Critique of Flow Cytometry usage in Environmental Microbiology - Research Paper Example

Critique of Flow Cytometry usage in Environmental Microbiology 17 November Flow cytometry is being used more in environmental microbiology. This paper will propose that flow cytometry is better than other used techniques. Environmental microbiology has become important worldwide. Flow cytometry is becoming a popular trend with microbiologists. The differences between flow cytometry and other processes will be examined and determined. In order to compare the flow cytometry with other techniques some factors will be take into consideration. Sensitivity, specificity, diverse environments, cost, and the ease of the technique will be equated. Microscopy is one of the other techniques. The use of antibodies is a third way to be compared. Magnetic beads is also compared against flow cytometry. Flow cytometry is of importance to an environmental microbiologist regarding the target microorganism like the morphology, physiological state, and cellular features. Microorganisms can be isolated, analyzed, and studied after being identified by these methods. Pros and cons are listed for each technique. All have benefits and detractions. After careful review the best process in analyzing environmental microbiology will be determined. Flow cytometry will be proven to be the best application in isolating and analyzing microorganisms. At the very least, flow cytometry needs to be further researched. The importance of flow cytometry will become clear as the trend to use this method grows. Introduction Environmental microbiology is using flow cytometry globally. It is a trend that shows the appreciation for this technique in comparison to other techniques that have been in use in environmental microbiology. This is due to the advantages of flow cytometry, along with the limitations of other techniques. Both flow cytometry and other techniques need to be examined to determine the difference. Flow cytometry is starting to make a mark in environmental microbiology. The applications of flow cytometry in environmental microbiology are several. These procedures include evaluation of bacteria involved in the degradation, quantifying heterotrophic bacteria, analysis of phytoplankton, and sorting of cells (Matsudaira, Wilson and Robinson 492). These are just a few uses of flow cytometry in environmental microbiology. This paper will utilize one application of flow cytometry to critique the method. The example to be used is the recovery of uncultivated microorganisms from the environment. These merits and demerits shall be compared with those of other techniques used in recovery of uncultivated microorganisms from the environment to determine if flow cytometry, is an appropriate technique or if the other processes are better. Material and Methods The method to be used in the recovery of microorganisms from the environment is determined by the intention. The purpose of recovering the microorganism is guided by the intention. A microbiologist can determine the method to utilize. In many instances, the necessity for qualitative or quantitative data is a factor in the choice of method. Research on microorganisms concerning the impacts to human beings and the environment as a whole has depended on their appropriate recovery and examination. In order to critique a method as this paper shall do, there are various considerations that have to be examined. These considerations or factors help determine the usefulness of the technique. The factors include, sensitivity, specificity, hindrances in using the technique in various environments, the cost of using the technique, and the simplicity of using the technique along with the duration needed to undertake the method (Porter 1779). The factors that will be used are crucial to the comparison of techniques. Sensitivity is the least detection level. It can be interpreted to mean the number of microorganisms required to be detected. Specificity is the capability of a method to differentiate between the microorganisms of interest and those that are not of interest. The hindrances in using the method in various environments consist of the extent to which the method is capable of detection of the microorganism in spite of its use in various types of environments. The cost determines the amount of money used to run the method each time. The simplicity of using the technique along with the duration needed to undertake the method determines how fast the method gives the results (Hurst and Crawford 1112). Results The factors listed previously shall be used in assessing the suitability of flow cytometry in comparison with other techniques. First and foremost, the principle that a technique uses is important. The principle is important because it determines how the technique functions. The principle of flow cytometry is pretty simple. It involves the utilization of the scattering, excitation of light, and the production of fluorochrome particles. Fluorochrome particles emission functions to produce particulate multi-parameter facts from the cells and particles that have a diameter lying in the 0.5 to 40 micrometer size range. The basic components of a flow cytometer are, a hydraulic fluidic system, a flow cell, a source of light, optical filters, data assessing instrument and photomultiplier tubes (Prorot, Chazal and Leprat 44). The scattering of light in a flow cytometer takes place when the light from the laser is deflected by the microorganisms or cell. The