Selection of species: Anabas testudineus (Bloch, 1792) is a fairly common fish species in Bangladesh that was selected as host species for conducting this study. A special characteristic of the fish is that it has additional respiratory organs, other than gills, that is known as accessory respiratory organs and by which it can receive oxygen directly from air and can survive long time in water with less oxygen or even without water. Simultaneously, our concerned protozoan parasites are very sensitive and cannot survive so prolonged except live fishes. This is the reason to select host fish with protracted survival capacity having accessory respiratory organ for collecting and transporting it from distant sampling place.
Collection of host sample: According to the experimental design of the research, a total of 473 host fish species, Anabas testudineus were collected alive from the freshwater bodies of Kishoreganj (Kuliar char-24°10'40" N, 90°50"57" E and Pakundia-24°30'07"N, 90°67'71"E), Mymensingh (Ishawrganj 24°41'16" N, 90°35"58" E and Trishal- 24°57'18"N, 90°43'84"E), Faridpur (Modhukhali-23°32'50"N, 89°31'22"E and Boalmari-23°44'04"N, 89°66'84"E), Jashore (Purondorpur, Jhikorgacha Upazila-23°5"51" N, 89°5'53" E and Monirampur-22°59'32"N, 89°11'53"E), Manikganj (Singair-23°81'45"N, 90°12'47"E and Ghior-23°93'74"N, 89°86'05"E) and Bogura (Sherpur24°68'21"N, 89°41'47"E and Sadar-24°87'45"N, 89°38'34"E) with the help of fishermen during mid of the April 2018 to end of the March 2019. Sample size of fish collected from each area was not sharply equal.
Sample preparation: The fish were examined as soon as possible after capture. Immediately after collection, the external surface of the fish was observed carefully using a magnifying glass. External surface of the fish were examined and recorded for any abnormalities. After collecting the samples, their total length and weight were measured. Evidences were collected from the body slime, gill slime and blood of host fish which are the best suited micro-habited for protozoan parasites to get harbor. Smears of body slime, gill slime and blood were made on glass slides on the spot and fixed them in ethanol for further observation in the laboratory.
Klein’s dry silver impregnation method: Klein's dry silver impregnation method was used for staining mobiline peritrichs and other ciliates from the surface of fish. Mucous was scraped gently off gills and skin with a scalped, spread thinly on a grease-free slide, and dry rapidly. The slide was covered with a 2% aqueous solution of silver nitrate (AgNO3) for 8 min. After that they were rinsed thoroughly with distilled water and were placed facing up in a dish of distilled water and expose to bright sunlight for 1-2 hours. Finally they were allowed to dry and mount with a neutral medium, Canada balsam.
Giemsa’s stain after acid hydrolysis: To detect the parasites in blood sample, the slides were stained using Giemsa stain and cover slipped by DPX mountant. During this process smears were fixed in Schaudinn's fluid and rinsed well in distilled water. After that they were hydrolysed for 8 min in 1N HCL at >60oC. Again they were rinsed for several times in distilled water and stained with stocked Giemsa's stain (diluted 1:20 with water at PH 7.0-7.2) for about 20 min and rinsed with tap water. Then they were allowed to dry directly and finally mounted with a neutral medium, Canada balsam. Giemsa’s stain technique was used for rapid demonstration of nuclei in ciliates and in microsporidian spores.
Count: The slides were observed under microscope to note the presence or absence of protozoan parasites. Counts of parasites found in selective organs were recorded. The numbers of observed parasites were counted for statistical analysis and microscopic photographs were captured for the identification of species with the help of 10-megapixel digital camera. Protozoans were identified according to the description of Lom and Dyková (1992), Sarkar (1985), Eiras (2002), Kalavati and Nandi (2007), Bash' and Abdullah (2010), Kibria et al. (2010). Some parasites could not be identified up to species level because these were not got matched with any of the available published description.