Isolation and Identification of Bacterial Strains from Skin of Terrestrial Amphibians

This research aimed to develop an efficient protocol for collecting skin microbiota from wild terrestrial amphibians and to ensure microbial integrity during storage and handling.

Key challenges include preventing sample contamination, maintaining microbial viability during storage, and culturing slow growth to fully assess amphibian skin microbiota diversity.

It was established that glycerol-enhanced saline preserves amphibian skin microbiota integrity during storage and handling, consistent bacterial culturing, and improving microbial conservation strategies for ecological and biotechnological research.

This protocol addresses the lack of standardized sampling methods, preserves and handles culturable amphibian skin bacteria, and ensures community integrity.

[Narrator] To begin, sterilize the medium by autoclaving at 121 to 134 degrees Celsius for 15 to 30 minutes. After autoclaving, allow the agar medium to cool to approximately 45 to 50 degrees Celsius. Under a previously sterilized laminar flow hood, pour the cooled medium into sterile Petri dishes. Once the agar has solidified, label the Petri dishes with the medium type and preparation date. Mix the sample obtained from the field with the solution by pushing the swab against the tube walls while squeezing the tube and rotating the swab thoroughly, 10 times clockwise and 10 times counterclockwise. Thoroughly mix the microbiological sample before processing. Prepare serial dilution of the cutaneous sample with saline solution ranging from 10 to the power of -1 to 10 to the power of -6. Inoculate 100 microliters of the undiluted sample and serial dilution onto the prepared agar plates. Using a sterile L-shaped loop, streak the sample across the surface of the agar medium, ensuring even distribution without applying excessive pressure. Invert the inoculated plates and place them inside an incubator set at 20 to 25 degrees Celsius for three to five days to support the growth of mesophilic and psychotropic bacteria on amphibian skin. After incubation, select bacterial colonies with distinct morphologies for further analysis. Ensure that the colonies are well isolated and exhibit different appearances, such as variations in color, size, shape, edge, surface, or texture. To subculture the selected colonies, transfer a well-isolated colony from the original agar plate to a fresh agar plate using an inoculating loop under aseptic conditions. Streak the colony to isolate it. And then incubate it appropriately. The coordinated sampling procedure of a speleomantes italicus individual using sterile tools in a field setting is shown here. Sampling was performed by two operators using sterile gloves and swabs to minimize stress on the amphibian and ensure precision during handling. The swab was immediately transferred into a saline solution with 20% glycerol to preserve microbial integrity during transport to the laboratory. A comparison of the microbial community density kept in saline solution with and without glycerol addition is shown here. The bacterial load in saline storage without glycerol was significantly lower than the control and saline with glycerol. The microbial community stored in saline with glycerol showed a similar bacterial load to the control, indicating the preservation of microbial integrity. The best preservation of the microbial community was observed in saline with glycerol stored at four degrees Celsius, showing the most stable bacterial load over time. Storage in saline alone at room temperature resulted in the highest bacterial growth, indicating microbial contamination or selective promotion of certain bacterial groups. Freezing at minus 20 degrees Celsius reduced the bacterial load, suggesting that low temperatures negatively impacted microbial survival.