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EFFECTS OF ANTIBIOTICS ON THE CONCENTRATION OF BACTERIA IN BIOFILMS
AND ON THE GROWTH OF HALIOTIS RUFESCENS POSTLARVAE
MATERIALS AND METHODS

Two assays were performed to evaluate the effects of
different concentrations of the antibiotics: chloramphenicol
(experiment 1) and a mixture of penicillin G sodium/dihydrostreptomycin
(experiment 2), on bacterial counts in biofilms
associated with Haliotis rufescens culture and on the growth
of postlarvae.
The Microalgae Laboratory of the Instituto de Investigaciones
Oceanolo´ gicas provided the diatom Navicula incerta
used to form the biofilm to feed abalone postlarvae. The farm
‘‘Abulones Cultivados S. A. de C. V.’’ (Ejido Ere´ndira, B.C.,
Me´xico) donated the veliger larvae of Haliotis rufescens. Larvae
were reared in 1-mmfiltered,UVtreated seawater changed every
day. Competent larvae (7–8 days old) were induced to metamorphose
in rectangular plastic vessels (1 L) with gammaaminobutyric
acid (GABA) at 1.5-mM (Searcy-Bernal &
Anguiano-Beltra´n 1998).
Sterile 12-well tissue culture plates (Corning, 3.8 cm2 bottom
area, and 6 mL volume) were used as experimental units (EU).
Diatoms were inoculated at 250-cell mm–2 one day before the
beginning of the trials and 4–5 postlarvae (5-d old in experiment
1 and 3-d old in experiment 2) were transferred to each EU the
next day. All antibiotics were tested at four concentrations with
three replicates per treatment following a completely randomized
design. Stock solutions of antibiotics were prepared with
sterile distilled water and work solutions were prepared with sterile seawater both of them at the moment of use.
Before adding the antibiotics to the EU samples (1-mL) from
the biofilm were collected with an automatic sterilized pipette
from a previously standardized area, to estimate the initial
number of bacteria. These samples were suspended in a test tube
with 9 mL of saline solution (0.9% NaCl), and immersed in an
ultrasound bath (Fisher Scientific) for 3 min to disaggregate
diatoms and to detach bacteria from diatoms (Liza´ rraga et al.
1998, Gorrostieta-Hurtado & Searcy-Bernal 2004). Second and
third dilutions of these sonicated test tubes were prepared and
samples of these (100 mL) were plated in triplicate onto Zobell
agar media and incubated at 24C to 25C for 5 days. Bacterial
counts at 5 days after incubation were made to determine CFU
mL–1. Experiments were conducted at 17 ± 1C under constant
fluorescent illumination (ca. 50 mE s m–2).
For experiment 1, concentrations of chloramphenicol were
0, 5, 10, and 20 mg L–1 and biofilm samples were taken at 1, 2, 7,
15, and 22 days to evaluate bacterial concentrations. In experiment
2, 0/0, 50/50, 100/100, and 150/150 mg L–1 of penicillin G
sodium/dihydrostreptomycin were used and biofilm samples
were taken at days 1, 2, 7, 14, 21, 28, and 35. Antibiotics were
replaced every other day during the water changes.
To measure shell length, video-images of 2–4 postlarvae
from each EU were recorded with a Sony SSC-C374 highresolution
camera on an inverted microscope (Meiji Techno)
and analyzed digitally (NIH image 1.59 Power PC). Survival
was evaluated counting live and dead postlarvae under an
inverted microscope only in the experiment 2. The statistical
significance of the differences between antibiotic concentrations
was determined by one-way analyses of variance. Percent
survival data were subjected to the arcsine square-root transformation
before the analyses (Zar 1999).