Sep 15, 2024
Determination of Clostridium sp. contamination level
- 1Soil and Water Research Infrastructure
Protocol Citation: Eva Petrova, Roey Angel 2024. Determination of Clostridium sp. contamination level. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l64q2rvqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 23, 2019
Last Modified: September 15, 2024
Protocol Integer ID: 26100
Abstract
adopted from Dr Tomasz Grenda (2018)
Guidelines
Example of serial dilutions preparation
Prepare 10g of sample and resuspend it in 90ml of saline solution (0.85% NaCl).
10 g soil sample
90 mL saline solution
Subject suspension to decimal dilutions in saline solution (see Guidelines), prepare at least 5 levels.
After sample preparation, dispense 10ml of TPGY broth into tubes (number of tubes depends on the number of samples multiplied by prepared dilutions, each tube corresponds to examined level). Transfer 1ml of each dilution to tubes with dispensed and preregenerated TPGY broth.
10 mL TPGY media 1 mL diluted sample
Subject the samples to pasteurization process at 70°C for 15 min.
70 °C pasteurization
00:15:00
Incubate inoculated tubes at 30°C or 37°C for 48h, under anaerobic conditions.
37 °C anaerobic incubation
48:00:00
After incubation check the turbidity of TPGY and the ability to gas production.
Spread 10µl of liquid culture onto surfaces of FAA and Willis–Hobbs agar media. Incubate inoculated plates at 37°C for 48h, under anaerobic conditions.
10 µL liguid culture
37 °C anaerobic incubation
48:00:00
After incubation evaluate characteristic phenotypic features of obtained cultures and determine the contamination level with clostridia adequately to dilution at which their growth was noticed.