Review Article Copyright © 2014 JABS CELL COUNTING: HOW

CELL COUNTING: HOW RELIABLE (ARE THESE METHODS?)

Franca Nneka Alaribe (PhD)

Biomedical Consulting Agency Via Pontisette 3, 44124 Ferrara, Italy Phone; +393292229258, Email; [email protected]

Received for Publication: July 24, 2014 Accepted: August 18, 2014

Abstract Cell counting is an inevitable step involved in experiments of many cell–based research studies. The technique allows standardization of cell concentration between samples with minimized error and variation in downstream results. Manual cell counting technique has been the most popular in use. Recently, technological improvement in cell imaging has lead to the manufacture of automated cell counters. The advancements in imaging technology, enabled by automated cell counters such as Countess™ and LUNA-FLTM, are improving experimental accuracy and reliability, with much less time and effort. This review explores different types of cell counting methods with more emphasis on the most recent counting method—the automated cell counters. The methodology, accuracy, precision, benefits and drawbacks of these methods are also highlighted.

Keywords: counting techniques, cell counting, reliability, mammalian cell, white blood cells.

INTRODUCTION Cell counting is an inevitable step involved in experiments Coulter Counter counted and measured only red blood of many cell–based research studies. Scientists are faced cells, however, with the enhancement in technology and with the problem of knowing the exact cell density in evolving instrumentation, it enabled clinicians to also many common procedures and assays. Cell counting count and measure white blood cells. enhances their work in the determination of viability in Recent studies have observed that modern cell analyzers microbial samples, error reduction, variation in down are multi-channeled and perform automated haematology streams results and standardization of cell concentration measurements simultaneously using different techniques. between samples. The procedures range from cell splitting The Cell-Dyn.RTM 3000 instrument, uses "impedance" in cell culture maintenance, to proliferation assays and measurements to count and size red blood cells (RBCs), quantitative experiments such as quantitative polymerase plateletcrit and platelets (PLTs),"absorption" measurements chain reaction (qPCR ) [1]. to for mean corpuscular haemoglobin (MCH). Furthmore, Furthermore, cell counting enhances microbiological "optical scatter" measurements to count and classify white methods in environmental samples. Despite limitations, blood cells (WBCs) and the five part differential are used account of viable bacteria provide information, on soil and [3,4]. water quality, environmental contamination, and Cells can be counted using methods such as haemocytometers, bioremediation [2] spectrophotometers, plate counting, flow cytometers, and To date, the haemocytometer is the most commonly used automated cell counters. The article reviews the types of cell counting method. Recently, advancements in imaging cell counting methods with more emphasis, on the most technology has facilitated the invention of the automated recent counting method namely, the automated cell cell counting techniques, which are capable of providing counters. The methodology, accuracy, precision, benefits less time and effort with improved experimental accuracy and drawbacks of these methods are highlighted. and reliability [3]. For example, previously, the original

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HAEMOCYTOMETERS/COUNTING CHAMBERS METHODS Haemocytometers, were the first method developed focused on an area of the counting chamber and the cells specifically for obtaining accurate cell counts. The are counted using a tally counter. It is always preferable to haemocytometer cell counting method uses a specially use a clean haemocytometer (70% ethanol) and lens. To crafted gridded-glass slide consisting of two chambers, determine the viability of the cells, trypan blue is added to each divided into nine 1 x 1 mm squares. A glass cover is the cell suspension, in the ratio of 1:1, before loading it to supported by the grid, and the cell suspension is loaded via the counting chamber (Fig.1) [5]. capillary action into the chamber. The microscope is then

Figure 1: Haemocytometer with counting chambers

Generally, cells are counted manually and are usually and the dilution factors are used. To determine cell placed over the grid occupying a volume of 0.1 μl for each viability, the number of dead and live cells, are separately 1 x 1 mm square. This process is usually repeated using determined. The dead cells are permeable with trypan blue four different 1 mm2 areas with an average result. To due to their damaged membranes hence the cells always determine the original concentration in cells/ml, the appear blue when viewed under the microscope [3, 6, 9, number of cells counted in a specific number of squares 10].

Table 1: Merits and Demerits of Haemocytometer Method Merits of Haemocytometers Method Demerits of Haemocytometers Method

In the haemocytometer technique, results can be obtained in Where there are many samples to process, the microscopy is labour minutes rather than days as required for plate counts, thereby intensive and can lead to operator fatigue. To date, new technology creating room for immediate judgments and decisions about the is gradually replaced with image analysis and flow cytometers. sample analysis.

