Improved neubauer

Growth pattern for Blastocystis sp. was done based on different thermal conditions. The groups were categorized as in vitro and in vivo, where the in vitro was represented by Blastocystis sp. from non-dengue isolates while the in vivo was parasite isolated from dengue patients. Parasite culture of the first group was incubated at 37°C (in vitro control), the second group was incubated at 41°C for 24 hours and re-cultured at 37°C (in vitro thermal stress), and the third group was parasite isolated from dengue patients (in vivo thermal stress). The parasites of each isolates were pooled together from day three cultures to make a final concentration of 1×10⁵ cells/ml in 3ml Jones’ medium supplemented with 10% horse serum. A temperature of 41°C was used as it previously showed an increase in parasite count in vitro [10 (link)]. Moreover 41°C was chosen intentionally as it was a degree more than the usual high fever temperature (40°C) dengue patients often experience during infection [22 ,23 ]. Each set was prepared in triplicates and all cultures were kept in airtight tubes. Parasite count was carried out after 24 hours and at every three days interval for up to 13 days. This was done using haemocytometer chamber (Improved Neubauer, Hausser Scientific) with 0.5% Trypan blue solution as viability indicator. Only viable cells which did not take up Trypan blue stain were counted.

The parasites of each isolates were pooled together from day 3 cultures to make a final concentration of 1×10⁵ cells/ml in 3 ml Jones' medium supplemented with 10% horse serum. Thermal stress was introduced by incubating two sets of each isolate at 39°C and 41°C respectively. After 24 hours stressing the parasites at these temperatures, the parasites from all the culture tubes were re-cultured at 37°C to resume optimal growth condition and this was continuously maintained for 13 days. One set which consist of 3 culture tubes of each isolate was continuously incubated at 37°C which was used as the control. Each set was prepared in triplicate and all the cultures were kept in airtight tubes. Parasite count was done after 24 hours of heat exposure and at every 3 days interval for up to 13 days. This was done using haemocytometer chamber (Improved Neubauer, Hausser Scientific) with 0.5% Trypan blue solution as viability indicator. Only viable cells which did not take up Trypan blue stain were counted. Statistical analyses were carried out using SPSS version 20.

Microalgae growth was determined by their cell density using the bright line heamacytometer (Improved Neubauer, 0.1 mm deep, Hausser Scientific, USA) at two-day intervals and duplicate counts were performed for each culture flask by light microscope (CME, Leica, Wetzlar, Germany). Growth rate (r) during exponential phase was determined using the following equation.
where, r is the growth rate per day. Δ_t is the length of the time interval (t₀–t₁), t₀ is time at the starts of the exponential phase (day), while t₁ is time at the end of the exponential phase (day). N₀ is the number of microalgae cells at the start of exponential phase (cell/mL), and N_t is the number of cells at the end (cell/mL) (Wood et al. 2005 (link)).