ExperimentQuantifyColonies
ExperimentQuantifyColonies[Samples]⟹Protocol
creates a Protocol object to prepare solid media plates designed to measure cell concentration of the provided Samples. Once the solid media plate is constructed by spreading cells with colony handler, it is placed in an incubator for a long period of time to allow for colony growth, then imaged with a colony handler. The total colony count of each prepared solid media plate is recorded and the result can be passed back to the original samples to calculate the Colony-Forming Unit (CFU). If the provided input Samples are already plated on solid media, the Protocol object is to count the number of colonies and update composition of the plated cells.
ExperimentQuantifyColonies measures the cell concentration in the provided samples. If the provided samples are already plated on solid media, the result is the count of colonies. If the samples are in suspension, they are spread onto solid media plates using a colony handler. The plates are then incubated for a period to allow colony growth. After incubation, the colonies are imaged with a colony handler, and the result is the Colony-Forming Unit (CFU) of the input samples. Specific applications of colony quantification include evaluating colony growth, screening recombinant bacteria, calculating Colony-Forming Units (CFU), and quantitating Phage Titers, among others.
Experimental Principles
Figure 1.1: Procedural overview of a QuantifyColonies experiment with PreparedSample set to True. Step 1: Samples are imaged using various ImagingStrategies. For ImagingStrategies including BlueWhiteScreen, an absorbance filter that blocks blue light is placed between the sample and the light source, eliminating blue colonies from the image, even when they appear powder blue. For ImagingStrategies including VioletFluorescence, GreenFluorescence, OrangeFluorescence, RedFluorescence, or DarkRedFluorescence, an LED-based light source with a specific fluorescence excitation wavelength illuminates the sample, and emission is filtered before reaching the CCD camera. Step 2: The images are analyzed by AnalyzeColonies, and cell concentration is updated.
Figure 1.2: Procedural overview of a QuantifyColonies experiment with PreparedSample set to False. Step 1: Samples are diluted and spread into solid media plates. Step 2: Newly prepared solid media samples are incubated. Step 3: Newly prepared solid media samples are imaged. Step 4: The images are analyzed by the function AnalyzeColonies and cell concentration from experimental data is updated if either NumberOfStableIntervals, MaxReliableColonyCount or MaxIncubationTime is reached, otherwise repeat step 2-4.
Instrumentation
QPix 420 HT
Figure 2.1.1: Overview of the ColonyHandler WorkCell integrations that are attached to the colony handler instrument deck.
Figure 2.1.2: Overview of the ColonyHandler WorkCell. This figure provides a comprehensive view of the ColonyHandler, showcasing its layout and main components. The QPix instrument is designed to perform multiple tasks including imaging solid media plates, picking colonies from solid media, and plating suspended colonies onto solid media. Its bed features a deck with designated positions for multi-well and single-well SBS plates, a light table, and wash baths for cleaning the pins before and during the process. Above the deck, a robotic gantry moves and is equipped with several components: a pipette nozzle, an LED-based light source, preset filter pairs for fluorescence excitation and emission wavelengths, a monochrome CCD camera, and a position to accept a ColonyHandlerHeadCassette. The robotic gantry moves seamlessly between tasks, and have the capacity to capture multiple types of images.
Diameter
diameterRules
Characterization
Select
The method used to group colonies into a population. Colonies are first ordered by their Diameter, defined as the diameter of a colony is the diameter of a disk with the same area as the colony. If the Select option is Min or Max, the ordered colonies are divided into partitions based on the NumberOfDivisions. From there, the smallest or largest partition is grouped into a population depending on if the Select option is Min or Max. For example, if the Select option is set to Max and the NumberOfDivisions is 10, the colonies will be divided into 10 partitions sorted from smallest to largest and the partition with the largest colonies will be selected as the population. Alternatively, if the Select option is AboveThreshold or BelowThreshold, an absolute cutoff is used to group ordered colonies into a population. The threshold is set by the ThresholdDiameter option. For example, if the Select option is BelowThreshold and the ThresholdDiameter is 0.8 Millimeter, all colonies smaller than 0.8 Millimeter in Diameter are grouped into a population.
