形態変化を特徴付けるプロトコル

The overall goal of this microbiological imaging method is to provide a more descriptive means to distinguish the phenotypic effects of pharmacological treatment. So this method can really help in the area of infective diseases. Specifically looking at the morphological effects of certain antibiotics and how it kills C.Difficile.

The main advantage of this technique is that it combines high resolution imaging with in vitro cell culture techniques to provide detailed outlook of pharmaceutical killing action. The implication of this technique may extend towards therapy for Clostridium difficile infection, or CDI in short. The reason is this technique may provide a mean for identifying how an antibiotic may be beneficial in treating CDI.

Though this method can provide insight into pharmacological action, it can also be applied to other systems, such as mixed culture model or in vivo animal studies. Generally, individuals struggle with this new method because culturing C.difficile and operating a scanning electron microscope is challenging. We first had the idea for this method when we were trying to distinguish between different antibiotics'modes of actions.

I'd like to introduce you to the research team that you'll meet today. Jahangir Alam is a professor and microbiologist. He coordinates all the lab activities that you'll see today.

Tasnuva Rashid is a graduate student in the lab. She does a lot of the day-to-day microbiology. Eugenie Bassere is a post doc fellow.

She really has taught Tasnuva many of the skills and also assists in the lab. Brad Endres is another post doc in the lab. He'll do a lot of microscopy with the help of Long Chang.

To prepare environment isolates while wearing sterile gloves, use a pre-sterilized cotton gauze to swab the surface of any area of interest, such as a floor, door, handle, or shelf. Then place the swab into a sterilized tube. Change gloves between samples.

To prepare clinical isolates, use an inoculation loop to plate 10 to 100 milligrams of clinical stool samples onto cefoxitin cycloserine fructose agar, or CCFA, and intubate the samples under strict anaerobic conditions for 48 to 72 hours. Store isolated colonies of the C.Difficile stock in cryo vials at negative 80 degrees Celsius for further analyses. Enrich the environmental swab samples in brain heart infusion, or BHI broth, with 05%sodium taurocholate and place the samples in an anaerobic chamber at 37 degrees Celsius for five days.

Centrifuge one milliliter of the culture at 10, 000 times G, and use 100 microliters of ethanol to re-suspend the pellet. Plate 50 microliters of the re-suspended cells onto CCFA plates and incubate the cultures in an anaerobic chamber at 37 degrees Celsius for 40 to 48 hours. Store the isolated colonies of C.difficile stock in cryo vials at negative 80 degrees Celsius for further analyses.

Use the latex agglutination reagent, or PCR, to test the suspected C.difficile colonies. Grow purified environmental or clinical C.difficile strains on blood agar plates in an anaerobic chamber at 37 degrees Celsius for 48 hours. Use an inoculation loop to take one isolated colony and transfer it into five milliliters of BHI medium in a 15 milliliter tube.

Then grow the culture in an anaerobic chamber at 37 degrees Celsius for 24 hours. Using fresh, pre-reduced BHIS supplemented with sodium taurocholate and the appropriate concentration of antibiotic to dilute the pre-cultures 1 to 100 to approximately 10 to the sixth CFU per milliliter. With a pipette, collect a one milliliter sample at each time point and plate or spread a small aliquot onto a blood agar plate.

Incubate the plate for 48 hours in an anaerobic chamber at 37 degrees Celsius and count the resulting number of colonies to determine the CFUs. Collect one milliliter of cells from each time point in microcentrifuge tubes and centrifuge at 10, 000 times G for 10 minutes. Discard the supernatant and use PBS to wash the cells.

Spin the samples again and discard the supernatant. Then use one milliliter of 4%paraformaldehyde to re-dilute the cells and incubate the tubes at room temperature for one hour. After spinning the samples again and discarding the supernatant, use distilled water to wash the cells twice before re-diluting the cells in 100 microliters of distilled water.

Adjust the volume depending on the turbidity of the solution. After labeling cover slips, add 40 microliters of the sample onto them. Under a flow hood, incubate the cover slips for 15 minutes to evaporate the liquid and allow the cells to adhere to the glass.

