Flow Cytometry Populations of the Micro Biota

Question: Discuss about theFlow Cytometryfor Populations of the Micro Biota. Answer: Application of Flow Cytometry The relationship between the populations of the micro biota in the mid-gut of the mosquito vector has drawn a lot of interest among the scientists. Since the culture-based and molecular techniques had their own drawbacks (Mayle et al., 2015). Therefore the Flow cytometry was used to enumerate the number of cells in the mid-gut. This was in an attempt to determine the variation in population across the feeding pattern and relate this information to the infectivity rates. Therefore single cells were analyzed. The flow cytometry, therefore, enumerates single cells from a population of cells. In this review, the cells were stained using different dyes, for instance, the PI, SGIU and a combination of the two dyes (Habtewold et al., 2016). Initially, the cells are harvested from the mid-gut of the mosquitoes. The homogenate is brought to 1.2ml by resuspending with a buffer. A third of the homogenate is removed and stained by use of the SYBR dye in order to be able to count the micro biota cell population. Approximately 25 l of the bead suspension is added to the homogenate and the Flow cytometry assay run. The total micro biota count in the sample is calculated and reported as the total number of events in cell suspension and the number of events containing the beads (Aghaeepour et al., 2013). The figure obtained is then multiplied by the ratio of the total of beads that were used for the assay as well as the final sample volume. The flow cytometry method then is able to enumerate the live versus the dead micro biota cells based on the different dye binding properties. Strength and Weaknesses In this review, different models f the flow cytometry equipment was used to enhance reliability. Strengths Fixing of the samples in the field before being sent to the laboratory ensures that the cells as intact as possible. The efficiency of the flow cytometry method is neither affected by the use of fixing reagents nor the prolonged period of sample storage (Graves et al., 2015). The use of the flow cytometry and SGI and PI dyes is able to make discrimination between live and dead micro biota in the parasite gut. This means that the method can be able to separate the live and dead cells from the debris which autofloresces. The flow cytometry has a high level of precision and reliability. This method is rapid and inexpensive as compared to the DNA extraction methods in micro biota enumeration. This is because this method bypasses some common steps like DNA extraction and conversion to cDNA which is time-consuming and expensive. When the species-specific antibodies are available, the flow cytometry can perform quantification and differential detection of micro flora in the gut of the mosqu ito vector. It is worth noting that the results obtained from the use of flow cytometry were comparable to those of qPCR indicating that the methods are both sensitive. Weaknesses The flow cytometer gives the results in the form of dot plots on the SSC and SYBR. However, more fluorescence was produced for SYBR as compared to the SSC. It is also important to note that the materials which had appeared to be more in SSC and lower in SYBR were confirmed by use of a microscope and they were confirmed to be debris. Therefore the fluorescence was found to be auto fluorescence as a result development of heme aggregates. (Demishtein et al., 2015). Since the samples are collected in the field and later transported to the laboratory for analysis. therefore a relapse in the population could due to this form of sampling. Reference List Mayle, A., Luo, M., Jeong, M. and Goodell, M.A., 2013. Flow cytometry analysis of murine hematopoietic stem cells. Cytometry Part A, 83(1), pp.27-37. Aghaeepour, N., Finak, G., Hoos, H., Mosmann, T.R., Brinkman, R., Gottardo, R., Scheuermann, R.H., FlowCAP Consortium and DREAM Consortium, 2013. Critical assessment of automated flow cytometry data analysis techniques. Nature methods, 10(3), pp.228-238. Graves, S.W., Applegate Jr, R.W., Lopez, G.P. and Piyasena, M.E., Stc. Unm, 2015. Multinode acoustic focusing for parallel flow cytometry analysis applications. U.S. Patent 9,074,977. Demishtein, A., Porat, Z., Elazar, Z. and Shvets, E., 2015. Applications of flow cytometry for measurement of autophagy. Methods, 75, pp.87-95. Habtewold, T., Duchateau, L. and Christophides, G.K., 2016. Flow cytometry analysis of the microbiota associated with the midguts of vector mosquitoes. Parasites vectors, 9(1), p.167. Habtewold, T., Duchateau, L. and Christophides, G.K., 2016. Flow cytometry analysis of the microbiota associated with the midguts of vector mosquitoes. Parasites vectors, 9(1), p.167.