ThresholdDiameter
The min or max possible diameter for a colony to be included in the population. When the Select option is AboveThreshold or BelowThreshold, all colonies with a diameter larger or smaller than the ThresholdDiameter, respectively, are grouped into a population. The colony diameter is defined as the diameter of a disk with the same area as the colony.
Pattern Description: Greater than or equal to 0.2 millimeters and less than or equal to 10 millimeters or Null.
NumberOfColonies
Default Calculation: If Select is Min or Max, up to 10 colonies are selected, otherwise All colonies are selected.
NumberOfDivisions
The number of partitions to group colonies into. Partitions are ordered by the Unit Operation feature. If the Select option is Min or Max, the lowest or highest partition will be used, respectively.
PopulationName
Default Calculation: By default, each population is labeled as ColonySelection with sequential integers.
Include
Exclude
Isolation
isolationRules
Characterization
Select
The method used to group colonies into a population. Colonies are first ordered by their Isolation, the shortest distance from the boundary of the colony to the boundary of another. If the Select option is Min or Max, the ordered colonies are divided into partitions based on the NumberOfDivisions. From there, the least isolated or most isolated partition is grouped into a population depending on if the Select option is Min or Max. For example, if the Select option is set to Max and the NumberOfDivisions is 10, the colonies will be divided into 10 partitions sorted from least isolated to most isolated and the partition with the most isolated colonies will be selected as the population. Alternatively, if the Select option is AboveThreshold or BelowThreshold, an absolute cutoff is used to group ordered colonies into a population. The threshold is set by the ThresholdDistance option. For example, if the Select option is AboveThreshold and the ThresholdDistance is 1 Millimeter, all colonies with separation greater than 1 Millimeter are grouped into a population.
ThresholdDistance
The min or max possible isolation for a colony to be included in the population. When the Select option is AboveThreshold or BelowThreshold, all colonies with an isolation distance larger or smaller than the ThresholdDistance, respectively, are grouped into a population. The isolation is the shortest distance from the boundary of the colony to the boundary of another.
NumberOfColonies
Default Calculation: If Select is Min or Max, up to 10 colonies are selected, otherwise All colonies are selected.
NumberOfDivisions
The number of partitions to group colonies into. Partitions are ordered by the Unit Operation feature. If the Select option is Min or Max, the lowest or highest partition will be used, respectively.
PopulationName
Default Calculation: By default, each population is labeled as ColonySelection with sequential integers.
Include
Exclude
Circularity
circularityRules
Characterization
Select
The method used to group colonies into a population. Colonies are first ordered by their Circularity, the ratio of the minor axis to the major axis of the best fit ellipse. If the Select option is Min or Max, the ordered colonies are divided into partitions based on the NumberOfDivisions. From there, the least circular or most circular partition is grouped into a population depending on if the Select option is Min or Max. For example, if the Select option is set to Max and the NumberOfDivisions is 10, the colonies will be divided into 10 partitions sorted from least circular to most circular and the partition with the most circular colonies will be selected as the population. Alternatively, if the Select option is AboveThreshold or BelowThreshold, an absolute cutoff is used to group ordered colonies into a population. The threshold is set by the ThresholdCircularity option. For example, if the Select option is AboveThreshold and the ThresholdCircularity is 0.9, all colonies with a circularity ratio greater than 0.9 are grouped into a population.
ThresholdCircularity
The min or max possible circularity ratio for a colony to be included in the population. When the Select option is AboveThreshold or BelowThreshold, all colonies with a circularity ratio larger or smaller than the ThresholdCircularity, respectively, are grouped into a population. The colony circularity ratio is the ratio of the minor axis to the major axis of the best fit ellipse.
NumberOfColonies
Default Calculation: If Select is Min or Max, up to 10 colonies are selected, otherwise All colonies are selected.
