Microbial Cell Quantification and Characterization

Quantifying microbial cells is challenging since microbes vary in size (often < 1 µm in diameter), shape (rod, sphere, etc.) and morphology (single cells, doublets, chains, clusters, etc.), and typically average more than one genome copy per cell. Additionally, microbial samples encompass an enormous range of complexity depending on the number of different microbes, acellular environmental particulates, and sample preparation or preservation. We are establishing standards, measurement methods, and RMs to enable stakeholders to assess performance and comparability across measurements and products.

Total Cell Count Measurements

Total or “absolute” microbial cell count is a fundamental measurement of microbial cell samples and facilitates accurate interpretation of other types of measurements (e.g., viability, genome copy number, genetic sequencing). NIST works to improve microbial cell counting in numerous ways, including:

• Advancing metrology for microbial cell counting with measurement quality metrics and international standards and methods development (BIPM-CAWG)
• Developing live, whole cell microbial Reference Materials (RMs) to meet demonstrated stakeholder needs – RM 8230 (Yeast), RM 8048 (Human Gut Microbiome)
• Leading stakeholder groups that bring together Government, Industry, and Academic partners to address shared challenges and develop consensus – NIST RMTM Consortium, IMMSA – Cell Enumeration and Viability Working Group
• Generating highly characterized microbial cell samples for a wide range of projects to facilitate interlaboratory studies and provide measurement assurance via inter-sample controls (e.g., “spike-ins,” process controls, etc.)

Genome Equivalent Count Measurements

Metagenomic sequencing is a critical technology applied across microbiology, including among Biosecurity and Biotechnology stakeholders. Likewise, digital polymerase chain reaction (dPCR) is often used to quantify, detect, or identify microbial cells. For molecular methods such as these, data interpretation often relies on the assumption that every microbial cell contains a single genome copy. However, microbial cells often contain two or more genome copies, such that a single cellular aggregate could have 10s of copies, and the correlation could change depending on the growth phase, nutrient conditions, and other environmental factors. NIST is developing tools to help stakeholders address these measurement biases, including:

• Assessing methods for genome equivalent count in intact microbial cells across a range of microbial cell types using fluorescence flow cytometry based on proof-of-concept studies in E. coli
• Evaluating orthogonal genome copy measurements (e.g., digital droplet PCR) to determine biases with a focus on the associated nucleic acid extraction method efficiency
• Developing live, whole cell microbial Reference Materials (RMs) with genome equivalent count values - RGTM 10199 (E. coli)

Viable Cell Count Measurements

For decades, CFU (colony forming unit) assays have been the definitive measurement for the viability of a microbial sample and have been written into regulations and technical standards including those related to food and pharmaceutical safety, antimicrobial susceptibility, and water quality. Yet, CFU measurements are imprecise, relatively slow, and difficult to correlate with viable cell count or nucleic acid-based measurements such as metagenomic sequencing. Newer viable cell count methods rely on a range of cell characteristics (e.g., heat generated, ATP production, membrane permeability) and measurement modalities (e.g., calorimetry, plate readers, flow cytometry) to paint a fuller picture of microbial cell health and activity. NIST supports cell viability measurements via:

• Developing complex microbial cell health characterizations based on per-cell measurements of metabolic activity, membrane permeability, and membrane potential
• Advancing metrology for population growth-based microbial cell count measurements (e.g., optical density, microcalorimetry) with new experimental and analysis frameworks
• Imaging fluorescence lifetime of membrane voltage probes (completed) as detailed in this publication 

Capabilities

NIST’s microbial cell enumeration and characterization activities are underpinned by advanced technical capabilities and microbiology expertise, including:

• Microbial flow cytometry
• Coulter counting
• Advanced imaging
• Digital droplet PCR (polymerase chain reaction)
• Next generation sequencing and metagenomics
• The NIST Microbial Strain Collection
• NIST LabCAS: Data Driven Science Architecture and Resource