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6.8.2: Viable Cell Counting - Biology

6.8.2: Viable Cell Counting - Biology


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Plate counting is used to estimate the number of viable cells that are present in a sample.

LEARNING OBJECTIVES

Explain viable cell counting

Key Points

  • The spread plate relies on bacteria growing a colony on a nutrient medium so that the colony becomes visible to the naked eye and the number of colonies on a plate can be counted.
  • Selective media can be used to restrict the growth of non-target bacteria.
  • The pour plate method is used when the analysis is looking for bacterial species that grow poorly in air, for example water samples.

Key Terms

  • plate count: A means to identify the number of actively growing cells in a sample.

Viable Cell Counting

There are a variety of ways to enumerate the number of bacteria in a sample. A viable cell count allows one to identify the number of actively growing/dividing cells in a sample. The plate count method or spread plate relies on bacteria growing a colony on a nutrient medium. The colony becomes visible to the naked eye and the number of colonies on a plate can be counted. To be effective, the dilution of the original sample must be arranged so that on average between 30 and 300 colonies of the target bacterium are grown. Fewer than 30 colonies makes the interpretation statistically unsound and greater than 300 colonies often results in overlapping colonies and imprecision in the count. To ensure that an appropriate number of colonies will be generated several dilutions are normally cultured. The laboratory procedure involves making serial dilutions of the sample (1:10, 1:100, 1:1000 etc. ) in sterile water and cultivating these on nutrient agar in a dish that is sealed and incubated. Typical media include Plate count agar for a general count or MacConkey agar to count gram-negative bacteria such as E. coli. Typically one set of plates is incubated at 22°C and for 24 hours and a second set at 37°C for 24 hours. The composition of the nutrient usually includes reagents that resist the growth of non-target organisms and make the target organism easily identified, often by a color change in the medium. Some recent methods include a fluorescent agent so that counting of the colonies can be automated. At the end of the incubation period the colonies are counted by eye, a procedure that takes a few moments and does not require a microscope as the colonies are typically a few millimeters across.

The pour plate method is used when the analysis is looking for bacterial species that grow poorly in air. The initial analysis is done by mixing serial dilutions of the sample in liquid nutrient agar which is then poured into bottles. The bottles are then sealed and laid on their sides to produce a sloping agar surface. Colonies that develop in the body of the medium can be counted by eye after incubation. The total number of colonies is referred to as the Total Viable Count (TVC). The unit of measurement is cfu/ml (or colony forming units per milliliter) and relates to the original sample. Calculation of this is a multiple of the counted number of colonies multiplied by the dilution used. Examples of a viable cell count are spread plates from a serial dilution of a liquid culture and pour plates. With a spread plate one makes serial dilutions in liquid media and then spreads a known volume from the last tube in the dilution series. The colonies on the plate can then be counted and the concentration of bacteria in the original culture can be calculated. In the pour plate method a diluted bacterial sample is mixed with melted agar and then that mixture is poured into a petri dish. Again the colonies would be counted and the viable cell count calculated.


Techniques for Oral Microbiology

2.4.1.2 Methods

Turbidimetry and viable count methods are commonly used to determine the growth curve.

Turbidimetry: After inoculation, measure the optical density (OD) of the cell culture during cultivation. Graph the growth curve using the OD value as the Y-axis and the cultivation time as the X-axis.

Viable count method: In microbial ecology research, the number of viable cells reflects dynamic changes in bacterial growth. Generally, the cells are plated and colonies are counted (more details in “methods for measuring colony forming units” below) to measure the number of viable cells, which are the only cells in the culture that can undergo cell division and reproduce. After inoculation and a certain period of cultivation, inoculate a certain volume of the cell culture onto the agar plate. Graph the growth curve using the logarithm of the number of colonies as the Y-axis and the growth time as the X-axis.


Cell Biology

In cell biology research, controlling cell counting procedures at the earliest stage encourages reproducible results. Using an automated cell counter is recommended to ensure equal numbers of cells are used in downstream analysis, and to determine cell concentration in an accurate and precise manner.

Solutions

In cell biology research, whether you need cell count and viability, or to perform advanced fluorescence-based cell analysis with high reproducibility, we have a solution to suit your needs. Our instruments are automated fluorescence image cytometers designed for ease of use, precision and accuracy.

Obtaining high-quality research depends on your data’s reproducibility and consistency. NucleoCounter ® instruments perform a wide range of functions, from precise cell count and viability determinations to advanced cell sample analysis, with minimal user interference.

Plug-and-play assays include five different apoptosis assays, a cell cycle analysis and a GFP-transfection assay. In addition, a platform for user-definable assays is also available. Our instruments are designed for dedicated cell biology research and development laboratories.

