Wednesday, September 30, 2015

Lab Series# 7: O&P examination of Stool Samples

Greetings,

In terms of parasitic diseases, an awful lot of parasites effect the gut. The diagnosis often requires nothing more than a simple stool examination. This methodology is in use for detection of cysts or trophozoite forms in case of protozoa infections, and eggs or proglottids of helminthic infections.

* I have previously posted this material in my website as notes. However, due to personal requirements, I had taken down the website domain. Though this is still available in parent webpage, many are not able to access it. So I decided that this post will lie down here so, as to enable access. 

Specimen collection and transport of stool:

The specimen is collected in a sterile container, or in a clean wax paper for which a portion most likely to contain parasitic stages (Bloody, mucoid regions and areas of loose or watery consistency.). A loose stool needs to be collected in containers only and labeled accordingly. The patient is advised not to contaminate the fecal matter with urine or water as it can be lead to false results. Ideally, the formed and semisolid stools should be examined within 1 hour of stool collection and loose stools within 30 min. If the patient has ingested materials such as antacids, oily laxatives, barium etc then stool should be collected after clearing.

If delay is expected in stool examination, then the sample can be preserved in fixatives depending on the examination required. Polyvinyl alcohol is a colorless, water soluble synthetic resin. It is used as preservative for stool collection by 2 vial technique. The method employs collecting of stool in 2 vials. The first portion is fixed with 10% formalin and the other in 3 parts of PVA. A modified LV (Low viscosity)- PVA is recommended for preserving fecal specimens for permanent staining procedures. The low viscosity formulation allows for better fixation due to faster penetration and drying. However PVA and its derivatives contain mercuric chloride which is hazardous. Another derivative- ZS PVA (Zinc sulfate based PVA) is also in commercial use.

Incomplete removal of mercuric chloride (liquid Schaudinn’s fixative) may cause smear to contain highly refractive crystals or granules, which may prevent detection or identification of any organisms present. The 70% ethanol-iodine solution removes the mercury from the slide; the subsequent alcohol rinses then removes the iodine Other preservation systems include MIF and SAF. SAF has an advantage of usability in wet mounts, concentration and permanent staining. The fixative capacity is however less compared to that of others.

Photo 1: SAF containers.
100 ml of SAF contains
  • Sodium acetate - 1.5 grams 
  • Glacial Acetic acid - 2.0 ml 
  • Formaldehyde, 40% - 4.0 ml 
  • Distilled water – Make up to 100ml 
Examination of minimum of 3 specimens, collected every alternate day is considered as necessary for an adequate O and P evaluation. In addition, if purgatives (such as sodium sulfate and buffered phosphosoda) are administered, it should be rapidly processed.

Macroscopic examination of stool sample:

The stool sample is checked for its labeling and identified. Macroscopic features such as consistency, evidence for blood and mucus, larval or adult forms is noted. Stool samples with watery consistency are more likely to contain trophozoite and formed stools predominantly contain cystic forms of protozoa.


Microscopic examination of stool sample:

A) Direct wet mount

It is the simplest and easiest technique. A wet mount can be prepared directly from fecal material or from the concentrated specimens. The basic types of wet mounts that should be made from each sample include:

1. Saline wet mount:
Photo 2: Iodine wet mount of stool
showing oocyst of Cyclospora spp.
in unconcentrated stool.

It is used to detect worm eggs or larvae, protozoan trophozoite and cysts. In addition it can reveal the presence of RBCs and WBCs.

2. Iodine wet mount:

It is used to stain glycogen and nuclei of the cysts.

Procedure
  • Place a drop of saline on one half of the slide and one drop of iodine on the other half. 
  • With an applicator stick pickup a small portion of the specimen and mix with saline drop 
  • Similarly pickup similar amount and mix with a drop of iodine 
  • Put the cover slip separately on both and examine under the microscope.
Advantages:
  • Easy to perform and reveals all types of parasitic forms 
  • Also gives an estimate of WBC and cellular contents in the sample.

