Name: quantifying microorganisms at low concentrations using digital holographic microscopy (dhm)
Uploaded: 2017-11-01T15:00:00
Description: Watch this Scientific Journal Video about Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy DHM at JoVE.com

The authors acknowledge the Gordon and Betty Moore Foundation Grants 4037 and 4038 to the California Institute of Technology for funding this work.

1. Growth and Enumeration of Bacteria
NOTE: This is applicable to almost any bacterial strain grown in the appropriate medium36. In our example, we use three strains: Serratia marcescens as a common, easy identifiable lab strain; and a smaller, highly motile environmental strain, Shewanella oneidensis MR-1. To compare detection of motile vs. non-motile cells, a non-motile Shewanella mutant, &#916; FlgM, is also used for comparison<sup class="xre...

<ol>
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Numerical reconstruction of holograms: For the numerical reconstruction of holograms, the angular spectrum method (ASM) is used. This involves the convolution of the hologram with the Green&#39;s Function for the DHM. The complex wavefront of the image at a particular focal plane can be calculated by employing the Fourier Convolution Theorem as follows:
<img alt="Equation 2" src="/files/ftp_upload/56343/56343eq2.jpg" /> (1)
Where&#160;<img alt="Equa...

Determination of accurate bacterial counts in very dilute samples is crucial in many applications: a few examples are water and food quality analysis1,2,3; detection of pathogens in blood, cerebrospinal fluid, or sputum4,5; production of pharmaceutical products, including sterile water6; and environmental community analysis in oligotrophic environments such as the open ocean and sediments7,8,9. There is also increasing interest in detection of possible extant microbial life on the icy moons of Jupiter and Saturn, particularly Europa10,11 and Enceladus12,13,14, which are known to have subsurface liquid oceans. Because no mission since Viking in 1978 has attempted to find extant life on another planet, there has been limited development of technologies and instruments for bacterial identification and counting during space missions15.
Traditional methods of plate count find only culturable cells, which can represent a minority of species in environmental strains, sometimes &#60;1%16. Plates require days or weeks of incubation for maximum success, depending upon the strain. Epifluorescence microscopy has largely replaced plate counts as the gold standard for rapid and accurate microbial enumeration. Nucleic-acid-labeling fluorescent dyes such as 4&#8242;,6-diamidino-2-phenylindole dihydrochloride (DAPI), SYBR Green, or acridine orange that bind to nucleic acids are the typical dyes used17,18,19, though many studies use fluorescent indicators of Gram sign20,21,22,23,24. Using these methods without pre-concentration steps leads to limits of detection (LoDs) of ~105 cells per mL. Improvements in LoD are possible using filtration. A liquid sample is vacuum-filtered onto a membrane, usually polycarbonate and ideally black to reduce background. Low-background dyes such as the DNA stains mentioned above may be applied directly to the filter25. For accurate counting by eye, ~105 cells are required per filter, which means that for samples more dilute than ~105 cells per mL, significant sample volumes must be collected and filtered. Laser-scanning devices have been developed in order to systematically explore all regions of the filter and thus reduce the number of cells required for counting, pushing limits of detection down to ~102 cells per mL26. However, these are not available in most laboratories, and require sophisticated hardware as well as software that permit expert confirmation that observed particles are bacteria and not debris.
For reference, adults with sepsis usually begin showing symptoms at &#60;100 cells/mL of blood, and infants at &#60;10 cells/mL. A blood draw from an adult takes 10 mL, and from an infant, 1 mL. PCR-based methods are inhibited by the presence of human and non-pathogenic flora DNA and by PCR-inhibiting components in the blood27,28. Despite a variety of emerging techniques, cultures remain the gold standard for the diagnosis of bloodstream infections, especially in more rural areas or developing nations. For detection of life on other planets, thermodynamic calculations can estimate the energy budget for life and thus the expected possible biomass. 1 - 100 cells/mL are expected to be thermodynamically reasonable on Europa29. It can be readily seen from these numbers that detection of very small numbers of cells in large amounts of aqueous solution is an important unsolved problem.
In this paper, we demonstrate detection of Serratia marcescens and Shewanella oneidensis (wild-type and non-motile mutant) at concentrations of 105, 104, 103, and 100 cells/mL using an off-axis digital holographic microscope (DHM). The key advantage of DHM over traditional light microscopy is the simultaneous imaging of a thick sample volume at high resolution&#8212;in this implementation, the sample chamber was 0.8 mm thick. These sample chambers were constructed by the soft-lithography of polydimethylsiloxane (PDMS) from a precision-machined aluminum mold with a tolerance of &#177; 50 &#181;m. This represents an approximately 100-fold improvement in depth of field over high-power light microscopy. DHM also provides quantitative phase information, allowing for measurements of optical path length (product of refractive index and thickness). DHM and similar techniques have been used for monitoring bacterial and yeast cell cycle and calculation of bacterial dry mass30,31,32; scattering differences may even be used to differentiate bacterial strains33.
The instrument we use is custom-built specifically for use with microorganisms, as previously published34,35, and its design and construction are demonstrated and discussed. Aqueous solutions are continuously supplied to a 0.25 &#181;L volume sample chamber via syringe pump; the flow rate is determined by camera frame rate in order to ensure imaging of the entire sample volume. A statistical calculation predicts the number of sample volumes that must be imaged in order to detect a significant number of cells at a given concentration.
For cell-detection applications, reconstruction of the holograms into amplitude and phase images was not required; analysis was performed on the raw hologram. This saves significant computational resources and disk space: a 500 Mb hologram video will be 1 - 2 Tb when reconstructed. However, we do discuss reconstruction through depth of the sample to confirm that the holograms represent the desired species. An important feature of DHM is its ability to monitor both intensity and phase of the images. Organisms that are nearly transparent in intensity (such as most biological cells) appear clearly in phase. As it is a label-free technique, no dyes are used. This is an advantage for possible space flight applications, since dyes may not survive the conditions of a mission and&#8212;more importantly&#8212;cannot be assumed to work with extraterrestrial organisms, which may not use DNA or RNA for encoding. It is also an advantage for work in extreme environments such as the Arctic and Antarctic, where dyes may be difficult to bring to the remote location and may degrade upon storage. Reconstruction of images into phase and amplitude is performed using an open-source software package that we have made available on GitHub (SHAMPOO) or using ImageJ.