Unbiased Cell Count & Viability Determinations

Manual cell counting using a hemocytometer and trypan blue exclusion method is time-consuming and depends heavily on the user’s perception and pipetting skills.

To determine cell count and viability using the NucleoCounter ® NC-202™ and NC-3000™ instruments, first load the cell suspension into the Via2-Cassette™ or Via1-Cassette™, respectively. Cells are automatically stained by the two fluorophores in the cassette, acridine orange and DAPI, staining total cells and dead cells, respectively. The volume of the counting chamber is pre-calibrated, ensuring high-precision and reproducibility.

High-speed Cell Viability & Counts

The multi-chamber NC-Slide A8™ enables high-speed viability and cell count determinations of insect and mammalian cells, measuring up to eight samples in less than three minutes. Mix the cell suspension with acridine orange and DAPI to stain all cells and the dead cells, respectively. Then, load the A8-slide™ chambers with the sample and insert the slide into the NucleoCounter ® NC-3000™.

We offer optimized counting protocols for aggregated cells, cells growing on microcarriers and in spheroids. Read more about cell count and viability using the NC-Slide A8™ for mammalian cells.

Superior Data Visuals & Analysis

The NucleoView™ software is the user interface for the NucleoCounter ® NC-200™ and NC-3000™. It handles data acquisition and presentation, along with image analysis. NucleoView™ includes a variety of data handling features that are perfectly suited to regulated environments.

The software enables you to visually inspect the fluorescence image and gives you the opportunity to verify the counting. You can select specific event populations in the scatter plots and examine and determine the validity of their inclusion or exclusion from the final counting results. Gating can be adjusted and you can save this adapted protocol for future measurements.

Counting Aggregated Cells

The NucleoCounter ® NC-202™ (and NC-200™) includes a wide range of specialized assays making it the perfect cell counter. Sphere cultures or highly aggregated cell types pose great challenges in cell counting, but with our specialized assays, these instruments can count even the most aggregated cell culture samples.

Standardized Apoptosis Assays

For advanced research into cell death, it is essential to study the mechanisms of apoptosis. Using the NucleoCounter ® NC-3000™, a series of five plug-and-play assays including Annexin V, mitochondrial potential with JC-1, caspase signaling, DNA fragmentation and the unique one-minute vitality assay, enabling you to fully investigate cell death mechanisms.

APOPTOSIS ASSAY PHYSIOLOGICAL CHANGES DETECTED STAGE
Mitochondrial potential Assay (JC-1) Collapse of the mitochondrial membrane potential Early
Annexin V Assay Collapse of plasma membrane lipid asymmetry Early-mid
Caspase Assay Caspase activation signals downstream apoptotic events Early-mid
Vitality (VB48™) Assay Decrease in cellular levels of reduced thiols e.g. GSH Late
DNA Fragmentation Assay Break-down and fragmentation of DNA Late

Covering early to late stages of apoptosis, these assays help you to carry out in-depth studies of apoptosis progression in mammalian cells. You can perform advanced data analysis effortlessly with NucleoView™. The platform presents links between images, histograms and scatter plots, giving you access to detailed and precise data analysis that identifies percentages of apoptotic, necrotic and living cells.

Monitoring Viability & GFP Transfection Efficiency

When transfecting cells with genes of interest, one way to determine the transfection efficiency is to co-express a fluorescent protein, such as green fluorescent protein (GFP) under the same promoter and determine the amount of GFP.

The NucleoCounter ® NC-3000™ offers a fast and easy to use assay to test GFP transfection efficiency and expression levels. By staining cells with Hoechst 33342 and propidium iodide (PI), you can define the total cell population and the dead cell population together with the population expressing GFP.

Cell staining in NucleoView™ software using GFP transfection efficiency assay with the NucleoCounter ® NC-3000™

A – Cells are located using Hoechst 33342 (blue) and the percentage of GFP expressing cells (green) can easily be determined. Non-viable cells are stained with propidium iodide (PI red).
B – All cells stained with Hoechst 33342 (blue) are identified.
C – GFP expressing cells are identified in green and non-viable cells in red by PI staining.

Easy coupling between the image obtained and the scatter plots or histograms allow you to determine the precise gate settings used to investigate the staining pattern for specific cell populations. The NucleoCounter ® NC-3000™ offers an all-in-one platform for evaluating GFP expression efficiency.

Non-viable cells stained with PI can be easily located with the image overlay function in the NucleoView™ software (A). GFP-transfected cells can be immediately identified (B).

Fast Cell Cycle Analysis

Investigating the impact of a treatment on cell division is one of the most powerful tools within cell biology. The NucleoCounter ® NC-3000™ provides fast and easy cell cycle analysis in under five minutes.

After adding a lysis buffer, all cell nuclei are stained, and the sample is measured using the NC-3000™. A cell cycle profile displays in the accompanying Plot Manager in the NucleoView™ software. Events in the sub-G1-phase, G0/G1-phase, S-phase and G2/M-phase are identified.

