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A comparison between total viable count by spread plating and AquaPlak® for enumeration of bacteria in water from a shrimp farm
ELSEVIER
Journal of Microbiological
Methods
30 (1997) 217-220
Journal ofMicrobiological Methods
A comparison between total viable count by spread plating and AquaPlakB for enumeration of bacteria in water from a shrimp farm Ana L. Guerra-Floresa’*,
Albert0 Abreu-Groboisb,
Bruno Gomez-Gil”
“Universidad Autbnoma de Sinaloa, Paseo Clausen sin, Mazatldn, Sin. 82LXW,Mt!xico blnstifuto de Ciencias de1 Mar y Limnologia, Universidad National Autdnoma de Mixico, AP. 811, Mazatla’n, Sin. 82oo0, M6xico ‘Department of Pathology, CIADIMazatldn Unit for Aquaculture and Environmental Management. AP. 711, Mazatldn, Sin. 82010, M&co Received
13 January
1997; received
in revised form 16 July 1997; accepted
16 July 1997
Abstract A new system named AquaPlakB designed for evaluating heterotrophic bacteria and those belonging to the genus Vibrio was compared to the standard method of total viable count by spread plating. Water samples from a shrimp farm were evaluated by the two procedures over a 3-month period. The spread plating technique gave significantly higher viable counts for both Vibrio and heterotrophic bacteria than the AquaPlakB system. 0 1997 Elsevier Science B.V. Keywords:
AquaPlak; Bacteria; Shrimp; Vibrio
1. Introduction While shrimp farming accounts for a significant portion of the annual world production (28% of the total [ 1]), important production decreases have occurred in recent years due to various diseases (some caused by bacteria): a 16% drop in the world annual production of head-on shrimp in 1993; in China a 70% production loss was reported [2]. Gram-negative rods and specially Vibrio spp. are commonly reported as the principal bacterial patho-
*Corresponding author. CIADIMazatlkr Unit for Aquaculture and Environmental Management, AI? 711, Mazatlln, Sin., Mexico 82000. Tel.: +52 69 880157; fax: +52 69 880159; e-mail: [email protected]
0167-7012/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO1 67-7012(97)00068-7
gen infecting shrimp at all stages of growth [3-51, with the latter as opportunistic bacteria. It is therefore important to evaluate the bacterial load of both heterotrophic bacteria and vibrios in the larviculture and grow-out phases of shrimp aquaculture. Traditionally, total viable count by spread plating on marine agar [7] and thiosulphate citrate bile sucrose agar (TCBS), a medium employed for isolating bacteria of the genus Vibrio [6], have been used for enumeration of bacteria in water samples. However, spread plating is time consuming and not well suited for field use. A rapid procedure capable of determining bacterial loads in the water of marine aquaculture facilities AquaPlakB has been introduced recently by Frippak Sanofi [8]. This system is based on a 96-microwell plate with lyophilized
218
A.L. Guerra-Flares et al. / Journal of Microbiological Methods 30 (1997) 217-220
media, which allows quantification of total heterotrophic bacteria and presumptive Vibrio spp. In this study both methodologies were analyzed and compared by testing water sampled from a shrimp farm.
