It is important to develop an easy and convenient technique for maintaining the viability, function, morphology and genetic traits of pure isolates to allow maximum restoration after storage¹¹. The present study found that the best temperature of preservation for S. aureus and E. coli was -20°C for a year, in all preservation methods used in our study except G100% and FO methods in E. coli, Fig. 6 and 9. To date, cryopreservation is considered the best method for storing bacteria, and the best survival rates are recorded using this method¹. In the cryopreservation method, bacteria are stored at a frozen temperature to minimize or prevent metabolism and physical change¹².

The current study found that S. aureus bacteria survived for one year in normal saline, while E. coli survived in sterile DW in the same period at (4, 25 and -20)°C. This is confirmed by researchers from Wyndmoor, PA, USA⁸, they were observed that bacteria could remain alive in water and PBS for 2 years at room temperature range between (20-25)°C, and showed that G+ bacteria can be maintained them in PBS better than DW, while the study⁴ conducted at University of California, USA too, confirmed that the bacteria they studied could survive in DW more than 20 years. The present study does not agree with researchers⁸, that S. aureus was unable to give golden endo-pigmentation at 4°C by NS preserving method. This may be due to a lack of continuous supply or fluctuations in electricity during the period under study, due to the city’s conditions after liberation. Researchers from the USA (Liao et al.) and Australia (Banning et al.)^8,13, reported that E. coli survived in sterile water and groundwater for several months respectively. This is the conclusion from the current study using DW as a preservative, the tested bacteria survived until the end of the study period. In another study by Ji and Jin of China¹⁴, found that the best option for bacterial preservation, is to use DW alone without any addition at 4°C, which agrees with the present study on E. coli, but it is not comparable to our study of S. aureus, where it could not live for a year in DW at 4°C, Fig. 5 and 8. Current findings contrast with those of researchers in the last century, where they pointed out that most microorganisms die if suspended with DW, except Pseudomonas solanacearum, they remained alive for 10 years in the water at room temperature while dying when stored in the refrigerator¹⁵. Despite the simplicity of using DW as a preservation liquid, it has not been widely used by microbiologists, due to insufficient data and studies related to it to verify the feasibility of this method or not⁸.

The present study is the first to use food oil alone or with normal saline as a preservation liquid for the conservation of bacteria, where no other studies have preceded. The FO+NS proved effectiveness in the preservation of E. coli at three temperatures used, Fig. 7-9, but S. aureus could not survive at 25°C Fig. (4), while using FO alone preserve S. aureus for a year at -20°C, Fig. 6, this may be due to the different composition of the cell wall in G+ve from G-ve bacteria. The use of FO alone or with NS maintained the phenotypic patterns of preserved bacteria. Many studies successfully used liquid paraffin or mineral oil, which prolongs the microbial growth life, prevents evaporation and reduces the rate of metabolism of the medium by reducing oxygen. This is a simple and economical method for maintaining pure cultures of bacteria. In this method, the oil or paraffin is poured over the slant of culture and stored at room temperature. This condition helps pure culture to remain in a dormant state and the culture can be preserved from months to years¹⁶.

There was a recorded report that Pseudomonas, Bacillus, and E. coli remained alive below the oil for 3 years and that their phenotypic characteristics did not change¹⁴. In a study conducted by Yamasato et al., from Japan¹⁷, they observed that 75-85% of the strains tested, remained viable under liquid paraffin at 10°C for 2 years and at -53°C for 3.5 years.

The researcher classified the Cryoprotectants as penetrating or non-penetrating the cell, Glycerol belongs to the latter one³. It is one of the most important types of cell-conserving substances¹⁸, that act to surround the cells, delay the freezing of intracellular substances in these cells and reduce the solution effects¹. It prevents ice crystallization and as a result, bacteria remain alive longer³, it is a strong substance that prevents water from entering the cell¹⁹. Frozen culture is the most commonly used preservation method²⁰. We can maintain Microorganisms by freezing broth cultures at -5 to -20°C for 1-2 years in suitable containers²¹. Chen and Jin’s study from China²² indicated that cryopreservation is easier and cheaper compared to lyophilization methods, and if freezing processes are successful, the bacterial mass survives for up to 35 years.

In this study, glycerol was used in many concentrations alone or with NS and found that these concentrations were good for preserving bacteria at -20°C, this does not correlate with the researchers’ study from India³, they may be due to the concentration of glycerol used in their study by 10-15% or without normal saline. An expanded study¹⁷ indicated that the use of a slanted agar method then covered aerobic bacteria growth by 10% glycerol, maintained the bacteria at -53°C for 16 months. In another study¹², the researchers reported that the bacteria kept in 20% glycerol including Staphylococcus that managed to survive for 3 months and there are no changes in biochemical properties, this is the result of the current study when using glycerol by 15% and 30% for the same period at 25°C.

Our study contradicts with Laurin et al., from Canada²³, where they found that the use of 15% glycerol at -20°C was sufficient to keep the bacteria for more than a week, while in this study, glycerol managed to keep the bacteria alive for a year at this temperature except for G100% method for preserving E. coli, Fig. 6 and 9.

In this study the Precipitation method was also used, the optimum temperature of preservation in this method is -20°C as well, but at a concentration of G50% and G100% in both S. aureus and E. coli, the bacteria couldn’t survive for a year, the preservation using FO at 4°C gave a result of S. aureus and remained alive for a year, while using G30% and G15%, E. coli also survived for a year at 4°C.

The difference in the result between the previous method and the Precipitation method in the use of FO or glycerol as a preservative, maybe due to the mechanical deposition of the suspended bacteria and then the oil is added, while in the first method the bacterial inoculation was added to the preservation liquid, so the FO or glycerol surrounding from all direction and maintaining it.

According to the data collected indicate that the preservation liquid consisting of one or both of these substances DW, NS, G, and FO, will provide protection against loss of viability and may increase the long-term preservation when maintained at -20°C.