TCBS Selection Agar Limitations

Shrimp farms can be stressful environments that have high organic matter and experience fluctuations of dissolved oxygen.  Changes in the environment in the shrimp farm will influence the number of bacteria and the species present.  For example, as the temperature increases and the salinity increases due to evaporation V. parahaemolyticus will thrive and become predominate. Shrimp farmers recognize the relevance of Vibrio species, and the safety level of heterotrophic bacteria and Vibrio to the health of their ponds.  Relative counts of bacterial groups are monitored and routine determination of Vibrio species are used to help in pond management.  Farmers realize that a high prevalence of green colonies on Thiosulfate Citrate Bile Salts Sucrose Agar (TCBS) are indicative of diseased ponds. However, not all farmers may be aware of the pitfalls of the TCBS selection system or the availability of the improved HiCrome chromogenic Vibrio detection system.

TCBS Selection Agar.  TCBS is a selective and differential culture media for Vibrio species based on sucrose fermentation.  Sucrose fermenting bacterial colonies produce acid, and the pH indicators in TCBS turn yellow in the acidic media. Those bacteria colonies that do not ferment sucrose remain green. Table 1 contains the formulation for 1 liter of TCBS and the functions of the different ingredients are noted. Table 2 indicates which bacteria are sucrose fermenters based on the colony colors indicated.

Limitations of TCBS Selection Agar:

1.    May not support good growth of all Vibrio. (Table 2.)

2.    Cultures should be visualized right after removal from the incubator as some yellow colonies might revert to green at room temperature.

3.    Dense plates with a lot of sucrose fermenting colonies might obscure the green colonies because the acid from the sucrose fermenting bacteria can diffuse through the agar.

4.    There is no correlation between sucrose fermentation and virulence.  Not all green colonies are pathogenic and not all yellow colonies are harmless. Detection of the PirA/PirB gene using PCR techniques would indicate the presence of APHND.  However high numbers of APHND- can still cause disease in shrimp.

5.    Accompanying sucrose fermenting bacteria can pose a problem with this selection methodology

6.    Further identification of green colonies would be suggested.  In lab, API-20 NFT (www.biomerieux-usa.com) or Hi-Vibrio™ Identification kit (www.himedialabs.com) would be helpful. Sequencing of 16S RNA gene is not sufficient to identify all Vibrio.  MLSA (multi-locus sequence analysis would be more sensitive.

 HiCrome™ Vibrio Selection Agar.  HiCrome was formulated to be an improved method for the detection of Vibrio parahaemolyticus.  The formulation for 1 liter is located in Table 3.  This agar medium detects beta-galactosidase using a chromogenic substrate.  This detection system eliminated a number of limitations the TCBS Agar had due to issues from the sucrose fermenting selection. 

Advantages of HiCrome Vibrio Agar

1.    The color changes on HiCrome™ Vibrio Agar do not change when kept at room temperature.

2.    Color is not effected by other bacteria.

3.    HiCrome™ Vibrio Agar is 2.5% salt compared to TCBS which is 1% salt.  The ocean is 3.5% salt therefore HiCrome™ is closer to the natural environment for Vibrio’s. 

4.    V. parahaemolyticus turns violet growing on HiCrome™ Vibrio Agar and is distinguishable from other non-sucrose fermenting bacteria and Vibrios commonly isolated from the same environment (Table 4).

Limitation

1.    Beta-galactosidase activity has not been associated to virulence.  Detection of the PirA/PirB gene using PCR techniques would indicate the presence of APHND.  However high numbers of APHND- can still cause disease in shrimp

Summary.  There are two methods that can be used to select for V. parahaemolyticus.  HiCrome™ Vibrio Agar appears to have the better selection system with potential for less false positives.  Pathogenic Vibrios occur naturally in ocean and are probably present in low levels in the beginning of the season.  As the growing season progresses the population of V. parahaemolyticus is likely to increase.  With a good selection and differential system these changes in population dynamic can easily be detected and signal the need for more probiotic intervention.

References:

Kudo.H.Y. et al, 2001. Improved Method for Detection of Vibrio parahaemolyticus in Seafood. ASM. Vol.67, 12 pg 5819-5823.

HiMedia HiCrome™ Vibrio Agar Technical Data

Hardy Diagnostics TCBS Technical Data