Improved Isolation of Uncultured Anaerobic Bacteria using Medium Prepared with Separate Sterilization of Agar and Phosphate

Abstract

We previously demonstrated that a simple modification in the preparation of agar media, i.e., autoclaving phosphate and agar separately (termed the "PS protocol"), improved the culturability of aerobic microorganisms by reducing the generation of reactive oxygen species. We herein investigated the effects of the PS protocol on the cultivation of anaerobic microorganisms using sludge from a wastewater treatment system as a microbial source. The application of the PS protocol increased colony numbers and the frequency of phylogenetically novel isolates under aerobic, nitrate reduction, and fermentation conditions. The PS protocol is useful for isolating both aerobic and anaerobic microorganisms.

Comparative Analysis of the Cultured and Total Bacterial Community in the Wheat Rhizosphere Microbiome Using Culture-Dependent and Culture-Independent Approaches

“…This could be due to the inhibitory effect of H 2 O 2 generated from the autoclaving of the gelling agents and phosphate together ( 20 ), and indeed separate autoclaving of the gelling agents and phosphate buffer resulted in much lower H 2 O 2 concentrations and increased culturability of microorganisms ( 20 , 28 ). Our data are also in agreement with previous publications, which reported that the PS technique is very effective for cultivating recalcitrant and novel bacterial taxa from various environments ( 8 , 17 , 21 ). Remarkably, isolates classified into Ochrobactrum and Neorhizobium were recovered only on Jensen medium solidified with agar following the PT protocol.…”

“…Many factors influence the culturability of microbes, such as the culture medium (e.g., medium composition and solidifying agents), growth conditions, and other cultivation techniques ( 21 , 32 , 33 ). Any modifications of these approaches may promote the growth of taxa that did not grow under the original conditions ( 34 ).…”

“…Historically, agar has been the predominant gelling agent in microbiological research, but a shortcoming in the preparation of agar-solidified medium is the difficulty in culturing microorganisms from diverse habitats because of how the agar is prepared ( 17 , 20 ). Autoclaving agar together with phosphate generates hydrogen peroxide (H 2 O 2 ), which limits both growth and colony formation of many microorganisms ( 20 ), whereas the separate sterilization of agar and phosphate improves the culturability of a wide range of microorganisms, including slow-growing bacteria ( 17 ), anaerobic bacteria ( 21 ), and rare actinobacteria ( 22 ), due to reduced H 2 O 2 production. In addition to the inhibitory effect of the peroxides, agarase produced by some bacteria can interfere with agar solidification and reduce the number of discrete colonies formed on a plate ( 23 ).…”

GVPC Medium Manufactured without Oxygen Improves the Growth of Legionella spp. and Exhibits Enhanced Selectivity Properties

“…As previously reported, L. pneumophila does not grow in autoclaved yeast extract medium in the absence of charcoal ( 20 , 26 ), unless it is sterilized by filtration ( 20 , 28 ). The importance of reducing the formation of reactive oxygen species (ROS) during autoclaving was also demonstrated in a recent study of a medium designed for the growth of environmental bacteria, in which ROS generation was minimized by separately autoclaving the phosphate and agar, thereby improving the culturability of microorganisms ( 29 ).…”

Plant Broth- (Not Bovine-) Based Culture Media Provide the Most Compatible Vegan Nutrition for In Vitro Culturing and In Situ Probing of Plant Microbiota

“…In contrast, the widely used standard culture media of designed and/or defined composition contain prescribed components, possibly not related, required and/or essential nutrients. They are of varying complexities, and may contain concentrated pure chemicals that might interact upon sterilization, resulting in chemical intermediates probably suppressing the growth of certain members of microbiota [56]. Probably, this explains why plant-based culture media supports better in situ bacterial recovery than designed/synthetic media.…”