AN ASSIGNMENT ON SAMPLE COLLECTION, OBSERVATION & TESTS RELATED TO COPROSCOPY

Collection of fecal sample: Collection site: Directly form rectum Freshly voided feces Fresh drooping (individual bird) or pooled samples (Whole flock)

Amount of faeces: 5g/ large animal (approximately the size of an adult man's thumb) Collection equipment: Specimen vial/Container /Screw cap bottles/ Plastic containers with lids Gloves Plastic bags Wooden tongue blades / spatula/ stick. Cool box (for transportation) Labeling: Owners name Animal ID Age, Sex Date and time, etc Pen marker for labeling

Precautions: Fresh faeces should be collected. Sample should be packed and dispatched in a cool box Filling the container to capacity or tightening the sleeve/gloves as close to the faeces as possible Add 3% or 10% formalin (5-20ml depending on the volume of the sample)

Refrigerate the sample (4 0C) for no more than 24 hours until they are processed. Best result will be found if sample is examined within 3wks of time.

Formalin fixed faeces cant be used for feceal culture.

Sample should never be kept in the freezer. Pooled sample: Samples form pigs, feedlot cattle, poultry or other grouped animals are often pooled samples. In this case several fecal samples are collected from pen without the specific animal of origin being known. This samples will represent the whole group.

If a herd is being examined samples should be collected from several different randomly selected or worm affected animals, depending on circumstances. Faeces may contain hazardous pathogens (bacteria, viruses etc). Appropriate hygiene and safety procedures should be employed during collection.

Gross examination of feces:

1. Consistency: (Condition of the feces) 2. Color: 3. 4. Mucus: 5. It indicates intestinal parasitism (Entamoeba, Balantidium, Gierdia) or other metabolic diseases. Gross parasites: Gross parasites/ portions of parasites/ larvae /proglottids of cestode/ Larval arthropods (bots) Gastrophilus sp./ maggots can be found. Light gray feces may indicate excessive fat in feces (A sign of poor intestinal absorption) Dark red or black feces indicate the presence of blood. Blood may indicate different parasitism along with other pathological conditions. Blood in fresh feces Soft, watery (Diarrheic) Very hard (Constipation)

Microscopic Examination of Faces:

Fecal sample

Qualitative test

Quantitative test

Direct Method Direct Smear Flotation

Indirect Method McMaster Technique Sedimentation

Simple Flotation

Test tube Flotation

Centrifugal Flotation

Simple Sedimentation

Centrifugal Sedimentation

Qualitative Tests: Equipment required for Qualitative tests:

Equipment:

Beakers or plastic containers Tea strainer or double layer of cheesecloth Measuring cylinder Fork, tongue blades or stirring rod Test tubes Test tube rack Methylene blue 1% solution or Malachite green 1% solution Microscope slides and coverslips Pipettes Balance or calibrated teaspoon Microscope Flotation fluids 1. Saturated salt solution Specific gravity: 1.18 - 1.20 Formula: Sodium chloride: 400 g Water: 1000 ml

2. Salt/sugar solution Specific gravity: 1.28 Formula: Sl. No. 01. 02. 03. 04. 05. 06. Sodium chloride: 400g Sugar: 500 g Water: 1000 ml Floatation Solution Saturated Salt Solution Salt Sugar Solution Sodium Nitrate Solution Saturated Sugar Solution Magnesium Sulfate Solution Zinc Sulfate Solution Specific Gravity 1.18-1.20 1.28 1.18 1.27 1.2 1.364

1. Direct Method Principle: Helminth eggs and larvae can be identified in a thin smear of faeces on a microscope slide. Fecal smear Equipment: Microscope slides Cover slips Saline solution (0.85%) or water Procedure: 1. Smear a small quantity of faeces on a clean microscope slide. 2. Mix with a few drops of water or physiological saline until it being homogenized. 3. Place a coverslip over the smear. 4. Observe under microscope (first at 10X and then 40X magnification) .

Wrong!

Right

Figure: Pattern of movement of microscopic field for thorough examination of area under cover slip. Advantages: If very small samples are available/ lack of equipments/ lack of time. Rapid examination of large number of samples.

Disadvantages: Fail to detect low grade infection & missing eggs are high. It reduces the chance of finding parasite eggs or larvae or protozoon cysts as scanty faeces are used.

