YCFA Medium, Full Recipe | For 12.7 L

YCFA Medium - Full Recipe | For 12.7 Litre

YCFA Medium is an enriched nonselective media for cultivating most anaerobic bacteria and other fastidious microorganisms. It can be used for microbial culturomics & mimicking the gut environment to obtain gut microbiota isolates, specifically anaerobic strains.

We recommend YCFA Medium - Modified, which is cheaper (without expensive component - Cellobiose) and easier to prepare.

YCFA Blood Media, Prepared with sterile defibrinated Sheep/Horse Blood is available. Please contact us via support@gmexpression.com if required.

Use

For microbiological culture

Per 980mL of YCFA Medium - Full Recipe contains

Below is a simplified preparation (Please see the YCFA medium Full Recipe - Instructions for detailed protocol)

It is strongly recommended to operate within a fume cupboard when preparing the solution.

-       Dissolve 21.44 g of mixture A, 5 g of mixture B, and 2.05 ml of stock F in 800 ml of MQ water in a closed container. Add 3.1 ml of stock C  to the mixture and make it 980 ml with MQ water. Seal the container and shake it well. Adjust the pH to 7.4-7.5 using 1M KOH or 1M HCl.

-       Autoclave at 121°C for 15 minutes (Nitrogen blanket autoclave recommended ^[1][2]; if the condition does not allow, see [3]).

-       Aseptically add 2 ml of vitamin stock D  and 2 ml of vitamin stock E solution into 980 ml autoclaved stock and transfer the stock to 10 ml anaerobic tubes before use (operate on a clean bench).

[1]     Inert gas blanketing, also known as nitrogen gas protection, is a safe and reliable method of preserving volatile or oxidatively sensitive substances.

[2]     A simple method of inert gas blanketing in the laboratory:

-   GL45-cap reagent bottles or anaerobic tubes filled with the liquid to be treated are placed in an airtight PP autoclave bag - provided in the pack (the bag is best placed on top of a stainless steel tray or in a steel mesh basket)

-   Squeeze as much air out of the bag as possible, then introduce carbon dioxide or nitrogen into the bag using a flexible tube and seal the bag (by tying a knot at the neck of the bag or using a heat-resistant rubber band - provided in the pack) when it is inflated to about half its maximum volume.

-   Place the tray in a microwave oven to boil the liquid or in an autoclave chamber to sterilise.

-   Be aware of the risk of asphyxiation from high concentrations of carbon dioxide or nitrogen and ensure that the operation is carried out in a ventilated room.

[3]     If inert gas protection is not intended,

-  GL45-cap reagent bottles or anaerobic tubes filled with the liquid to be treated are placed in an airtight PP autoclave bag - provided in the pack (the bag is best placed on top of a stainless steel tray or in a steel mesh basket).

-  Squeeze as much air out of the bag as possible, then seal the bag (by tying a knot at the neck of the bag or using a heat-resistant rubber band - provided in the pack) immediately.

-  Place the tray in a microwave oven to boil the liquid or in an autoclave chamber to sterilise.

Note:

An appropriate amount of agar (1~2% w/w) may also be added to produce a semi-solid or solid culture medium.

Freshly prepared YCFA media before PH adjusted, will generally be acidic, with a pH in the range of 5 to 6. The use of 5M KOH for rough adjustment and then 1M KOH for fine adjustment can save preparation time.

Caution:

During the preparation, after adding Mixture B, the solution will quickly produce a lot of foam (carbon dioxide produced by sodium bicarbonate when it meets acidic liquid), which is normal and the foam will disappear after a few minutes of resting.

After adding Mixture B, there will be a small amount of insolubles, which is protein impurity from Hemin. After adjusting the pH to 6.7~6.8, the insolubles will decrease significantly, and the remaining Insolubles will not affect the bacterial culture. In case of removing it, rest stock and let Insolubles precipitate, then filtrate the supernatant, or pour the supernatant directly into another clean reagent bottle.

When the liquid YCFA Medium is stored at low temperature and when the pH is lowered during the incubation process, a tiny amount of insolubles may precipitate slowly, which is protein impurity from Hemin with low solubility, and only needs to be left to precipitate to the bottom of the bottle or the bottom of the tube, which is normal and will not affect the growth of bacteria.

Storage and Expiry Date:

This product can be shipped at ambient temperature. Upon receipt of the product, please store Stock D and Stock E at 4°C.

When stored at room temperature (15~25°C), Mixture A, Mixture B, Stock C and Stock F can be used for three years without losing their performance; when stored at 2~6°C, Stock D and Stock E can be valid for one year.

Prepared liquid media and agar plates are suggested to be vacuum sealed together with the oxygen absorber and stored at 4°C and the shelf life will be one year.

Safety Notes:

Mixture B is not vacuum-packed as the content may release a small quantity of carbon dioxide which will not affect the use of the product.

Stock C may emit an irritant vapour. The walls of the square bottle may fog over time. This is due to oxidation of the PET by the small amount of anhydride in the carboxylic acid and will not affect the normal use of the liquid.

The isobutyric and isovaleric acids contained in the liquid may emit an unpleasant odour, please operate in a fume hood. Keep the bottle sealed tightly after use.

The Valeric acid contained is harmful to aquatic organisms and it is strictly prohibited to pour the liquid directly into the sewer. Wear latex gloves and goggles to prevent skin/eye contamination and inhalation.