Khan, K Z

Zaman, V

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eCommons@AKUDepartment of Pathology and Laboratory Medicine Medical College, PakistanSeptember 1994A concentration technique for obtaining viablecysts of blastocystis hominis from faecesV ZamanAga Khan University, viqar.zaman@aku.eduK Z. KhanAga Khan UniversityFollow this and additional works at: https://ecommons.aku.edu/pakistan_fhs_mc_pathol_microbiolPart of the Microbiology CommonsRecommended CitationZaman, V., Khan, K. Z. (1994). A concentration technique for obtaining viable cysts of blastocystis hominis from faeces. Journal ofPakistan Medical Association, 44(9), 220-221.Available at: https://ecommons.aku.edu/pakistan_fhs_mc_pathol_microbiol/750A Concentration Technique for Obtaining Viable Cysts ofBlastocystis hominis from FaecesPages with reference to book, From 220 To 221 V Zuman, K.Z. Khan ( Department of Microbiology, Aga Khan University, Karachi. ) Introduction Blastocystis hominisis a common parasite implicated in a variety of intestinal problems1. Oral-faecalroute of transmission has been postulated but the cystic phase which is presumably the infective stageof the parasite, has only recently been described using electron microscope2. However, littleinformation is available on the light microscopic morphology of the cyst. There are two possiblereasons for this. The first is the relatively small size of the cyst, as compared to some other intestinalprotozoa and the difficulty in separating it from the non-cystic stages of the parasite. The concentrationtechnique described in this paper ovemomes this problem and separates the cysts from the non-cysticstages, there by enabling its easy recognition in faecal samples.Materials and Method The concentration technique used is as follows:1. Approximately 5 gm of faeces was thoroughly mixed in 15-20 ml of distilled water and sievedthrough gauze to remove coarse particles.2. Sediment was re-suspended in 15-20 ml of distilled water and centrifuged at 300g for 20 minutes.This was repeated 3 times.3. The centrifuged deposit was mixed in 1 nil of distilled water and layered on 10 ml of Ficoll-Paque(Pharmacia) column. This was centrifuged at 2000g for 20 minutes.4. Blastocystis cysts banded about 0.5 cm below the top along with other protozoan cysts such asEntamoeba, Jodamoeba and Giardia. Most, but not all, of the bacteria and other faecal debris gotpelleted at the bottom of the tube.5. The concentrated material in the band was carefully removed with a pasteur pipette and re-centrifuged in 20 ml of distilled water to remove Ficoll-Paque. Suspension of cysts was now availablefor establishing cultures and studying morphology in electron and light microscope. The essence of thetechnique is the use of distilled water which breaks up all the non-cystic stages of the parasite. Theseinclude the vacuolar, the granular and the ainoeboid forms.Results The main morphological features of the cyst in the light microscope are as following:1.Shape - Using phase-contrast the cyst appeared as a sharply demarcated polymorphic, but mostly ovalor circular dense body, surrounded by an outer membranous layer (Figure).Without phase- contrast the membranous layer was not easily visible. The space between themembranous layer and the cyst usually showed fine particulate matter.2. Size -20 cysts were measured with and without the membranous layer. With the membranous layerthe mean diameter was 12.65gm (SD=l .14). Without the membranous layer (cyst only) the meandiameter was 6.65um (SD=2.32).3. Cyst contents - A number of large and small circular bodies were visible inside the cyst The electron-microscopy of the parasite reveals large mitochondria, so some of these were probably mitochondriaand others nuclei. Their relative numbers could not be assessed accurately.Discussion One of the diagnostic features of the cyst under the electron microscope is the fibrillar layer whichloosely surrounds the cyst. The term fibrillar layer was originally used by Boreham and Stenzel1, as itappeared to be made up of fibre like material. The outer membranous layer seen in the light microscopeis the same as the fibrillar layer and confirms the observation that we were dealing with the cyst andnot any other stage of the parasite. The particulate matter seen between the cyst and the outermembranous layer is also seen in the electron microscope. The size of the cyst in the light microscopeis larger than that seen in the electron microscope (3-5um). This is probably due to the flattening of thespecimen because of the pressure of the cover glass during light microscopy. The viability of the cystsis maintained in this technique which is very useful as the cysts can be cultured for various otherstudies.References 1.Boreharn, P.F.L. and Stenzel, D.J. Blastocystis in humans and animals. Adv. Parasitol., 1993;32: 1-70.2. Stenzel, D.J. and Boreham, PF.L A cyst like stage of Blastocystis hominis. Int.J.Parasitol.,1991;21:613-15.

A concentration technique for obtaining viable cysts of blastocystis hominis from faeces

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A concentration technique for obtaining viable cysts of blastocystis hominis from faeces

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