavy to float in tap water. This is the result we use floatation solution than tap water.So that parasite eggs can float on liquid and other large particle of feces sink at the bottom of floatation solution.

9. Indirect Method 1)-Simple Floatation 2)-Centrifugal Floatation 1) Simple Floatation Materials: Test tube or cylinder Floatation solution Applicator stick Tea stainer/Cloth Beakers

10. Indirect Method 1) Simple Floatation Procedure: About 1g of faces taken in beaker and mixed with few ml (15-20ml) of distilled water. Then this mixture is filter with cloth. The filterate is then mixed with 4-5ml of saturated salt. Then placed in test tube and filled up to the top . A clean glass slide is placed over the test tube left for 30-60mint at room temp with out any disturbance. After that,Cover slip or glass slide is removed and examine under microscope for the presence of parasitic ova.

11. Indirect Method 2)Centrifugal Floatation Materials: Centrifuge Machine Centifuge tubes Patinum loop or glass rounded-end rod.

12. Indirect Method 2)Centrifugal Floatation Procedure: 1g of feses mixed with distilled water in beaker. Filter through muslin cloth. filtered material is then mixed with sugar solotion in a ratio of in test tube. Mix the content and centrifuge at 1500rpm for 5 mint. The tube is then out and placed on stand without any disturbance in test rack. Transfer small amount of superficial contents in clean and dry glass slide. Examine under microscope for parasitic ova.

13. Indirect Method Sedimentation Method Principle:- This is based on the difference in specific gravity of parasitic eggs,cysts,larvae,faecal debris and that of sedimentation solution to make the eggs settle down, a liquid with lower specific gravity than that of eggs must be used e.g water Sedimentation is of two types 1) Simple sedimentation 2) Centrifugal sedimentation

14. Indirect Method 1) Simple Sedimentation Materials:- Same as for simple floatation Procedure:- 5g of faeces is mixed with small amount of tap water in beaker(250ml). Fill the beaker with tap water and strain suspension through cheesecloth in conical flask. Let settle it for 20-30 mint. Decant supernatant and continue washing process until supernatant is clear. Take a drop from top layer of sediment with Pasteur’s pipette on microscopic slide. Apply a cover slip to the drop and examine microscopically.

15. Indirect Method 2) Centrifugal Sedimentation. Material:- Same as for centrifugal floatation Procedure:- Same as for simple sedimentation but centrifuge the sample at 1500-2000 rpm for 5mint.

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17. Quantitative Faecal Examination To obtain more accurate information with regard to the severity of an infection,egg-counting method have been devised. 1) MC Master Technique 2) Stoll’s egg counting technique

18. McMaster egg method Objective:- It is a method used for determining the no. of eggs per gram of faeces in order to estimate the worm burden in an animal. Materials:- Mc Master counting chamber slide Beakers Applicator Test tube Test tube holder Sugar Flotation solution Graduated cylinder 1ml pipettes

19. McMaster egg method Procedure:- Weight out 2gram of faeces. Pass the faeces through a sieve into a dish containing 60ml of ZnSo4 saturated salt solution left the sieve and hold over the dish.Push out any remaining solution from the faeces. While stirring vigorously take a sample of mixture with a pipette and transfer it to one of the chamber of Mc Master slide. Repeat the procedure and fill the other chamber. Wait 30 sec and then count the total no.of eggs under both of the etched area of slide. Multiply total no.of egg in 2 chambers by 100, this is EPG(egg per gram).

20. McMaster egg method Mathematics:- Volume of each chamber = 0.15ml Volume of slide = 0.3ml Since you started with 2g of feces and the multiply by 100, The final result is EPG of faeces. Uses:- Count done before and after arthalmetic treatment allow you to monitor for drug resistance. Counts done between scheduled treatments allows you to monitor the worm lead and thus allow to change the treatment if necessory.

21. Stoll’s Egg counting Method Objective:- The stoll’s egg counting technique is a method for determining the number of trematode and nematode eggs per gram of feces in order to estimate the worm burden in an animal. Materials:- Petri dishes Beaker Distilled water Sieve (strainer)(chalni in urdu) Stirrer (vibrator) Glass slide Tongue depressor (medical instument depress the tongue)

22. Stoll’s Egg counting Method Procedure:- Weight out 3g of feaces. Measure out 42ml of water and place it into a dish using a tongue depressor, push the 3g feaces through a sieve into the water lift the sieve and hold over the dish push out remaining water from the feaces. While stirring the water feaces mixture ,take 0.15ml of suspension and spread over 2 slides. cover each slide with a long coverslip . Examine both slides for worm eggs.The total amount of eggs per gram of feaces.

23. Stoll’s Egg counting Method Mathematics:- 0.15ml is 1/300 of 45ml(42 ml water + 3g feaces) so no.of eggs in 0.15ml*100 is equal to 1/3 of total no.of eggs in orignal 3gram.