3. Results
3.1. Bacteriology of Artemia cysts In Table 1, we presented the enumeration of VHB and TCBSB in 26 samples of
Artemia cysts, their age and hatching percentage. The VHB has presented concentrations comprised between 10
6
to 10
8
CFU per milliliter of Artemia homogenate, without Ž
2
. Ž
2
correlation with cyst’s age R s 0.1939, a s 0.05 or hatch percentage R s 0.06954, .
a s 0.05 . The TCBSB show lower numbers than the VHB, with values comprised between 10
1 7
Ž
2
and 10 . This population present an inverse correlation with cyst’s age R s 0.57955 .
Ž
2
. a s 0.05 , but no correlation with hatch percentage R s 0.24126, a s 0.05 .
Concerning the hatching percentage, we have not found statistical correlation with Ž
2
. Ž
. their age R s 0.38668, a s 0.05 , but the oldest ones SFBB1, SFBB2 and SAL have
shown the lower percentages.
Table 1 Ž
. Artemia cysts brands, age, viable heterotrophic bacteria
VHBrml , bacteria growing on TCBS media Ž
. TCBSBrml and hatched percentage
Commercial brand Brand
Lot Cysts age
VHBrml TCBSBrml
Cysts Ž
. of Artemia cysts
code number
months hatched
in S.E.
7 6
Great Lake Artemia GLA2
GLA 90 1
5.4=10 3.0=10
95.81.4
7 6
Ž . Salt Creek 1
SAC1 00016002
2 6.3=10
1.8=10 81.31.7
8 6
Ž . Salt Creek 2
SAC2 00016002
2 2.9=10
5.3=10 93.62.1
8 6
Ž . Salt Creek 3
SAC3 00016002
2 2.9=10
5.3=10 93.03.0
7 6
Prime USA PRIME
971020 2
1.1=10 7.2=10
99.60.6
7 5
Biomarine BIO1
no register 2
4.6=10 4.0=10
53.91.2
8 6
Biomarine BIO4
AA05127 3
2.3=10 6.0=10
5.20.6
7 6
Great Lake Artemia GLA3
no register 3
8.0=10 1.3=10
81.71.2
8 6
Inve INVE
06335 3
1.2=10 2.7=10
49.81.7
8 7
Biomarine BIO3
AA03177 4
2.5=10 1.4=10
85.33.5
8 6
Zion’s Artemia ZION
no register 4
2.3=10 1.8=10
75.01.9
7 3
Sanders Grade B SAND1
SP1352 5
4.5=10 8.5=10
79.01.9
8 5
Ž . Georgia Packaying ?
GEOP FR2175-B
9 2.0=10
5.0=10 75.83.3
8 6
Sanders Grade B SAND2
SP1547 9
1.3=10 1.6=10
49.31.5
8 7
Great Lake Artemia GLA1
no register 10
1.3=10 6.8=10
67.54.3
8 6
Argentemia Grade III ARGGIII
B32003K 15
1.3=10 8.8=10
29.92.7
7 6
Argentemia Grade II ARGGII
B52308K 24
7.3=10 3.4=10
93.62.5
7 6
Biomarine BIO2
no register 24
8.2=10 1.2=10
38.81.5
8 5
Aurum 90q A90
769 30
3.5=10 7.1=10
86.91.9
7 1
Argentemia Grade I ARGGI1
900103 60
3.2=10 -
1=10 87.93.0
7 1
Argentemia Grade I ARGGI2
900103 60
3.7=10 -
1=10 74.32.2
8 6
Utah Pacific 90 UP1
no register 60
2.8=10 1.3=10
83.61.5
7 6
Utah Pacific 85 UP2
no register 60
9.2=10 6.0=10
82.92.5
7 4
San Francisco Bay Brand SFBB1
1342 84
5.5=10 1.1=10
27.71.0
7 1
San Francisco Bay Brand SFBB2
1739 84
6.6=10 9.0=10
0.0
6 2
Salt Lake SAL
1689 84
3.5=10 -
1=10 3.50.4
3.2. Taxonomic tests From the 617 strains analyzed for their morphological and basic physiological
Ž .
characteristics Table 2 , 94 of them were Gram-positive and only 6 Gram-negative. Ž
. The potentials Vibrio spp. Gram-negative rods or coccoid bodies represented only
Ž .
