The overall goal of this procedure is to isolate c difficile from fecal samples to culture C difficile for preparation of glycerol stocks for long-term storage and to prepare and enumerate spore stocks for a variety of downstream applications. This is accomplished by first isolating C difficile from stool samples by plating on selective medium. The second step is to grow C difficile in liquid medium and maintain long-term C difficile stocks in glycerol.
Next C difficile is retrieved from frozen glycerol stocks and streaked onto agar plates. In the final step, C Difficile spore stocks are prepared and enumerated ultimately by following these protocols. C difficile can be grown on various media to verify and confirm its identity and be successfully cultured and maintained.
These glycerol stalks and cultures can be further used in numerous downstream applications, including animal studies and additional molecular biology studies. The main advantage to using a vinyl anaerobic chamber to cultivate c difficile over other methods like using candle jars, is that a strict anaerobic environment can be maintained, allowing for consistent and reliable growth of c difficile. Generally, people who are new to these techniques may encounter problems due to the lack of dexterity within the chamber because of the gloves, and also because of the critical need to plan their experiments ahead of time.
Demonstrating these procedures will be a technician from my lab, Jose Suarez In the beginning. This procedure may be performed in aerobic conditions. First, weigh the stool sample.
This is necessary for enumerating c difficile from the stool. Then resuspend the stool sample in one XPBS and vortex to ensure that the stool sample is fully resuspended. Next, make serial dilution of the Resuspended stool sample in one XPBS for appropriate isolation or enumeration of colony forming units or CFU pergram of stool from here on.
Perform the procedure in anaerobic conditions to ensure that vegetative cells of C difficile are not exposed to any oxygen. The anaerobic chamber used in this experiment is kept at 37 degrees Celsius. Using aseptic technique.
Apply 100 microliters of each serial dilution to Toro collate oxetane, cyclo Seine, fructose agar, or T-C-C-F-A plates. These plates have been pred reduced for at least one to two hours in the anaerobic chamber to ensure the removal of residual oxygen. To isolate colonies for accurate enumeration or for other downstream applications.
Use a sterile inoculating loop to evenly spread the applied culture on the surface of the T-C-C-F-A plate. Incubate the plates anaerobically for 48 hours at 37 degrees Celsius 48 hours later. Identify c difficile colonies based on their flat, irregular ground glass appearance to ensure vegetative c difficile are not killed by oxygen exposure.
Plates for isolating colonies for downstream applications must be kept inside the anaerobic chamber at all times. For enumeration of colonies, the plates can be brought outside the anaerobic chamber. Note that once a plate is brought outside the chamber, vegetative c difficile will be exposed to oxygen and will die.
These C difficile colonies cannot be rescued even if the plate is returned to the chamber. Count the C difficile colonies of each dilution and calculate the number of CFU per gram of stool sample to culture and make glycerol stalks of c difficile. Use a sterile inoculating loop to pick an individual colony and streak onto a pre reduced brain heart infusion.
Supplemented with yeast extract or BHIS agar plate, supplemented with 0.03%cysteine streak individual colonies and quadrants using a new sterile inoculating loop for each quadrant. Incubate the plates anaerobically at 37 degrees Celsius for 16 to 24 hours. Once isolated colonies appear on the BHIS agar plates, pick an individual isolated colony and resus suspend the colony in 10 milliliters of pre reduced BHIS liquid medium, supplemented with 0.03%L cysteine incubate overnight.
Anaerobically at 37 degrees Celsius or until the culture becomes turbid. On the following day at 333 microliters of 50%glycerol and 666 microliters of the c difficile culture to a 1.8 milliliter cryogenic tube. To create a 15%glycerol stock of the isolated strain tightly cap the cryogenic tube and mix.
Well immediately remove the stock from the anaerobic chamber and place it in a negative 80 degree Celsius freezer for long-term storage. To begin this procedure, remove the frozen glycerol stock of c difficile from the negative 80 degrees Celsius freezer. To prevent thawing.
Place the frozen glycerol stock immediately in a cooling rack that has been stored at negative 80 degrees Celsius prior to use. Bring the frozen glycerol stock in the cooling rack into the anaerobic chamber using aseptic technique and a sterile inoculating loop. Place a small amount of the stock onto the plate and streak across one quadrant of the plate.
Rotate the plate 90 degrees and using a new sterile inoculating loop streak across the second quadrant. Repeat for the third and fourth quadrants to ensure the isolation of individual colonies. Immediately remove the frozen glycerol stock from the anaerobic chamber and return to the negative 80 degrees Celsius freezer.
Incubate the plate anaerobically overnight at 37 degrees Celsius to purify spores from c difficile culture. Strains from frozen glycerol stock onto pre reduced BHIS plates. Following the procedure demonstrated in the previous segment.
Incubate anaerobically overnight at 37 degrees Celsius on the following day. Rere individual colonies into several pred reduced 70 30 plates. Incubate anaerobically at 37 degrees Celsius for 24 to 48 hours.
Once spores have formed, remove the plates from the anaerobic chamber. Scrape the plates using a sterile inoculating loop and resuspend the cells in 10 milliliters of sterile one XPBS. Discard the plates, pellet the cells at 3000 G for 15 minutes.
Remove the supernatant, add one milliliter of one XPBS resus, suspend and pellet again in this manner. Wash the cells twice in PBS incubate at four degrees Celsius overnight to aid in lysis of vegetative and mother cells on the following day, incubate at 70 degrees Celsius for 20 minutes to kill any residual vegetative cells. To determine CFU per milliliter, serially dilute each spore preparation in one XPBS and plate onto pre reduced BHIS plus 0.1%Sodium Toro collate incubate plates at 37 degrees Celsius for minimum of 24 hours before enumerating colonies.
Spores can be stored in one XPBS at either room, temperature for short-term storage or four degrees Celsius for long-term storage. A.An example of c difficile grown on BHIS and Columbia anaerobic sheep. Blood agar media can be seen here.
C difficile forms irregular colonies that are flat and possess a ground glass appearance that is evident on both media. Panel A shows an erythromycin sensitive clinical is isolate c difficile 630 E grown on BHIS agar, an enriched non-selective medium for 24 hours. At 37 degrees Celsius colonies on Columbia anaerobic sheep Blood agar shown in panel B appear similar to those grown on BHIS under white light.
However, the use of this medium also provides for detection of the greenish or citrus fluorescence exhibited by C difficile under long wave ultraviolet light shown in panel C Following this procedure. Other methods like growth curves in liquid medium, DNA and RNA, isolation colony, PCR and other molecular biology techniques and manipulation suitable for c difficile as well as utilizing c difficile spores for animal studies can be performed in order to answer additional molecular biology questions. After watching this video, you should have a good understanding of how to isolate culture and maintain c difficile vegetative cells and spores within an anaerobic environment.
Don't forget that c difficile is a human and animal pathogen that can cause gastrointestinal disease. Experiments with c difficile must be performed under appropriate biosafety level two conditions.