With the FlexiCyte™ software package, the NucleoCounter ® NC-3000™ can even be used to study cell proliferation with BrdU and EdU incorporation, detected with fluorescently labeled antibodies allowing for advanced studies of cell proliferation.

Illustration: Two-step cell cycle assay of untreated and camptothecin-treated (CPT) Jurkat cells. The histograms display intensity of the DNA-stain DAPI and can be used to define cell cycle events in the sub-G1-phase, G0/G1-phase, S-phase and G2/M-phase. After CPT treatment, the cell cycle is arrested in the G2/M-phase.

Advanced Cell Analysis

The FlexiCyte™ module available for the NucleoCounter ® NC-3000™ enables users to perform detailed advanced cell analysis of their own choice in mammalian and insect cells. The combination of LEDs, from UV to far red, together with a carefully chosen set of emission filters, allows you to detect a broad range of fluorescent antibodies and proteins.

The Protocol Adaptation Wizard feature guides the user through a selection of optimal settings. After image acquisition, in the Plot Manager, cell data is presented beside the fluorescent image as either scatter plots, histograms, or both. By linking images with plots, you are armed with everything you need to perform detailed data analyses.

The FlexiCyte™ software package enables detailed biomarker analysis of a broad range of fluorescent antibodies and proteins.


Procedure

    Obtain a uniform suspension of cells: Follow the typsinization/trypsin neutralization protocol for the specific cell type. Place the cell suspension in a suitably-sized conical centrifuge tube. For an accurate cell count to be obtained, a uniform suspension containing single cells is necessary. Pipette the cell suspension up and down in the tube 5-7 times using a pipette with a small bore (5 ml or 10 ml pipette). For cells thawed from cryopreservation (in 1ml cryopreservation medium), pipette up and down 7-10 times using a one ml pipette.

For an accurate determination, the total number of cells overlying one 1 mm 2 should be between 15 and 50. If the number of cells per 1 mm 2 exceeds 50, dilute the sample and count again. If the number of cells per 1 mm 2 is less than 15, use a less diluted sample. If less dilute samples are not available, count cells on both sides of the hemocytometer (8 x 1 mm 2 areas).

Keep a separate count of viable and non-viable cells. If more than 25% of cells are non-viable, the culture is not being maintained on the appropriate amount of media. Reincubate the culture and adjust the volume of media according to the confluency of the cells and the appearance of the media. Include cells on top and left touching middle line. The cells touching middle line at bottom and right are not counted.

i. Trypan Blue is the "vital stain" excluded from live cells.
ii. Live cells appear colourless and bright (refractile) under phase contrast.
iii. Dead cells stain blue and are non-refractile.

  • %Cell Viability = [Total Viable cells (Unstained) / Total cells (Viable +Dead)] X 100.
  • Viable Cells/ml = Average viable cell count per square x Dilution Factor x 10 4 /
  • Average viable cell count per square = Total number of viable cells in 4 squares / 4.
  • Dilution Factor = Total Volume (Volume of sample + Volume of diluting liquid) / Volume of sample.
  • Total viable cells/Sample = Viable Cells/ml x The original volume of fluid from which the cell sample was removed.
  • Volume of media needed = (Number of cells needed/Total number of viable cells) x 1000.

  • Personal protective equipment (sterile gloves, laboratory coat, safety visor)
  • Waterbath set to appropriate temperature
  • Microbiological safety cabinet at appropriate containment level
  • Centrifuge
  • CO2 incubator
  • Hemocytometer
  • Inverted phase contrast microscope
  • Pre-labelled flasks
  1. Bring adherent and semi-adherent cells into suspension using trypsin/EDTA as described previously and resuspend in a volume of fresh medium at least equivalent to the volume of trypsin. For cells that grow in clumps centrifuge and resuspend in a small volume and gently pipette to break up clumps.
  2. Under sterile conditions remove 100-200 μL of cell suspension.
  3. Add an equal volume of Trypan Blue (dilution factor =2) and mix by gentle pipetting.
  4. Clean the hemocytometer.
  5. Moisten the coverslip with water or exhaled breath. Slide the coverslip over the chamber back and forth using slight pressure until Newton’s refraction rings appear (Newton’s refraction rings are seen as rainbow-like rings under the coverslip).
  6. Fill both sides of the chamber with cell suspension (approximately 5-10 μL) and view under an inverted phase contrast microscope using x20 magnification.
  7. Count the number of viable (seen as bright cells) and non-viable cells (stained blue). Ideally >100 cells should be counted in order to increase the accuracy of the cell count (see notes below). Note the number of squares counted to obtain your count of >100.
  8. Calculate the concentration of viable and non-viable cells and the percentage of viable cells using the equations below.

