Revisiting soil bacterial counting methods: Optimal soil storage and pretreatment methods and comparison of culture-dependent and -independent methods

Abstract

Although a number of different methods have been used to quantify soil bacteria, identifying the optimal method(s) for soil bacterial abundance is still in question. No single method exists for undertaking an absolute microbial count using culture-dependent methods (CDMs) or even culture-independent methods (CIMs). This study investigated soil storage and pretreatment methods for optimal bacterial counts. Appropriate storage temperature (4°C) and optimal pretreatment methods (sonication time for 3 min and centrifugation at 1400 g) were necessary to preserve bacterial cell viability and eliminate interference from soil particles. To better estimate soil bacterial numbers under various cellular state and respiration, this study also evaluated three CDMs (i.e., colony forming unit, spotting, and most probable number (MPN) and three CIMs (i.e., flow cytometry (FCM), epifluorescence microscopy (EM) count, and DNA quantitation). Each counting method was tested using 72 soil samples collected from a local arable farm site at three different depths (i.e., 10-20, 90-100, and 180-190 cm). Among all CDMs, MPN was found to be rapid, simple, and reliable. However, the number of bacteria quantified by MPN was 1-2 orders lower than that quantified by CIMs, likely due to the inability of MPN to count anaerobic bacteria. The DNA quantitation method appeared to overestimate soil bacterial numbers, which may be attributed to DNA from dead bacteria and free DNA in the soil matrix. FCM was found to be ineffective in counting soil bacteria as it was difficult to separate the bacterial cells from the soil particles. Dyes used in FCM stained the bacterial DNA and clay particles. The EM count was deemed a highly effective method as it provided information on soil mineral particles, live bacteria, and dead bacteria; however, it was a time-consuming and labor-intensive process. Combining both types of methods was considered the best approach to acquire better information on the characteristics of indigenous soil microorganisms (aerobic versus anaerobic, live versus dead).

Fig 1. Study area and sampling points (garden and farm soil) for soil bacterial counts.

Fig 2

Experimental steps and methods for bacterial counts from garden (A) and farmland (B) soil samples.

Fig 3. Effects of storage temperature on bacterial numbers.

Results were obtained by epifluorescence microscopy (EM) using garden soil samples collected from Korea University. The green lines indicate the initial cell numbers in the soil sample counted within 1 h. All other pretreatment conditions were the same except the storage temperature; vortexed at maximum speed for 5 min, sonication at 300 W for 3 min, centrifugation at 1400 × g for 15 min, and filtration through 10 μm filters. Experiments were conducted in triplicate and data are shown as the mean ± standard deviation.

Fig 4. Contour and bar plots showing the distribution of bacterial numbers at three depths of farmland soil samples.

The numbers were determined by five different methods (CFU, spotting, MPN, EM, DNA quantification). These soil samples were stored at 4°C and pretreated by vortexation at maximum speed for 5 min, sonication at 300 W for 3 min, centrifugation at 1400 × g for 15 min, and filtration through 10 μm filters. Experiments of bacterial counts were conducted in triplicate. *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig 5

Scattered graphs (A and C) and pie charts (B and D) of bacterial numbers determined by FCM. The results of A and B indicate the bacterial numbers in the farmland soil sample (A1 at 10–20 cm), and those of C and D show the bacterial numbers in the single cell cultured media (Shewanella sp. in R2A media). Live cells, dead cells, and soil mineral and organic matter particles are shown in green, red, and brown, respectively. The samples were stored at 4°C and pretreated by vortexation at maximum speed for 5 min, sonication at 300 W for 3 min, centrifugation at 1400 × g for 15 min, and filtration through 10 μm filters.

Fig 6. Photographs of bacteria and soil particles under EM.

The photographs of A and B show bacterial cells in the garden soil samples filtered through 10 μm filters, and those of C and D show bacterial cells attached on the soil microaggregate in the soil samples without filtration. The photograph of C was taken under differential interference contrast (DIC) mode, whereas those of A, B, and D were taken under fluorescence mode. Live cells, dead cells, and damaged cells are shown in green, red, and yellow, respectively. The samples were stored at 4°C and pretreated by vortexation at maximum speed for 5 min, sonication at 300 W for 3 min, and centrifugation at 1400 × g for 15 min.