I‘m going to prepare yeast suspension of saccharomyces boulardii,and a group of mouse will be given about 10^9 CFU/ml by oral administration every day. I would like to know how long can yeast suspension keep in 4 and whether the living-yeast will reduce significantly in 4℃ or must I prepare a fresh yeast suspension.
Furthermore,to prepare the suspension,choose which solution to resuspend the pellet after centrifugation:PBS or physiological saline ?what is the difference between?
We use to subculture yeast in plate kept at 4 degree after three months, in liquid culture one can keep four week without reduction of viabiliyty
Depends on type of suspension media used, but generally yeasts can be kept in liquid media for few months, there is no data available regarding viability.Also I recommend using 5% sterilized glycerol for suspension of the stock yeast to be stored for long time at 4o from which you can dilute another suspension for your experiment
You could check the viability of yeasts by cells count in Thoma or Burker chambers, after staining with methylene blue. Blue yeasts are death.
Concerning solution: it could be up to you, because an oral administration is envisaged.
yeast suspension can be stored at 4 c for about a month.
more than a month is not advisable.
still before administration, it is advisable to check viability of yeast cell by taking sample and going through the procedure of viable staining. in viable staining methylene blue is used. here is method:
Principle of Procedure used:
The dead cells have increased porosity of the membrane, which allows easy penetration of dyes, and thus they get intensely stained. On the other hand, live cells resist diffusion of the dye because of controlled porosity, integrity and permeability of the plasma membrane. This principle is used here to differentiate live cells from dead cells with the help of dye like Methylene blue or neutral red at a concentration that is non-toxic to the cells. Here, dead cells appear stained blue in color, whereas, live cells appear faintly stained or colorless.
Demonstration of viable cells.
Requirements:
yeast culture suspension
0.5 % Methylene blue solution
Glass slide
Cover slip
Sterile pipettes and test tubes
Procedure:
1. In a sterile tube, transfer 5.0 ml of yeast suspension aseptically.
2. To this, add one drop of 0.5 % Methylene blue solution under aseptic condition.
3. Set the tube aside for five minutes at room temperature.
4. Transfer 3 to 4 loopful of stained suspension from tube on a glass slide.
5. Place coverslip and observe under high power.
6. Record your results.
Best to store fresh compressed yeast loosely wrapped in the refrigerator at 30-40F. It's a living organism -- or a colony of living organisms -- so allow it to breathe and get rid of excess moisture without drying out. I use fresh yeast almost exclusively and find that it will stay healthy for 2-3 months when stored this way.
As for dry yeast, either active dry or instant, it's still best not to freeze. Again, the cells are still living organisms, although their activity is severely reduced because of the drying, which gets out most, but not all, of the moisture. As with fresh, freezing causes crystallization of the remaining water, which destroys cell walls, further reducing the power of the yeast, which is only at about 40-50% of the fresh yeast equivalent under the best of circumstances. Keep it refrigerated in an airtight container and it should keep for over a year.
Yeast suspension of saccharomyces boulardii, acn be made in ethanol or in distilled water.yeast suspension can be stored at 4 c for about a month.Saccharomyces boulardii is a nonpathogenic yeast with proven health benefits, some of them depending on its viability. Yeasts strains is grown and kept in YPD medium (1% yeast extract, 2% polypeptone and 2% dextrose). Yeast Nitrogen Base with ammonium sulphate and without amino acids (YNB medium; Sigma-Aldrich) is prepared at a concentration of 0.67% and is supplemented with 2% glucose, 10 mM uridine and uracil, and 0.1% 5-FOA (Sigma) to use in screening of auxotrophs.
For me, the pichia plates I made remained viable for about 4 months at 4-7 degrees Celsius in a normal refrigerator. It started to die afterwards. I usually use a plate for about 1 month.
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