degree of light scattering is determined by the features of the microorganisms or the cells. These features are the physical features such as size and internal constitutions. The degree of light scattered is influenced by various aspects like the nucleus, or the cell membrane. Light that is scattered to the front avails details about the size of the microorganisms or the cells, while the light that is scattered to the sides can be used to determine the morphology (Prorot, Chazal and Leprat 45). Figure 1: Picture of a flow cytometer depicting its principles (Prorot, Chazal and Leprat 46). Parametric measurements of the microorganism can be utilized in the quantification of the fluorescent dyes that have been absorbed by the microorganisms. These fluorescent dyes are utilized in the differentiation of microorganisms in accordance with the characteristics that are examined. By evaluating microorganisms at their distinct levels many of their characteristics can be assessed. The assessment leads to finding and identifying the microorganism of interest that can then be retrieved (Prorot, Chazal and Leprat 47). Having looked at the principle behind flow cytometry, it is important that the principles behind other methods used in the recovery of uncultivated microorganisms from the environment. The essence of this is to avail a know-how of the functioning of these other methods so that their functionality can be used to evaluate that of flow cytometry. Microscopy is a technique utilized in the recovery of uncultivated microorganisms from the environment. The principle behind the method is that it makes use of staining with dyes. The dyes that are commonly used are dyes like fluorescent dyes. After staining, epifluorescence microscopy can be utilized to detect the microorganisms. Hybridization techniques can be utilized along with microscopy to distinguish microorganisms. The combination makes use of dyes like fluorescein isothiocyanate that is tagged onto probes made up of oligonucleotides (Porter 1781). Microscopy has been extensively used in recovery of microorganisms from the environment due to the availability of solutions that solve issues that interfere with the method (Sen and Ashbolt 52). The use of antibodies is another method that is also used in the recovery of microorganisms from the environment. Usually, antibodies that have been developed against certain microorganism strains are utilized to evaluate microorganisms for particulate sub-populations. The technique is plagued by the presence of signals from the background organisms. To eradicate these background signals, it must be ensured that the antibodies are particulate for the microorganism that has been targeted. Samples from the environment can also show binding that is not specific to the antibodies. The reason being that there may be organic substances and compounds comprising phenol. Nevertheless, the non-specific binding can be prevented through counterstaining techniques. The use of magnetic beads is another method that aids in the recovery of uncultured microorganisms from the environment. It is normally referred to as the capturing of antibodies. This method needs the presence of monoclonal antibodies that are linked to the microorganism of interest. The monoclonal antibodies are connected to a magnetic bead made of polystyrene. These beads are then put into the sample containing the microorganisms. The organisms having the right antigen bind to these beads and they are recovered using a magnet. However, the method is plagued by the lack of specificity in binding and little recovery of the microorganisms of interest (Gadd and Sariaslani 15). Discussion The method of interest, flow cytometry, is utilized along with fluorescence-activated cell sorting that permits the fast recognition, counting, sorting, and the recovery of microorganisms. However, the technique has not been in wide use, but is gaining popularity. When compared with other methods that include microscopy, magnetic beads, and the utilization of antibodies, flow cytometry proves to be a superior method due to its rapid assessment of the environmental sample. The fast assessment reduces the time needed to carry out the procedure. The ability of flow cytometry to be used along with other methods gives it an upper hand by availing it with a diverseness. The use of light or laser to scan microorganisms is in itself a better means of recovering microorganisms. It is because it avails many characteristics of the target microorganisms such as the morphology, number, the size of the microorganisms and internal composition. These are important characteristics that aid in the recovery of the specific microorganisms without interference by the unwanted microorganisms (Matsudaira, Wilson and Robinson 495). Further examination of the principle of flow cytometry reveals some hindrances that have caused concern of larger uses for recovery of uncultivated microorganisms from the environment. One of the hindrances is that, in spite of the type of fluorescent dye utilized, it requires some form of regulation. The regulation is undertaken on the protocol. Why is this so? Protocol regulation is not an abnormal thing because due to the existences of dissimilarities in the structure of the microorganisms. Also, it could be that the knowledge of certain uncultivated microorganisms at present is limited (Porter 1784). In other cases, the dyes that are utilized may not function as is expected especially with the presence of a multitude