Enumeration Procedure for S.

Boulardii Scope
An enumeration method to determine the number of viable cells in freeze-dried S. boulardii and blended products containing S. boulardii.

Purpose
Use of this SOP is recommended for freeze-dried yeast. Following these guidelines may allow recovery of more viable cells by reducing cell stress and maximizing cell repair.

Reagents/Equipment
Osterizer blender or equivalent Sterile blender jar or equivalent Sterile peptone (0.1% w/v, pH 6.8) pre-wamed to 37oC Micro pipettors and sterile tips Sterile petri dishes PDA Agar (Becton Dickinson 214971 or equivalent) 47oC+ 2oC Water Bath 37oC+ 2oC Incubator Incubator/Shaker set at 37oC, 150 rpm Sterile stir bar (ca. 1 long) Magnetic stir plate

*NOTE: Use of Butterfields Buffer or other low ionic strength diluent may result in significantly lower recovery of viable bacteria.

Procedure
1. 2. 3. 4. 5. At least one hour before plating, pre-warm the needed 99 ml peptone diluent bottles in a 37oC incubator. For each sample, weigh 11 g of product and 99 ml of diluent into a sterile blender jar to make a 1:10 initial dilution. Shake gently to mix; place on incubator/shaker and allow to rehydrate for 15 minutes. Blend for 30 seconds on low speed (stir setting on the Osterizer). Remove jar, aseptically open lid and insert sterile stir bar (optional). Place on magnetic stirrer. Mix at moderate speed to create a vortex about deep while initial aliquot is removed. (Mixing is needed to obtain a representative sample as yeast cells tend to settle out of suspension rapidly).

S. Boulardii Enumeration.docx/Aug 2012/Page: 1(2) Chr. Hansen A/S -10-12 Bge All DK-2970 Hrsholm, Denmark - Phone: +45 45 74 74 74 - Fax: +45 45 74 88 88 - www.chr-hansen.com
The information contained herein is to the best of our knowledge true and correct and presented in good faith. It may be subject to change without further notice. This information is offered solely for your consideration and verification. Copyright Chr. Hansen A/S. All rights reserved.

Using aseptic technique, remove 1 ml of the 1:10 dilution and add to 99 ml of diluent to obtain the 1:1000 dilution. Shake and continue subsequent serial dilutions until the required VPC dilutions are obtained. 7. Pipet 1 ml of final dilution into sterile petri plate. Prepare a minimum of 3 plates per weighing. 8. Pour 12-15 ml of liquefied and tempered PDA into each inoculated plate. Avoid pouring the medium directly onto the inoculum. Mix the plate and contents thoroughly by first rotating in one direction then in the opposite direction, taking care not to splash the medium on the outside of the plate or inside of the lid. Allow the agar to cool and solidify on a level surface. 9. Also pour an agar control plate. 10. Invert and incubate plates aerobically. 11. Incubate at 37oC for 3 days.

6.

Results
1. 2. Count the colonies on the plates having between 30 and 300 colony forming units. If plates contain <30 or >300 colonies, retest using the appropriate dilution. Compute the average number of colonies of the triplicate plates, multiply by the dilution factor, and report as Colony Forming Units per gram (CFU/g). Also report the standard deviation.

S. Boulardii Enumeration.docx/Aug 2012/Page: 2(2) Chr. Hansen A/S -10-12 Bge All DK-2970 Hrsholm, Denmark - Phone: +45 45 74 74 74 - Fax: +45 45 74 88 88 - www.chr-hansen.com
The information contained herein is to the best of our knowledge true and correct and presented in good faith. It may be subject to change without further notice. This information is offered solely for your consideration and verification. Copyright Chr. Hansen A/S. All rights reserved.