Cell counting

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Cell counting is any of various methods for the counting or similar quantification of cells in the life sciences, including medical diagnosis and treatment. It is an important subset of cytometry, with applications in research and clinical practice. For example, the complete blood count can help a physician to determine why a patient feels unwell and what to do to help. Cell counts within liquid media (such as blood, plasma, lymph, or laboratory rinsate) are usually expressed as a number of cells per unit of volume, thus expressing a concentration (for example, 5,000 cells per milliliter).

Contents

Uses

Numerous procedures in biology and medicine require the counting of cells. By the counting of cells in a known small volume, the concentration can be mediated. Examples of the need for cell counting include:

Manual cell counting

There are several methods for cell counting. Some are primitive and do not require special equipment, thus can be done in any biological laboratory, whereas others rely on sophisticated electronic appliances.

Counting chamber

A counting chamber Hemocytometer.jpg
A counting chamber

A counting chamber, is a microscope slide that is especially designed to enable cell counting. Hemocytometers and Sedgewick Rafter counting chambers are two types of counting chambers. The hemocytometer has two gridded chambers in its middle, which are covered with a special glass slide when counting. A drop of cell culture is placed in the space between the chamber and the glass cover, filling it via capillary action. [1] Looking at the sample under the microscope, the researcher uses the grid to manually count the number of cells in a certain area of known size. The separating distance between the chamber and the cover is predefined, thus the volume of the counted culture can be calculated and with it the concentration of cells. Cell viability can also be determined if viability dyes are added to the fluid.

Their advantage is being cheap and fast; this makes them the preferred counting method in fast biological experiments where it is only necessary to determine if a cell culture has grown as expected. Usually the culture examined needs to be diluted, otherwise the high density of cells would make counting impossible. The need for dilution is a disadvantage as every dilution adds inaccuracy to the measurement. [2]

Plating and CFU counting

A picture of Staphylococcus aureus colonies growing on an agar plate (photographed in transmitted light). Such homogeneously spread colonies are suitable for CFU enumeration. S.aureusAgar.jpg
A picture of Staphylococcus aureus colonies growing on an agar plate (photographed in transmitted light). Such homogeneously spread colonies are suitable for CFU enumeration.

To quantify the number of cells in a culture, the cells can be simply plated on a petri dish with growth medium. If the cells are efficiently distributed on the plate, it can be generally assumed that each cell will give rise to a single colony or Colony Forming Unit (CFU). The colonies can then be counted, and based on the known volume of culture that was spread on the plate, the cell concentration can be calculated. This is often carried out following the ASTM D5465 standard. [3]

As is with counting chambers, cultures usually need to be heavily diluted prior to plating; otherwise, instead of obtaining single colonies that can be counted, a so-called "lawn" will form: thousands of colonies lying over each other. Additionally, plating is the slowest method of all: most microorganisms need at least 12 hours to form visible colonies.

Although this method can be time-consuming, it gives an accurate estimate of the number of viable cells (because only they will be able to grow and form visible colonies). It is therefore extensively used in experiments aiming to quantify the number of cells resisting drugs or other external conditions (for instance the Luria–Delbrück experiment or the gentamicin protection assay). In addition, the enumeration of colonies on agar plates can be greatly facilitated by using colony counters.

Automated Cell Counting

Electrical resistance

The electrode of a Coulter counter Coultercounter.jpg
The electrode of a Coulter counter

A Coulter counter is an appliance that can count cells as well as measure their volume. It is based on the fact that cells show great electrical resistance; in other words, they conduct almost no electricity. In a Coulter counter the cells, swimming in a solution that conducts electricity, are sucked one by one into a tiny gap. Flanking the gap are two electrodes that conduct electricity. When no cell is in the gap, electricity flows unabated, but when a cell is sucked into the gap the current is resisted. The Coulter counter counts the number of such events and also measures the current (and hence the resistance), which directly correlates to the volume of the cell trapped. A similar system is the CASY cell counting technology.

