2.1. Inoculum preparation
The cultivated strain L. lactis subsp. lactis IL1403was provided by
Dr. Ogier from INRA (French National Institute for Agricultural
Research, Jouy-en-Josas, France). Frozen storage cultures of L. lactis
IL1403 were thawn and pregrown on Petri dishes with M17 Agar
(LAB M, UK) for 24 h at 35 C. One colony from a pregrown Petri dish
was used as an inoculum for a 10 mL culture in liquid M17 Broth
(FLUKA) at 35 C. 1 mL of bacterial suspension grown overnight
(exactly 24 h) was used as inoculum for the next liquid 10 mL M17
broth culture and grown 12e14 h which allowed the L. lactis IL1403
bacteria to reach the middle of the exponential growth phase. The
number of bacteria was determined by plating on M17 Agar and
incubating for 3 days at 35 C, and average number of bacteria in the
mid-exponential culturewas determined (4.15þ/0.15)*108 cfu/mL.
The inoculum needed for the experimentswas prepared by diluting
samples of the mid-exponential culture in peptone water. The
calculated inoculum concentrations in calorimetric and in parallel
samples were varied from 106 to 100 cfu/mL with the 10-fold
increment. It was important to have the correct desired number of
colonies in the samples as special attentionwas paid to study growth
of individual colonies. From the practical point of view it was the
most difficult to get up to about three colonies, and not more, in
a sample (inoculation rate 100 cfu/mL). These colonies are separated
from each other sufficiently and grow thus as independent individual
colonies. True, in these cases direct visual inspection of
samples after the completion of the experiments together with the
analysis of poweretime curves enabled to reach an unequivocal
verdict on the success of the experiments. However, taking into
account that the length of the experiments is typically about one
month, the pure trial and error strategy was not the most feasible.
Thus dilution procedures were developed and carefully implemented
to assure that serial dilutions end with the desired inoculation
rates. The exact following of the procedures of preparation of
bacterial cultures described above which ensured high reproducibility
at lower inoculation rates validated by direct counting of the
colonies in the samples after the experiments, allowed to be confident
that the number of bacteria in samples was also as assumed at
inoculation rates higher than 102 cfu/mL.
2.1. Inoculum preparation
The cultivated strain L. lactis subsp. lactis IL1403was provided by
Dr. Ogier from INRA (French National Institute for Agricultural
Research, Jouy-en-Josas, France). Frozen storage cultures of L. lactis
IL1403 were thawn and pregrown on Petri dishes with M17 Agar
(LAB M, UK) for 24 h at 35 C. One colony from a pregrown Petri dish
was used as an inoculum for a 10 mL culture in liquid M17 Broth
(FLUKA) at 35 C. 1 mL of bacterial suspension grown overnight
(exactly 24 h) was used as inoculum for the next liquid 10 mL M17
broth culture and grown 12e14 h which allowed the L. lactis IL1403
bacteria to reach the middle of the exponential growth phase. The
number of bacteria was determined by plating on M17 Agar and
incubating for 3 days at 35 C, and average number of bacteria in the
mid-exponential culturewas determined (4.15þ/0.15)*108 cfu/mL.
The inoculum needed for the experimentswas prepared by diluting
samples of the mid-exponential culture in peptone water. The
calculated inoculum concentrations in calorimetric and in parallel
samples were varied from 106 to 100 cfu/mL with the 10-fold
increment. It was important to have the correct desired number of
colonies in the samples as special attentionwas paid to study growth
of individual colonies. From the practical point of view it was the
most difficult to get up to about three colonies, and not more, in
a sample (inoculation rate 100 cfu/mL). These colonies are separated
from each other sufficiently and grow thus as independent individual
colonies. True, in these cases direct visual inspection of
samples after the completion of the experiments together with the
analysis of poweretime curves enabled to reach an unequivocal
verdict on the success of the experiments. However, taking into
account that the length of the experiments is typically about one
month, the pure trial and error strategy was not the most feasible.
Thus dilution procedures were developed and carefully implemented
to assure that serial dilutions end with the desired inoculation
rates. The exact following of the procedures of preparation of
bacterial cultures described above which ensured high reproducibility
at lower inoculation rates validated by direct counting of the
colonies in the samples after the experiments, allowed to be confident
that the number of bacteria in samples was also as assumed at
inoculation rates higher than 102 cfu/mL.
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