Long-term Storage of Bacterial Isolates by Using Tryptic Soy Broth with 15% Glycerol in The Deep Freezer (-70 to -80 °C)

For different bacterial preservation techniques, there is no single method applicable for all bacteria. This study aimed to assess the viability of seven species/species groups of clinical bacteria isolates on the long-term storage (more than 5 years) by using Tryptic Soy Broth with 15% glycerol in the deep freezer (-70 to -80°C). A total 10,654 clinical bacteria isolates used as samples in this study. The isolates consisted of seven species/species groups (i.e. Escherichia coli, Campylobacter spp, Shigella spp, Vibrio spp, Salmonella spp, Aeromonas hydrophila, and Neisseria gonorhoeae). The isolates were collected from some previous studies and preserved in the Tryptic Soy Broth (TSB) with 15% glycerol and stored in the deep freezer (-70 to -80°C) for more than five years. The samples were revived on the suitable medium to evaluate the viability of bacteria. Identification conducted by microscopic examination, biochemical test, and latex agglutination. The study showed that the viability of Salmonella spp, Shigella spp, Aeromonas hydrophila and E. coli was 100%, while Campylobacter spp, Vibrio spp, and N. gonorhoeae were 66.7%, 66.4%, and 52.5% respectively. We concluded that viability of Salmonella spp, Shigella spp, A. hydrophila, and E. coli was optimum thus better than Campylobacter spp, Vibrio spp, and N. gonorhoeae for more than 5 years storage by using TSB with 15% glycerol in the deep freezer (-70 to -80 °C).


1.
Introduction Indonesia is a tropical country with mega biodiversity, ranging from plants, animals, and also microbes. The diversity of microbes can further increase through their mutation into a different strain [1], adding to the sum of total biodiversity. This high level of biodiversity is a precious asset, we find it as useful resources for research and the development of science and technology [2,3]. The development in science and technology allows humankind to obtain many benefits from microbes that makes life easier and more convenient, e.g., vaccine production in medicine [4], enzyme production in biochemistry [5], and probiotic microorganism addition in biotechnology of food production [6]. Polymerase Chain Reaction (PCR) assessment method is becoming easier and faster using the Taq polymerase enzyme, which was isolated from the bacterium Thermus aquaticus [7]. The development of DNA vaccine is more effective using cloning method with Escherichia coli as vector [8]. Various fermented foods (e.g., yogurt, cheese) were manufactured using microorganisms such as bacteria [9]. Nevertheless, several microbes could be misused as a threat, such as bioterrorism that can threaten the safety and health of humankind. Since the first world war, there were several microorganisms utilized as biological weapons [10]. The Center for Diseases Control and Prevention (CDC) has assigned three different categories of microorganism-based biological weapons. The most harmful one is the category A which consisted of bacteria and virus, such as Bacillus anthracis, Francisella tularensis, Smallpox virus, Clostridium botulinum, Yersinia pestis, and Viral hemorrhagic virus (Ebola, Marburg, Lassa, and Machupo) [10,11].
In order to gain benefits and avoid the harmful possibilities of various microorganisms, special attention and specific efforts are needed for storage and maintaining them. The microorganism preservation and maintenance techniques are customized based on their intended utilization, i.e., daily, short-term, or long-term. Short-term utilization is usually related to research purposes that were daily needed. Long-term utilization is related to collection and conservation purposes [2]. There are several microorganism preservation methods and each of it has its own advantages and/or disadvantages. Thus, there is no single method applicable for all kinds of microorganisms. Some microorganisms can be easily stored and be able to survive with various preservation methods. Meanwhile, any other microorganisms have preservation limitations, for instance, their sensitivity for changes in temperature, pH, or moisture [12,13]. This study aimed to assess the viability of seven species/species groups of clinical bacteria isolates on the long-term storage (more than 5 years) by using TSB with 15% glycerol in the deep freezer (-70 to -80 o C).  Table 1). The isolates collected from some previous studies. The isolates were preserved in 1.5 ml tubes containing Tryptic Soy Broth (TSB) with 15% glycerol and stored in the deep freezer (-70 to -80 o C) for more than five years. Sample selections were conducted based on inclusion criteria, including their packagings and label were still intact and not disintegrate.

