CRM-LPN.BH analysis according to ISO 11731

A property value is given on the Certificate of analysis for 5 different analyses using the following methods:

Without filtration (direct plating)

0,1 ml RM is diluted in 4,9 ml PFZ
0,1 ml is plated on the media

With filtration, without treatment (WT)

Bring 50 ml sterile water on the filter
Mix 0,1 ml RM in this water and filtrate
Add another 50 ml of sterile water and filtrate to rinse the funnel
Place the filter faced-down in a cup with glass beads and 5 ml Phosphate Buffered Saline (PBS)
Use the ultrasonic bath for 1 minute
Plate 0,1 ml of this sample on the media

With filtration, with acid treatment (diluting 1 ml in 9 ml acid) (ADT)

Bring 50 ml sterile water on the filter
Mix 1,0 ml RM in this water and filtrate
Add another 50 ml of sterile water and filtrate to rinse the funnel
Place the filter faced-down in a cup with glass beads and 5 ml PBS
Use the ultrasonic bath for 1 minute
Mix 1 ml of this sample with 9 ml acid solution
Incubate for 5 minutes
Plate 0,1 ml of this sample on the media

With filtration, with acid treatment (30 ml acid on filter) (AFT)

Bring 50 ml sterile water on the filter
Mix 0,1 ml RM in this water and filtrate
Add another 50 ml of sterile water and filtrate to rinse the funnel
Add 30 ml acid solution on the filter and incubate for 5 minutes
Filtrate and rinse with 50 ml sterile water
Place the filter faced-down in a cup with glass beads and 5 ml PBS
Use the ultrasonic bath for 1 minute
Plate 0,1 ml of this sample on the media

With filtration, with heat treatment (HT)

Bring 50 ml sterile water on the filter
Mix 0,1 ml RM in this water and filtrate
Add another 50 ml of sterile water and filtrate to rinse the funnel
Place the filter faced-down in a cup with glass beads and 5 ml PBS
Use the ultrasonic bath for 1 minute
Place the cups with glass beads and filter in a water bath for 30 min at 50°C
Cool the cups in water (room temperature)
Plate 0,1 ml of this sample on the media

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