Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy (DHM)

The overall goal of this experiment is to demonstrate the detection of microorganisms at very low levels using digital holographic microscopy. This technique is more sensitive than traditional light microscopy and provides real-time information about bacterial behaviors. This method can help answer key questions in the biological and cosmological fields such as searching for pathogenic organisms in water or life within our solar system on icy moons.

The main advantage of this technique is that DHM is a volumetric technique which means we can capture three-dimensional information on our sample instantaneously without any need to physically refocus. The implications of this technique extend toward the diagnosis of blood stream or cerebrospinal fluid infections because of the ability to detect low bacterial concentrations. To begin this procedure, on the day of the experiment, take a spectrophotometric reading of the bacterial master culture which is expected to be in the range of 0.6 to 0.7.

Then take a sample of the master culture and count the cells directly using a Petroff-Hausser counting chamber to enumerate both live and dead cells. Transfer a 10 microliter sample of the undiluted culture with a micropipette to the chamber. Image it under a high dry objective microscope using phase contrast.

Subsequently, count the bacteria in at least 20 squares and average them. Calculate the concentration as the average of 20 squares times the dilution factor. To enumerate only live cells, make a serial dilution of each of the selected bacterial samples with sterile 0.9%saline solution by transferring 20 microliters of the bacterial solution to another well and diluting it with 180 microliters of saline.

Repeat the procedure until the lowest concentration is approximately 10 to the three cells per milliliter. Next, take 100 microliters of the samples from at least two dilutions and plate them on the appropriate solid media plates. Spread them with a sterile spreader and perform at least three replicates of each dilution.

After that, incubate them at an appropriate temperature overnight or until the colonies grow. Then count the colonies. Calculate the colony-forming units and average it over the replicates.

In this procedure, make serial dilutions of the master culture for DHM and post-DHM counting of CFU on Lysogeny broth media plates. Dilute the bacteria into 25 milliliters of a minimal medium that will encourage motility but inhibit cell division so that the concentration of cells does not change appreciably during the experiment. To record DHM videos, using a sterile syringe, draw in about 10 milliliters of the dilution of interest.

Then connect the syringe to the DHM sample chamber using sterile fittings and tubing. Flow the sample from the syringe through the sample chamber continuously using a syringe pump. As the sample flows through the sample chamber, acquire holograms consecutively at an appropriate frame rate.

Allow sufficient time for the entire 10 milliliters of sample to flow through the DHM. To ensure that bacteria are not growing or dying during the experiments, inoculate the enriched media plates with 100 microliters of the spent media post-DHM image capture by spreading it with a sterile spreader. Then incubate them at the appropriate temperature for 24 hours before counting the colonies.

Having accurate cell counts is essential to determine the detection limits quantitatively. It is important to make all dilutions with great care using calibrated micropipettes and confirm all counts by optical density, plating, and direct counting in the Petroff-Hausser chamber. To analyze the data for bacterial presence in the acquired hologram videos, conduct medium subtracted filtering by first converting the images to a 32-bit format.

Then calculate the median pixel value. Finally, subtract this median value from each respective pixel to eliminate stationary artifacts in the hologram. After that, count the number of visible area rings and in-focus cells manually.

Each series of holograms will be accompanied by a timestamp file. Use the timestamp file to calculate the total amount of sample pumped at the time the image was captured. Subsequently, calculate the cell density by dividing the total number of cells detected by the total volume of sample imaged.

Average five to 10 frames for accurate statistics. This plot shows the predicted cells per field of view based upon a sample volume of 365 micrometers by 365 micrometers by one millimeter. For concentrations where the number of cells per field of view falls below one, multiple images are required in order to achieve detection.

This is a DHM hologram sequence of a Serratia marcescens culture at 2, 100 cells per milliliter measured by plate count. The sequence shows one cell roughly every one to two seconds which is an excellent fit to the prediction. And this is another DHM hologram sequence of a Serratia marcescens culture at 620 cells per milliliter measured by plate count.

This sequence shows one cell roughly every six to seven seconds. When using digital holographic microscopy to image cells, remember to use filter sterilization techniques in preparation of all solutions. This will avoid introducing particulate matter into the instrument.

While attempting this procedure, it is important to remember to prepare healthy cell cultures and have extra growth medium, plates, and motility medium on hand. Following this procedure, other methods like fluorescent staining can be performed in order to answer additional questions such as the percentage of cells that are alive versus dead. After its development, this technique paved the way for researchers in the field of microbiology to explore three-dimensional motility in cultured cells and environmental samples.

After watching this video, you should have a good understanding of how to enumerate bacteria using holographic microscopy and validate the results using other counting techniques. Don't forget that working with bacterial cultures can be extremely hazardous. Appropriate biosafety precautions should always be taken when growing and handling live organisms.