2. Indirect methods: Purpose: To concentrate eggs so that light infection can be detected To save time by eliminating the troubles caused by large undigested fecal particles. Floatation Techniques : Purpose: To float the ova or cyst having higher specific gravity For the detection of Nematode & cestode eggs. Mostly coccidial oocysts

Simple floatation: low no of eggs. Procedure: 1. 2. 3. 4. 5. 6. 7. Put approximately 3g of feces into container 1 Pour 50ml of floatation fluid into container 1 Mix (Stir) the contents thoroughly with a stirring device. Pour the resultant fecal suspension through a tea strainer or double layer of cheese cloth in to container 2 Leave the container to stand for 10 minutes. Press a test tube to the bottom of the filtrate, lift it quickly & transfer a few drops adhering to the surface to a micro slide. Mount the cover slip on the micro slide for microscopic examination. This is also a good technique for the initial survey. it can also be used in conjunction with McMaster technique to detect

B. Test tube floatation: Procedure: 1. 2. 3. 4. 5. 6. Approximately 2 to 5g of feces is put in to a suitable container. 50ml of floatation fluid put on directly to the specimen (fecal sample) Mix thoroughly by a stirring device (tongue blade/ fork). Pour the resulting suspension through a tea strainer in to another container. Pour the fecal suspension into a test tube until a meniscus is formed. A glass cover slip is placed over the meniscus &

7. Allowed to stand for 15 - 30 minutes depending on the of floatation fluid 8. Carefully lift off the cover slip from the tube, immediately place the cover slip on a microscope slide. 9. Observe under microscope. C. Centrifugal flotation: The centrifugal floatation procedure more efficiently recovers parasite eggs, cyst & requires less time than the floatation procedure.

It does require a centrifugal capable of holding 15ml test tubes & producing a centrifugal force of 400X to 650Xg.

Procedure: 1. 2. 3. 4. 5. Take 1 tsp of feces with enough water to make a semisolid suspension. Pour the resultant fecal suspension through a tea strainer. Transfer the contents into a 15ml centrifuge tube (Test tube). Centrifuge for 5 minutes at 400X to 650Xg. or 1500 rpm. (Rotation/ revolution per min). Add concentrated floatation solution within 1/2 to 3/4 inch of the top of the tube & resuspend

5. Decant the supernatant the sediment using a stirring action with a wooden applicator stick. Insert a rubber stopper & mix by four or more invasions, so that the solution is thoroughly mixed with the sediment.

Sedimentation technique:
Principle: The majority of trematode eggs are too large and heavy to float reliably in the flotation fluids normally used for nematode eggs. They do however sink rapidly to the bottom of a faecal/water suspension and this is the basis of the faecal sedimentation technique. Objective: For identifying trematode eggs

Fig: Sedimentation Technique Procedure: 1. 2. 3. 4. Weigh or measure approximately 2 - 3 gm of feces into a container. Pour 40- 50ml of tap water into the container. Mix (Stir) thoroughly with a stirring device. Filter the fecal suspension through a tea strainer

5. 6. 7. 8. 9.

Pour the filtered material into a test tube. Allow to sediment for 5 minutes / 20-30 minutes or centrifuge at 1500 rpm. (5 minutes) Discard the supernatant very carefully. Resuspend the sediment in 5ml to water & allow to sediment for 5 minutes. Remove the supernatant very carefully.

10. Transfer a small amount of the top layer of sediment to a microscope slide. If the drop is too thick dilute it with a drop of water. ( Lugol's iodine solution diluted 1 : 5 in water) may be used for dilution instead of water, to aid in identification of protozoan cyst. 11. Apply a cover slip & observe under microscope. Note: Sedimentation is primarily used to detect eggs or cysts that have too high a specific gravity to float or they would be severely distorted by floatation solution. Mainly used for trematodes egg detection.

Quantitative Tests:
Objectives: For Determination of degree of infection. McMaster technique: Materials: Beaker or Plastic container Floatation Solutions McMaster counting chamber 0.15mm wire mesh screen/Tea strainer Pasteur pipette

Procedure: 1. 3gm of feces was taken and mixed with 45ml of water in a plastic jar. 2. Shaking the mixture for making an even suspension. 3. Filtration with a filter having aperture of about 0.15-0.25mm. 4. 15 ml of above mixture is taken in a centrifuge tube. 5. Centrifugation at 1500rpm for 3 minutes. 6. Discard or pour off of supernatant. 7. Flotation fluid (saturated salt solution) was added up to 15ml mark.

8. Mixing by inverting the tube 3-6 times. 9. Filling the chamber of McMaster slide with the suspension with the Pasteur pipette. 10. Repeating the process of inversion and filling another chamber. 11. Leaving the slide for 3-5 minutes. 12. Mounting the McMaster slide on microscope and counting the eggs.

