Slide Culture Technique
Slide culture technique can adequately be used for the determination of sessile and nocturnal microbial such as fungi population size. Fungi are eukaryotic and are known to grow in damp and moist places yet are non-motile thus can be referred to as sessile and nocturnal. In this laboratory technique, a slide culture is prepared by use of a fungal culture and Sabouraud agar as a medium for the culture. After preparation, the culture is left to incubate for 48 hours at room temperature, then observations made under the low power objective lens of a microscope (Jeffries et al., 2015). From the observation, the presence of spores and growth of hyphae indicate growth; if no observations are made, then the culture should be left for 48 more hours.
After observation, a stain is applied using lactophenol cotton blue stain which serves as a mounting fluid and kills all suspended organisms as well as deactivating the lytic cellular enzymes to prevent lysing by the cells. Application of this mounting fluid ensures that chitin within the cell walls is stained hence visible. This method is advantageous as it is an easy and quick way of preparing colonies for identification and examination with as little disturbance as possible (Jeffries, 2015). In the same way, sessile and nocturnal organisms are identified by careful study of their morphological characteristics. And since the cultures are grown from the slides, there is no need to remove segments of the fungus; thus all the main features are observed as no damages take place.
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Pan-Genome Growth Curve
Pan-genome can be defined as the description of every genetic variation together with the genes within a selected species under observation. This method has been used effectively by scientists to determine the population sizes of eukaryotes such as bacteria (mobile and diurnal). The pan-genome or supra-genome analysis focuses on using identified genomes to determine the niche dynamics and population sizes of selected organisms under study. In this study method, the pan-genome is the complete set of genes present in species under study (Khamchatra et al., 2016). The supra-genome includes the “core genome” which is genes present in all the species and the accessory genome which is the genes present in only two or more of the species.
Scientists have used this method to develop vaccines against bacteria that are pathogenic. Furthermore, scientists have used the supra-genome method to track, identify, and detect new strains in meta-genomics models based on their separate gene subset of the complete gene set of the species. Moreover, researchers have used the method to explore strain diversity in studies that involve genomics of environmental populations (Rengefors, Kremp, Reusch, & Wood, 2017). Similarly, pan-genomes have been used to study the horizontal transfer of genes in a species and the characterization of species based on their specific gene set. The characterization is vital as it helps in detecting aspects like the virulence features that might be present in only one species and lacking in the rest.
Antibody Labeling
This method is useful in the determination of both eukaryotic and prokaryotic population sizes since they both contain proteins. This technique involves the connection of a particular mark to an antibody to assist in recognition or separation of a protein molecule. The marked antibodies are essential for immune-based assessments such as immunofluorescence, immunohistochemistry, flow cytometry, and many others. Antibodies with marks can also be used to separate and cleanse a single strand of protein from a compound blend of proteins (Shaw, Hoffmann, & Horne, 2016). Radioisotopes, small molecules, fluorescent dyes, and enzymatic proteins have been used to label antibodies just like they have been used on other protein molecules.
Two traditional methods have been employed to label antibodies with a variety of chemistries covalently with small particles. First, the mark is made sensitive to essential amines and employed to mark lysine remains in the antibody particle. Secondly, the disulfide ties within the antibody series are decreased and later countered with a thiol-reactive mark. Labels that are often used include biotin, fluorescent tags, and enzyme reporters (Shaw, Hoffmann, & Horne, 2016). Biotin marks are preferable over the other two since it makes one of the most robust non-covalent bonds present in nature with its attaching partners streptavidin and avidin. Owing to its miniature size, biotin seldom disturbs the activity of antibodies, making it the best option for a mark.
Capture-recapture Method
This method can easily be adapted for mobile and either nocturnal or diurnal organisms but not sessile organisms. The technique has been used actively in macro-organisms but can also be employed in the determination of population sizes in prokaryotic and eukaryotic organisms. In the capture-recapture method, this is also known by different names including capture-mark-recapture, the organism is captured and a mark put on the organism for identification then it is released (Weir et al., 2016). After some time, the samples within the same niche are captured, and those with marks noted while those without the tags are tagged.
For micro-organisms, this method could prove tedious and might not be very accurate. Instead of tags, permanent ink can be used on the specimen even though this might impact on the results as the samples marked usually behave differently from others of the same kind as they have a different outlook. The marks which are made of chemicals could further lead to genetic mutations or cause genetic disorders to the samples under investigations (Weir et al., 2016). The other challenge posed by this method is in the calculation of the population size. Furthermore, the capture-recapture method cannot be used in large ecological niches in estimating the population size of microbial.
Spore Tracer Method
This technique is best for sessile and nocturnal spore-forming prokaryotes since the method uses spores to analyze various DNA extractions of the organisms under study. Micro-Organisms in this technique are seeded with spores obtained from another micro-organism referred to as a tracer. An example of an organism that is used as a tracer organism is the Bacillussubtilis . To extract the DNA, a variety of DNA extraction methods are employed using soil microcosms sowed with spores of the tracer organism (Shaw, Hoffmann, & Horne, 2016). After extraction, the raw DNA is cleansed and stored using appropriate storage techniques and later analyzed accordingly for obtaining relevant data.
This technique is essential in studies meant to assess the composition of microbes in the soil especially after application of toxic chemicals to the soil sample. The composition of spore-forming bacteria or fungi in a soil sample can be used to determine the quality of the soil for such activities as farming. The composition of microbial in soil has been used extensively to assess the fertility after elongated use of herbicides, pesticides, or fungicides. The spore tracer method can also be used in different research apart from the one discussed in this paper.
References
Bentkowski, P., Van Oosterhout, C., & Mock, T. (2015).A model of genome size evolution for prokaryotes in stable and fluctuating environments. Genome biology and evolution , 7 (8), 2344-2351.
Jeffries, T. C., Ostrowski, M., Williams, R. B., Xie, C., Jensen, R. M., Grzymski, J. J.,& Neches, R. Y. (2015). Spatially extensive microbial biogeography of the Indian Ocean provides insights into the unique community structure of a pristine coral atoll. Scientific reports , 5 , 15383.
Khamchatra, N. M., Dixon, K., Chayamarit, K., Apisitwanich, S., &Tantiwiwat, S. (2016). Using in situ seed baiting technique to isolate and identify endophytic and mycorrhizal fungi from seeds of a threatened epiphytic orchid, DendrobiumfriedericksianumRchb. f.(Orchidaceae). Agriculture and Natural Resources , 50 (1), 8-13.
Rengefors, K., Kremp, A., Reusch, T. B., & Wood, A. M. (2017). Genetic diversity and evolution in eukaryotic phytoplankton: revelations from population genetic studies. Journal of Plankton Research , 39 (2), 165-179.
Shaw, M. P., Hoffmann, H., & Horne, M. (2016, May). Case study: comparison of microbial monitoring techniques used in the field and how their complementarity can be harnessed to build a full picture of the microbial life in the field. In SPE International Oilfield Corrosion Conference and Exhibition .Society of Petroleum Engineers.
Weir, M. H., Shibata, T., Masago, Y., Cologgi, D. L., & Rose, J. B. (2016).Effect of surface sampling and recovery of viruses and non-spore-forming bacteria on a Quantitative Microbial Risk Assessment model for fomites. Environmental science & technology , 50 (11), 5945-5952.