2. Material and methods Thirty-two water samples were collected weekly over a 2-month period from a semi-intensive shrimp farm located in the Southern part of Sinaloa State (Mexico). The sampling sites were the estuary (E), the intake water channel (C), and six shrimp ponds (nos. 1, 4, 3, 3a, 8, 10). Only one pond (no. 4) was sampled on all occasions, three sites were sampled on six occasions (channel, ponds 1 and 8), two sites only twice (estuary and pond 3a) and two sites only once (ponds 3 and 10). The stocking and harvesting calendar of the farm management necessitated the sampling periodicity. The sampled water was refrigerated and taken to the laboratory for analysis not more than 3 h after collection. pH, temperature, salinity and dissolved oxygen were measured on site. The samples were serially diluted in sterile seawater (aged and filtered through a 0.45pm filter) and spread plated onto marine agar and TCBS agar by duplicate (Difco Laboratories, Detroit, MI, USA). The AquaPlakB microwell plate (Sanofi, Aquaculture, Paris, France) was inoculated according to the manufacturer’s instructions. Serial dilutions from samples were performed in a 96-well microtiter plate containing lyophilized media; four columns of 12 wells each with marine agar for total heterotrophic bacteria, and four other columns with TCBS medium to enumerate Vibrio spp. Each plate thus served for four samples, The first row of wells was inoculated with 200 ~1 of the sample and the rest of the wells in that column were re-hydrated with 100 ~1 from an ampoule containing sterile, reconstituted seawater. From the first well, 100 p+l were transferred into the adjacent well, diluting the concentration in the second well by half. From this well another 100 pl were transferred to the third well. The process was continued down the wells, until all 12 wells were filled. From well number 12, 100 ~1 were removed
and discarded to leave all wells with 100~cl,1 volumes. Petri dishes and microtiter plates were incubated at 30°C and the colony forming units (cfu) were counted after 24 and 48 h in the petri dishes. The wells in which color changed to yellow and/or those that showed turbidity were considered as positives. The cfu ml-’ were calculated according to a table provided with the AquaPlakB system. The concentration of bacteria ml-’ was estimated on the basis of the number of positive wells in each column. The manufacturer suggests using dilutions depending of the expected bacterial concentration of the sample. In this study, the dilution used meant that the first positive well was equivalent to 5 cfu ml-‘, the next well would have 10 cfu ml-‘, and so on. The results of both procedures were logarithmic base lo-transformed and analyzed for normality (Levene’s test) and homogeneity of variances (Bartlett’s test). Individual statistical analyses were performed for every sampling site with three or more data. Their means were compared by paired-sample t-tests (if they showed a normal distribution) or by Wilcoxon paired-sample tests (if not normally distributed). Concordance correlations (r,) [9] were used to assess the linear relationship between the two methodologies using pooled data from all samples.
3. Results and discussion A total of 32 samples were analyzed for total heterotrophic bacteria and for presumed Vibrio spp. grown on TCBS with both methodologies (Table 1). 3.1. Total heterotrophic
bacteria
Heterotrophic bacterial estimates were usually lOOO-fold lower with the AquaPlakB method than with total viable count (Table 1). When all samples were pooled together, a very low correlation was encountered between both methodologies for this bacterial group (r,=0.05971) which was not statistically different from zero (t-test=0.30498, P>O.O5, rt=28). However, heterotrophic bacterial estimates for the channel, El, E4, E6 and ES sampling sites (Table 2) were significantly different (PcO.05) for both methodologies.
219
A.L. Guerra-Flares et al. I Journal of Microbiological Methods 30 (1997) 217-220 Table 1 Comparison
of bacterial loads in shrimp farm samples, estimated by two methodologies:
total heterotrophic
counts (marine agar) and Vibrio
spp. (TCBS agar)
1
8/4/93
4 3a 8 Channel 4 3a 8 Channel 4 3 Estuary Channel 4 1 8 Channel 4 Estuary 10 Channel 4 1 8 Channel 4 8 1 Channel 4
1414193
2914193
1415193
2115193
2815193
216193
916193
(*) of bacterial
Spread plate
AquaPlak@
Spread plate
