Sep 09, 2020

Public workspaceWILLIS -HOBBS AGAR

DOI
dx.doi.org/10.17504/protocols.io.3exgjfn
  • 1Soil and Water Research Infrastructure
  • Anaerobic and Molecular Microbiology Lab, Biology Centre CAS
    Tech. support email: eva.petrova@bc.cas.cz
Ana Lara
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.3exgjfn
External link: http://himedialabs.com/TD/M1375.pdf
Protocol Citation: Roey Angel, Ana Lara-Rodriguez 2020. WILLIS -HOBBS AGAR. protocols.io https://dx.doi.org/10.17504/protocols.io.3exgjfn
Manuscript citation:

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: May 28, 2019
Last Modified: September 09, 2020
Protocol Integer ID: 23735
Abstract
For the diferential isolation of Clostridium sp.
Guidelines
Materials
MATERIALS
ReagentAgar
ReagentPeptone A FROM MEATBio Basic Inc.Catalog #G213.SIZE.500g
ReagentD-Lactose, monohydrateBio Basic Inc.Catalog #LC4280.SIZE.500g
ReagentNeutral redBio Basic Inc.Catalog #NB0652.SIZE.25g
ReagentSodium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S3014
ReagentMeat ExtractMerck Millipore (EMD Millipore)Catalog #70164-100G
ReagentEgg Yolk Emulsion Catalog #414722,1608
Safety warnings
Use temperature resistant gloves for taking the media out of the autoclave and during pouring of the petri dishes.
Before start
  • Make a 10% skim milk solution and sterilize it by autoclaving at 15 lbs pressure Temperature121 °C for Duration00:15:00 .
  • Turn ON the water bath to Temperature50 °C
  • Before taking the media from the autoclave, open the petri dish bags and organice them on the flow box. Mark the petri dishes as W-H agar.
For the preparation of 1L of media (50 to 66 petri dishes depending of depth) weigh:


Amount10 g Peptic Digest from Animal tissue
Amount10 g Meat Extract
Amount5 g NaCl
Amount12 g Lactose
Amount0.032 g Neutral Red
Amount10 g Agar
Amount30 mL Egg yolk Emulsion

Final pH ( at 25°C) 7.0±0.2


Heat to boiling to dissolve the medium completely.
Sterilize by autoclaving at 15 lbs pressure Temperature121 °C for Duration00:15:00 .
Cool to Temperature50 °C
Add Amount120 mL sterile skimmed milk 10% solution.

Aseptically addAmount30 mL Egg Yolk Emulsion.

Pour Amount15 mL or Amount20 mL of media in petri dishes depending on desired depth .
Let cool the open petri dishes under UV light for Duration00:10:00 to Duration00:15:00 .
Close petri dishes and keep at Temperature4 °C until use. (2 weeks max)