Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

The overall goal of this technique is to provide a collection of protocols that are simple to carry out and can help any investigator characterize a given clinical or environmental isolate of Vibrio cholerae. This method can help answer key questions in the bacteriology field such as the specific genotypic and phenotypic characteristics of any Vibrio cholerae isolate, especially those of clinical relevance. The main advantage of this technique is that it allows any investigator to use this collection of assays to reliably characterize and distinguish clinical or environmental Vibrio cholerae isolates.

Together, these assays provide a systematic approach to be used as an alternative or in addition to costly labor intensive experiments and the characterization of Vibrio cholerae clinical and environmental isolates. Though this method can provide insight into the characterization of Vibrio cholerae biotypes, it can also be applied to characterize the biochemical and metabolic capabilities of other enteric bacteria. Generally, individuals new to this method will struggle with time management as the incubation times necessary to ensure optimal results differ between biochemical assays.

Visual demonstrations of the spotting and stabbing protocols are critical as splatter can often lead to cross-contamination and maximal spacing is required to prevent merging between cultures. Begin the protocol by pelleting 1.8 mL of liquid overnight culture for two minutes by centrifugation at a minimum of 8, 600 xG in a sterile 2 mL microcentrifuge tube. After centrifugation, remove the supernatant and re-suspend the cell pellet in 900 mcL of fresh Luria-Bertani or LB broth by pipetting.

Next, add 900 mcL of sterile 60%volume-to-volume glycerol to the culture and mix by vortexing. Transfer the mixture to a sterile 2 mL cryogenic tube and store the stock culture at 80 degrees Celsius. To use the stock, with a sterile inoculating loop, remove a small amount of frozen stock and streak the bacteria to obtain single colonies on LB agar plates.

Immediately return the frozen stock to 80 degrees Celsius after use to prevent cultures from completely thawing. Incubate the plates lid-side down for 12-16 hours at 37 degrees Celsius. Obtain previously prepared plate.

Inoculate 4 mL of liquid LB broth in a sterile 10 mL culture tube with a single colony by touching the surface of the single colony with a sterilized loop and transferring the colony into the liquid broth. Then, incubate the culture with aeration in a shaker incubator at 225 rpm for 12-16 hours at 37 degrees Celsius. Prepare overnight culture in liquid LB broth.

The next day, pellet the culture and wash the cells by re-suspending the cell pellet in 1.8 mL of 1x phosphate-buffered saline or PBS. Repeat the washing procedure three times. Follow the third wash with a final re-suspension in 1.8 mL of 1x PBS.

Then, use a pipette tip to spot 1 mcL of washed culture on the respective medium. Wash the cell pellet obtained from overnight culture. Inoculate motility agar plates by inserting an inoculating stab into the washed liquid culture and stab vertically into the media.

Ensure the inoculating stab does not bend while stabbing the agar. Between each inoculation, sterilize the wire stab using a Bunsen burner. Incubate the plates lid side up for 14-24 hours at 37 degrees Celsius.

After 14 hours, monitor the motility plates closely to prevent the overgrowth of cultures. Inoculate 4 mL of Methyl Red Voges-Proskauer or MR-VP broth by pipetting 10 mcL of previously prepared overnight culture into 4 mL of MR-VP broth in a sterile culture tube and incubate with aeration in a shaker incubator at 225 rpm for 12-16 hours at 37 degrees Celsius. Next, add 150 mcL of 5%weight per volume of Alpha-naphthol and 50 mcL of 40%weight per volume potassium hydroxide to 1 mL aliquots of the MR-VP overnight culture in sterile culture tubes respectively.

Then, briefly vortex the tubes. Allow the tubes to stand at room temperature for up to four hours until color change develops. Biotype reference strains and representative El Tor variants were included in PCR base genetic screens and phenotypic assays to distinguish between biotype backgrounds of V.Cholerae clinical and environmental isolates.

Wild-type classical O395 demonstrated classical ctxB and tcpA sequences. Conversely, wild-type El Tor strains N16961 and C6706 demonstrated El Tor ctxB and tcpA sequences. MQ1795 and BAA-2163 contains the classical biotype ctxB subunit comparable to 0395.

Yet both El Tor variants contained the tcpA indicative of the El Tor biotype background. When grown on LB agar plates supplemented with polymyxin B, the wild-type classical biotype strain 0395 showed sensitivity to polymyxin B while wild-type El Tor biotype strain showed resistance. The representative El Tor variant strains demonstrated similar resistance to the antibiotic.

After watching this video, you should have a good understanding of how to distinguish between Vibrio cholerae biotypes using the multiple assay identification system outlined in this manuscript. Don't forget that working with BSL-2 pathogens can be extremely hazardous and proper handling and disposal of all materials and waste products must be enforced according to institutional, local, state, and federal regulations. In addition to the protocols listed in this manuscript, further characterization of Vibrio cholerae isolates can be achieved through investigating other phenotypic properties such as biofilm formation and autoagglutination.