Culture Methods to Determine the Limit of Detection and Survival in Transport Media of Campylobacter Jejuni in Human Fecal Specimens

The difficulty of Campylobacter culture has inhibited fundamental studies such as how few bacteria are needed to produce illness. These combined analytical and contrived samples used here can address this issue. This technique allows clinical relevant patient symptoms to be correlated with a positive fecal culture result and for the first time, an actual number of bacteria.

Learning how many bacteria correlate with illness will provide diagnostics, an important goal for detection and a solid method for comparing virulence between strains or species of Campylobacter. Campylobacter culture is straightforward but it requires quick and organized preparation to maintain quantitative viability. Considerable judgment is also needed to identify the Campylobacter colonies amongst competing fecal flora on a plate.

Before starting a C.jejuni or C.coli culture, put on gloves, a lab coat, and safety glasses, and place a disposable protective sheet within a disinfected laminar flow safety hood. Using sterile technique streak every hydrated or thawed bacterial stock onto a Campylobacter specific agar plate and place the plate at 37 degrees Celsius for 48 hours in an anaerobic jar containing a microaerobic atmosphere gas generating sachet. 24 hours later, loosely cover a flask of BHI broth and place the flask into a new anaerobic jar with a sachet that will produce a microaerobic environment for an overnight incubation at 37 degree Celsius.

The next morning use three milliliters of the pre-reduced broth and an inoculating loop to scrape the starter plate of Campylobacter culture. Transfer the slurry to a sterile tube, and inoculate the remaining 97 milliliters of the pre-reduced broth with the three milliliters of scraped slurry. Place the broth in the jar with the gas sachet and incubate the culture at 37 degrees Celsius and 115 revolutions per minute on a shaking incubator for 48 to 72 hours, until the culture reaches an optical density at 600 nanometers of no greater than 4.

To establish the number of bacteria in this pure stock culture perform eight 10-fold dilution series of 100 microliters of broth in 900 microliters of dilution. Using sterile plating beads, spread 100 microliters of the one times 10 to the negative fifth to the 10 to the negative seventh dilutions on duplicate pre-reduced Campylobacter specific plates and label plates with the dilution concentration before placing the plates into a second anaerobic jar with a gas generating sachet for 48 to 72 hours at 37 degrees Celsius. At the end of the growth period select plates with between 30 to 300 colonies for counting.

After counting use the counts to determine the colony forming units per milliliter of the stock broth culture according to the formula. It is critical that the analytical colony counts are accurate as this underpins all of the steps using spiked fecal pool. Immediately after plating for counting as demonstrated, mix equal volumes of broth and Campylobacter negative fecal pool and make nine subsequent two-fold dilutions in negative fecal pool, adding a control plate with broth containing no Campylobacter to the fecal pool to help identify the non-Campylobacter colonies.

Streak 10 microliters of each Campylobacter stool dilution on duplicate pre-reduced Campylobacter specific agar plates and incubate the plates in anaerobic jar with a gas generating sachet at 37 degrees Celsius for approximately 48 hours. At the end of the incubation visually examine the streaked plates for colonies resembling those from pure Campylobacter cultures. To confirm that the colonies are actually Campylobacter select multiple Campylobacter like colonies for gram staining and visualize a thinly streaked area by light microscopy using an oil immersion lens to identify gram-negative curved, spiral, or cigar shaped small bacteria.

A negative control plate without added Campylobacter is important for helping to identify other fecal flora. Consider the last dilution that contains a visual Campylobacter-like gram-negative colony the limit of culture detection. And use the formula to calculate the colony forming units per milliliter of the positive dilution in contrived clinical fecal specimen.

To determine the viability of Campylobacter stored in transport medium mix one milliliter of Campylobacter broth culture with one milliliter of negative fecal pool and prepare 10 duplicate two-fold serial dilutions in negative fecal pool. Further dilute each dilution at an additional one to four ratio in Cary-Blair medium and store the 20 dilution tubes and a negative control in Cary-Blair medium in cap tubes at two to eight degrees Celsius for 96 hours. Starting at time zero and every 24 hours thereafter streak 10 microliter aliquots of each dilution onto Campylobacter selective agar plates and incubate the plates at 37 degrees Celsius for 48 hours.

As there is no independent method for establishing accurate bacterial numbers from patient samples two simultaneous measurements can be made with one pure bacterial stock. One test is used for the visual detection of Campylobacter colonies from serial dilutions of the stock bacteria in a fecal matrix, simulating clinical specimens. The other test is used analytically, quantify the colony forming units per milliliter present in the same bacteria stock culture used for spiking.

A key parameter for success is identifying the pinpoint sized colonies among the competing fecal flora, as illustrated by this representative plate of spiked stool culture. Here, typical data from seven independent experiments can be observed. Simply identifying colonies that resemble Campylobacter within the fecal cultures is not enough.

All of the colonies should be confirmed with gram stain or more advanced methods. We use an enzyme immunoassay that is more accurate than culture to detect uncommon species such as C.upsaliensis or C.lari, which grow poorly on standard antibiotic containing agar. This technique lays a necessary foundation for studying things like the non-symptomatic carriage of Campylobacter and whether high bacterial loads put a patient at higher risk for serious consequences of Campylobacter infection.

Always wear PPE when working with bacteria, which are often infectious and can be dangerous, and with the negative fecal pool which may contain unknown pathogens even when collected from healthy donors.