ZINC SULFATE CENTRIFUGAL FLOTATION METHOD
This technique is predominantly used in veterinary laboratories. By exploiting the density of the parasites, particularly eggs, it allows the parasites to float to the top of a dense solution (final specific gravity of about 1.20) and can then be skimmed from the top of the tube. The most commonly used reagent is zinc sulphate. Operculated eggs as well as schistosome and infertile Ascaris eggs are not easily recovered by this method. Also trophozoites are killed due to the high specific gravity and certain other fragile eggs such as Hymenolepis nana become distorted. Mechanism
1. Fill a 15 ml centrifuge tube with ZnSO4 solution (1.18 specific gravity) and pour into a glass dish. 2. Using a tongue depressor, push the feces (2 to 3 grams, a piece the size of a grape) through the strainer into the ZnSO4 solution in the dish. TIPS: 1. The sieve must be in the liquid in order for the feces to be passed through. 2. The more feces you use, the more likely you will be able to find eggs which are present in low numbers. 3. Using a funnel, pour the ZnSO4-fecal mixture back into the centrifuge tube. 4. Centrifuge for 2 min at high speed (1500 - 2000 rpm). 5. Using a headed-rod or loop, remove a sample from the surface of the solution and place on a microscope slide. (You may have to take several samples with the rod or loop to get enough material to examine, you want the equivalent of a large drop on the slide.) Add a drop of iodine of iodine (to stain the cysts and ova) and a coverslip.
the floated eggs will be on top of the surface of the solution and therefore anything which goes under the surface (such as a pipette) will not recover them.
To
increase the sensitivity of this technique do the following: After
removing the tube from the centrifuge, fill the tube with ZnSO4 to just over the top of the tube, place a coverslip over the top of the tube and wait 5 to 10 min. Place a drop of iodine on a slide and place the coverslip onto the drop of iodine and examine at 10X. This modification also allows you to skip using the loop or headed rod to obtain your sample, and thus may be easier to do at a veterinary practice.
If
the sample contains a large amount of fat or other material that floats in water , you may want to wash the sample before doing
the flotation. To do this, start at step 1 but use water instead of ZnSO4. When you centrifuge the water-fecal mixture the eggs ,being heavier than water, will sink but the fat and other material will float. After centrifugation pour off the supernatant, add the ZnSO4 solution and mix well. Centrifuge as in step 4 and examine as in step 5. Pr
eparation of the ZnSO4 Solution
ZnSO4 solution (1.18 sp. Gr.) is made by adding 386 grams of ZnSO4 to 1 liter of water. The mixture should be checked with a hydrometer and adjusted to 1.18. The ZnSO4 solution should be stored tightly capped to prevent evaporation (and the resulting change in the specific gravity of the solution). Pr
eparation of the Iodine Solution
Iodine solution: 10 gms Potassium Iodide (KI) is added to 1 liter of distilled H2O. Shake to dissolve. Add 10 gms of Iodine (I2) to the above solution. Allow to stand over-night with stirring, at this time you may still have Iodine crystals at the bottom, this is OK, just leave them there. This solution will stain (and kill) most parasite eggs and cysts (coccidialoocysts are an exception, they do not take in the iodine) ARASEP Ò METHOD
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Parasep Ò is a commercial kit for faecal parasite concentration, it is a rapid, single use, disposable module for the clean and efficient concentration of helminth ova, protozoal cysts and oocysts. The methodology is a modification of the Ridley-Allen method.
The ParasepÒ incorporates into the device a patented filter thimble presenting a large surface area. The 3-dimensional structure prevents blocking of the filtration surface, therefore, large particles are prevented from passing through the filter and are held by the horizontal bars of the primary screen. The vertical orientation of the second screen means that the filtration is tangential, again preventing build up of rejected particles on the filter mesh. At the base of the filter thimble is a debris trap, where the rejected particles gather and hence are prevented from occluding the pores, (pore size 425m m) allowing for more ova and cysts to filter through. The large surface area retains more faecal exudate and hence yields very high clarity microscopic mounts. (Diagram 2)
a asep Ò Inst ructions for use
P r
Advantages of Parasep Ò , compa red with conventional method
An in-house study was carried out where, the conventional Ridley-Allen method, was compared with the Parasep Ò , which is an enclosed, single use, disposable system. During the study the observations that they took into consideration included parasite recovery, the density of the deposit, ease of handling, health and safety aspects and cost. 100 faecal samples were carried out in duplicate by both techniques to allow for comparable results. The parasite recovery by both techniques was comparable. Although the Ridley-Allen technique is cheaper it is labour intensive and has inherent health and safety hazards.