B) Stool concentration technique:

Low concentrations of parasitic forms are often undetected by direct microscopy. In such cases a concentration technique is employed. It is classified into 2 types
  1. Flotation technique 
  2. Sedimentation technique
Fecal flotation technique:

The principle of flotation technique is to mix the eggs with a solution whose specific gravity is more than that of the eggs. Hence the eggs float in the solution.

Table 1: Parasitic eggs and
their specific gravity.
1. Sheather’s Sucrose method (Specific gravity- 1.275)

Sheather’s sucrose solution consists of 454 grams sucrose sugar, dissolved in 355 ml water. 6 ml of formalin is added to prevent mold growth. The method is used preferentially for identification of infections with Toxocara sp, Ancylostoma sp., Trichuris sp., Capillaria sp. and Isospora sp.

2. Zinc Sulfate method (Specific gravity- 1.18)

33% Zinc Sulfate solution contains 33 grams of granular zinc sulfate, dissolved in 100 ml of water. The method is used preferentially for identification of infections with Giardia, Trichuris and Ascaris eggs.

3. Saturated salt solution (Specific gravity- 1.20)

Saturated salt solution is prepared by adding excess of NaCl to water and removing the un-dissolved contents by filtration.

Procedure for flotation technique:
  • A small amount of feces (Aprox 1 gram) is mixed with about 10 ml of flotation medium 
  • The solution is poured slowly into tube so that the liquid forms a convex shape. 
  • The mixture is allowed to sit for about 15 min while the eggs float to the top and the rest of the fecal matter sinks to the bottom. 
  • A cover slip is placed on the top of the tube before the incubation period starts or can be applied at the end. 
  • The cover slip is then transferred to a microscope slide and observed
Fecal sedimentation technique:

The principle of sedimentation technique is to mix the eggs with a solution whose specific gravity is less than that of the eggs. In contrast to flotation, here the eggs sediment in the solution. The most commonly used method is Formalin- Ether Concentration Technique.

1. Formalin- Ether Concentration Technique

Formalin acts both a fixative and preservative of protozoan eggs, larvae and cysts. The specific gravity of protozoan cysts and helminth eggs is greater than that of water. Fecal debris is extracted into the ether phase so that the parasitic forms can be separated and then sedimented by centrifugation.

Procedure:
  • Mix a small portion of the stool, about the size of a marble, in 10 ml 5% - 10% formalin or saline in a flat-bottomed test tube. Allow to stand 30 minutes for fixation. 
  • Strain about 10 ml of this suspension through a small funnel (or pointed paper cup with end cut off) containing wet gauze or cheesecloth into a 15 ml centrifuge tube. Use two layers of wide- mesh gauze or one layer of narrow- mesh gauze. 
  • Add physiological saline or 5%- 10% formalin to within ½ inch of the top of the test tube. Centrifuge for 10 minutes at 500g. 
  • Decant supernatant, leaving 0.5- 1.5 ml of sedimented material 
  • Resuspend the sediment in saline to within ½ inch of the top of the tube. Centrifuge again for 10 minutes at 500g. This second wash is not needed if the first supernatant wash is light tan or clear in color. 
  • At the end of washing step, nearly 1 ml of sediment should remain. 
  • Add approximately 3 ml ethyl acetate, insert stopper, and shake vigorously for a minimum of 30 seconds. 
  • Centrifuge at 500g for 10 minutes.
Fig 1: Formal Ether Sedimentation
Technique. Source
At the end of procedure four layers should result:

a. Top layer of ethyl acetate
b. Plug of fecal debris
c. Layer of formalin
d. Sediment with parasitic eggs, cysts or larvae.
  • Free the plug of debris from the sides of the tube with an applicator stick. Carefully decant the three layers.
  • Use a cotton swab to clean debris from the walls of the tube to prevent it from settling down into the sediment. 
  • Using a pipette, mix the remaining sediment with a small amount of the fluid that drains from the sides of the tube. 
  • Prepare a wet mount for examination.
It should noted that not all techniques are applicable in every scenario. Depending on the organism expected based on clinical history, different technique needs to be applied at different scenario. There are several methodologies in alternate such as Acid fast staining for stool samples when for example Cryptosporidium is expected. Several new kits have been approved such as immunofluorescence for specific cases. I have not discussed them here. Maybe another time.

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