Add Dilution Method for McMaster Counting Technique:

Solution: 3gm of feces = 45ml of water 1gm of feces = 45/3ml of water = 15ml of water Volume of McMaster chamber: Volume of 1 chamber = 1.5mm (height) 10mm (length) 10mm (width) = 150cmm = 150/1000cc =0.15cc = 0.15ml So, Volume of 1 chamber = 0.15ml Volume of 2 chambers = (0.15 2)ml = 0.30ml Multiple Factor/ Dilution Factor: For 1 chamber = 15/0.15 = 100 For 2 chambers = 15/0.30 = 50 (1cc = 1000cmm)

Calculation of counting:
If count 1 chamber, EPG = Total egg 100 If count 2 chamber, EPG = Total egg 50

Fig: Eggs on McMaster Slide (Schematic)

Fecal culture:
Fecal culture is used in diagnostic parasitology to differentiate parasites whose eggs and cysts cannot be distinguished by examination of a fresh fecal sample. To distinguish between then feces containing eggs are allowed to incubate at room temperature for several days while the larvae hatch from the eggs. Objectives: To provide suitable conditions for the hatching of eggs and larval development of the infective third stage (L3). The third stage larvae can be recovered by the Baermann technique and identified to genus level. Equipment Fork, spoon, tongue depressor, spatula Water Jars, containers Charcoal (dried (heat at 70oC), sterile bovine faeces may be used if charcoal is not available). Once sterilized the sample is ground to make fine powder. Incubator

Fig: Fecal Culture Technique Procedure: 1. 2. 3. 4. 5. Placing 20-30gm of fresh fecal sample in a jar. Break up the feces with a tongue depressor and moistened slightly with tape water. The mixture should not be so weight as to appear soupy. Placing the far on a shelf away from direct sunlight and allow it to incubate at room temperature for 7 days. The moisture of the sample should be maintained by adding water routinely.

6. Isolation of larvae through Baermann Apparatus technique.

Baermann technique:
The Baermann technique is used to recover the larvae of nematodes (roundworms) from feces, soil or animal tissues.

Principle:
The Baermann technique is based on the active migration or movement of larvae. Faeces are suspended in water, the larvae move into the water. They sink to the bottom and can be collected for identification

Purpose:
The Baermann technique is used to separate larvae from faecal material. For example: o Diagnosing lungworm infection o The identification of third stage larvae [L3] from a faecal culture. Funnel size according to need Dropper Funnel stand Small petri dishes Equipment List: Rubber or plastic tubing Scissors Clamp or spring clip Spoon or spatula Cheesecloth or dental napkin/Gauge Rubber band or length of cloth string Thin stick or metal rod Jug or flask Strainer Microscope slides and Microscope coverslips Test tube Iodine

Procedure: 1. 2. 3. 4. 5. 6. 7. Spread a piece of cheese cloth or a gauge Placing 5 -15 gm of the faeces/fecal culture/ fecal soil/tissue sample on the above. Make a pouch showing in the figure and hang it in the funnel with the help of a stick. The funnel is then filled with water or physiologic saline at about 300C to a level 1-3 cm above the sample. Allow the apparatus to remain undisturbed overnight. Hold a glass microscope slide under the cutoff pipette and open the pinch clam long enough to allow a large drop of fluid to fall on the slide. Apply a cover slip to the slide and examine it microscopically for the presence of larvae.

8. 9.

OR, Collect the fluid from the funnel and centrifuge to collect the sediment. Add iodine in the sediment and examine under microscope as above.

Fig: Procedure of Baermann Technique

add

Micrometry:
Micrometry is the microscopic measurement (length & width) of parasite, parasitic eggs, oocyst, larvae as well as virus, bacteria, spore etc. There are 2 scales: Ocular micrometer: It is used for measuring parasite, parasitic eggs, oocyst, larvae as well as virus, bacteria, spore etc. It has 05-100 calibrations. Stage micrometer: It helps Ocular micrometer to calculate a constant for measuring. It has 100 divisions.

Procedure:
Place the an ocular micrometer on eye piece and stage micrometer on stage of a compound microscope and focus on the scale. Adjust the stage micrometer so that the 0 line on the ocular micrometer is exactly lined up on top of the 0 line on the stage micrometer.

When these two 0 lines are lined up and look to the right of the 0 lines for another set of lines superimposed on each other. Now, Calculate the distance of 1 ocular mark/unit. Remove the stage micrometer and set desirable slides containing egg, oocyst, parasites etc.

Calculate the factors as follows:

100 divisions of stage micrometer = 1 mm 1 ,, ,, ,, ,, = 0.01 mm We know that, 1 mm = 1000m 0.01mm = (10000.01)m = 10m So, 1 division of stage micrometer = 10m Imagine that, 83 divisions of Ocular micrometer = 21 divisions of Stage micrometer 1 ,, ,, ,, ,, = 21/83 ,, = (21/83 10)m = 2.53m Now placing of suspected slide under microscope and measuring by following calculation: Length of egg/ parasite/ oocyst/ larva = [X line ( No. of calibration)2.53]m Width of ,, ,, ,, ,, = [Y line ( No. of calibration)2.53]m ,, ,, ,,

Fig: Measuring Technique of an Oocyst