2004). Lactic acid bacteria isolates were grown (10%: v/v inoculum)
in 10 ml MRS broth for 48 h at 37 C under anaerobic conditions.
Cultures were centrifuged at 10,000 rpm for 10 min at 4 C,
supernatants aseptically collected, and pelleted cells resuspended
in 10 ml MRD. For the agar spot method,10 ml (107 cfu ml1), of each
LAB suspension was spotted on the surface of MRS agar plates in
duplicate. The plates were left at 4 C for 1 h to allow cultures to
diffuse and incubated at 37 C for 24 h before overlaying with 20 ml
of soft nutrient agar made up of nutrient broth (NB, Oxoid CM001)
with added 0.8% (w/v) agar (Oxoid LP0013) containing 200 ml of a
24 h indicator bacteria culture (108 cfu ml1) that had been grown
aerobically at 37 C. The 200 ml inoculum was added to the 20 ml
volumes of soft nutrient agar after tempering at 55 C. Uninoculated
MRS agar plates were overlaid with soft nutrient agar inoculated
with the same volume of indicator bacteria, as controls. The
agar was allowed to set for 2 h, then incubated aerobically at 37 C
for 48 h. The zones of inhibition around individual colonies of indicator
bacteria were measured to give an estimate of antimicrobial
activity by LAB isolates. For the well diffusion method,
20 ml of nutrient agar (NA, Oxoid CM003) was inoculated with
200 ml of a 24 h culture of the indicator organism and poured into a
Petri dish. When the agar had set, wells were made and filled with
100 ml of supernatants from 24 h cultures of test organisms (LAB).
Plates were held at 4 C for about 3e4 h to aid radial diffusion,
before being incubated at 37 C for 24e48 h, depending on indicator
organism. After incubation, clear zones around the wells
containing LAB were observed as inhibitory reactions and recorded
in mm
2004). Lactic acid bacteria isolates were grown (10%: v/v inoculum)
in 10 ml MRS broth for 48 h at 37 C under anaerobic conditions.
Cultures were centrifuged at 10,000 rpm for 10 min at 4 C,
supernatants aseptically collected, and pelleted cells resuspended
in 10 ml MRD. For the agar spot method,10 ml (107 cfu ml1), of each
LAB suspension was spotted on the surface of MRS agar plates in
duplicate. The plates were left at 4 C for 1 h to allow cultures to
diffuse and incubated at 37 C for 24 h before overlaying with 20 ml
of soft nutrient agar made up of nutrient broth (NB, Oxoid CM001)
with added 0.8% (w/v) agar (Oxoid LP0013) containing 200 ml of a
24 h indicator bacteria culture (108 cfu ml1) that had been grown
aerobically at 37 C. The 200 ml inoculum was added to the 20 ml
volumes of soft nutrient agar after tempering at 55 C. Uninoculated
MRS agar plates were overlaid with soft nutrient agar inoculated
with the same volume of indicator bacteria, as controls. The
agar was allowed to set for 2 h, then incubated aerobically at 37 C
for 48 h. The zones of inhibition around individual colonies of indicator
bacteria were measured to give an estimate of antimicrobial
activity by LAB isolates. For the well diffusion method,
20 ml of nutrient agar (NA, Oxoid CM003) was inoculated with
200 ml of a 24 h culture of the indicator organism and poured into a
Petri dish. When the agar had set, wells were made and filled with
100 ml of supernatants from 24 h cultures of test organisms (LAB).
Plates were held at 4 C for about 3e4 h to aid radial diffusion,
before being incubated at 37 C for 24e48 h, depending on indicator
organism. After incubation, clear zones around the wells
containing LAB were observed as inhibitory reactions and recorded
in mm
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