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).

Soil bacterial biodiversity characterization by flow cytometry: The bottleneck of cell extraction from soil

“…type of cells and extraction protocols) and conditioned by a trade‐off with cell viability. These results agree with recent publications highlighting that step repetitions (re‐extractions) or stronger dispersion conditions could increase recovery but decrease cell viability (Lee et al, 2021; Ouyang et al, 2021). Our analysis of recovery efficiency was based on microscopy count, as this was the method most frequently used.…”

“…Therefore, we could not rule out the use of soil pre‐treatments before bacterial counts. All these factors could also contribute to the high variation detected for recovery efficiency estimates (Lee et al, 2021). However, even considering these constraints, our review revealed that recovery efficiency of native bacteria from soils is low (i.e.…”

“…This protocol had the highest cell viability and yield among the alternatives evaluated; however, blending is time‐consuming and the use of Nycodenz or Tween 20 should also be validated. Recently, Lee et al (2021) comparing culture dependent and independent quantification methods reported that FCM was ineffective in counting soil bacteria due to low purity of bacteria extracted from soils and staining of clay particles by the dyes used. However, the purification step of the protocol used was inadequate according to our review.…”

“…Recovery efficiency is actually very hard to estimate. No single method exists for absolute microbial count in soil due to its complex nature and, therefore, most of the available methods use a pre‐treatment of soil (Lee et al, 2021). According to our review, recovery efficiency showed high variation but was in general below 25% for native cells.…”

Biodegradation of cellulose fibers functionalized with CuO/Cu2O nanoparticles in combination with polycarboxylic acids

“…To determine the number of microbial cells per gram of garden soil and model compost, aliquots (1 g) before and after the soil burial of textile samples were taken and the standard culture-dependent enumeration procedure was applied (Lawlor et al 2000;Lee et al 2021). Soil micro-organisms were also cultured at 30 °C on three types of agar media: Luria-Bertani agar (LA, Oxoid, UK) for heterotrophic bacteria; mannitol soy flower agar (MSF) (Hobbs et al 1989) for spore-forming bacteria; and Sabouraud dextrose agar (SAB, Difco, UK) for fungi.…”