of microorganisms. Hence, without the dye getting to the microorganism of interest, failure to properly label each microorganisms. The microorganisms may also have intricate cell walls that hinder the dye from properly functioning. Another structural characteristic that hinder the proper functioning of the dyes is the active ion pumps that may thwart the dye from the correct function. Hindrances can cause wrong reading, analysis, and conclusion of the results that are supposed to be negative (Hurst and Crawford 1116). For starters, the need for regulation could be a factor in their non-interest in the method. It is especially so when a protocol that is well outlined in the provided kit needs adjustment. Rather than enhance the usage of the flow cytometry in the recovery of uncultured microorganisms, the adjustment works to reduce its usage. It must be remembered that adjusting the given protocol is undertaken to factor in the specific software and hardware in use for the flow cytometry technique. It also factors in the source of the microorganisms, the species these microorganisms belong to, and the conditions that are required for their growth (Hurst and Crawford 1118). The utilization of flow cytometry as a method to recover uncultivated microorganisms from the environment is also hampered by the type or types of death that microorganisms may undergo. The preparation of the control is affected by the type of death. Flow cytometry might not be able to identify other uncultivated microorganisms due to the type or types of death. As stated earlier, the duration that a method requires in order to complete it and obtain results determines the usefulness of the technique. For flow cytometry, when the development of the protocol enters the second stage, time is the limiting factor. The second stage uses more time. It takes more time as the kit has to be adjusted to function with the flow cytometer that is accessible. Also, the microorganism that is off target has to be considered in the adjustments. The trial and error method is used at times when the differentiation between the control samples of the dead and live microorganisms is poor (Prorot, Chazal and Leprat 48). Normally, the concentration of the dye is changed to enhance control differentiation. However, in some cases, the microorganisms may need prior treatment. Prior treatment may enhance the absorption of the dye. Nevertheless, this process is tedious and consumes more time. The time consumption and the tediousness of the process decrease the use and the usefulness of the method (Prorot, Chazal and Leprat 50). However, flow cytometry is not bad off when compared with a method like microscopy. The reason being that microscopy is itself more time-consuming and tiresome. The reason holds true mainly when the number of microorganisms to be recovered is a lot. The higher the number, the more time-consuming and tiresome the method gets. The other hindrance that affects the use of flow cytometry is the large number of likelihoods that are there in regards to; the selection of the dye to be used, the duration of dying, and the concentration of the dye. There is a large number of likelihoods because there is no specific dye or dyeing method that has been stated to be appropriate for all the microorganisms. The model of action for the various fluorescent stains that are in use currently has been the subject of thorough analysis in many studies (Sen and Ashbolt 55). The large number of likelihoods or choices to choose from in the staining part can be looked at from two perspectives. The positive and the negative aspects. On the negative aspect, the array of choices may cause confusion. Nevertheless, this confusion can be negated by using numerous dyes at the same time. The concept behind using numerous dyes at the same time is to permit feasibility-associated considerations to be analyzed for each microorganism that is recovered. Thus, numerous staining dyes shall avail an enhanced outlook of the physiological variations. The enhanced outlook will be similar to that obtained when utilizing one stain (Sen and Ashbolt 56). The use of numerous stains eases the process of flow cytometry. However, in cases where these multiple stains are limited in number flow cytometry might not be the right option. Some situations do not a merit this the method. It calls for the development of ways to negate this hindrance. Hence, the development of ways to negate the hindrance has been in the process. One of the developed ways is the production of ready-made kits that have reagents in the right mixture so that a number of microorganisms can be stained. The reagents are added to the sample containing uncultured microorganisms from the environment. After being placed in an incubator, the samples are evaluated. All this is done in one evaluation procedure. Hence, it enhances the ease of use factor to flow cytometry. In cases where the ready-made commercial kits are not available, then the issue is a major hurdle to flow cytometry. It makes it cumbersome to perform especially if the environmental sample is large (Gadd and Sariaslani 18). Some kits have also been developed to check for the feasibility of the microorganisms in the environmental sample. These kits are of an essence to recovering microorganisms from the environment. Evaluating the necessity of the use of kits in flow cytometry leads to questioning the cost of using the method. If there is a large sample size, it means that the number of kits required is