Time-Lapse Epifluorescence Microscopy Imaging of Pseudomonas aeruginosa and Staphylococcus aureus Heterogeneous Phenotypes

Our lab's research focuses on why antibiotics fail. In particular, we're interested in the few cells called persisters in the bacterial populations that are supposedly to be killed by the antibiotic, but they can survive antibiotic treatment. And it's thought that these persisters cause infections to relapse after antibiotic treatment is over.

So we're interested in looking at the gene expression and metabolism of these persisters to better understand their survival strategies. The current challenge for the persister field is to balance throughput with single cell sensitivity. We need to match a single cell's ability to divide after treatment, that will qualify it as a persister, with some measurable phenotype that can help us understand the cell's inner workings.

And we may have to do this for thousands of cells to capture even a single persister. Our protocol was developed to be easily implemented compared to imprecise agar sandwich methods or fabrication of microfluidic devices, which can be technically challenging. We found that our protocol yields reproducible and stable imaging over time with minimal materials, meaning it's both cost-effective and minimally wasteful for quality data collection.

Our future research will use a combination of omics and single cell approaches to study how pathogens respond to and recover from treatment with different classes of antibiotics. Our ultimate goal is to leverage this understanding to develop novel strategies for improving the treatment of clinical infections. To begin, place a sterile 30 millimeter cover slip into the bottom of the stainless steel base of an interchangeable cover slip dish.

Gently thread the polycarbonate insert into the base, ensuring the cover slip forms the base of the chamber and seal it in place by compressing the attached silicone O-ring. Place a custom 3D printed divider against the 30 millimeter cover slip to prevent modal cells from cross contaminating. Prepare 1.5%agarose in a 50 milliliter conical tube using the media of choice and swirl gently to mix.

Loosen the cap of the conical tube and place it into a glass beaker or microwave-safe holder. Microwave the agarose mixture on high, stopping every three to four seconds to swirl and mix to prevent bubbling over. After one minute, check if any unmelted agarose remains in the mixture.

If uniformly clear, allow the agarose to cool briefly to around 60 degrees Celsius. Add the relevant antibiotics or dies and swirl gently to mix, avoiding bubble formation. Pipette two milliliters of the agarose into the chamber with the 30 millimeter cover slip.

Gently lay a sterile 25 millimeter cover slip over the agarose in the top chamber opening. Allow the pad to solidify for one to two hours. When the pad is solidified, use a fine tip permanent marker to mark the locations and identities of each sample on the 25 millimeter cover slip.

Invert the chamber so that the stainless steel base ring is facing upwards and carefully hold the polycarbonate insert underneath while unthreading the base. Set the stainless steel base aside. Slide the 30 millimeter cover slip off the agarose pad and discard it.

Dilute the Pseudomonas aeruginosa or Staphylococcus aureus cell cultures in PBS to a sparse density suitable for visualizing single cells. Pipette vigorously up and down to evenly distribute the cells. Spot five microliters of the diluted cell suspensions at the marked locations on the agarose pad.

Once the spots are dried, place a new sterile 30 millimeter cover slip over the pad. Then hold the polycarbonate insert with one hand and slowly rethread the stainless steel base over the cover slip. Once the chamber is sealed, check if the agarose is making contact with the surface of the 30 millimeter cover slip.

Using the blunt end of tweezers, gently press against the 25 millimeter cover slip until the agarose touches the 30 millimeter cover slip evenly across its surface. To begin, prepare the microscope and imaging chamber environmental controls by setting the stage top incubator and large chamber incubator temperatures to 37 degrees Celsius. Next, turn on the chamber humidifier.

Place one agarose pad preparation into the stage top incubator and close the chamber to allow equilibration. Prepare the MetaMorph software for image acquisition by configuring the built-in autofocus algorithm to use the phase channel during multidimensional acquisition. In the Stage tab, set multiple stage positions for each sample, targeting fields of view with uniformly distributed cell density.

Then in the Time-Lapse tab, configure the duration and frequency for image acquisition. In the Wavelengths tab, set the desired channels for acquisition and adjust the exposure times based on the signal intensity of the sample. Once the agarose pad has warmed up in the chamber, place the humidifying lid on the stage top incubator.

Next, on the microscope, switch from phase contrast to differential interference contrast mode. Adjust the condenser and aperture diaphragm to achieve proper Kohler illumination. After completing the adjustments, switch back to phase contrast mode.

Navigate to each stage position, adjust the focus and reset the stage position to the new focal plane. Click on the Acquire button to start the multidimensional acquisition. After the acquisition, to compile images, open MetaMorph and use the Review Multidimensional Data app to select the desired channels or wavelengths and include all time points for each stage position.

Click Load Images and save each compilation with its respective channel name as a TIFF file. Time-lapse imaging of Staphylococcus aureus cells was conducted showing propidium iodide staining as antibiotic treatment progressed, which indicated membrane damage. Pseudomonas aeruginosa imaging during antibiotic treatment showed cells that undergo spheroplast formation, filamentation, and cytoplasmic condensation, accompanied by propidium iodide staining.

Successful agarose pad preparation and imaging conditions enabled observation of Staphylococcus aureus recovering from antibiotic treatment and Pseudomonas aeruginosa recovering from antibiotic treatment to form large clusters derived from single cells.