with Bacillus coagulans (A + BC), Artemia with
commercial food and Bacillus coagulans (A + AL +
BC), Artemia with Bacillus subtilis (A + BS), Artemia
with commercial food and Bacillus subtilis (A + AL +
BS), Artemia with benefi cial bacteria and pathogenic
Vibrio (A + BC + VA, A + BS + VA), and Artemia
with commercial food, benefi cial bacteria, and
pathogenic Vibrio (A + AL + BC + VA, A + AL + BS
+ VA). Treatment of Artemia with commercial food
(A + AL) was chosen as a control treatment because
larvae survival showed a higher percentage at the
end of the experimentation period. Th e duration of
the tests was 6 days. Commercial food and potential
probiotic bacterial strains were added during the fi rst
3 days. Th e pathogenic bacterial strain was provided
only at day 3.
Survival and growth of Artemia and methods
used to verify axenity
During the challenge tests, the number of
swimming larvae (survival percentage) was
determined daily and the percentage of survival
was calculated. At the end of each experiment, live
Artemia were fi xed with Lugol’s solution to measure
their individual length (IL) under a binocular
magnifying glass containing a graduated micrometer
(Nikon Eclipse 50i, Japan).
At the beginning and end of each run of the
procedure, the absence of bacteria was monitored by
transferring larvae and 100 μL of water to petri plates
in triplicate with marine agar.
Statistical analysis
Th e values of survival larvae (percentage)
were arcsine transformed, while the values of IL
were square-root transformed to satisfy normal
distribution and homoscedasticity requirements.
Diff erences in survival, individual length, and
total length of the Artemia larvae under diff erent
treatments were examined by analysis of variances
(ANOVA) and Duncan’s test using Statistica 5.5
soft ware.
Results
Bacterial strain identifi cation, antimicrobial
activity, and adherence assay
Bacterial strains isolated from the Artemia culture
were identifi ed as B. subtilis and B. coagulans using
the Api 50 CHB and 20 E systems.
with Bacillus coagulans (A + BC), Artemia withcommercial food and Bacillus coagulans (A + AL +BC), Artemia with Bacillus subtilis (A + BS), Artemiawith commercial food and Bacillus subtilis (A + AL +BS), Artemia with benefi cial bacteria and pathogenicVibrio (A + BC + VA, A + BS + VA), and Artemiawith commercial food, benefi cial bacteria, andpathogenic Vibrio (A + AL + BC + VA, A + AL + BS+ VA). Treatment of Artemia with commercial food(A + AL) was chosen as a control treatment becauselarvae survival showed a higher percentage at theend of the experimentation period. Th e duration ofthe tests was 6 days. Commercial food and potentialprobiotic bacterial strains were added during the fi rst3 days. Th e pathogenic bacterial strain was providedonly at day 3.Survival and growth of Artemia and methodsused to verify axenityDuring the challenge tests, the number ofswimming larvae (survival percentage) wasdetermined daily and the percentage of survivalwas calculated. At the end of each experiment, liveArtemia were fi xed with Lugol’s solution to measuretheir individual length (IL) under a binocularmagnifying glass containing a graduated micrometer(Nikon Eclipse 50i, Japan).At the beginning and end of each run of theprocedure, the absence of bacteria was monitored bytransferring larvae and 100 μL of water to petri platesin triplicate with marine agar.Statistical analysisTh e values of survival larvae (percentage)were arcsine transformed, while the values of ILwere square-root transformed to satisfy normaldistribution and homoscedasticity requirements.Diff erences in survival, individual length, andtotal length of the Artemia larvae under diff erenttreatments were examined by analysis of variances(ANOVA) and Duncan’s test using Statistica 5.5soft ware.ResultsBacterial strain identifi cation, antimicrobialactivity, and adherence assayBacterial strains isolated from the Artemia culturewere identifi ed as B. subtilis and B. coagulans usingthe Api 50 CHB and 20 E systems.
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