Microscopy for the Winery

There are essentials tasks that every winemaker should be able to do with a microscope. First you should be able to distinguish between bacteria, yeast, and fungi under the microscope. Second you should be able to tell living organisms from debris (plant cells, crystals, filter and fining agents, etc.). Third you should be able to identify the most common and easily identifiable organisms by sight so that you can tell when you are heading for a problem. With some experience you will know enough to tell that you don’t know what is growing in the wine and take action or get help quickly. There are images of the most common of the wine yeast, bacteria, and mold with their descriptions on this web site. Lastly you should be able to count yeast cells and distinguish between live and dead cells. These are some of the goals we strive for in the Wine Microbiology lab class taught at UC Davis.

Types of microscopy: There are different types of microscopes available ranging in price from a few hundred dollars to many thousands of dollars. Typically for a winery a good quality optical scope is desirable, one with phase contrast is helpful when looking at bacteria. Most microscopes have 10x oculars and objectives from 10x to 100x. This will give you a range of magnification from 100x to 1000x. Sometimes you can get oculars that are 15x or 20x that can increase your magnification without having to use a 100x objective. Any 100x objective requires oil (oil immersion) to decrease the refraction from the light traveling through air between the sample and the objective. This effectively increases the resolution of objects under the microscope. A 20x ocular with a 40x objective will give you a total magnification of 800x without the use of oil immersion. However the resolution is not as good. Besides objectives at 10x, 40x, and 100x some scopes may have 4x, 16x, or 20x objectives as well. The figure below shows Saccharomyces cerevisiae visualized at different magnifications (100x, 400x, 1000x).

Saccharomyces cerevisiae

100X 400X 1000X

There are now very inexpensive digital microscopes on the market that replace the ocular with a digital screen similar to what you find in digital cameras. The objectives are the same as a standard microscope but some people find it easier to view the screen than look through the oculars. The digital scope allows for a digital zoom of up to 4x but no optical zoom. The digital zoom can result in pixilation of the image. As with any microscope the quality of the optics determines the quality of the image and most digital microscopes do not have the best quality optics. The best optical microscopes now have a third optical port for the insertion of a digital camera lens. This allows images to be viewed on a computer screen. Use of a digital camera makes it easy to share images with colleagues and some users find the screen easier to use than the traditional ocular.

There are also different types of microscopy for specific purposes. The one you are most familiar with is probably bright field microscopy, which uses light to illuminate an object that absorbs light in denser areas to give contrast for visualization. Dark field microscopy excludes the non-scattered light from the field of view leaving the background dark and the object in the field of view light. Fluorescent microscopy uses a mercury lamp and a filter to illuminate the viewing field with light of a specific wavelength. A second filter filters out that wavelength but allows the light emitted from an excited molecule to pass through the ocular and be visualized. Many biological specimens, especially photosynthetic ones, have a great deal of natural fluorescence, but more typically samples are stained or genetically tagged with fluorescent dyes or labels to allow visualization of structures or specific compounds in cells. Phase contrast and differential interference contrast (DIC) microscopy are techniques that are used to enhance the contrast of low contrast objects especially in unfixed specimens. Use of a phase contrast or DIC microscope for viewing bacteria makes it far easier for an inexperienced user to visualize small, low contrast bacteria. In phase contrast microscopy phase shifts in light are converted to brightness in the field of view allowing normally transparent structures to be seen. DIC works in a similar fashion but uses interferometry rather than phase shifts to allow visualization of invisible structures in cells. Phase contrast is more common and less expensive than DIC but DIC visualized samples have less light halos than phase contrast samples. Below are photographs of Oenococcus oeniusing bright field at 400x, phase contrast, and DIC at 1000x.

Oenococcus oeni

400X Phase Contrast 1000X DIC 1000X

Differentiating yeast, bacteria, and mold: The easiest way to differentiate bacteria, yeast (single celled fungi), and mold (filamentous fungi) is generally by size. Molds are easy to see at 100x magnification, yeast at 400x magnification, and bacteria are usually hard to see unless you go to 1000x magnification. However comparing the size of these organisms can be difficult without a reference. It is often easier if you mix cultures on a single microscope slide or if they are already mixed in a fermentation sample. But there are some commonly seen organisms in the winery that are not as easy to distinguish. Below is a mixture of the common soil bacterium Bacillus megaterium and a Lactobacillus from a wine sample. Bacillus megateriumis the most common non-wine Bacillus that we find in California wines and as its name implies it is a very large bacterium.