Coulter and CASY counters are much cheaper than flow cytometers, and for applications that require cell numbers and sizes, such as cell-cycle research, they are the method of choice. Its advantage over the methods above is the large number of cells that can be processed in a short time, namely: thousands of cells per second. This offers great accuracy and statistical significance.

Flow cytometry

Flow cytometry is by far the most sophisticated and expensive method for cell counting. In a flow cytometer the cells flow in a narrow stream in front of a laser beam. The beam hits them one by one, and a light detector picks up the light that is reflected from the cells.

Flow cytometers have many other abilities, such as analyzing the shape of cells and their internal and external structures, as well as measuring the amount of specific proteins and other biochemicals in the cells. Therefore, flow cytometers are rarely purchased for the sole purpose of counting cells.[ citation needed ]

Image analysis

Recent approaches consider the use of high-quality microscopy images over which a statistical classification algorithm is used to perform automated cell detection and counting as an image analysis task. [4] Generally performs with a constant error rate as an off-line (batch) type process. A range of image classification techniques can be employed for this purpose. [5]

Stereologic cell counting

At present, stereologic cell counting with manual decision for object inclusion according to unbiased stereologic counting rules remains the only adequate method for unbiased cell quantification in histologic tissue sections, thus it's not adequate enough to be fully automated. [6]

Indirect Cell Counting

Spectrophotometry

A spectrophotometer Spektrofotometri.jpg
A spectrophotometer

Cell suspensions are turbid. Cells absorb and scatter the light. The higher the cell concentration, the higher the turbidity. Spectrophotometers can measure intensity of light very accurately. The cell culture is placed in a transparent cuvette and the absorption is measured relative to medium alone. Optical density (OD) is directly proportional to the biomass in the cell suspension in a given range that is specific to the cell type. Using spectrophotometry for measuring the turbidity of cultures is known as turbidometry.

This has made spectrophotometry the methods of choice for measurements of bacterial growth and related applications. Spectrophotometry's drawback is its inability to provide an absolute count or distinguish between living and dead cells.

Impedance microbiology

Impedance microbiology is a rapid microbiological technique used to measure the microbial concentration (mainly bacteria but also yeasts) of a sample by monitoring the electrical parameters of the growth medium. It is based on the fact that bacteria metabolism transforms uncharged (or weakly charged) compounds into highly charged compounds thus changing the growth medium electrical properties. The microbial concentration is estimated on the time required for the monitored electrical parameters to deviate from the initial baseline value.

Different instruments (either built in a laboratory or commercially available) to measure the bacterial concentration using Impedance Microbiology are available. [7] [8] [9] [10] [11]

Related Research Articles

<span class="mw-page-title-main">Complete blood count</span> Routine laboratory test of blood cells

A complete blood count (CBC), also known as a full blood count (FBC), is a set of medical laboratory tests that provide information about the cells in a person's blood. The CBC indicates the counts of white blood cells, red blood cells and platelets, the concentration of hemoglobin, and the hematocrit. The red blood cell indices, which indicate the average size and hemoglobin content of red blood cells, are also reported, and a white blood cell differential, which counts the different types of white blood cells, may be included.

<span class="mw-page-title-main">Agar plate</span> Petri dish with agar used to culture microbes

An agar plate is a Petri dish that contains a growth medium solidified with agar, used to culture microorganisms. Sometimes selective compounds are added to influence growth, such as antibiotics.

<span class="mw-page-title-main">Automated analyser</span>

An automated analyser is a medical laboratory instrument designed to measure different chemicals and other characteristics in a number of biological samples quickly, with minimal human assistance. These measured properties of blood and other fluids may be useful in the diagnosis of disease.