more. Hence, because these kits are commercially produced, they have to be bought. Buying them is an expense that costs money. It pushes the cost of using flow cytometry in the recovery of microorganisms from the environment higher. A good recovery method should not be expensive to use. Thus, the flow cytometry method’s cost is impacted by the cost of using it if kits are required. If they are not required, using numerous stains could be tedious in the long-run despite the advantages it avails (Gadd and Sariaslani 19). Assessing the cost of the other methods used in the recovery of microorganisms from the environment so as to determine how they compare with that of flow cytometry gives a clearer picture. A clearer picture of whether the cost of using flow cytometry is sustainable. Microscopy is one such method. The cost of using microscopy is comparatively low as the instruments and equipment in use are largely available. The antibody technique is another method. The method is largely expensive as it requires the buying of antibodies that are expensive. These antibodies are also not bought in large quantities as their sale is done in minute quantities. Hence, if large samples are under assessment, then more of them are needed. It raises the cost up. Using magnetic beads is also expensive due to its utilization of monoclonal antibodies (Hurst and Crawford 1120). Conclusion Evaluation of these techniques indicates that flow cytometry falls within the same range as the other methods that are considered costly. Hence, it cannot be disregarded on the cost factor. Flow cytometry has proved to be a fifty-fifty method as the merits and demerits, based on the principle and the described factors, have not yielded any considerable demerit that warrants it stop being used. As there are measures to mitigate the hurdles and hindrances encountered during its use, the method’s usefulness is worthwhile. Flow cytometry as a method to recover uncultured microorganisms from the environment, in spite of it not been widely used, proves to be an important method just like any other method. In fact, it avails some diversity when it comes to the use of stains. It also provides various details that are of importance to an environmental microbiologist regarding the target microorganism like the morphology, physiological state, and cellular features. Flow cytometry can be developed and used as efficiently as other techniques such as microscopy, antibodies, or magnetic beads. Flow cytometry is being used further in regards to environmental microbiology. This paper has proposed that flow cytometry is better than other used techniques. Environmental microbiology is benefited from flow cytometry. The differences between flow cytometry and other processes were examined and determined. The factors of sensitivity, specificity, diverse environments, cost, and the ease of the technique were equated. Microscopy is inexpensive. The solutions used in this method are readily available. However, it is hard to process many cells at once. The use of antibodies is expensive. Antibodies must be bought. In some environments the antibody might not stick to the microorganism intended. Magnetic beads is relatively cheap. The use of dye is used with the magnetic beads. When using magnetic beads expense is a hindrance. The drawbacks of microscopy is also the drawback of magnetic beads. Too many elements cannot be examined efficiently. Flow cytometry is of significance to an environmental microbiologist concerning the target microorganism like the morphology, physiological state, and cellular features. Microorganisms can be isolated, analyzed, and studied after being identified by these methods. Flow cytometry might be expensive due to the individual kits needed to complete the process. A great number of samples cannot be processed. Dyes are also used in flow cytometry. The needed solutions are regulated. The adjustment of the solutions might also be a problem. Flow cytometry was not proven to be the best application in isolating and analyzing microorganisms, but shows as much promise as the other methods. At the very least, flow cytometry needs to be further researched. Flow cytometry might be the next trend. As the microbiology field advances, flow cytometry might evolve to be the best method. Only time will tell. All of the methods, flow cytometry, microscopy, antibodies, and magnetic beads are important. These applications can be used together or separately. Flow cytometry should be developed and studied more. Works Cited Gadd, Geoffrey and Sima Sariaslani. Advances in Applied Microbiology. Vol. 83. London: Academic Press, 2013. Print. Hurst, Christon, J and Ronald, L Crawford. Manual of environmental microbiology. 2. ASM Press, 2002. Print. Matsudaira, Paul, T, et al. Flow Cytometry, Part 2. 2nd. Elsevier, 1994. Print. Porter, Jonathan. "Section 8 Update - Physical separation of active bacteria from natural environments using flow cytometry cell-sorting." Kowalchuk, G, A, et al. Molecular Microbial Ecology Manual. Springer Netherlands, 2004. 1775-1788. Print. Prorot, Audrey, Philippe Chazal and Patrick Leprat. "Flow Cytometry as a Powerful Tool for Monitoring Microbial Population Dynamics in Sludge." Schmid, Ingrid. Flow Cytometry-Recent Perspectives. InTech, 2012. 43-68. Web. 17 November 2014. . Sen, Keya and Nicholas, J Ashbolt. Environmental Microbiology: Current Technology and Water Applications. Ed. Keya Sen and Nicholas, J Ashbolt. Norfolk: Horizon Scientific Press, 2011. Print. Read More