It is a good method for samples full of debris and contains multiple Only capable of analyzing small number of cells (100-250 cells object shapes and sizes. can be counted) and statistically unreliable.

The instrument does not require as such trained operators/ Sometimes, there is no uniform distribution of cells which gives professionals as in the case of flow cytometers. imprecise results. Coefficients of Variation (CV) ranging from 30 – 110 %.

The glass haemocytometers, are subject to over-filling and with high error in cell counting. This leads to unreliable cell counts with a high standard deviation. At times, monocytes tend to be underestimated and the lymphocytes tend to be overestimated. The procedure is tedious and requires careful cleaning and handling of the haemocytometer. Validity of the practice has been questioned because of low specificity, accuracy, and precision of counts.

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SPECTROPHOTOMETER METHOD A cell counting technique seldom used for obtaining estimation because absorbance is liable to be affected by relative estimates of cell density. With the method, other variable components of cell suspensions [8]. increase in cell density leads to passage of less light through the cuvette. The action is possible due to the turbid PLATE COUNTING METHOD nature of cells. With the spectrometer, the optical density Cell plating is another common method of counting cells (OD) of a culture is proportional to the biomass in the cell in for example bacterial colonies. A sample to be counted suspension. The process generates an immediate result. is diluted in a solution rendering it harmless to the Using spectrophotometry for measuring the turbidity of microbe. The most used methods of plate counting are cultures (turbidometry) has its advantages and viable plate count and pour plate count techniques. This disadvantages [7]. enables the cells to be counted by plating the cells and Advantages: Some spectrophotometers are characterized streaking the diluted cells onto a plate. After sufficient with many curvette spaces in which several cuvettes can be time is allowed for growth, colonies are counted. Based on inserted, thereby further reducing work time. Additionally, the dilution and the known volume of suspension, the there are spectrophotometers that require extremely small density of the original suspension can be determined. volumes of culture, such as 1 u/l thereby reducing waste. Using a Pour plate technique involves mixing a portion of Disadvantage: The spectrophotometer’s drawback is its the dilution with molten agar, after which the mixture is limited accuracy. It is the only method in which the cells poured into a petri plate. According to the sample dilution, are not directly counted. The machine measures the individual cells are dropped on the agar to form colonies. amount of light rather than cells. Combined with the To determine the colony forming units (CFUs) on the stochastic nature of liquid cultures, the methodology plate, the total number of colony is counted (Fig 2). To enables only an estimation of cell numbers. Thus find the total number of CFUs in the original sample, the spectrophotometer seems to be unreliable in cell density total number of CFUs is multiplied by the total dilution of the solution [9].

-1 - 2 -3 Figure 2: Illustration of series of pour plates showing the appearance of viable plate count. The four plates show a 10 , 10 , 10 and 10-4 of a natural sample and the number of colony forming units which decreases 10 folds between the plates. No of bacterial/ml = number of colonies X dilution of sample.

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Table 2: Advantages and Disadvantages of Plate Count Methods Advantages of Plate counting Method Disadvantages of Pate counting Method The plate count is a popular method for determining cell number. Despite its widespread use, it cannot, be considered a universal The technique is sensitive and has the advantage of counting living approach as 95% of all cultivated and published species belong to 5 bacteria, often the important issue. of the 53 recognized bacterial phyla [1].

Concentration of microorganism can be easily counted, if the Microbial cells tend to exist in dormant states and cannot be able appropriate dilution is plated. to form colonies on nutrient media. However, the method may have other measureable activity, such as in disease or economic loss.

The equipment for performing plate counts is readily available and There is the assumption that “each colony arises from one cell” this is cheap in comparison to other methods. leads to gross underestimation of the true population in species, especially where cells grow together in clusters [9].

Use of a selective medium makes it possible to determine the This technique is time consuming thereby, delaying experiments or number of bacteria of a certain class, even in mixed populations. information. In plate count method, organisms with 1% visible Hence its application in food, research laboratories, medical and with the microscope are scored as viable. aquatic experiments for the usual cell number determination.