An assay is an investigative (analytic) procedure in laboratory medicine, mining, pharmacology, environmental biology and molecular biology for qualitatively assessing or quantitatively measuring the presence, amount, or functional activity of a target entity. The measured entity is often called the analyte, the measurand, or the target of the assay. The analyte can be a drug, biochemical substance, chemical element or compound, or cell in an organism or organic sample. An assay usually aims to measure an analyte's intensive property and express it in the relevant measurement unit.

<span class="mw-page-title-main">Flow cytometry</span> Lab technique in biology and chemistry

Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles.

<span class="mw-page-title-main">Hemocytometer</span> Medical device used to count cells

The hemocytometer is a counting-chamber device originally designed and usually used for counting blood cells.

<span class="mw-page-title-main">Microbiological culture</span> Method of allowing microorganisms to multiply in a controlled medium

A microbiological culture, or microbial culture, is a method of multiplying microbial organisms by letting them reproduce in predetermined culture medium under controlled laboratory conditions. Microbial cultures are foundational and basic diagnostic methods used as a research tool in molecular biology.

<span class="mw-page-title-main">Bacteriological water analysis</span>

Bacteriological water analysis is a method of analysing water to estimate the numbers of bacteria present and, if needed, to find out what sort of bacteria they are. It represents one aspect of water quality. It is a microbiological analytical procedure which uses samples of water and from these samples determines the concentration of bacteria. It is then possible to draw inferences about the suitability of the water for use from these concentrations. This process is used, for example, to routinely confirm that water is safe for human consumption or that bathing and recreational waters are safe to use.

<span class="mw-page-title-main">Viral plaque</span>

A viral plaque is a visible structure formed after introducing a viral sample to a cell culture grown on some nutrient medium. The virus will replicate and spread, generating regions of cell destruction known as plaques. For example, Vero cell or other tissue cultures may be used to investigate an influenza virus or coronavirus, while various bacterial cultures would be used for bacteriophages.

<span class="mw-page-title-main">Coulter counter</span> Device to count and size particles

A Coulter counter is an apparatus for counting and sizing particles suspended in electrolytes. The Coulter counter is the commercial term for the technique known as resistive pulse sensing or electrical zone sensing. The apparatus is based on the Coulter principle named after its inventor, Wallace H. Coulter.

In microbiology, colony-forming unit is a unit which estimates the number of microbial cells in a sample that are viable, able to multiply via binary fission under the controlled conditions. Counting with colony-forming units requires culturing the microbes and counts only viable cells, in contrast with microscopic examination which counts all cells, living or dead. The visual appearance of a colony in a cell culture requires significant growth, and when counting colonies, it is uncertain if the colony arose from one cell or a group of cells. Expressing results as colony-forming units reflects this uncertainty.

<span class="mw-page-title-main">Streaking (microbiology)</span> Method for isolation of bacterial strains

In microbiology, streaking is a technique used to isolate a pure strain from a single species of microorganism, often bacteria. Samples can then be taken from the resulting colonies and a microbiological culture can be grown on a new plate so that the organism can be identified, studied, or tested.

Plate Count Agar (PCA), also called Standard Methods Agar (SMA), is a microbiological growth medium commonly used to assess or to monitor "total" or viable bacterial growth of a sample. PCA is not a selective medium.

CASY technology is an electric field multi-channel cell counting system. It was first marketed by Schärfe System GmbH in 1987 under the name CASY1. The first systems were sold with an ATARI computer and a rectangular chassis. In the 1990s the ATARI computer got replaced by a common PC and the chassis changed into cylinders. In 2006, Schärfe System was acquired by Innovatis AG, a company focused on cell culture analysis. CASY utilizes the techniques of electric current exclusion and pulse area analysis, the cells can be analyzed and counted in an efficient and precise manner. This technology can be applied for cell counting, cell culture analysis at a certain time interval, or even a period of time.

