# The Latest Practices in Culture‐Free Detection of Bacteria in Water, From Sampling to Membrane Filtration and DNA Extraction: A Systematic Review

## TL;DR

This paper reviews methods for detecting bacteria in water without culturing, covering sampling, filtration, and DNA extraction techniques.

## Contribution

The paper systematically reviews and compares current culture-free bacterial DNA detection methods in water research.

## Key findings

- Most studies used 1000 mL containers for sampling and Polyethersulfone (PES) membranes with 0.22 µm pores for filtration.
- Common DNA extraction methods included commercial kits and in-house protocols, often preceded by enzymatic or physical pretreatment.
- The review highlights the lack of standardization and the need for method adaptation based on research goals.

## Abstract

The advancement of molecular biology in water research, combined with the lack of standardization in this area of research, exposed the need for presenting the different methodological approaches available to researchers. The aim of the article is to identify and critically discuss the water filtration methods for culture‐free bacterial DNA extraction. A systematic review was conducted on PubMed and Web of Science, according to the 3Cochrane Handbook recommendations and PRISMA 2020 Checklist. The initial search retrieved 513 articles, and 53 were included with a multi‐step approach screening (title, abstract, full text). Outcomes of interest involved details about sampling, filtration methods and DNA extraction. The most reported sampling methods were using containers, especially of 1000 mL. Filtration was performed using membrane filters, the majority of the studies using Polyethersulfone (PES) membranes with the pore size of 0.22 µm. Samples were prepared for extraction either with the help of an enzymatic pretreatment (protease, proteinase K, lysozyme, or lysostaphin), physical methods (bead‐beating, centrifugation, vortexing, sonication, heating, freeze‐thaw cycles), or detergent pretreatment. The preferred method for extraction was using commercially available kits, although some of the studies described in‐house protocols. The methods must be adapted to the scientific scope. The review summarizes the existing methods and critically appraises their utility and promotes advancement in the field of environmental molecular biology.

Multiple methodologies are available for researchers for culture‐free bacterial DNA detection from water. Sampling can be performed by using containers, pumps or specialized devices. Filtration can done by using various membrane filters materials and pore sizes. Bacterial DNA extraction with commercial kits or in‐house protocols.