Bacteriophages are a type of virus that infects bacteria (Pizarro-Bauerle and Ando, 2020). They are made up of proteins and either DNA or RNA (Paez-Espino et al., 2016, Williamson et al., 2017). They can be classified based on their morphological characteristics, type of nucleic acid, biological cycle (lytic and lysogenic or virulent and temperate), their host bacteria, and the location where they are found (Principi et al., 2019). Bacteriophages are biological entities with a length generally raging between 20 and 200 nm. They consist of various structures such as a head with nucleic acid, fibers, tail, collar, spikes, and a base plate (Adriaenssens and Brister, 2017, Aiewsakun and Simmonds, 2018, Simmonds and Aiewsakun, 2018, Tolstoy et al., 2018). They are detectable in different environments such as soil, animal secretions, swage, seawater, ocean, and extreme environments with very high or low temperature, and are considered as the most abundant biological entity (Clokie et al., 2011, Zhan et al., 2015).

In recent years, there has been an increase in antibiotic-resistant bacteria and a slow development of new antibiotics. This has prompted scientists to search for new therapeutics methods (Lewis, 2020, Wei et al., 2020). The Centers for Disease Control and Prevention (CDC) reported in 2019 that there are more than 2.8 million infectious diseases resulting in approximately 35000 deaths each year in the United States (Becattini et al., 2016, Ianiro et al., 2016). According to reports, bacterial antimicrobial resistance (AMR) could lead to 10 million deaths annually by 2050 (Murray et al., 2022, O'neill, 2014), 2016). However bacteriophages are being considered as an alternative approach to overcome resistant bacteria and infectious diseases (Ács et al., 2020). The use of bacteriophages has several advantages, such as specificity, safety, easy administration, a narrow spectrum of host, low cost to isolate, limited side effects on the infection site, self-replication, and higher tolerability (Principi et al., 2019).

There are various ways to detect bacteriophages, and one such method is through the use of a transmission electron microscope (TEM). TEM can help in identifying families of bacteriophage (Ács et al., 2020) and it can also classify new viruses in novel families (Ackermann, 2012). The development of electron microscopy (EM) in the 1930s enabled the identification of objects that were not detectable by common light microscopy (Prebus and Hillier, 1939, Ruska, 1934). Early electron microscopes were able to detect tobacco mosaic virus, although the micrographs produced were of low quality compared to present-day instruments (Kausche et al., 1939, Stanley and Anderson, 1941). In the late 1950s, the standard preparation called negative staining was developed, which increased the sensitivity of the method to 106 viruses per ml (Brenner and Horne, 1959). EM identification has several advantages, such as high speed (10–15 minutes), the ability to analyze both liquid and tissue samples, and the ability to process large numbers of samples. Moreover, prior information is not necessary for this experiment (Goldsmith and Miller, 2009, Hazelton and Gelderblom, 2003). TEM is a rapid test used to detect disease outbreaks (Beniac et al., 2014) by generating a beam of electron that provides 1000x higher resolution than a light microscope. The resolution should be about 0.2 nm or lower for viruses to be visualized. To prepare TEM micrographs, it is necessary to concentrate the bacteriophage to almost 106 particles per ml (Goldsmith and Miller, 2009, Mann, 2005). TEM is not only suitable method for examining morphology but also for bacteriophage enumeration. However, the preparation step is expensive and time-consuming, requiring a skilled technician and complex instrument. This step can also be tedious and may have an effect on the quality of photograph (Ackermann, 2012).

Certain types of bacteriophages, such as those with tails and large capsids, tend to be more stable than others. The stability, occurrence, and viability of bacteriophages are influence by various physical and chemical factors, including ions, salinity, temperature and acidity. These factors can cause damage to bacteriophages by altering their lipids, nucleic acids, or their structures, such as their head, envelope and tail (Ackermann et al., 2004). There is no universal preservation method for all bacteriophages due to their varying sensitivity (Łobocka et al., 2018). Bacteriophages have different susceptibilities to various solution ingredients (Knezevic et al., 2011, Tovkach et al., 2012), particularly for long-term preservation (Jończyk-Matysiak et al., 2019). For instance, solutions with neutral pH and low level of ionic contents are suitable for preserving bacteriophages at 4°C and increasing their stability, but this varies for different types of bacteriophages (Briers et al., 2008, Duyvejonck et al., 2021).

Researchers often have to send their bacteriophage sample for TEM analysis multiple times because of low quality of TEM micrographs. It requires more money and time, particularly in laboratories without enough advanced equipped devices such as ultracentrifugation. Therefore, researchers need a simple and cost-effective way to increase TEM micrographs quality. In this study, we aimed to improve a simple and cost-effective way to promote the quality of TEM micrographs. Additionally, we determined the viability and stability of our bacteriophages in SM buffer and deionized water, which is crucial for further experiments like TEM observation.