Bacillus megaterium and Lactobacillus sp. 1000X magnification

While bacteria are usually much smaller than yeast, Bacillus megaterium is much larger than most bacteria. In the image below the B. megaterium is seen in comparison to Saccharomyces cerevisiae. Even though the sizes are similar, the bacteria are less refractile, more transparent and difficult to see. This is also typical of bacteria versus yeast. The second image is of a Schizosaccharomyces yeast which divides by fission rather then budding. Because bacteria also divide by fission, a large bacterium, like B. megaterium, could be mistaken for a fission yeast. In the case below both images are at the same magnification and you can see that the B. megaterium is smaller than the yeasts.

S. cerevisiae and B. megaterium Schizosaccharomyces pombe 1000X

Yeasts are also smaller then molds, which are filamentous, whereas yeasts are single celled. Usually this makes them easy to tell apart but there are exceptions. Most importantly in a wine environment are yeasts, such as Brettanomyces bruxellensis, that form pseudohyphae. Below is a direct comparison of pseudohyphae from Brettanomyces and hyphae fromBotrytis cinerea. The first panel is Brettanomyces at 1000x magnification and the second is Botrytis at 100x magnification. TheBotrytis is about 10 times as big as the Brettanomyces.

Brettanomyces bruxellensis 1000x Botrytis cinerea 100x

Another instance where it can be difficult to determine if you are seeing yeast or mold is when the mold has formed spores and you see loose spores. Below are some photos of yeast and mold spores for comparison.

Metschnikowia pulcherrima - 400X Aspergillus niger
Saccharomyces bayanus at 1000x Botrytis cinerea spores at 400x maginfication
Candida intermedia at 1000x Fusarium spores at 400x magnification

Counting viable and non-viable yeast: (See also “Cell Counting – Total and Viable”) A microscope and a few simple tools can be used to give you an idea of the cell number and viability in your Saccharomyces inoculum. A simple counting chamber used to count blood cells or sperm can be used to determine the number of cells in a given volume and methylene blue dye can be used to determine the percent viability of an inoculum. Below is a photograph of the grid as you would see it under the microscope.

The image above is at 100x magnification and the cells shown are yeast cells. The square bounded by the 3 bright lines is 0.04 mm square, 25 of them will be 0.1 mm square or 0.1 ml in volume. Count enough squares to give you about 100 cells and then divide the number of cells by the number of squares counted, multiply by 25 squares in 0.1 ml and again by 10 to give you the number of cells in 1 milliliter. You must also take into account any dilution that you made of your inoculum by multiplying by the dilution factor to give you the number of cells in your original sample.

Methylene blue can be used to estimate the percent viability of the cells in your inoculum. You can purchase a methylene blue solution at the correct concentration to do your count. Prepare a microscope slide with 5 ul of dye and 5ul of your solution. Count 100 cells keeping track of how many are blue and how many are clear. The blue cells are are not viable, they cannot pump the dye back out after it penetrates their cell wall, and the clear cells are viable. The time the cells are in the dye is important. Try to count the cells at about 5 minutes after mixing the dye and the cell suspension. If you count to soon the dye may not have entered all the cells. If you wait too long some of the viable cells may no longer be able to pump the dye out and may become non-viable. The image below shows the same field of view at 2, 5 and 10 minutes. (See also “Methylene Blue Staining”)

2 minutes 5 minutes 10 minutes

Distinguishing living cells from debri: The biggest clue to what is a biological organisms and what isn’t is symmetry. Debris tends to by asymmetric while living organisms are symetric. Of course many things that you will see while looking under the microscope are symetrical and not living, such as air bubbles, crystals, and dead cells. Crystals are easily identified by their geometric shape and sharp angles. Bubbles can be more difficult because they are rounded but they lack any internal structure and can be any size. They also tend to grow as the slide dries out. Dead cells can be harder or impossible to distinguish from live ones. Much of the debris in must is plant cells and will have cells that are joined together but ragged at the edges. Other particles you will see are often things that have been added to the wine such as fining or filtering agents. Some of these agents were once living and may be mistaken for living cells, such as the fossilized diatoms that make up diatomacious earth (DE). Below are some photographs of things you might see in fermentations and wine that are not alive.


Total cell count enumerates all living and dead microbial cells in a sample. In contrast, viable cell count enumerates only the living cells in a sample. So, this is the key difference between total cell count and viable cell count. Total cell count is independent of the growth of colonies on agar plates while viable cell count is a growth-based technique and it depends on the growth of microbial colonies on agar plates.

The below info-graphic summarizes the difference between total cell count and viable cell count.


Introduction to Cell Viability Assays

Cell viability assays use a variety of markers as indicators of metabolically active (living) cells. Examples of markers commonly used include measuring ATP levels, measuring the ability to reduce a substrate, and detecting enzymatic/protease activities unique to living cells.

