Some Techniques for Processing of Stool Sample

Simple flotation approach

Principle

The precept for the easy flotation approach is similar to for the easy check tube flotation method.

Utility

This is another right approach to be used in preliminary surveys. Similarly, it is able to be used along with the McMaster method (see phase three.four.1) to discover low numbers of eggs (when present beneath the McMaster sensitivity of 50 eggs according to the gram of faeces).

Equipment

• two beakers or plastic containers
• A tea strainer or cheesecloth
• Measuring cylinder or other container graded by way of extent
• Fork, tongue blades or different form of stirring rod
• check tube (dry)
• Microscope
• Microslides, coverslips
• balance or teaspoon
• Flotation fluid (see the Appendix to this guide for system)

(a) positioned about three g of faeces (weigh or measure the faeces with a precalibrated teaspoon) into box 1.

(b) Pour 50 ml of flotation fluid into field 1.

(c) mix (stir) the contents very well with a stirring device (tongue blade, fork).

(d) Pour the ensuing faecal suspension via a tea strainer or a double-layer of cheesecloth into container

(e) leave the field to face for 10 minutes.

(f) Press a check tube to the lowest of the filtrate, carry it quick and switch some drops adhering to the floor to a micro slide.

(g) The test tube needs to contact the micro slide for as a minimum 2-four seconds for the drops to run off.

(h) Mount the coverslip on the micro slide for microscopical examination.

Sedimentation method (for trematode eggs)

Precept

The sedimentation method is a qualitative technique for detecting trematode eggs (Paramphistomum) within the faeces. most trematode eggs are tremendously big and heavy as compared to nematode eggs. This approach concentrates them in a sediment.

Application

This is a process to assess the presence of trematode infections. It's miles generally run only whilst such infections are suspected (from preceding postmortem findings on other animals within the herd/flock location) and is not run routinely. The system may be used to stumble on the liver fluke (Fasciola) and Paramphistomum eggs.

Device

• Beakers or plastic containers
• A tea strainer or cheesecloth
• Measuring cylinder
• Stirring device (fork, tongue blade)
• take a look at tubes
• check tube rack
• Methylene blue
• Microslide, coverslips
• balance or teaspoon
• Microscope technique

(a) Weigh or measure approximately three g of faeces into field 1.

(b) Pour 40-50 ml of tap water into box 1.

(c) blend (stir) thoroughly with a stirring device (fork, tongue blade).

(d) clear out the faecal suspension through a tea strainer or double-layer of cheesecloth into field 2.

(e) Pour the filtered cloth right into a test tube.

(f) permit to sediment for 5 minutes.

(g) get rid of (pipette, decant) the supernatant very carefully.

(h) Resuspend the sediment in five ml of water.

(i) permit to sediment for five mins.

(j) Discard (pipette, decant) the supernatant very carefully.

(ok) Stain the sediment via including one drop of methylene blue.

(l) transfer the sediment to a micro slide. cover with a coverslip.

Microscopical examination of prepared samples

The prepared samples on micro slides from the simple check tube flotation approach, the easy flotation method, and the sedimentation method are examined under a microscope at the magnifications indexed.

Desk magnification levels for examining prepared samples

Magnification Parasites

10 x 10 Nematode and cestode eggs

10 x forty Coccidia oocysts

10 x four Trematode eggs

Caution: In a case of a time delay in processing the pattern and analyzing the be counted, egg numbers might also decline dramatically. additionally, eggs may additionally change their look, becoming crenated and "ghost-like". it is therefore really helpful to put together only some samples at a time. those modifications may be averted by way of keeping prepared samples within the fridge after blending. the usage of the salt-sugar solution as flotation fluid additionally reduces the morphological modifications.

Quantitative strategies for separating and concentrating eggs/larvae

The simplest and only method for determining the variety of eggs or oocysts consistent with the gram of faeces is the McMaster counting method defined under.

McMaster counting method

Principle

The McMaster counting method is a quantitative technique to determine the number of eggs gift per gram of faeces (e.p.g.). A flotation fluid is used to split eggs from faecal cloth in a counting chamber (McMaster) with compartments. The method described beneath will come across 50 or extra e.p.g. of faeces.

Application

This technique may be used to provide a quantitative estimate of egg output for nematodes, cestodes, and coccidia. Its use to quantify tiers of infection is constrained via the elements governing egg excretion.

Equipment

• Beakers or plastic bins
• balance
• A tea strainer or cheesecloth
• Measuring cylinder
• Stirring tool (fork, tongue depressor)
• Pasteur pipettes and (rubber) teats
• Flotation fluid (see the Appendix to this guide for components)
• McMaster counting chamber
• Microscope

Process

(a) Weigh 4 g of faeces and location into container 1.

(b) add fifty-six ml of flotation fluid.

(c) mix (stir) the contents thoroughly with a stirring tool (fork, tongue blade).

(d) clear out the faecal suspension thru a tea strainer or a double-layer of cheesecloth into container 2.

(e) whilst stirring the filtrate in container 2, take a sub-sample with a Pasteur pipette.

(f) Fill both sides of the McMaster counting chamber with the sub-sample.

(g) permit the counting chamber to stand for five mins (this is important)

(h) observe the sub-sample of the filtrate under a microscope at 10 x 10 magnification.

(i) be counted all eggs and coccidia oocytes within the engraved region of each chamber.

(j) The number of eggs in keeping with the gram of faeces may be calculated as follows: upload the egg counts of the two chambers together.

Multiply the whole with the aid of 50. This offers the e.p.g. of faeces. (instance: 12 eggs visible in chamber 1 and 15 eggs visible in chamber 2 = (12 + 15) x 50 = 1350 e.p.g.)

(k) inside the event that the McMaster is negative (no eggs visible), the filtrate in container 2 can be used for the easy flotation approach.

Caution: In a case of a time postpone between processing the sample and reading the rely upon, egg numbers may additionally decline dramatically. additionally, eggs may additionally trade their appearance, turning into crenated and "ghost-like". it's far consequently recommended to prepare just a few samples at a time. these changes can be averted through retaining prepared samples within the fridge after blending. the use of the salt-sugar solution as flotation fluid additionally reduces the morphological changes.

References:

D greenwood, Slack RCB and J Peutherer.Medical microbiology.2001.

JG College, AG Fraser and BP Marmion.Practical Medical microbiology.Fourteenth Edition. Churchill Livingstone, 1996.

JP Micheal, ECS Chan and NR Krieg.Microbiology.Fifth Edition. Delhi: Mcgraw-hill, 1993.

M Cheesbrugh.Medical laboratory manual for tropical countries.London, 2007.