2.2.
Laboratory Examinations All samples were revived on the suitable growth or selective medium. Salmonella spp, Shigella spp, Campylobacter spp, Vibrio spp, E. coli, and A. hydrophila, were revived on the MacConkey agar and blood agar. Meanwhile, N. gonorhoeae were revived on the chocolate agar. The inoculated medium was incubated at 37°C for 24 hours. The isolates of three bacteria species/species groups (Campylobacter spp, A. hydrophila, and N. gonorhoeae) were incubated in the CO2 incubator. The bacterial colonies on culture medium were identified by microscopic examination dan biochemical test. Latex agglutination was used to determine serotype of some bacteria. The conclusion of bacterial viability was determined in the presence or the absence of bacterial colonies on the culture medium by ignoring number of colonies. When we found at least one bacterial colony and identified correctly by microscopic and biochemical test, it mean the isolate viable. Hence, if there was no bacterial colony found on the culture medium, the samples were re-cultured. And when there was still no bacterial colony for second reviving, it concluded that isolates were non-viable.

3.
Results There were 9,920 (93%) out of a total 10,654 samples containing viable bacteria. Description of the viable bacteria by species/species groups were shown in Table 2. Compared to the other six bacteria in this study, N. gonorhoeae has the most proportion of non-viable isolates ( Table 2). The other bacteria showing a relatively sensitive were Campylobacter spp and Vibrio spp ( Table 2). On the other hand, viability of aerobic bacteria better than anaerobic bacteria relatively as well as diarrhea-causing bacteria better than sexually transmitted infection-causing bacteria (Table  3).

Discussion
Based on the theory, all methods of preserving bacteria are carried out by limiting the metabolism of bacterial isolate. Bacterial isolate preservation using medium TSB + 15% glycerol in the ultra-low temperature freezer has several advantages, e.g., relatively easy, space-saving, and applicable for aerobic and anaerobic bacteria. However, some bacteria cannot be stored using this medium. For instance, Neisseria meningitidis has shown better compatibility with Greaves medium and Mycobacterium tuberculosis that was better stored in medium containing skim milk [14,15]. Therefore, the preservation technique needs to be customized based on the bacterial isolates since the bacterial viability depends on the preservation method and bacterial species. Table 2 showed that the viability of bacterial isolates may vary. One factor that influences the viability of bacteria during ultra-low temperature preservation is the temperature instability [16]. A broken or less-functional freezer and the high frequency of opening and closing the freezer door can cause freeze-thawing and temperature instability [17]. Furthermore, an electrical problem can also cause temperature loss. Several bacteria, e.g., Vibrio spp, Campylobacter spp, and Neisseria spp were showing a sign of sensitivity towards the temperature changes. A study by Mils & Gherna showed the optimum viability of Campylobacter spp was gained with the liquid-nitrogen preservation method [18]. One limitation of this study was the absence of information regarding the freezer history condition (whether it was broken before or not), the frequency of opening the freezer door, freeze-thawing, and electrical instability during the five years of preservation. Even though there was an electricity generator available in the laboratory where samples were kept, this generator needs some times to start generates electricity. On the other hand, qualitative data of the bacterial viability (presence or absence of bacterial colony) only observed in this study is another limitation.
All bacterial isolates were Gram-negative bacteria in this study and all samples (94.4%), but N. gonorhoeae were bacteria causing gastroenteritis diseases (Table 3). The Gram-negative bacteria has a relatively thin and fragile cell wall compared to the Gram-positive bacteria. This characteristic can influence bacterial viability during preservation. A study by Mai-Prochnow, et al showed that the thickness of the cell wall influenced the sensitivity and resistance towards cold atmospheric-pressure plasma (CAP). Another study by Miyamoto-Shinohara, et al showed a higher survival rate of Gramnegative bacteria compared to Gram-positive bacteria during preservation with freeze-drying [19,20].
Based on the results of this study, we concluded that viability of Salmonella spp, Shigella spp, A. hydrophila, and E. coli was optimum thus better than Campylobacter spp, Vibrio spp, and N. gonorhoeae for more than 5 years storage by using TSB with 15% glycerol in the deep freezer (-70 to -80 o C). Laboratories storing bacterial isolates, especially Vibrio spp, Campylobacter spp, N. gonorhoeae, and other bacteria that were sensitive to temperature changes need to pay attention to the factors affecting the bacterial viability, especially related to the freezer and indoor temperature stability [17]. The scheduled monitoring of freezer and indoor temperature is required to be done, so it can be used as early warning system to detect the risk of damage or death of bacterial isolates.