1.94 of the isolates Table 2 , nevertheless, they were oxidase-negative. The cocci Gram-positive and Gram-negative account almost 22. In TCBS media, almost 72
Ž .
present a yellow colony in TCBS agar. Also, most of the isolates in TCBS 96.6 , when subculturing in ZoBell media, show a pigmented colony. All of the 617 strains
Ž were oxidase-negative and catalase positive. All of the 52 selected strains 15 cocci and
. Ž
37 bacillircoccobacilli, Gram-positive were strict aerobic do not ferment glucose on .
O–F media . From the 15 cocci, 46 of them grow in 15 NaCl. The isolated Gram-positive cocci with the capacity to grow in T N
has been
1 15
Ž .
identified as Staphylococcus spp. brands A90, BIO2, ARGGII and those without this Ž
. capacity has been identified as Micrococcus spp. brands A90, BIO1, SFBB1 . The
remainder strains were regular or irregular rods, nonsporing, Gram-positive, catalase- positive strictly aerobic, but they have not been assigned to any genus.
In Table 3, we presented the comparative enumeration of TCBSB and basic physio- logical tests obtained of isolates from laboratory and commercial cysts hatching condi-
tions. Enumeration fluctuated from 10
6
to 10
7
, with a mean order of magnitude of 10
7
for both conditions. Great differences are recorder in the presence of Gram-negative and
Gram-positive bacteria. Sterile conditions, which reflect the bacteria associated with Artemia cysts, show that Gram-positive, oxidase-negative organisms are the dominant
population, at the instant of nonsterility commercial hatchery condition where Gram- negative, oxidase-positive bacteria constitute 100 of the isolates; those results corre-
spond to Vibrio spp. basic taxonomy. Identification of 34 strains of VLO with the API
Table 2 Characteristics of bacteria isolated of hatched Artemia cysts, in relation to Gram stain and TCBS colony color
Ž .
Morphology Gram
Color in N ,
TCBS N s617
Group Totals
Ž .
Yellow 39.71 245
Ž .
RODS q
Green 12.31 76
52.03 53.00
Ž . Yellow
0.65 4 Ž .
y Green
0.32 2 0.97
Ž .
Yellow 15.72 97
Ž .
COCCOID q
Green 8.75 54
24.47 25.44
Ž . Yellow
0.16 1 Ž .
y Green
0.81 5 0.97
Ž .
Yellow 14.26 88
Ž .
COCCI q
Green 3.24 20
17.50 21.56
Ž . Yellow
1.46 9 Ž
. y
Green 2.60 16
4.06 Total gram positive 94; Total gram negative 6.
Table 3 Ž
. Ž
. Bacteria isolated of Artemia cysts Salt Creek, lot 00016002 hatched in laboratory sterile and at shrimp
Ž .
hatchery nonsterile conditions Hatching conditions
Characteristics Laboratory 1
Shrimp Laboratory 2
Shrimp hatchery 1
hatchery 2 Density nauplirml
1249 1633
1253 237
6 7
7 7
TCBSBrml 5.3=10
1.7=10 8.7=10
2.0=10 Gramq
90 100
Gramy 10
100 100
Oxidaseq 100
100
system indicate that 24 correspond to V. alginolyticus, 1 to V. Õulnificus, 1 to V. pelagius and 8 to Vibrio spp.
For the primary isolation, the TCBS media that we use was DIFCO, but 47 isolates of our Gram-positive bacteria collection has been cultivated in TCBS brands other than
Difco, obtained a grow of 95 in Oxoid, 95 in Merck and 4 in Eiken.
4. Discussion
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