<table>
	<tbody>
		<tr>
			<td>Bacto Yeast</td>
			<td>BD Biosciences</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Tryptone</td>
			<td>BD Biosciences</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7710</td>
			<td>Many options for purchase</td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>BD Biosciences</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm Petri dishes</td>
			<td>VWR</td>
			<td>10053-704</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL culture tubes</td>
			<td>Falcon (Corning Life Sciences)</td>
			<td>352002</td>
			<td>Loose-capped</td>
		</tr>
		<tr>
			<td>Petroff-Hauser chamber</td>
			<td>Electron Microscopy Sciences</td>
			<td>3920</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL syringes</td>
			<td>BD Biosciences</td>
			<td>309604</td>
			<td>Luer-Lok tip not necessary</td>
		</tr>
		<tr>
			<td>Male Luer to 1/16&#8221; barbed fitting</td>
			<td>McMaster-Carr</td>
			<td>51525K291</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclavable 1/16&#8221; ID PVC tubing for flow</td>
			<td>Nalgene</td>
			<td>8000-0004</td>
			<td>Sold by length, purchase accordingly</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>PHD 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample Chamber</td>
			<td>Custom</td>
			<td>n/a</td>
			<td>See Materials Section</td>
		</tr>
		<tr>
			<td>Holographic Microscope</td>
			<td>Custom</td>
			<td>n/a</td>
			<td>See Wallace et al.</td>
		</tr>
		<tr>
			<td>Open-source software</td>
			<td>Custom</td>
			<td>https://github.com/bmorris3/shampoo</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantifying microorganisms at low concentrations using digital holographic microscopy (dhm)

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in medical diagnostics, and for life detection by robotic missions to other planets and moons of the solar system. Currently, sparse bacterial samples are counted by culture plating or epifluorescence microscopy. Culture plates require long incubation times (days to weeks), and epifluorescence microscopy requires extensive staining and concentration of the sample. Here, we demonstrate how to use off-axis digital holographic microscopy (DHM) to enumerate bacteria in very dilute cultures (100-104 cells/mL). First, the construction of the custom DHM is discussed, along with detailed instructions on building a low-cost instrument. The principles of holography are discussed, and a statistical model is used to estimate how long videos should be to detect cells, based on the optical performance characteristics of the instrument and the concentration of the bacterial solution (Table 2). Video detection of cells at 105, 104, 103, and 100 cells/mL is demonstrated in real time using un-reconstructed holograms. Reconstruction of amplitude and phase images is demonstrated using an open-source software package.

The results should indicate the ability to detect living and dead bacteria at very low levels by DHM. The number of bacteria counted should be consistent with the results obtained using the Petroff-Hauser counting chamber and plate counts. Standard statistical methods provide information about the accuracy of the different detection methods at various bacterial concentrations.
Figure 1 shows the Pet...

Watch this Scientific Journal Video about Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy DHM at JoVE.com

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy DHM

Digital holographic microscopy (DHM) is a volumetric technique that allows imaging samples 50-100X thicker than brightfield microscopy at comparable resolution, with focusing performed post-processing. Here DHM is used for identifying, counting, and tracking microorganisms at very low densities and compared with optical density measurements, plate count, and direct count.

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

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in ...

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.

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