Concentration Techniques

Light Infection

Number of parasites present in the sample is low

Concentrate

To gather or aggregate the sample to increase the chances of recovering parasites

Aggregate parasites present into a small volume of the sample and to remove as much debris as possible

Purpose of Concentration techniques

Specific gravity

ratio or comparison between the density of a reference material and the density of water

• Flotation Methods

• Sedimentation Methods

Concentration Methods

• Brine Flotation Method

• Zinc Sulfate Flotation Technique

• Sheather’s Sugar Flotation Methods

Enumerate all Flotation Methods

Less dense

In Flotation technique, parasites are _____ than the solutions

• Zinc sulfate

• Magnesium sulfate

• Brine

• Sugar

Reagents use in Flotation techniques

1.18-1.20

Flotation techniques reagent Specific gravity

At the top or at the surface of the vial

Where do we recover the parasites under floatation techniques?

They have higher specific gravity

What is the common characteristics of all reagents in flotation techniques?

1.05 - 1.23

Usual specific gravity of parasites

distortion and shrinkage

Exposure of parasites to high specific gravity will cause ________ and ______ of protozoan cysts and thin-walled nematode eggs after 20 min.

Both have bipolar plugs

What is common between the eggs of Trichuris and Capillaria ?

Fasciola spp. and Fasciolopsis spp.

Examples of Large eggs that will not float?

Brine Flotation Method

Identify the Technique in the set up:

• Stool samples

• Clean glass slides

• Coverslips

• Microscope

• Applicator stick

• Marking pens,

• Vial

• Saturated brine solution

• NaCl - 40g

• Distilled water - 100 mL

• Check for SG (1.18-1.20)

Composition of Saturated brine solution

Low cost and simple

Advantage of Brine Flotation Methods

• Helminth eggs of Hookworm and Schistosoma become badly shrunken

• Not useful for operculated (Clonorchis, Opistrochis and Heterophyids)

Disadvantage of Brine Flotation Method

Recovery of both helminth eggs and larvae and protozoan cysts

Purpose of Zinc Sulfate Flotation Method

33% Zinc Sulfate Solution

Main reagent used for Zinc Sulfate Flotation Technique

1.18

Specific Gravity used for fresh stool

1.20

Specific gravity used fo preserved stool

Zinc Sulfate Flotation Method

Identify the technique in the set-up:

• Stool samples

• Clean glass slides

• Coverslips

• Marking pens

• Microscopic

• Applicator stick

• Centrifuge

• Test tubes

• Gauze

• Zinc Sulfate (dry crystal) - 330g

• Distilled water - 670 mL

Composition of Zinc Sulfate Solution

To remove the bigger clamps of stool samples

What is the purpose of straining fecal suspension?

Sugar boiled with phenol

What reagent does Sheather’s Sugar Flotation Technique use?

1.27

specific gravity for Sheather’s Sugar Flotation Technique

Sheather’s Sugar Flotation Technique

Considered best for the recovery of coccidian oocysts mainly Cryptosporidium

Sediment Techniques

Parasites are concentrated in the sediment of the tube following centrifugation.

Lower specific gravity

In Sediment Techniques, Reagents have ______ than parasites

• Simple Gravity Sedimentation Technique

• Acid Ether Concentration Technique (AECT)

• Formalin Ether Concentration Technique (FECT) / Formalin Ethyl Acetate Concentration Technique (FEACT)

• Merthiolate Iodine Formalin Ether Concentration Technique (MIFECT) / Methiolate Iodine Formaldehyde Concentration Technique (MIFCT)

• Baermann Concentration Technique

Enumerate Sedimentation Techniques

Stool + tap water —> mix —> allow parasite to settle down by gravity

Procedure of Simple Gravity Sedimentation Technique

Schistosomoma spp. eggs

Will hatch quicky in tap water

Add Normal Saline solution

Remedy to prevent Hatching of Eggs for Schistosoma

Glycerated water ( 0.5 % solution of glycerin in tao water)

Enhance sedimentation or settling of parasites

Advantage : Minimal use of glassware, reagents and equipment.

Disadvantage: Time consuming

Simple Gravity Sedimentation Technique Advantage and disadvantage

Hydrochloric Acid is used to dissolve albuminous material; recommended for the recovery of Trichuris, Capillaria, and Schistosoma

Principle of Acid Ether Concentration Technique

Acid Ether Concentration Technique

Method of choice if stool material comes from animals like cats and dogs

Acid Ether Concentration Technique

Identify the Technique in the set up:

• Stool Samples

• Clean glass slides

• Coverslips

• Marking pen

• Teat tube rack

• Microscope

• Applicator stick

• Centrifuge

• Gauze pad

• Rubber stopper

• Dropper/pipette

• Ether/ethyl acetate

• 40 % HCL

Ether

Dissolve neutral fats/lipids and carbohydrates

• Concentrated hydrochloric acid - 40mL

• Distilled water - 60 mL

Composition of 40% HCL

Identify the layers pointed

Recovery of both helminth eggs and protozoan cysts

Purpose of Formalin Ether Concentration Technique/ Formalin Ethyl Acetate Concentration Technique

Formalin-preserved samples

More parasites and be recovered from _________

Loss of parasite to plug of debris

FECT/FEACT disadvantage

Explosive and Flammable

Disadvantage of using ether

Formalin Ether Concentration Technique

Technique if reagent used is ether?

Formalin Ethyl acetate Concentration techniques

Technique if reagent used is ethyl acetate

• 10% Formalin

• Ether / Ethyl acetate

Main reagent for FECT / FEACT

Formalin Ether Concentration Technique/ Formalin Ethyl Acetate Concentration Technique

Identify the technique in the set up:

• Stool Samples

• Clean glass slides

• Coverslips

• Marking pen

• Teat tube rack

• Microscope

• Applicator stick

• Centrifuge

• Gauze pad

• Rubber stopper

• Dropper/pipette

• Ether/ethyl acetate

• 10% Formalin

• Concentrated formaldehyde - 10mL

• Distilled water 90mL

Composition of 10% Formalin

Identify the layers

Better Identification of protozoan cysts

What is Merthiolate Iodine Formaline Recommended for?

Merthiolate Iodine Formaldehyde concentration Technique (MIFCT) / Merthiolate Iodine Formalin Ether Concentration Technique (MIFECT)

Identify the technique in the set up:

• Stool Samples

• Clean glass slides

• Coverslips

• Marking pen

• Teat tube rack

• Microscope

• Applicator stick

• Centrifuge

• Gauze pad

• Rubber stopper

• Dropper/pipette

• Ether/ethyl acetate

• Lugol’s iodine solution

• Stock Merthiolate Formaldehyde solution

• Iodine powdered crystals - 5g

• Potassium iodine - 10g

• Distilled water - 90mL

Composition of Lugol’s idoine solution

• Distilled water - 50 mL

• Formaldehyde (USP) - 5 mL

• Tincure of Merthioate - 50 mL

• Glycerin - 1

Composition of Stock Merthiolate Formaldehyde solution

Merthiolate Iodine Formaldehyde concentration Technique (MIFCT) / Merthiolate Iodine Formalin Ether Concentration Technique (MIFECT)

Identify layers and What technique

Baermann Concentration Method

Using a funnel apparatus, active larvae migrate from a fresh specimen that has been placed on a wire mesh with several layers of gauze, which are in contact with tao water.

Examination of stool specimens suspected of having small numbers of Strongyloides larvae

Baemermann concentration technique is used for ?