NumberOfDivisions
The number of partitions to group colonies into. Partitions are ordered by the Unit Operation feature. If the Select option is Min or Max, the lowest or highest partition will be used, respectively.
PopulationName
Default Calculation: By default, each population is labeled as ColonySelection with sequential integers.
Include
Exclude
Regularity
regularityRules
Characterization
Select
The method used to group colonies into a population. Colonies are first ordered by their Regularity, the ratio of the area of the colony to the area of a circle with the same perimeter. If the Select option is Min or Max, the ordered colonies are divided into partitions based on the NumberOfDivisions. From there, the least regular or most regular partition is grouped into a population depending on if the Select option is Min or Max. For example, if the Select option is set to Max and the NumberOfDivisions is 10, the colonies will be divided into 10 partitions sorted from least regular to most regular and the partition with the most regular colonies will be selected as the population. Alternatively, if the Select option is AboveThreshold or BelowThreshold, an absolute cutoff is used to group ordered colonies into a population. The threshold is set by the ThresholdRegularity option. For example, if the Select option is AboveThreshold and the ThresholdRegularity is 0.9, all colonies with a regularity ratio greater than 0.9 are grouped into a population.
ThresholdRegularity
The min or max possible regularity ratio for a colony to be included in the population. When the Select option is AboveThreshold or BelowThreshold, all colonies with a regularity ratio larger or smaller than the ThresholdRegularity, respectively, are grouped into a population. The colony regularity ratio is the ratio of the area of the colony to the area of a circle with the same perimeter.
NumberOfColonies
Default Calculation: If Select is Min or Max, up to 10 colonies are selected, otherwise All colonies are selected.
NumberOfDivisions
The number of partitions to group colonies into. Partitions are ordered by the Unit Operation feature. If the Select option is Min or Max, the lowest or highest partition will be used, respectively.
PopulationName
Default Calculation: By default, each population is labeled as ColonySelection with sequential integers.
Include
Exclude
Fluorescence
fluorescenceRules
Characterization
Dye
The fluorophore used to detect colonies that fluoresce at an EmissionWavelength when exposed to an ExcitationWavelength.
Default Calculation: Automatically set to the fluorophore within the excitation/emission range. For VioletFluorescence, set to DAPI. For GreenFluorescence, set to GFP. For OrangeFluorescence, set to Cy3. For RedFluorescence, set to TxRed. For DarkRedFluorescence, set to Cy5.
Pattern Description: DAPI, UltraViolet, GFP, RGBColor[0, 0, 1], Cy3, TxRed, Cy5, or RGBColor[1, 0, 0] or Null.
Programmatic Pattern: ((DAPI | UltraViolet | GFP | Blue | Cy3 | TxRed | Cy5 | Red) | Automatic) | Null
ExcitationWavelength
The wavelength of light that adds energy to colonies and causes them to fluoresce. Pairs with an EmissionWavelength.
Default Calculation: Automatically set to the excitation wavelength specified in the FluorescentExcitationWavelength field of model cell in CellTypes.
Programmatic Pattern: ((377*Nanometer | 457*Nanometer | 531*Nanometer | 628*Nanometer) | Automatic) | Null
EmissionWavelength
The light that the instrument measures to detect colonies fluorescing at a particular wavelength. Pairs with an ExcitationWavelength.
Default Calculation: Automatically set to the emission wavelength specified in the FluorescentEmissionWavelength field of model cell in CellTypes.
Pattern Description: 447 nanometers, 536 nanometers, 593 nanometers, 624 nanometers, or 692 nanometers or Null.
