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

Summary

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.

Abstract

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.

Introduction

Determination of accurate bacterial counts in very dilute samples is crucial in many applications: a few examples are water and food quality analysis^1,2,3; detection of pathogens in blood, cerebrospinal fluid, or sputum^4,5; production of pharmaceutical products, including sterile water⁶; and environmental community analysis in oligotrophic environments such as the open ocean and sediments^7,8,9. There i....

Protocol

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, Δ FlgM, is also used for comparison³⁷. All strains are grown in lysogeny broth (LB).

1. Prepare sterile LB medium (per liter of distilled water: 10 g bacto tryptone,....

Results

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.......

Discussion

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'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:

Materials

List of materials used in this article

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