6.40X 10’ 1.20x lo1 6.40X lo2 1.60X lo2 3.20X lo2 3.20X 10’ 1.60X10* 8.00X 10 3.20X 10’ 8.00X 10’ 8.00X 10’ 1.00x 10’ 0.00 2.00x 10’ 0.00 5.00 1.60X 10’ 1.00x10’ 1.00x 10’ 1.60x102
7.10x los 5.70x lo5 1.50x105 1.60X105 7.50x 10” 2.50X 10’ 8.60X lo4 6.50X lo4 1.00x lo4 6.50X 10’ 9.30x lOA 2.28X loa 4.50x IO1 6.60X 10’ 6.05x 10’ 5.20x 10’ 4.40x lo3 1.05x loJ 1.55x lo3 2.60X 10J 1.74x IO4 9.63X IO4 1.04x IO’ 3.38x 10’ 1.59x lo4 1.35x105 3.24X IO* 5.80X lo2 1.40x104 3.60x lo4 1.33x10’ 4.47x lo4
1.00x lo4 2.50x lo1 6.40X 10’ -
100x10” l.Ooxlos 1.00x104 -
2.50x lo2 -
1.61x10’ -
8.00X 10’ 1.60X lo2 8.00X 10’ 1.00x 10’ 1.00x10’ 8.00X 10’ 4.00x 10’ -
3.60X IO2 1.96x10’ l.17X103 1.00x102 5.00x 10 8.50x lo2 3.50x 10’
1.00x 2.50X 6.40x 4.00x -
0.00 l.OOxlol 0.00 1.00x10 -
2.00x10’ 4.00x IO’ 8.00X 10’ 5.00 5.00 2.00x 10’ 3.20X lo*
1
differences
AquaPlak@
2.00x 10’ 1.00x10’ 2.00x 10’
8
Table 2 Statistical
TCBS agar (Vibrios)
Marine agar (total heterotrophs)
Location (pond)
Date
counts between the AquaPlak@
10’ lo2 lo2 10’
1.00x10’
1.00x103
3.20X IO’ 3.20X lo* 1.60x10* 1.20x102 2.00x 10 2.00x 10’ 4.00x lOI 3.20X lo2
8.00x 10’ 3.65x IO* 1.54x 10’ 7.50x 10’ 8.50X 10 9.00x 10’ 4.10x10* 3.75x 10’
system and spread plating from shrimp farm water
Location
Sample
n
Test
P
Channel
THB* Vibrio THB* Vibrio THB* Vibrio THB* Vibrio THB* Vibrio*
4 5 6 4 8 8 5 3 28 22
Paired r-test=3.48 Wilcoxon=5.00 Wilcoxon=21.00 Wilcoxon=4.00 Wilcoxon= Wilcoxon= 16.00 Paired t-test=2.91 Paired t-test=2.52 Wilcoxon= Paired r-test=3.76
0.0401 0.6250 0.0313 0.5000 0.0078 0.3125 0.0335 0.1279 ~0.0001 0.0012
El E4 E8 All samples pooled
Only sampling sites with more than three samples were considered. THB, total heterotrophic forming units grown in TCBS agar. Wilcoxon, signed rank paired-sample test.
bacteria grown in marine agar; Vibrio, colony
220
A.L. Guerra-Flares et al. I Journal of Microbiological Methods 30 (1997) 217-220
3.2. Vibrio spp.
Both methodologies for this group were poorly correlated (r, =0.47766), and not significantly different from zero (t-test=2.43145, FWO.05, n=22) when all samples were pooled together. No statistically significant differences (ZQO.05) between the methodologies were encountered for Vibrio spp. estimates for the channel, El, E4, E6 and E8 sampling sites (Table 2). As can be seen in Table 1, the AquaPlakB system consistently underestimates bacterial numbers compared to the spread plate method for total heterotrophic bacteria. In contrast, both procedures produced similar counts of bacteria growing on TCBS agar. The lower heterotrophic bacteria counts obtained from the AquaPlakB system demonstrated an unacceptable accuracy of the procedure, making it unsuitable for correct estimations of bacterial loads in shrimp aquaculture farms. Moreover, estimates of the bacterial count using AquaPlakB system plates were sometimes difficult to obtain since well reactions were unclear. The bacterial underestimation and the inconvenience mentioned above, contradicts the manufacturer’s claim that the system is easy to use in shrimp farming facilities as a tool for routine bacteriological analysis with no specially trained personnel. Im-
provements can be made to estimate more accurately total heterotrophic bacteria.
References 111B. Rosenberry, World shrimp farming
1994, Shrimp News Int., San Diego, 1994, 68 pp. 121B. Rosenbeny, World shrimp farming 1993, Shrimp News hit., San Diego, 1993, 52 pp. [31 D.V. Lightner, Diseases of cultured penaeid shrimps, in: J.P. McVey (Ed.), CRC Handbook of Mariculture, 2 ed., CRC, Boca Raton, FL, 1993 pp. 393-486. [41 J.A. Brock, D.V. Lightner, Diseases caused by microorganisms, in: 0. Kinne (Ed.), Diseases of Marine Animals, John Wiley and Sons. New York, 1990, pp. 390-399. [51 L.L. Mohney, D.V Lightner, T.A. Bell, An epizootic of vibriosis in ecuadorian pond-reared Penaeus vannamei Boone (Crustacea: Decapoda), J. World Aquacul. Sot. 25 (1994) 116-125. [61 K.M. Nicholls. J.V Lee, T.J. Donovan, An evaluation of commercial thiosulphate citrate bile salt sucrose agar (TCBS), J. Appl. Bacterial. 41 (1976) 265-269. [71 F.M. Patrick, The use of membrane filtration and marine agar 22168 to enumerate marine heterotrophic bacteria, Aquaculture 13 (1978) 369-372. VI T. Noel, E. Aubree, E. Miahle, B. Panatier, G. Flamion, H. Grizel, A new tool for bacteriological survey in aquaculture, Int. Aquaculture Conference, Bordeaux, 1989, pp. 193-194. [91 J.H. Zar, Biostatistical Analysis, Prentice Hall, New Jersey, 1996, pp. 662.