FLOW CYTOMETER METHOD Flow cytometry is another technology adopted to count energizing, RNA and/or DNA content, membrane fluorescently labeled cells. The technique uses a permeability [1, 3, 9]. fluorescence detection system. Flow cytometry technology Disadvantages: Notwithstanding the benefits of flow is basically made for passing cells singly through a sensing cytometric cell counting, the devices are expensive and are region of a flow cell. The cell counting and sizing is rarely used for general cell counting applications. The carried out by calculating the changes in the optical instrument requires professionals due to its complex and technique, Optical scattering of light (laser beam) by each complicated mechanical configuration. Additionally, for cell is measured [3]. acquisition of reliable data, constant maintenance is Flow cytometry has its origins in the analysis of required [1, 3, 9]. microorganisms. It has developed over the last 30 years as a technique primarily optimized for, and associated with, AUTOMATED CELL COUNTERS the analysis of clinical samples [10, 11, 12]. Previously, manual use of haemocytometers and counting Currently, specialized type of flow cytometry techniques chambers in blood counts were very common. Today such as the Fluorescence-activated cell sorting, are technological evolution and technological advancement available. These are based upon integrating certain has made it possible for the development of more substances known as “fluorophores” as labels into the sophisticated automated blood cell counters/analyzers in cells. Immunological analysis for cell antigens (eg research analysis. These automated counters has come a lymphocytes) is now possible by fluorescence flow long way in enhancing throughput, due to their multiple cytometry, using monoclonal antibodies [3, 9]. functions and high speed performance. Further studies have shown that only volumetric flow Almost two decades ago, a series of image-based cytometry can count the absolute number of cells in unit automated cell counters has been introduced to provide volumes of liquid to provide highly accurate cell numbers accurate cell number and viability data. Basically, these proved comparable to those obtained with cell counters use light microscopy and computer-based haemocytometers [13]. image analysis software. Instead of manual counting, Benefits/advantages: Despite the fact that Flow cytometers magnified cell images are automatically captured by a are most frequently used for more detailed cellular digital camera and transported to computer software. analysis, it is also capable of providing highly accurate cell Algorithms are used to accurately find cells and counts, and can discriminate cells based on factors, such as discriminate against non cellular objects such as cell debris protein expression, by using fluorescently labeled [13]. antibodies. Flow cytometers are very easy to use and Recently, these advancements in imaging technology has depend more on the experimental set-up. There is room for enabled inventions like the Countess™ Automated Cell measurement of higher cell concentrations than the manual Counter with the best promise for improving experimental method. Flow cytometry analyzes individual cells, which accuracy, reliability, and time-to-results, with much less means that their viability determination is assured. The time and effort[14]. viability measurements include but not limited to Unlike the haemocytometer, Countess™ Automated Cell measurement of metabolic activity, membrane Counter is a bench top instrument that does not involve

038 http://www.jabsdirect.com . Alaribe F. N. J. Advances in Biomedical Studies, 2014, 1 (2), 035-040 manual counting. The technique uses the standard trypan viewing and adjusting the image of the sample for optimal blue stain to determine cell viability. The digital image alignment and quickly acquire an image with the cell count capture, with a sophisticated analysis program, determine and viability results (Fig 3). There is also the possibility of the cell count, size and percentage viability of the cell the user to save the image or analyze it further using population. With its small microscope, digital camera and Countess™ PC Software, common spread sheet and other computer, the user can maneuver the instrument by image processing programs [14].

A B C Figure 3: The Bench Top Countess™ Automated Cell Counter A, Countess™ chamber slide; B, Countess™ instrument; C, Countess™ Image analyzer with sample images. (Image from Invitrogen Technical Note).