Real-time Cell Viability Assays

The RealTime-Glo&trade MT Cell Viability Assay (Cat.# G9711) measures cell viability in real-time. In this assay, an engineered luciferase and a prosubstrate (which is not a substrate of luciferase) are added directly to the culture medium. The prosubstrate can penetrate cell membranes and enter cells (Figure 1). However, only viable cells with active metabolism can reduce the prosubstrate into a substrate for luciferase. The substrate then exits the cell where it is used by luciferase in the detection reagent to generate a luminescent signal. The same wells can be measured repeatedly for 3 days. The main advantages of this method are that it allows simple kinetic monitoring to determine dose response using fewer plates and cells. Also, because the method does not require cell lysis, the same cells can be used in additional cell-based assays or downstream applications.

Figure 1. RealTime-Glo&trade MT Cell Viability Assay overview.

ATP Cell Viability Assays

ATP can be used to measure cell viability since only viable cells can synthesize ATP. ATP can be measured using the CellTiter-Glo ® Luminescent Cell Viability Assay (Cat.# G7570) with reagents containing detergent, stabilized luciferase and luciferin substrate. The detergent lyses viable cells, releasing ATP into the medium. In the presence of ATP, luciferase uses luciferin to generate luminescence, which can be detected within 10 minutes using a luminometer (Figure 2). The CellTiter-Glo ® 2.0 Assay (Cat.# G9241) is provided as a single solution that reduces reagent preparation time and provides the convenience of room temperature storage for easy implementation. These ATP assays are faster than other methods since they do not require long incubation times to convert a substrate into a colored product. They also have excellent sensitivity and broad linearity, making them highly compatible with high-throughput applications where low cell numbers are used. They are also less prone to artifacts than other methods.

Figure 2. The CellTiter-Glo® Assay detects ATP as an indicator of viable cells.

Live-Cell Protease Viability Assay

Live-cell protease activity disappears rapidly after cell death, so it is a useful marker of viable cells. Using the CellTiter-Fluor™ Cell Viability Assay (Cat.# G6080), live-cell protease activity can be measured using a cell-permeable fluorogenic protease substrate (GF-AFC). The substrate enters live cells where it is cleaved by live-cell protease to generate a fluorescent signal proportional to the number of viable cells (Figure 3). The incubation time for this method is 0.5–1 hour, which is shorter than tetrazolium assays (1–4 hours). Because this method does not lyse cells, it allows for multiplexing with many other assays in the same sample wells, including bioluminescent reporter cell-based assays.

Figure 3. The CellTiter-Fluor&trade Assay detects protease activity in living cells.

Tetrazolium Reduction Cell Viability Assays

Tetrazolium compounds used to detect viable cells fall into two basic categories:

Positively charged compounds (MTT) that readily penetrate viable cells:

Viable cells with active metabolism are able to convert MTT into a purple-colored formazan product. Thus, color formation can be a useful marker of viable cells. The CellTiter 96 ® Non-Radioactive Cell Proliferation Assay (MTT) (Cat.# G4000) uses this chemistry. However, the incubation time for this method is long (usually 4 hours). Also, the formazan product is insoluble, so a solubilizing reagent must be added prior to recording absorbance readings.

Negatively charged compounds (MTS, XTT, WST-1) that do not penetrate cells:

When using the CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (MTS) (Cat.# G3582), negatively charged compounds must be combined with intermediate electron coupling reagents, which can enter cells, be reduced and then exit the cell to convert tetrazolium to the soluble formazan product. The incubation time for this method is 1&ndash4 hours. There is no need to add a solubilizing reagent since the resulting formazan is soluble, making it more convenient.

Resazurin Reduction Cell Viability Assay

Resazurin is a cell-permeable indicator dye that is dark blue in color with little intrinsic fluorescence. The CellTiter-Blue ® Cell Viability Assay (Cat.# G8080) uses resazurin to measure cell viability. Only viable cells with active metabolism can reduce resazurin into resorufin, which is pink and fluorescent. After 1&ndash4 hours of incubation, the signal is quantified using a microplate spectrophotometer or fluorometer. This method is relatively inexpensive and more sensitive than tetrazolium assays. However, fluorescence from compounds being tested may interfere with resorufin readings.

A disadvantage of all tetrazolium or resazurin reduction assays is that they depend on the accumulation of colored or fluorescent products over time. Since the signal gradually increases over time, a decrease in cell viability during this long incubation cannot be detected.


Cell Counting

Automated cell counting uses optical methods to count cells in fluid samples for applications such as standardizing experiments and measuring assay impact. The TC20 &trade automated cell counter assesses total cell count and cell viability within 30 seconds with a broad range of cell types..

  • Accuracy and reproducibility with auto-focus technology
  • Total and viable cell counts within 30 seconds
  • User-defined gates for complex samples


Launch the System Tour

The TC20&trade automated cell counter has two counting modes. Depending on the sample type, the user-defined gates can be either enabled or disabled.

