YCFA Medium (Modified) | For 13L

YCFA Medium (Modified)   (Yeast Casitone Fatty Acids Broth) | For 13L

YCFA (Modified) 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 (Reference materials).

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

YCFA without Glucose, Please comment "without Glucose" when checking out if you would like to exclude glucose from mixture A.

Use

For microbiological culture

Per 1000mL of YCFA Modified Medium contains

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

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

-       Dissolve 19.44g of mixture A and 2.9 ml of stock C in 900ml MQ water.

-       Boil the prepared 900ml stock for 10 minutes, then cool to room temperature (it is recommended to work under nitrogen ^[1][3] or carbon dioxide blanket ^[2][3]; if the condition does not allow, see [4]).

-       Add 5g of mixture B into the 900 ml of prepared stock, make it 1000 ml with MQ water, and adjust pH to 6.7 ~ 6.8 with 1M KOH or 1M HCl.

-       Transfer the 1000 ml stock to 10 ml anaerobic tubes and autoclave at 121°C for 15 minutes (nitrogen blanket autoclave recommended ^[1][3]; if the condition does not allow, see [4]).

-       Aseptically add 100 µl of vitamin stock D solution per anaerobic tube 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]     Carbon dioxide blanketing can be easily achieved by placing a small amount of dry ice in a small beaker and evaporating at RT in a sealed container.

[3]     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.

[4]     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 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 at 4°C.

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

Prepared liquid media or 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.