Concentration techniques

Unit 2: Principle and application of concentration techniques of stool for demonstration of ova and cysts

Concentration technique

The concentration technique is a procedure to increase the number of specific substances, parasites ova, cysts, etc in the sample. Concentration techniques make their detection easy. Sedimentation and floating techniques are commonly used as concentration techniques for ova (eggs) and cysts.

Principle of concentration techniques of stool for demonstration of ova and cysts

Stool sample suspension is used in the concentration technique. The difference between ova and cyst-specific gravity and stool debris-specific gravity is the basis of their concentration techniques. There are two concentration techniques for stool sample concentration to detect eggs and cysts in stool samples. These are the sedimentation method and the floating method.

Sedimentation method:

Principle: If the specific gravity of the ova and cyst is higher than the specific gravity of stool debris, then the ova and cyst will settle down in at the bottom of the sample container. This concentration technique is called the sedimentation technique.

Procedure:

1. Collect stool samples in the container.

2. Mix the sample with the floating solution such as sodium nitrate solution. Make a homogeneous suspension.

3. Pour the mixture into a centrifuge tube or sedimentation container.

4. Set aside for 30 minutes to 60 minutes without any disturbance. This is sedimentation time.

5. Pour off the supernatant liquid. The concentrated sample will be at the bottom of the container as sediment.

Formalin-Ether Sedimentation Method for Stool Concentration

The formalin-ether sedimentation method is a concentration technique used to improve the detection of ova (eggs) and cysts of parasites in stool samples.

Principle:

• · Formalin acts as a fixative, preserving the ova and cysts.

• · Ether dissolves fat and debris in the stool sample.

• · Centrifugation forces the ova and cysts to settle at the bottom of the tube.

Materials:

• · Stool sample

• · 10% formalin solution in saline

• · Ether (flammable, handle with caution in a well-ventilated area)

• · Centrifuge tube

• · Funnel with gauze mesh

• · Microscope slide and coverslip

• · Saline solution

• · Iodine stain (optional)

Procedure:

1. Emulsification and Fixation:

• o Mix a small amount of stool (pea-sized) with 10 ml of 10% formalin saline in a test tube.

• o Set aside the mixture and stand for 10-30 minutes to fix the parasites.

2. Straining:

• o Put a wet gauze mesh in a funnel. Pour the mixture through a funnel lined with wet gauze mesh into a centrifuge tube.

• o This removes large debris from the sample.

3. Centrifugation:

• o Centrifuge the solution in a centrifuge tube at a specific speed for 10 minutes.

• o This will cause the ova and cysts at the bottom of the centrifuge tube.

4. Ether Layer Addition:

• o Carefully pour off the supernatant without disturbing the ova and cyst.

• o Add 3-4 ml of ether to the centrifuge tube.

5. Second Centrifugation:

• o Centrifuge the mixture again at the same speed and time as above stated.

• o Ether will dissolve the remaining fatty material and debris. It will leave the ova and cysts at the bottom of the centrifuge tube.

6. Removing the Ether Layer:

• o Carefully decant most of the supernatant. It will leave a small amount of fluid above the ova and cyst.

7. Preparing the Wet Mount:

• o Resuspend the ova and cyst with a drop of the remaining supernatant or saline solution.

• o Transfer a small drop of the suspension onto a microscope slide.

• o Add a coverslip and examine under the microscope.

8. Optional Staining:

• o To enhance visualization, a drop of iodine stain can be added to the edge of the coverslip.

9. Microscopic Examination:

• o Systematically scan the slide under low power (10x objective) to locate ova and cysts.

• o Use high power (40x objective) for confirmation and identification of specific parasites.

Advantages:

• · Increases the sensitivity of parasite detection compared to direct smear examination.

• · Relatively simple and inexpensive technique.

Disadvantages:

• · Ether is flammable and requires careful handling.

• · Does not detect motile stages of parasites (trophozoites).

• · May not be suitable for all stool consistencies.

Safety Precautions:

• · Work in a well-ventilated area when handling ether.

• · Wear gloves and eye protection.

• · Dispose of ether according to institutional guidelines.

Floating method

Principle: If the specific gravity of the ova and cyst is less than the particular gravity of stool debris, then the ova and cyst will float in the sample container. Stool debris will be in the bottom of the container. This concentration technique is called the floating technique.

Procedure:

1. Collect stool samples in the container.

2. Mix the sample with the floating solution such as zinc sulphate solution. Make a homogeneous suspension.

3. Strain the suspension through a fine sieve or gauze to remove large stool debris.

4. Transfer the strained suspension into a specialized floating device.

5. Add more floating solution into the floating device to make a convex meniscus.

6. Place a cover slip on the upper surface of the meniscus.

7. Set aside for 10 to 15 minutes without disturbance. This is floating time.

8. Remove the coverslip gently.

9. The cover slip will have a concentrated sample.

Sodium chloride floatation technique:

This technique is useful for concentrating hookworms or Ascaris.

Reagent: Saturated solution of sodium chloride: Prepare saturated solution of sodium chloride(NaCl). The solution should have a specific gravity of 1.2.

Procedure:

1. Collect stool samples in the container.

2. Mix the sample with the floating solution such as NaCl solution. Make a homogeneous suspension.

3. Strain the suspension through a fine sieve or gauze to remove large stool debris.

4. Transfer the strained suspension into a specialized floating device.

5. Add more floating solution into the floating device to make a convex meniscus.

6. Place a cover slip on the upper surface of the meniscus.

7. Set aside for 10m to 15 minutes without disturbance. This is floating time.

8. Remove the cover slip gently.

9. The coverslip will have a concentrated sample

Author Dr Pramila Singh