Virus quantification involves counting the number of viruses in a specific volume to determine the virus concentration. It is used in both research and development (R&D) in commercial and academic laboratories as well as production situations where the quantity of virus at various steps is an important variable. For example, the production of viral vaccines, recombinant proteins using viral vectors and viral antigens all require virus quantification to continually adapt and monitor the process in order to optimize production yields and respond to ever changing demands and applications. Examples of specific instances where known viruses need to be quantified include clone screening, multiplicity of infection (MOI) optimization and adaptation of methods to cell culture. This page discusses various techniques currently used to quantify viruses in liquid samples. These methods are separated into two categories, traditional vs. modern methods. Traditional methods are industry-standard methods that have been used for decades but are generally slow and labor-intensive. Modern methods are relatively new commercially available products and kits that greatly reduce quantification time. This is not meant to be an exhaustive review of all potential methods, but rather a representative cross-section of traditional methods and new, commercially available methods. While other published methods may exist for virus quantification, non-commercial methods are not discussed here.

<span class="mw-page-title-main">Cytometry</span> Measurement of number and characteristics of cells

Cytometry is the measurement of number and characteristics of cells. Variables that can be measured by cytometric methods include cell size, cell count, cell morphology, cell cycle phase, DNA content, and the existence or absence of specific proteins on the cell surface or in the cytoplasm. Cytometry is used to characterize and count blood cells in common blood tests such as the complete blood count. In a similar fashion, cytometry is also used in cell biology research and in medical diagnostics to characterize cells in a wide range of applications associated with diseases such as cancer and AIDS.

Virtual colony count (VCC) is a kinetic, 96-well microbiological assay originally developed to measure the activity of defensins. It has since been applied to other antimicrobial peptides including LL-37. It utilizes a method of enumerating bacteria called quantitative growth kinetics, which compares the time taken for a bacterial batch culture to reach a threshold optical density with that of a series of calibration curves. The name VCC has also been used to describe the application of quantitative growth kinetics to enumerate bacteria in cell culture infection models. Antimicrobial susceptibility testing (AST) can be done on 96-well plates by diluting the antimicrobial agent at varying concentrations in broth inoculated with bacteria and measuring the minimum inhibitory concentration that results in no growth. However, these methods cannot be used to study some membrane-active antimicrobial peptides, which are inhibited by the broth itself. The virtual colony count procedure takes advantage of this fact by first exposing bacterial cells to the active antimicrobial agent in a low-salt buffer for two hours, then simultaneously inhibiting antimicrobial activity and inducing exponential growth by adding broth. The growth kinetics of surviving cells can then be monitored using a temperature-controlled plate reader. The time taken for each growth curve to reach a threshold change in optical density is then converted into virtual survival values, which serve as a measure of antimicrobial activity.

Impedance microbiology is a microbiological technique used to measure the microbial number density of a sample by monitoring the electrical parameters of the growth medium. The ability of microbial metabolism to change the electrical conductivity of the growth medium was discovered by Stewart and further studied by other scientists such as Oker-Blom, Parson and Allison in the first half of 20th century. However, it was only in the late 1970s that, thanks to computer-controlled systems used to monitor impedance, the technique showed its full potential, as discussed in the works of Fistenberg-Eden & Eden, Ur & Brown and Cady.

Bacterioplankton counting is the estimation of the abundance of bacterioplankton in a specific body of water, which is useful information to marine microbiologists. Various counting methodologies have been developed over the years to determine the number present in the water being observed. Methods used for counting bacterioplankton include epifluorescence microscopy, flow cytometry, measures of productivity through frequency of dividing cells (FDC), thymidine incorporation, and leucine incorporation.

Hematology analyzers are used to count and identify blood cells at high speed with accuracy. During the 1950s, laboratory technicians counted each individual blood cell underneath a microscope. Tedious and inconsistent, this was replaced with the first, very basic hematology analyzer, engineered by Wallace H. Coulter. The early hematology analyzers relied on Coulter's Principle. However, they have evolved to encompass numerous techniques.

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