The microscopic examination of faeces is required for the recognition and identification of intestinal parasites

1. Direct Microscopy

Advantages

useful for the observation of motile protozoan trophozoites and the examination of cellular exudate

may not detect ova, cysts and larvae which are present in scant numbers

2. Concentration techniques

Advantages

maximises the numbers of organisms detected which may be too scanty to be seen by direct microscopy alone

Disadvantages

destroys trophozoite stages and distorts cellular exudate

Types of Concentration techniques

• 1. Sedimentation techniques

uses formalin as a preservative and ether or ethyl acetate as an extractor of fat and debris from faeces after filtration to leave the parasites in a sediment at the bottom of the tube after centrifugation.

Advantages

n Recovers most ova, cysts and larvae

n Morphology of most parasites is retained

n Less risk of infection from bacteria and viruses

• Disadvantages

n Preparation contains debris

n Some parasites do not concentrate well

n Ether is flammable and formalin is an irritant

n Liquid faeces do not concentrate well

• 2. Flotation

uses the high specific gravity of a solution to float the lighter ova and cysts

• Advantages

The concentrate is clear of debris

• Disadvantages

n Delay in examination can result in distortion

n Larvae and some fluke eggs do not concentrate

n Frequent checking of specific gravity

Risk of infection from unfixed material

The most widely used sedimentation technique in clinical laboratories is the Modified Ridley-Allen method

Materials.

a. Formalin water (100ml formaldehyde and 900ml distilled water)

b. Ether or ethyl acetate.

c. Mesh (425µm) brass wire filter, 3 inches in diameter (Endcott Sieves Ltd. Lombard Road, London SW19 3BR). Nylon tea strainers provide a low cost alternative.

d. Small 3inches porcelain or stainless steel dish.

Method.

a. Using orange sticks, select a quantity of faeces (approximately 1g or pea size) to include external and internal portions.

b. Place the faeces in a centrifuge tube containing 7 ml of 10% formalin.

c. Emulsify the faeces in the formalin and filter through the brass/nylon filter into the dish.

d. Wash the filter and discard any lumpy residue.

e. Transfer the filtrate to a boiling tube and add 3 ml of ether/ethyl acetate. Mix well on a vortex mixer for 15 seconds or by hand for 1 minute.

f. Transfer back to the centrifuge tube and centrifuge at 3,000 rpm. for 2 minute.

g. Loosen the fatty plug with an orange stick and pour the supernatant away by quickly inverting the tube.

h. Allow the fluid on the side of the tube to drain on to the deposit and mix well. Transfer a drop to a slide for examination under a coverslip.

i. Use the x10 and x40 objectives to examine the whole of the deposit for ova and cysts.

 The important points to be considered when performing a concentration technique are:

 1. In a specimen the whole of the sample (equivalent to 1 gram of faeces) should be concentrated and the whole of the deposit examined. This corresponds to good practice with clinical samples.

 2. It is important to vortex the sample for at least 15 seconds after the addition of ether or ethyl acetate, as failure to do so may result in excess deposit, thus obscuring ova and cysts.

 3. Adequate centrifugal force must be used because if this is below the required value, there may be insufficient gravitational force to sediment the ova and cysts. The centrifugal time is also critical, since the ova and cysts may remain in suspension if the sample is not centrifuged for the minimum required time. It is recommended that the sample be centrifuged at 1000g for 2 minute or 3000rpm for 2 minutes.

NEQAS Parasitology now performs all pre and post distribution concentrations on faecal samples using the Parasep Faecal Concentrator.

Ether versus Ethyl Acetate

Ethyl-acetate is a less flammable alternative to the use of ether and after an in-house study which compared these 2 lipid extraction agents, the following observations were noted

Ether

Advantages

n Cleaner deposit

n A very effective lipid extraction agent

 

Disadvantages

n Disposal procedure

n Very unstable as it forms explosive peroxidases on exposure to light

n Highly flammable

n Some ova become trapped in the fatty plug

Ethyl acetate

Advantages

n Less flammable and more stable than ether

n Less combustable than ether

n Greater numerical recovery of parasites (especially with Taena sp,  H. nana and  A. lumbricoides, and many cysts)

Disadvantages

n Thicker deposit

n A less effective lipid extraction agent therefore Triton-X must be added