Journal of Microbiological
Methods
30 (1997) 217-220
Journal ofMicrobiological Methods
A comparison between total viable count by spread plating and AquaPlakB for enumeration of bacteria in water from a shrimp farm Ana L. Guerra-Floresa’*,
Albert0 Abreu-Groboisb,
Bruno Gomez-Gil”
“Universidad Autbnoma de Sinaloa, Paseo Clausen sin, Mazatldn, Sin. 82LXW,Mt!xico blnstifuto de Ciencias de1 Mar y Limnologia, Universidad National Autdnoma de Mixico, AP. 811, Mazatla’n, Sin. 82oo0, M6xico ‘Department of Pathology, CIADIMazatldn Unit for Aquaculture and Environmental Management. AP. 711, Mazatldn, Sin. 82010, M&co Received
13 January
1997; received
in revised form 16 July 1997; accepted
16 July 1997
Abstract A new system named AquaPlakB designed for evaluating heterotrophic bacteria and those belonging to the genus Vibrio was compared to the standard method of total viable count by spread plating. Water samples from a shrimp farm were evaluated by the two procedures over a 3-month period. The spread plating technique gave significantly higher viable counts for both Vibrio and heterotrophic bacteria than the AquaPlakB system. 0 1997 Elsevier Science B.V. Keywords:
AquaPlak; Bacteria; Shrimp; Vibrio
1. Introduction While shrimp farming accounts for a significant portion of the annual world production (28% of the total [ 1]), important production decreases have occurred in recent years due to various diseases (some caused by bacteria): a 16% drop in the world annual production of head-on shrimp in 1993; in China a 70% production loss was reported [2]. Gram-negative rods and specially Vibrio spp. are commonly reported as the principal bacterial patho-
*Corresponding author. CIADIMazatlkr Unit for Aquaculture and Environmental Management, AI? 711, Mazatlln, Sin., Mexico 82000. Tel.: +52 69 880157; fax: +52 69 880159; e-mail: [email protected]
0167-7012/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO1 67-7012(97)00068-7
gen infecting shrimp at all stages of growth [3-51, with the latter as opportunistic bacteria. It is therefore important to evaluate the bacterial load of both heterotrophic bacteria and vibrios in the larviculture and grow-out phases of shrimp aquaculture. Traditionally, total viable count by spread plating on marine agar [7] and thiosulphate citrate bile sucrose agar (TCBS), a medium employed for isolating bacteria of the genus Vibrio [6], have been used for enumeration of bacteria in water samples. However, spread plating is time consuming and not well suited for field use. A rapid procedure capable of determining bacterial loads in the water of marine aquaculture facilities AquaPlakB has been introduced recently by Frippak Sanofi [8]. This system is based on a 96-microwell plate with lyophilized
218
A.L. Guerra-Flares et al. / Journal of Microbiological Methods 30 (1997) 217-220
media, which allows quantification of total heterotrophic bacteria and presumptive Vibrio spp. In this study both methodologies were analyzed and compared by testing water sampled from a shrimp farm.
2. Material and methods Thirty-two water samples were collected weekly over a 2-month period from a semi-intensive shrimp farm located in the Southern part of Sinaloa State (Mexico). The sampling sites were the estuary (E), the intake water channel (C), and six shrimp ponds (nos. 1, 4, 3, 3a, 8, 10). Only one pond (no. 4) was sampled on all occasions, three sites were sampled on six occasions (channel, ponds 1 and 8), two sites only twice (estuary and pond 3a) and two sites only once (ponds 3 and 10). The stocking and harvesting calendar of the farm management necessitated the sampling periodicity. The sampled water was refrigerated and taken to the laboratory for analysis not more than 3 h after collection. pH, temperature, salinity and dissolved oxygen were measured on site. The samples were serially diluted in sterile seawater (aged and filtered through a 0.45pm filter) and spread plated onto marine agar and TCBS agar by duplicate (Difco Laboratories, Detroit, MI, USA). The AquaPlakB microwell plate (Sanofi, Aquaculture, Paris, France) was inoculated according to the manufacturer’s instructions. Serial dilutions from samples were performed in a 96-well microtiter plate containing lyophilized media; four columns of 12 wells each with marine agar for total heterotrophic bacteria, and four other columns with TCBS medium to enumerate Vibrio spp. Each plate thus served for four samples, The first row of wells was inoculated with 200 ~1 of the sample and the rest of the wells in that column were re-hydrated with 100 ~1 from an ampoule containing sterile, reconstituted seawater. From the first well, 100 p+l were transferred into the adjacent well, diluting the concentration in the second well by half. From this well another 100 pl were transferred to the third well. The process was continued down the wells, until all 12 wells were filled. From well number 12, 100 ~1 were removed
and discarded to leave all wells with 100~cl,1 volumes. Petri dishes and microtiter plates were incubated at 30°C and the colony forming units (cfu) were counted after 24 and 48 h in the petri dishes. The wells in which color changed to yellow and/or those that showed turbidity were considered as positives. The cfu ml-’ were calculated according to a table provided with the AquaPlakB system. The concentration of bacteria ml-’ was estimated on the basis of the number of positive wells in each column. The manufacturer suggests using dilutions depending of the expected bacterial concentration of the sample. In this study, the dilution used meant that the first positive well was equivalent to 5 cfu ml-‘, the next well would have 10 cfu ml-‘, and so on. The results of both procedures were logarithmic base lo-transformed and analyzed for normality (Levene’s test) and homogeneity of variances (Bartlett’s test). Individual statistical analyses were performed for every sampling site with three or more data. Their means were compared by paired-sample t-tests (if they showed a normal distribution) or by Wilcoxon paired-sample tests (if not normally distributed). Concordance correlations (r,) [9] were used to assess the linear relationship between the two methodologies using pooled data from all samples.
3. Results and discussion A total of 32 samples were analyzed for total heterotrophic bacteria and for presumed Vibrio spp. grown on TCBS with both methodologies (Table 1). 3.1. Total heterotrophic
bacteria
Heterotrophic bacterial estimates were usually lOOO-fold lower with the AquaPlakB method than with total viable count (Table 1). When all samples were pooled together, a very low correlation was encountered between both methodologies for this bacterial group (r,=0.05971) which was not statistically different from zero (t-test=0.30498, P>O.O5, rt=28). However, heterotrophic bacterial estimates for the channel, El, E4, E6 and ES sampling sites (Table 2) were significantly different (PcO.05) for both methodologies.
219
A.L. Guerra-Flares et al. I Journal of Microbiological Methods 30 (1997) 217-220 Table 1 Comparison
of bacterial loads in shrimp farm samples, estimated by two methodologies:
total heterotrophic
counts (marine agar) and Vibrio
spp. (TCBS agar)
1
8/4/93
4 3a 8 Channel 4 3a 8 Channel 4 3 Estuary Channel 4 1 8 Channel 4 Estuary 10 Channel 4 1 8 Channel 4 8 1 Channel 4
1414193
2914193
1415193
2115193
2815193
216193
916193
(*) of bacterial
Spread plate
AquaPlak@
Spread plate
6.40X 10’ 1.20x lo1 6.40X lo2 1.60X lo2 3.20X lo2 3.20X 10’ 1.60X10* 8.00X 10 3.20X 10’ 8.00X 10’ 8.00X 10’ 1.00x 10’ 0.00 2.00x 10’ 0.00 5.00 1.60X 10’ 1.00x10’ 1.00x 10’ 1.60x102
7.10x los 5.70x lo5 1.50x105 1.60X105 7.50x 10” 2.50X 10’ 8.60X lo4 6.50X lo4 1.00x lo4 6.50X 10’ 9.30x lOA 2.28X loa 4.50x IO1 6.60X 10’ 6.05x 10’ 5.20x 10’ 4.40x lo3 1.05x loJ 1.55x lo3 2.60X 10J 1.74x IO4 9.63X IO4 1.04x IO’ 3.38x 10’ 1.59x lo4 1.35x105 3.24X IO* 5.80X lo2 1.40x104 3.60x lo4 1.33x10’ 4.47x lo4