Programmatic Pattern: ((447*Nanometer | 536*Nanometer | 593*Nanometer | 624*Nanometer | 692*Nanometer) | Automatic) | Null
Select
The method used to group colonies into a population. Colonies are first ordered by their Fluorescence, the average pixel value inside the colony boundary from an image generated using an excitation wavelength and emission filter. If the Select option is Min or Max, the ordered colonies are divided into partitions based on the NumberOfDivisions. From there, the least fluorescing or most fluorescing partition is grouped into a population depending on if the Select option is Min or Max. For example, if the Select option is set to Max and the NumberOfDivisions is 10, the colonies will be divided into 10 partitions sorted from least fluorescing to most fluorescing and the most fluorescing partition will be selected as the population. Alternatively, if the Select option is Positive or Negative, the ordered colonies are first clustered into two groups based on their Fluorescence above or below background intensity. Then, if the Select option is Positive, the group with the higher fluorescing values is selected as the population. However, if the Select option is Negative, the group without fluorescence values are chosen as the population.
NumberOfColonies
Default Calculation: If Select is Min or Max, up to 10 colonies are selected, otherwise All colonies are selected.
NumberOfDivisions
The number of partitions to group colonies into. Partitions are ordered by the Unit Operation feature. If the Select option is Min or Max, the lowest or highest partition will be used, respectively.
PopulationName
Default Calculation: By default, each population is labeled as ColonySelection with sequential integers.
Include
Exclude
BlueWhiteScreen
blueWhiteScreenRules
Key/value pairs that define a set of colonies into a population based on their BlueWhiteScreen feature.
Characterization
Color
The color to block with the absorbance filter which is inserted between the light source and the sample. Default to blue to eliminate blue colonies from the image, even when the colonies are newly formed and appear powder blue.
FilterWavelength
The mean wavelength of light blocked by the absorbance filter. Blue colonies, which have a wavelength around 450 nanometers, appear darker.
Select
The method used to group colonies into a population. Colonies are first ordered by their intensities from BlueWhiteScreen images, the average pixel value inside the colony boundary from an image generated using an absorbance filter. If the Select option is Positive or Negative, the ordered colonies are first clustered into two groups based on their Absorbance. Then, if the Select option is Positive, the group with the higher brightness is selected as the population, and in this case white colonies. However, if the Select option is Negative, the group with the lower brightness values are chosen as the population, and in this case blue colonies. Alternatively, if the Select option is Min or Max, the ordered colonies are divided into partitions based on the NumberOfDivisions. From there, the least bright or most bright partition is grouped into a population depending on if the Select option is Min or Max. For example, if the Select option is set to Max and the NumberOfDivisions is 10, the colonies will be divided into 10 partitions sorted from least bright to most bright and the most bright partition will be selected as the population.
NumberOfColonies
Default Calculation: If Select is Min or Max, up to 10 colonies are selected, otherwise All colonies are selected.
NumberOfDivisions
The number of partitions to group colonies into. Partitions are ordered by the Unit Operation feature. If the Select option is Min or Max, the lowest or highest partition will be used, respectively.
PopulationName
Default Calculation: By default, each population is labeled as ColonySelection with sequential integers.
Include
Exclude
AllColonies
MultiFeatured
multiFeaturedRules
Key/value pairs that define a set of colonies into a population by multiple features, including Diameter, Regularity, Isolation, Circularity, BlueWhiteScreen, and Fluorescence.
Characterization
Features
The characteristics selected from Diameter, Regularity, Isolation, Circularity, BlueWhiteScreen, and Fluorescence of the colony by which the population will be isolated. For example, if Features is set to {Isolation, Diameter}, the colonies that are both larger than the median colony and more fluorescing than the median colony will be grouped into a population. More than one feature must be specified, otherwise the individual feature Unit Operation should be used. Colonies that match all features in the MultiFeatured unit operation will be included in the population.
Pattern Description: Diameter, Isolation, Regularity, Circularity, Fluorescence, or BlueWhiteScreen or {Isolation, Diameter}
Select
The method used to group colonies into a population. Colonies are first ordered by their Feature. If the Select option is Min or Max, the ordered colonies are divided into partitions based on the NumberOfDivisions. From there, the smallest or largest partition is grouped into a population depending on if the Select option is Min or Max. For example, if the Select option is set to Max and the NumberOfDivisions is 10, the colonies will be divided into 10 partitions sorted by the Feature and the highest partition will become the selected population. If the Select option is AboveThreshold or BelowThreshold, an absolute cutoff is used to group ordered colonies into a population. The threshold values is set by the Threshold option. For example, if the Select option is BelowThreshold, the Feature is Diameter, and the Threshold is 0.8 Millimeter, all colonies with Diameter less than 0.8 Millimeter will be selected as the population. If the Select option is Positive or Negative, the colonies are first clustered into two groups based on their Fluorescence or BlueWhiteScreen image intensity compared with background. Then, if the Select option is Positive, the group with the higher intensity values is selected as the population. However, if the Select option is Negative, the group without Fluorescence or BlueWhiteScreen signal is chosen as the population.