Samples with Multiple Cell Populations (stem cells and other primary cells)

User-defined gates must be enabled. Once the user-defined gates are enabled, a histogram will be displayed at the beginning of each count and the user can select the population of interest by adjusting the position of the cell size gates. Only objects within the designated range will be analyzed as cells those with diameters outside of the size range will be excluded from the cell count.

If the user-defined gates are disabled, the cell counting algorithm will automatically determine the population to count without any user input. The algorithm will count the cells of the most predominate cell type that typically comprise the largest peak, which may not be the population the user wants to count.



Gating setup window. Cell size gating window.

To make counting of such samples easier, when counting multiple sample replicates, the TC20 cell counter can save the positions of gates and apply them to subsequent counts (choose Yes in the &ldquoUse saved gates&rdquo field in Gating setup).

If the Image preview is On, an image of cells will be displayed during gating. In the image, only cells with diameters between the selected size gates will be marked with a yellow dot.

Immortal Cell Lines

When counting immortal cell lines or other samples that are composed of cells with similar cell size, the user-defined gates can be disabled. The cell counting algorithm then automatically identifies the cell population of interest without requiring user input.

The data below show the TC20&trade automated cell counter is compatible with a broad range of cell types, such as cell lines, primary cells (from tissue or blood), and stem cells. The TC20 cell counter can count cells with a 6&ndash50 µm cell diameter and within a broad concentration range of 5 x 10 4 &ndash1 x 10 7 cells/ml, which eliminates the need to dilute cells, thus reducing the error associated with sample dilutions prior to counting.

The TC20 automated cell counter demonstrates accurate cell counts across a range of cell sizes. Small (PBMC, Jurkat), medium (HeLa), and large (MEF) cells were counted with a hemocytometer, a TC20 automated cell counter, and a competitor's image-based automated cell counter. The TC20 counter and hemocytometer cell counts showed no statistically significant differences. Precision is indicated by the standard deviations error bars represent average standard deviations. Cell counts on the TC20 counter were performed on one instrument with four sample replicates.

The TC20 automated cell counter demonstrates accurate counts of viable cells. Pan T cells mixed with trypan blue (1:1) were counted with a hemocytometer, a TC20 automated cell counter, and a competitor&rsquos image-based automated cell counter. The TC20 counter and hemocytometer cell counts showed no statistically significant differences. Precision is indicated by the standard deviations error bars represent average standard deviations. Cell counts on the TC20 counter were performed on four different instruments with five sample replicates.

The TC20&trade automated cell counter uses microscopy with auto-focus that analyzes multiple focal planes to identify the best plane. The cell counting algorithm then uses the image acquired from the best focal plane to identify cells and exclude debris, thereby calculating the total cell count.

Using auto-focus instead of subjective manual focusing is especially important when assessing cell viability because an incorrectly selected focal plane will lead to inaccurate results.

If trypan blue is detected along with the total cell count, the TC20 counter assesses cell viability. The conventional method of analyzing viability using a single focal plane can lead to inaccurate conclusions because light scattering and the alignment of cells at different heights in a counting chamber can change the appearance of cells &mdash live cells may appear to be dead and vice versa. To determine if cells are viable, the TC20 counter analyzes each cell using images acquired from multiple focal planes during the focusing step.


Cell Banking

VI Environmental Monitoring

The EM program is designed to ensure overall control of the production environment based on fully justified acceptance, alert, and action levels. The level of monitoring depends on site ISO class and risk of product contamination by the site’s environment (USP 〈 797 〉 ). The following EM activities must be performed in the manufacturing site.

Active air sampling and monitoring for nonviable particle count (APC). Under ISO 14644‐1, cleanliness classes are assigned to clean zones using particle size and levels detected by APC equipment. Federal Std. 209E, as applied in the pharmaceutical industry, is based on limits of all particles with size ≥0.5 μm. APC is measured using particle counters with more than one sizing channel, which provides better information about the nature of the counted particles. Both APC ≥0.5 μm and ≥5.0 μm (macroparticles) can be recorded simultaneously. The following technical activities can be considered for effective air particle monitoring:

Number of sampling locations (X): This is calculated according to ISO 14644‐1 specifications based on surface area in square meters (Y) of the tested site, where X is the square root of Y. Sampling locations are selected to be evenly distributed throughout the testing environment.

Sampling volumes: The total sampling air volume should be at least 2 liters, which can be collected over a short time (<3 min) using modern APC counters.

Sampling practice: The sampling probe should be positioned pointing into the airflow or be directed vertically upward if the direction of the airflow is not predictable. In the processing BioSafety Cabinets (BSCs), both static preprocessing sampling and dynamic in‐process sampling should be performed per production run.

