**7. The importance of culture-dependent and culture-independent studies of halophilic viruses**

Within the field of microbiology, there are two different generalized approaches that can be used to study the microorganism(s) of interest. These being the cultureindependent and culture-dependent approaches, both of which have their own advantages and limitations. The culture-independent approach is often used by environmental scientists that are interested in how microorganisms exist within their natural habitat. This type of information can tell us how these microbes respond to everyday natural elements that they are exposed to and help formulate questions as to how they are able to adapt to changes in environment. This approach can also give us insight into which microorganisms are able to coexist within the same

### *Bacteriophages in Therapeutics*

environment and hypothesize what roles they may play with one another. While this approach can give scientists a large amount of useful information, there are some limitations. For example, if a mutation were to be observed across a species of microorganisms within a specific habitat, it would be difficult to pinpoint the specific cause. This is because in nature, there are countless amounts of influential factors to be taken into consideration.

The culture-dependent approach on the other hand, is an approach used by scientists within a laboratory setting. The microorganisms used to conduct these experiments are ones that have been harvested from a type of medium within the lab. Using this method, scientists are able to understand and better identify the microorganism of interest as well as observe its ability to adapt to changes initiated by researchers. Another advantage to this type of approach is that within the lab, microorganisms can be replicated and used on a larger scale. The culture-dependent approach is also important because this allows for isolation and purification of an organism of interest. Once a purified culture is obtained, meaning a culture is not contaminated by the presence of other organisms, it is able to be sent for genetic sequencing. As previously mentioned, the ability to sequence an organism's genetic material not only gives scientists more information as to why a certain organism may have certain behaviors or characteristics but it also opens an infinite number of doors for future possible research avenues. Just like the culture-independent approach, the culture-dependent approach also has limitations. For example, when how a microorganism may respond to a certain change within the environment this does not necessarily represent how it would respond in its natural habitat.

Examples of scientists utilizing the culture independent approach would be the scientists previously mentioned Daniels and Wais. They used this method to evaluate the fluctuation in the microbial communities within salt ponds due to rain fall. This type of experiment would not be able to be carried out within a laboratory setting with the same amount of accuracy. Another example of this approach being utilized was by scientist Oren et al. when they were collecting water samples from the Dead Sea over a specific time period. The purpose of this was to see how the microbial population as well as the amount of viral like particles fluctuated throughout the year. This also would be nearly impossible to carry out accurately within a laboratory setting. Many of the initial discoveries made of halophilic viruses were done with this culture-independent approach.

Examples of scientists utilizing the culture-dependent approach include studies done by Guixa-Boixareu et al. when they evaluated samples taken from a Spanish saltern [31]. Within the laboratory they were able to assess how varying salt concentration percentages affected the causes of prokaryotic death. It was found that at higher salt concentrations, more prokaryotes were terminated by amoebas or protozoans while at lower salt concentrations prokaryotic death was more due to viral lysis [9]. Scientists Torsvik and Dundas also used this approach to help support the environmental findings previously mentioned by Daniels and Wais. While they were studying the halophile *Halobacterium Salinarum* within the laboratory they were able to observe that the virulence exhibited by the virus Hs-1 appeared to be influenced by the saline concentrations present [9]. Their findings also suggested that as salt concentration is increased, the rate at which viruses are able to infect a host decrease.

In conclusion, both the culture-dependent and culture-independent methods of research are of vital importance within the scientific community. These approaches allow us to examine microbes within their natural habitats and from there formulate hypotheses about why they behave in the ways that they do. In the field of halophilic viruses, it is important that scientists continue to utilize both methods to gain a better overall understanding. There is still so much information needing

to be collected and investigated to better understand these viruses and how they have influenced not only their environment but their possible influence on already known and studied bacteriophages.