TheC, Countess™ use of the ImageCountess™ analyzer Automated with sample Cell images.Counter (Image reliance from Invitrogenon manual Technical differential Note). counts [3, 6, 15, 16]. involves three steps: Finally, the techniques are more affordable, easier to learn 1. Prepared cells are suspended to a concentration of than the flow-based system and essentially maintenance approximately 1 x 106 cells/ml, aliquots are mixed with free. 0.4% trypan blue and 10 μl is suspended using a pipette More sensitive and accurate cell counting techniques have into Countess™ chamber slide. been introduced to overcome the shortcomings of bright 2. The slide is inserted into the instrument. field image based cell counting. Cell counters such as 3. Press the “Count cells” button and results are displayed. LUNA-FLTM and others, which are equipped with a The image that appears on the screen represents the same fluorescence microscope module, and an image area as four 1 x 1 mm squares on a haemocytometer. analyzing/counting algorithm, has been developed. The Adjusting zoom and focusing knob produces the desired LUNA-FLTM is a cell counter based on sensitive and image with total readout of cells/ml, live cells/ml, dead accurate fluorescence staining methods to provide cost- cells/ml and % viability of cells [3, 6, 13, 14]. effective and reliable cell counting solutions. Advantages of Countess™ Automated Cell Counter: The latest development in the instrument is that, unlike the Due to the use of digital image capture and computer cytometry, the LUNA-FLTM captures and analyzes three analysis for counting, Countess™ Automated Cell Counter different images: bright field, green fluorescence, and red always show significantly higher effective concentration fluorescence. Within 30 seconds, total cell numbers, range than the haemocytometer. viability, cell size and histogram data are displayed on a As an important technological advance in automated cell touch screen LCD monitor. In summary, it is designed in a counting, Countess™ Automated Cell Counter readily small, all in one configuration, that it can be applied in provides the researcher with high-quality cell count data in space limited environments and are used to count cells less time and effort. It also eliminates the tediousness and without the limitation of the cell types [12, 13]. subjectivity of manual cell counting. With the method of instant image generation of samples, the Countess™ CONCLUSION instrument generates an image of the actual cells, Despite the successful use of mammalian cells with increasing the researcher’s confidence in the results. In popular dyes such as trypan blue and methylene for cell automated cell counter, there is no slide distribution error. s viability and bright field imaging techniques, most Many studies have suggested that laboratory efficiency mammalian cell counting still remain challenging. Cells could be improved by relying more on instruments such as like the peripheral blood mononuclear cells (PBMCs), the Countess™ Automated Cell Counter. Due to its yeast cells , sperm cells and primary cells are not easily capability to perform accurate count on leukocyte and counted with the bright field image analysis [14]. monocyte cells used in diagnosis of infection with less Additionally, the accuracy of counting chambers seem to fall short and subjective means of counting are poor in 039 http://www.jabsdirect.com . Alaribe F. N. J. Advances in Biomedical Studies, 2014, 1 (2), 035-040 discriminating between multiple types of cells in a 9. Microbiology Laboratories. Microbes in our world and suspension. The technique is accomplished when the size what they do. Test book for Microbiology, or shape differences between the various cell types are http://inst.bact.wisc.edu/inst/index.php. structurally distinguishable. 10. lvarez-Barrientos A, Arroyo A J, Canto´n R., Nombela Whilst on the other hand, flow cytometry has many C, Sa´nchez- Pe´rez M. Applications of flow cytometry to advantages and exciting opportunities for research, it has clinical microbiology. Clin. Microbiol. Rev. 2000; 13:167– not yet become as widely used as cell counting potential 195. deserves. Instruments seems to be extremely expensive and 11. Gucker FT, O’Konski C, Pickard HB, Pitts JN. A sometimes challenging in cell counting. Commercial photoelectric counter for colloidal particles. Am. J. Chem. instruments lack the sensitivity required for the analysis of 1947; 69:2422–2431. certain cells such as microbial cells. 12. Steen HB, Boye E. Bacterial-growth studied by flow With the proliferation of automated cell counters like cytometry. Cytometry 1980; 1:32–36. Countess™ and LUNA-FLTM these shortcomings are 13. Biosystem. Application note in flourescence cell addressed. Currently, these alternatives has demonstrated counting. www.logosbio.com success for all the mammalian cell types, The 14. Invitrogen technique note on Comparism of Image methodology has a high ability of producing or generating based cell counting method. www.invitrogen.com an image of the actual cells rather than relying on 15. Novak RW. The beleaguered band count. Clin Lab representation on a dot plot. The outcome generally tends Med. 1993;14:895-903. to increase the researcher’s confidence in results obtained. 16. Wenz B, Gennis P, Canova C, et al. The clinical utility However, despite the efficiency of automated cell counting of the leukocyte differential in emergency medicine. Am J analyzers the instrument is still not efficient in detecting Clin Pathol. 1986;86:298-303. abnormal WBCs. Therefore, the manual cell counting techniques and automated cell counters still attend brighter prospects especially when haematological malignancy or rare hereditary disease is suspected.

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