Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

The overall goal of this procedure is to evaluate the sensitivity and specificity of a new chromogenic medium for the ability to detect and isolate Vibrio parahaemolyticus as compared to conventional media. This method can help answer key questions in the food and environmental microbiology field, such as what is the prevalence of Vibrio species in food and harvesting environments? The main advantage of our procedure is that many bacterial isolates can be used, including in the presence of a food matrix.

Before culturing the bacteria, autoclave all of the agar media. Then cool the agar to 45 to 50 degrees Celsius in a water bath. When the agar is ready, arrange empty petri plates in stacks of five to six plates.

Then, starting from the bottom of the stack, pour the molten agar into each plate until about half full, replacing the lids after pouring. Allow the agar to solidy at room temperature for at least 12 hours. To setup the microbial strain cultures, when the agar is ready, use a sterile inoculating loop to transfer the cultures onto the appropriate non-selective agar plates, streaking the bacteria in a pattern that will allow the observation of isolated colonies.

When all of the colonies have been plated, incubate the cultures upside down at 35 to 37 degrees Celsius for up to 48 hours, except for Campylobacter species, which should be incubated in a closed-lid jar containing a gas pouch to produce a microaerophilic environment. Observe the colony morphology at the end of the incubation. Pure cultures should yield colonies that exhibit a similar colony morphology.

To grow out the colonies of interest on selective and differential media, transfer a few isolated colonies into five milliliters of the appropriate broth and reincubate the cultures at 35 to 37 degrees Celsius for 16 to 24 hours. The next day, streak a loopful of the overnight cultures onto at least one Thiosulfate-Citrate-Bile-Salts-Sucrose, or TCBS, agar plate, and at least one chromogenic agar plate for up to 96 hours of incubation at 35 to 37 degrees Celsius. At the end of the incubation, examine both the culture density on the plate and the size of the isolated colonies to determine the overall growth of the strains, recording the color of the colonies under ambient or UV light, as appropriate, and noting any other important characteristics of the colonies.

To perform a recovery assay, inoculate young cultures of Vibrio parahaemolyticus in five milliliters of tryptic soy broth, supplemented with two percent sodium chloride, or TSBS, and place the tubes at 35 to 37 degrees Celsius for 16 to 24 hours. The next day, vortex the overnight cultures and setup one times 10 to the negative one, to one times 10 to the negative seven dilutions of the bacteria in PBS. Using the one times 10 to the negative four to one times 10 to the negative seven dilutions, evenly plate 100 microliters of each dilution onto individual chromogenic, TCBS, and tryptic soy agar supplemented with two percent sodium chloride agar, or TSAS, plates.

Incubate all of the plates at 35 to 37 degrees Celsius for up to 96 hours. Then count the colonies, ignoring the plates bearing colonies too numerous to count or containing fewer than 25 colonies. To perform a competition assay, select a V.Parahaemolyticus strain that yields the expected turqouise colonies on TCBS and cyan colonies on chromogenic agar, and a non-V.

Parahaemolytius species that does not grow on either media or exhibits a different colony color. Transfer a few isolated V.Parahaemolyticus and V.Metschnikovii colonies from a TSAS agriculture, to five milliliters of TSBS for 16 to 24 hour incubation at 35 to 37 degrees Celsius. Transfer a few isolated Shigella sonnei colonies from Brain Heart Infusion, or BHI, agar to five milliliters of BHI broth for 16 to 24 hours at 35 to 37 degrees Celsius.

The next day, perform a serial dilution for each strain, plating the dilutions onto non-selective agar plates to determine the colony-forming units per milliliter of the overnight cultures. Next, use the overnight cultures and dilution tubes to mix different volumes of a V.Parahaemolyticus strain and a non-V. Parahaemolyticus species and spread 100 microliters of the bacterial mixture onto individual chromogenic, TCBS and TSAS agar plates.

After up to 96 hours at 35 to 37 degrees Celsius, count the colonies based on their differences in growth and morphology on the chromogenic and TCBS plates. To assess the effects of oyster homogenates on bacterial growth on selective and differential media, first weigh at least 50 grams of oyster meat from at least 12 molluscan shell fish, including the meat and liquor. Then add an equal mass of PBS to the oyster tissues and blend the mixture at a high speed for 90 seconds.

Next, add 100 grams of the diluted oyster homogenate to 400 grams of PBS and blend the mixture at high speed for one minute. Then autoclave the homogenate. After cooling, add 100 microliters of each overnight V.Parahaemolyticus and non-V.

Parahaemolyticus culture to the oyster slurry, and use a homogenizer to mix the bacterial cells with the oyster homogenate. Make one times 10 to the negative one to one times 10 to the negative three dilutions of the spiked oyster homogenate in PBS as demonstrated, spreading 100 microliters of each dilution onto individual chromogenic, TCBS and TSAS plates. After 96 hours at 35 to 37 degrees Celsius, compare the actual colony counts on the chromogenic and TCBS agars with the expected colony counts deduced from the standard plate count conducted on the overnight cultures.

To determine the growth and colony morphology of the strains used in this study, the bacteria were grown on selective and differential media. TCBS is the conventional medium used for the isolation of some Vibrio species, including turquoise V.Parahaemolyticus and yellow V.Cholerae. The use of chromogenic medium to select for clinically relevant Vibrio species from food and environmental samples results in the generation of cyan V.Parahaemolyticus and magenta V.Cholerae.

Colonies of V.Parahaemolyticus grown on chromogenic agar plates in the presence of oyster homogenate are observed in similar numbers to those on non-selective media, suggesting that the detection limit of the chromogenic medium is similar to that of non-selective media, even in the presence of a food matrix. Nor is the growth and recovery of V.Parahaemolyticus affected by the presence of a non-V. Parahaemolyticus species, an essential attribute for differential and selective media, as environmental samples contain various Vibrio species at high numbers, especially after an enrichment procedure.

Further, the comparison of TCBS and chromogenic agars by thermolabile hemolysin PCR reveals a higher sensitivity and specificity for the chromogenic agar, indicating a better performance overall for this differential and selective medium for detecting and identifying V.Parahaemolyticus. Once mastered, the entire procedure including the testing and incubation times of approximately 50 strains can be completed within 30 days when performed properly. While attempting this procedure, it is important to remember to diligently label all cultures and to use aseptic technique at all times.

Following this procedure, other methods like colony PCR can be performed to answer additional questions, like, Does this specific isolate carry a virulence gene? After its development, this technique paved the way for researchers in the field of food safety and public health to explore Vibrio species detection in shellfish and the estuarine environment. After watching this video, you should have a good understanding of how to conduct a standard plate count and how to evaluate the sensitivity, specificity and detection limit of a growth medium.

Don't forget that working with pathogens and hot medium can be extremely hazardous, and precautions such as wearing protective clothing, covering any open wounds, and segregating biohazardous waste should always be taken while performing this procedure.