Enumeration of probiotic bacilli spores in animal feed: interlaboratory study

Fourteen out of 17 laboratories completed an interlaboratory study comparing 2 pretreatment protocols of feed samples containing authorized probiotic bacilli spores. Both methods used tryptone soy agar for enumeration. Pretreatment A involved preparation of a suspension of the feed sample in 50% ethanol. For pretreatment B, the sample was suspended in peptone salt solution and heated at 80 degrees C for 10 min. Each laboratory analyzed 12 samples (6 per pretreatment), which represented duplicates of a high (10(9) colony-forming units [CFU]/g) and low (10(5) CFU/g) level of bacilli spores or a blank that contained vegetative probiotic bacteria only. For pretreatment A, the repeatability relative standard deviation (RSD(r)) was 2.9% for the low level and 2.5% for the high. The reproducibility relative standard deviation (RSDR) values were 7.8 and 5.9%, respectively. Pretreatment B revealed RSD(r) values of 1.1 and 1.0%, and RSDR values of 5.8 and 3.4%, respectively. The heat treatment (pretreatment B) of feed samples had better precision data, resulted in higher viable bacilli counts, and was more effective in deactivating vegetative background flora. It is therefore recommended for adoption for official control purposes and for CEN and ISO standards.