Nematode community dynamics in Johnson Su compost

Characterising microscopic communities is important for both ecological research and diagnostic applications. In these areas, light microscopy is still a fundamental tool due to its accessibility and real-time imaging capabilities. However, the higher magnifications required for accurate identifications typically necessitates extensive sample preparation. Therefore, this study aimed to design and validate a bright field light microscopy-based platform with magnification up to 1000x without the need for extensive sample preparation. An adapted version of the Peter’s slide was developed, which allows the use of specialised long-working distance objectives without the need for oil immersion. Image quality at higher magnifications were evaluated using nematodes as test organisms. Additionally, the use of the adapted Peter’s slide in characterising microscopic communities was evaluated by studying the temporal dynamics in nematode functional diversity from Johnson Su compost. The adapted Peter’s slide facilitated the identification and counting of nematodes to genus level, demonstrating sufficient image clarity even at 1000x magnification. My results confirmed that this platform overcomes the limitations of standard approaches by eliminating the need for extensive sample preparation and mounting on microscope slides, which are typically required for high-magnification observations. This platform reduces sample processing time, while also facilitating live viewing under high magnification. These features also make it a valuable educational tool in classroom settings. Future enhancements could include the integration of AI-based imaging software to automate and refine identification and counting processes. However, a current limitation is the cost of the long-working objectives, which may limit widespread adoption.

Methods
To validate the adapted Peter’s slide and microscope setup as a platform for characterising microscopic communities, the temporal changes in nematode functional diversity within Johnson Su composting were investigated. This static composting method does not involve any turning after startup and aeration is facilitated via vertical shafts in the compost reactor. Six Johnson Su compost reactors serving as replicates were constructed and maintained following Johnson and DeSimio (2017). The feedstock material consisted of 60% dried grass, 30% wood shavings, and 10% horse manure. Compost samples were collected at three intervals, namely the 1st, 3rd, and 5th months after startup. Using a 20cm stainless steel corer (5 cm diameter) attached to a battery drill, five randomly located cores were extracted per reactor per interval and homogenised to acquire one composite sample. From each composite sample, nematodes were extracted from 200 ml of compost using the decanting and sieving followed by the sugar centrifugal flotation methods (Marais et al., 2017). The nematodes were heat killed and fixed using hot/cold 6% formaldehyde solutions (final sample concentration of 4% formaldehyde) (Van Bezooijen, 2006).

Prior to counting, each nematode community sample was rinsed with clean water using a 20 µm sieve to remove the carcinogenic formaldehyde. The nematodes were transferred to sample bottles with a final sample volume of 20 ml. Before counting, each sample was vigorously shaken to suspend and homogenise the nematode community. Without delay, a 1 ml aliquot was abstracted and transferred to the adapted Peter’s slide. All the nematodes in the 1 ml aliquot were identified and counted using the described microscope setup. The number of nematodes per taxon per 1 ml aliquot were extrapolated to the total volume (20 ml) of the original sample.