Alert, action, or failure of site compliance is documented when the average calculated concentration APC is above the expected limits. Exceeding alert levels is not necessarily grounds for definitive corrective actions, but prompts a documented follow‐up investigation and possibly a transient modification of the sampling plan. A reported failure in a controlled environment requires a defined sequence of actions. Processing should not be resumed if failure involves a critical processing site such as a BSC until a resolution of the condition has been accomplished.

Active air sampling and monitoring for microbial viable particle count (AVPC): Environmental AVPC is the enumeration and identification (as needed) of microbial (bacterial/fungal) particles in air. The AVPC is determined after air flowing across the media of a culture plate is sampled using an automated microbial air sampler. Viable particle samplers are highly efficient in the collection of particles containing microbial cells in the range of 2.1–14 μm (bacterial spore size and larger). Typically, Tryptic Soy Agar (TSA) medium is used, and supports both bacterial and fungal growth if incubated at 30–35 °C for 2 to 3 days and then at 20–25 °C for 5 to 7 days. Although the medium is validated by the vendor for sterility and high growth performance, different shipments of medium should initially be verified on-site for both sterility and growth promotion activity. Frequency, sampling time, and sampling size depend on the site’s production activities and its ISO class. For sampling purposes, monitored areas are divided into equal quarters. Sampling in the BSC is taken within 1 foot downstream of the active working site. Sampling volume, time and frequency, and alert/action levels need to be decided and enacted ( Table 3 ). Corrective actions must be dictated by the identification of microorganisms recovered mainly in the BSCs. If highly pathogenic microbes and product’s common contaminants are found, an investigation of the microbial source should be performed. The investigation should ascertain whether the isolated environmental microbe was introduced into the CT product and that appropriate corrective actions were enforced.

Table 3 . Air Sampling for AVPC and Alert/Action Levels—Proposed Example

Manufacturing SiteNumber of Sampling QuartersInitial Frequency of MonitoringAlert Level CFU count/m 3 Action Level CFU count/m 3
BSCs, ISO-51Daily (during each processing run)1–2≥3
Processing room: ISO-74Weekly10–20≥20
Ante-processing room: ISO-82Bi-weekly30–60≥60
Qualification laboratory: ISO-83Monthly50–80≥80

Surface microbial sampling and monitoring for viable particle count: Sampling of hard working surfaces, equipment, and personnel is best accomplished using contact plates and/or a swabbing method. Sterile TSA medium 25-cm 2 plates are used when sampling regular surfaces and cultured to monitor bacterial and fungal growth. Samples from irregular surfaces are obtained with swabs that are placed in appropriate diluents before plating on TSA plates or by directly rolling them onto the plate. Swabbing should cover a defined area of about 25 cm 2 . The microbial colony forming unit (CFU) counts are reported per contact plate or per swab. For microbial EM, sampling is done routinely at the conclusion of processing operations. The number of samples (contact plates) used per surface is dependent on total surface area, production activities performed on the surface, and classification of the environment around the surface. For critical benches, at least 1 sample/m 2 should be taken within 1 foot of the active working site. For other benches, shelves, cabinets, sinks, doors, windows, floors, partitions, and ceiling, at least 1 sample/4 m 2 is taken in a random homogenous pattern. For BSC internal surfaces, 4 samples are taken (middle of work surface, each side, and rear panel). Table 4 shows a proposed surface microbial sampling activities and alert/action levels for production sites.

Table 4 . Frequency of Microbial Sampling a of Various Surfaces and Alert/Action Levels—Proposed Example

SurfaceFrequency of SamplingAlert Level of CFU Count/PlateAction Level of CFU Count/Plate
Biosafety Cabinets (ISO-5)
Internal surfaces of all BSCsDaily, if used1–3&gt3
Processing Room (ISO-7)
All benches and sinksBi-weekly5–10&gt10
Floors, ceilings, partitions, windows, doors, shelves, and cabinetsMonthly10–20&gt20
Other Sites (Controlled Unclassified ISO-8)
Critical benches and sinksMonthly20–30&gt30
Noncritical benchesQuarterly50–80&gt80
Floors, ceilings, partitions, windows, doors, shelves, and cabinetsQuarterly70–100&gt100

The development of an adequate EM program is not free of challenges. It is not clear what can be adequate, what levels can be acceptable (mainly for viable counts), and how to perform EM in various types of CT facilities. Therefore, we advise that CT facilities obtain FDA approval for the EM plan prior to implementation. The plan should consist of following:

Monitoring of all surfaces, including facility and equipment, and personnel.

Sample timing, method, frequency, and location.

Nonviable and viable (microbial) particles acceptable, alert and action levels. ( Tables 3 and 4 suggests acceptable/alert/action levels for air viable counts, and for surface viable counts in critical manufacturing sites including Class 5 cabinets.)

Results analysis for trending, investigations, and corrective actions.

Periodic review and introduction of changes related to cleaning/disinfection, and EC/EM.


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