Sep 09, 2020

Public workspaceFAA MEDIA (FASTIDIOUS ANAEROBES AGAR)

This protocol is a draft, published without a DOI.
  • 1Soil and Water Research Infrastructure
  • Anaerobic and Molecular Microbiology Lab, Biology Centre CAS
    Tech. support email: eva.petrova@bc.cas.cz
Ana Lara
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External link: https://foodsafety.neogen.com/pdf/acumedia_pi/7531_pi.pdf
Protocol Citation: Roey Angel, Ana Lara-Rodriguez, Eva Petrova 2020. FAA MEDIA (FASTIDIOUS ANAEROBES AGAR). protocols.io https://protocols.io/view/faa-media-fastidious-anaerobes-agar-3gngjve
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: In development
We are still developing and optimizing this protocol
Created: May 29, 2019
Last Modified: September 09, 2020
Protocol Integer ID: 23790
Abstract
For the growing and maintance of Clostridium sp.

Fastidious Anaerobe Agar is used for the cultivation of anaerobic microorganisms and is not intended for use in the diagnosis of disease or other conditions in humans. A primary isolation medium capable of growing most clinically significant anaerobes. Developed by Lab M (Neogen® Corporation), comparisons have shown this medium to be superior to other formulations as a primary isolation medium for fastidious organisms. The peptones included have been chosen for maximum growth stimulation. Starch and sodium bicarbonate act as de-toxification agents while hemin encourages pigment production in Porphyromonas melaninogenicus. Specific growth promoting agents are Cysteine for Fusobacterium necrophorum, Propionibacterium acne and Bacteriodes fragilis, arginine for Eubacterium spp. soluble pyrophosphate for Porph. gingivalis and Porph. asaccharolyticus. Pyruvate helps neutralize hydrogen peroxide and is also utilized by Veillionella spp. as an energy source. Vitamin K and sodium succinate provide essential growth factors for some anaerobes as does the 0.1% glucose. The low level of glucose prevents the production of high levels of acids and alcohols which would inhibit colonial development.

Guidelines
Taken from: https://foodsafety.neogen.com/pdf/acumedia_pi/7531_pi.pdf

Medium final pH: 7.2 ± 0.2 at 25°C


























Before start
Make the stock solutions for Hemin, L-Cysteine HClxH2O and vitamin K1.
VITAMIN K1 SOLUTION NEEDS TO BE DONE AT LEAST 3 DAYS IN ADVANCE!!!

Prepare stock solutions:

L-cystein hydrochloride solution (50 g/l):
Dissolve Amount0.5 g of Cysteine HCl Monohydrate in a Amount10 mL distilled water and filter sterilize. Store refrigerated.

HEMIN solution (10 g/l) :
Dissolve Amount0.1 g Hemin in Amount100 µL Concentration1 Molarity (M) NaOH ; make up to Amount10 mL with distilled water and filter sterilize. Store refrigerated.
VITAMIN K1 solution (10 g/l):
Dissolve Amount0.1 g of vitamin K1 in Amount10 mL Concentration95 % volume Ethanol and filter sterilize. Store refrigerated in a brown bottle.

For the preparation of 1L of media (50 to 66 petri dishes depending of depth) dilute in 1000 ml of distilled water:

Amount23 g Peptone
Amount5 g Sodium Chloride
Amount1 g Soluble Starch
Amount0.4 g Sodium Bicarbonate
Amount1 g Glucose
Amount1 g Sodium Pyruvate
Amount0.25 g Sodium Pyrophosphate
Amount1 g L-Arginine
Amount0.5 g Sodium Succinate
Amount12 g Agar

Final pH: 7.2 ± 0.2 at 25°C

Heat to boiling to dissolve the medium completely.
Sterilize by autoclaving at 15 lbs pressure Temperature121 °C for Duration00:15:00 minutes.
Cool down to Temperature50 °C -Temperature55 °C and aseptically add the Cysteine HCl Monohydrate, Hemin and the Vitamin K .

Amount1 mL Hemin stock solution
Amount1 mL L-Cysteine HClxH20 stock solution
Amount100 µL Vitamin K1 stock solution

Refrigerate the sterile medium until use (no more than 2 weeks).