Practical Report: Determination of a Bacteriophage Titer Report

Summary

Viruses are microorganisms that can only survive and reproduce in the cells of other organisms and, as such, can infect a wide range of living entities, from bacteria to plants, animals, and humans. It is suggested that all the different viruses account for the quantity exceeding the number of stars in the universe (Wu, 2020). For this reason, the scientists developed various typologies that would help them to organize these microorganisms in groups based on common features and functions.

David Baltimore suggested one such classification, which sorted viruses based on how they produced messenger RNAs (Ryu, 2016). The first two groups include double-stranded DNA viruses and single-stranded DNA viruses. The next three groups encompass double-stranded RNA viruses, positive-sense single-stranded RNA viruses, and negative-sense single-stranded RNA viruses. Finally, single-stranded RNA viruses with a DNA intermediate and double-stranded DNA viruses with an RNA intermediate form the last two groups.

According to the other classification, viruses are grouped by the kind of cells that they infect. In this regard, bacteriophages are known as viral organisms that attach solely to bacteria. These viruses have received increased attention from the academic community since understanding the bacteriophage functioning can lead to their usage as human diseases treatment as an alternative to antibiotics. Considering that, all the current and future specialists in the spheres of biology and immunology need to have knowledge in how to examine these microorganisms in the laboratories.

The first step in this process is learning how to determine bacteriophage titer or phage concentration. To achieve that, the bacterial cells should be mixed with bacteriophage in soft agar in order to examine how the former would create colonies and the latter would form the plaque by destroying bacterial cells. Then, the plaque-forming units are determined based on the received plaque assay. Therefore, the aim of this report is to present the results of the experiment that followed the abovementioned procedure.

Results

Table 1: Dilution of the bacteriophage and plaque number

Appendices

1. Gram-negative bacteria’s outer membrane consists of various proteins, lipids, porins, and lipopolysaccharides (Vergalli et al., 2020; Breijyeh, Jubeh, and Karaman, 2020). In this regard, different bacteriophages may attach to different parts of the bacteria. For instance, T4 bacteriophage attaches either to OmpC protein or lipopolysaccharide on the surface of Escherichia coli bacteria (Brzozowka et al.,2020). On the other hand, the host receptor for the lambda phage in the same bacteria is found to be the FhuA protein (Ge et al., 2020).
2. Viruses are called obligate intracellular parasites as they need the host cell in order to survive and multiply (Durmuş and Ülgen, 2017). Nevertheless, such definitions may be confusing as viruses and parasites are different because the latter sustain their lives by consuming the living parts of the cells. On the contrary, the former infects the host organism with its RNA or DNA (Durmuş and Ülgen, 2017).
3. Endospores are dormant forms of bacteria that are quite challenging to destroy. However, there are still some sterilization methods that can help to effectively eliminate those organisms. As such, Yoo (2018) names three options, namely the usage of high temperatures, ethylene oxide, and hydrogen peroxide vapor or plasma. For instance, autoclaves can be used to destroy endospores with high-temperature steam.
4. As for humans, the type of viral infection that almost certainly alters the host cell genetics is the group of retroviruses. Even though some other viruses, such as, for instance, cell fusing agent virus, can sometimes lead to viral integration, only for retroviruses is this step mandatory (Engelman and Cherepanov, 2017). For this reason, such diseases as HIV can be transmitted from the infected mother to a child during pregnancy. As for bacteria, those bacteriophages that follow the lysogenic life cycle can impact the cell genome in case lysogenic conversion occurs.
5. Lysogenic conversion is the process that occurs when a bacteriophage accidentally gains part of the bacterial genome when it exits the bacterial chromosome to start the lytic cycle. As a result, when a phage infects the next microorganism, the genetic structure of the latter would change as it will receive part of the previous bacteria genome.
6. The conducted experiment revealed that when plated, Escherichia coli colonies are presented as dark creamy areas, whereas bacteriophage plaques are transparent due to the lysis of the bacterial colony.
7. The lytic cycle consists of four steps, namely bacteriophage attachment, bacterial cell penetration, replication, and release. After the bacteriophage attaches to the cell, it makes a hole in the protection wall and inserts its genome inside the bacterium. Next, the virus starts multiplying by using the nucleic acid found in the host genome, which leads to the degradation of the latter (Stewart, 2021). As a consequence, the newly formed bacteriophages leave the bacterium to continue infecting the healthy cells.
8. After the phage nucleic enters the host cell, the bacterium’s genome starts degrading (Stewart, 2021).
9. Lysozymes were found to play an important role in the lysis process. Ragland and Criss (2017) maintain that this enzyme causes the cell wall peptidoglycan destruction through its hydrolysis. Therefore, many bacteriophages have lysozyme encoded in their genome. This chemical not only helps with cell penetration and infection spread process but also facilitates lysis which is associated with the release of newly formed viruses.

Reference List

Breijyeh, Z., Jubeh, B. and Karaman, R. (2020) ‘Resistance of gram-negative bacteria to current antibacterial agents and approaches to resolve it. Molecules, 25(6), p.1340.

Brzozowska, E. et al. (2018) ‘Interactions of bacteriophage T4 adhesin with selected lipopolysaccharides studied using atomic force microscopy, Scientific Reports, 8, pp. 1-7.

Durmuş, S. and Ülgen, K. (2017) ‘Comparative interactomics for virus–human protein–protein interactions: DNA viruses versus RNA viruses’, FEBS Open Bio, 7(1), pp. 96-107.

Engelman, A. and Cherepanov, P. (2017) ‘Retroviral intasomes arising’, Current Opinion in Structural Biology, 47, pp. 23-29.

Ge, H. et al. (2020) ‘The” fighting wisdom and bravery” of tailed phage and host in the process of adsorption’, Microbiological Research, 230, p.126344.

Ragland, S. A., and Criss, A. K. (2017). ‘From bacterial killing to immune modulation: recent insights into the functions of lysozyme’. PLoS Pathogens, 13(9), p. e1006512.

Ryu, W. S. (2016). Molecular virology of human pathogenic viruses. Cambridge: Academic Press.

Steward, K. (2021) ‘Lytic vs. lysogenic – understanding bacteriophage life cycles’, Technology Networks, Web.

Vergalli, J. et al. (2020) ‘Porin-mediated small-molecule traffic across the outer membrane of Gram negative bacteria’, Nature Reviews Microbiology, 18(3), pp.164-176.

Wu, K. J. (2020) ‘There are more viruses than stars in the universe. Why do only some infect us?’, National Geographic, Web.

Yoo, J. H. (2018) ‘Review of disinfection and sterilization – back to the basics, Infection & Chemotherapy, 50(2), pp.101-109.