If liquid is still present, use a blower to remove it. Place the cover slips in a desk sputtering machine and tape them down. Secure pure gold in the sputtering machine.

Then turn on the machine and commence sputtering at low pressure. Coat the cells for 30 seconds at 80 microamperes, which translates to 20 nanometers of gold coating. To begin the imaging, first vent the SEM properly in the computer software by pressing the Vent button.

After coating the cells, transfer them to the scanning electron microscope, or SEM. Using the carbon tape, secure the coated cover slips onto the metal stage. Once the SEM is vented, the door should easily open.

Lock the metal stage into the SEM chamber by screwing it in. Next, click the Pump button in the software. When the system reads back okay, the SEM will be ready to use.

Under the Detector's tab, click on the SE detector. Turn on the beam by clicking on the button that displays the voltage. Start imaging at a lower voltage before increasing the voltage.

An image will appear after the beam is turned on. Using the tracking function, find an area on the coated cover slips to image. Zoom into the region and find rod-shaped structures, which represent Clostridium difficile.

To calibrate the system, zoom in, coarsely focus the image, and link Z to the free working distance. This should be done at multiple working distances, such as at 15, 9, and 5 millimeters. Then switch to ultrahigh resolution imaging mode.

Use the coarse and fine focus toggles to begin to focus at high magnification. Adjust the astigmatism toggles for a clearer image, and check the image for clarity by using the computer software to digitally zoom in on the image. Use the slow scan to collect a high-quality image.

Save the collected image as a tiff file for later analysis, ensuring that the data bar is selected if measurements will be made during analysis. Collect images at different angles to reveal more depth information by tilting the stage on the SEM. Optimize focus and astigmatism before collecting a slow scan image.

After imaging is completed, turn the beam off and raise the working distance to 20 millimeters. Then vent the chamber and remove the stage. Process the images, open the image files in Fiji.

Using the line function, precisely trace the scale bar. Click on the Analyze tab;then choose the Select Scale function. A window will appear that will require the setting of known distance based on the scale bar.

Change the unit of length as well, and click Okay. Finally, to measure cell length, use the line function to trace the cell in its entirety. Select the Analyze tab again, and then click on measure.

The length should appear in the units denoted previously. These images show C.difficile vegetative cells that were captured during the exponential phase of the growth curve, as well as spore cells. Vegetative cells are long, smooth, rod-shaped structures;whereas, spores are small oval structures that have a rough exterior.

As shown in this figure, the control cells grow and reach a plateau;whereas, the cells treated with the antibiotics vancomycin and metronidazole decrease in total CFUs to the limit of detection, indicating a bactericidal effect. As demonstrated, vancomycin and metronidazole are effective at killing C.difficile at super MIC concentrations. To demonstrate the utility of using SEM for imaging C.difficile, cells were imaged before and after drug treatment to determine how the morphology changed.

In the case of vancomycin, some of the cells'walls were affected and some metronidazole-treated cells were smaller in size. To test whether cell size was effected, cell length was analyzed using the program Fiji. As demonstrated in this image, vegetative cell size can vary some in the control case;but most are roughly 6 micrometers in length.

However, as shown in this graph, cell length was effected in metronidazole-treated cells, but not in vancomycin-treated cells. Once mastered, this technique can be performed in under two days. While attempting this procedure, it is important to remember that your sample is fixed and completely dry before coating and imaging.

Following this procedure, we can perform additional methods to answer additional questions, such as how bacteria will responds to antibiotic treatments;and this can give us some more insight into understanding the mechanics of action of antibiotics, for example. This technique has immense potential and may pave the way for researchers in the field of microbiology to further explore the physiology and pharmacology in Clostridium difficile. I hope you've enjoyed watching this video today.

I hope what you've gained from this is how to grow and characterize C.difficile cells, how to look at the killing kinetics of antibiotics against C.Difficile, and then how to assess the morphological changes associated with that killing patterns using high-level microscopy. While we are working with Clostridium difficile, we must take extra precautions and use all protective