1.00x lo4 2.50x lo1 6.40X 10’ -
100x10” l.Ooxlos 1.00x104 -
2.50x lo2 -
1.61x10’ -
8.00X 10’ 1.60X lo2 8.00X 10’ 1.00x 10’ 1.00x10’ 8.00X 10’ 4.00x 10’ -
3.60X IO2 1.96x10’ l.17X103 1.00x102 5.00x 10 8.50x lo2 3.50x 10’
1.00x 2.50X 6.40x 4.00x -
0.00 l.OOxlol 0.00 1.00x10 -
2.00x10’ 4.00x IO’ 8.00X 10’ 5.00 5.00 2.00x 10’ 3.20X lo*
1
differences
AquaPlak@
2.00x 10’ 1.00x10’ 2.00x 10’
8
Table 2 Statistical
TCBS agar (Vibrios)
Marine agar (total heterotrophs)
Location (pond)
Date
counts between the AquaPlak@
10’ lo2 lo2 10’
1.00x10’
1.00x103
3.20X IO’ 3.20X lo* 1.60x10* 1.20x102 2.00x 10 2.00x 10’ 4.00x lOI 3.20X lo2
8.00x 10’ 3.65x IO* 1.54x 10’ 7.50x 10’ 8.50X 10 9.00x 10’ 4.10x10* 3.75x 10’
system and spread plating from shrimp farm water
Location
Sample
n
Test
P
Channel
THB* Vibrio THB* Vibrio THB* Vibrio THB* Vibrio THB* Vibrio*
4 5 6 4 8 8 5 3 28 22
Paired r-test=3.48 Wilcoxon=5.00 Wilcoxon=21.00 Wilcoxon=4.00 Wilcoxon= Wilcoxon= 16.00 Paired t-test=2.91 Paired t-test=2.52 Wilcoxon= Paired r-test=3.76
0.0401 0.6250 0.0313 0.5000 0.0078 0.3125 0.0335 0.1279 ~0.0001 0.0012
El E4 E8 All samples pooled
Only sampling sites with more than three samples were considered. THB, total heterotrophic forming units grown in TCBS agar. Wilcoxon, signed rank paired-sample test.
bacteria grown in marine agar; Vibrio, colony
220
A.L. Guerra-Flares et al. I Journal of Microbiological Methods 30 (1997) 217-220
3.2. Vibrio spp.
Both methodologies for this group were poorly correlated (r, =0.47766), and not significantly different from zero (t-test=2.43145, FWO.05, n=22) when all samples were pooled together. No statistically significant differences (ZQO.05) between the methodologies were encountered for Vibrio spp. estimates for the channel, El, E4, E6 and E8 sampling sites (Table 2). As can be seen in Table 1, the AquaPlakB system consistently underestimates bacterial numbers compared to the spread plate method for total heterotrophic bacteria. In contrast, both procedures produced similar counts of bacteria growing on TCBS agar. The lower heterotrophic bacteria counts obtained from the AquaPlakB system demonstrated an unacceptable accuracy of the procedure, making it unsuitable for correct estimations of bacterial loads in shrimp aquaculture farms. Moreover, estimates of the bacterial count using AquaPlakB system plates were sometimes difficult to obtain since well reactions were unclear. The bacterial underestimation and the inconvenience mentioned above, contradicts the manufacturer’s claim that the system is easy to use in shrimp farming facilities as a tool for routine bacteriological analysis with no specially trained personnel. Im-
provements can be made to estimate more accurately total heterotrophic bacteria.
References 111B. Rosenberry, World shrimp farming
1994, Shrimp News Int., San Diego, 1994, 68 pp. 121B. Rosenbeny, World shrimp farming 1993, Shrimp News hit., San Diego, 1993, 52 pp. [31 D.V. Lightner, Diseases of cultured penaeid shrimps, in: J.P. McVey (Ed.), CRC Handbook of Mariculture, 2 ed., CRC, Boca Raton, FL, 1993 pp. 393-486. [41 J.A. Brock, D.V. Lightner, Diseases caused by microorganisms, in: 0. Kinne (Ed.), Diseases of Marine Animals, John Wiley and Sons. New York, 1990, pp. 390-399. [51 L.L. Mohney, D.V Lightner, T.A. Bell, An epizootic of vibriosis in ecuadorian pond-reared Penaeus vannamei Boone (Crustacea: Decapoda), J. World Aquacul. Sot. 25 (1994) 116-125. [61 K.M. Nicholls. J.V Lee, T.J. Donovan, An evaluation of commercial thiosulphate citrate bile salt sucrose agar (TCBS), J. Appl. Bacterial. 41 (1976) 265-269. [71 F.M. Patrick, The use of membrane filtration and marine agar 22168 to enumerate marine heterotrophic bacteria, Aquaculture 13 (1978) 369-372. VI T. Noel, E. Aubree, E. Miahle, B. Panatier, G. Flamion, H. Grizel, A new tool for bacteriological survey in aquaculture, Int. Aquaculture Conference, Bordeaux, 1989, pp. 193-194. [91 J.H. Zar, Biostatistical Analysis, Prentice Hall, New Jersey, 1996, pp. 662.