Default Calculation: For Diameter, Isolation, Regularity, and Circularity features the default is Max unless a Threshold is specified, in which case the default is AboveThreshold. For Fluorescence and BlueWhiteScreen the default is Positive, unless the NumberOfDivisions is specified, in which case the default is Max.
Programmatic Pattern: (Min | Max | AboveThreshold | BelowThreshold | Positive | Negative) | Automatic
NumberOfDivisions
The number of partitions to group colonies into. Partitions are ordered by the Features. If the Select option is Min or Max, the lowest or highest partition will be used, respectively.
Threshold
The min or max possible feature value (e.g., Diameter or Regularity) for a colony to be included in the population. When the Select option is AboveThreshold or BelowThreshold, all colonies with a feature value larger or smaller than the Threshold, respectively, are grouped into a population.
Pattern Description: Greater than or equal to 0 and less than or equal to 1 or greater than or equal to 0 millimeters or Null.
Color
The color to block with the absorbance filter which is inserted between the light source and the sample. Default to blue to eliminate blue colonies from the image, even when the colonies are newly formed and appear powder blue.
FilterWavelength
The mean wavelength of light blocked by the absorbance filter. Blue colonies, which have a wavelength around 450 nanometers, appear darker.
Dye
The coloring used to detect colonies that fluoresce at an EmissionWavelength when exposed to an ExcitationWavelength.
Default Calculation: Automatically set to the fluorophore within the excitation/emission range. For VioletFluorescence, set to DAPI. For GreenFluorescence, set to GFP. For OrangeFluorescence, set to Cy3. For RedFluorescence, set to TxRed. For DarkRedFluorescence, set to Cy5.
Pattern Description: DAPI, UltraViolet, GFP, RGBColor[0, 0, 1], Cy3, TxRed, Cy5, or RGBColor[1, 0, 0] or Null.
Programmatic Pattern: ((DAPI | UltraViolet | GFP | Blue | Cy3 | TxRed | Cy5 | Red) | Automatic) | Null
ExcitationWavelength
The wavelength of light that adds energy to colonies and causes them to fluoresce. Pairs with an EmissionWavelength.
Default Calculation: Automatically set to the excitation wavelength specified in the FluorescentExcitationWavelength field of model cell in CellTypes.
Programmatic Pattern: ((377*Nanometer | 457*Nanometer | 531*Nanometer | 628*Nanometer) | Automatic) | Null
EmissionWavelength
The light that the instrument measures to detect colonies fluorescing at a particular wavelength. Pairs with an ExcitationWavelength.
Default Calculation: Automatically set to the emission wavelength specified in the FluorescentEmissionWavelength field of model cell in CellTypes.
Pattern Description: 447 nanometers, 536 nanometers, 593 nanometers, 624 nanometers, or 692 nanometers or Null.
Programmatic Pattern: ((447*Nanometer | 536*Nanometer | 593*Nanometer | 624*Nanometer | 692*Nanometer) | Automatic) | Null
PopulationName
Default Calculation: By default, each population is labeled as ColonySelection with sequential integers.
NumberOfColonies
Default Calculation: If Select is Min or Max, up to 10 colonies are selected, otherwise All colonies are selected.
Include
Exclude
Experiment Options
General
ImagingInstrument
Figure 3.1: Diagram of the deck layout of the ColonyHandler. The ColonyHandler is equipped with both transmitted white light and fluorescence imaging capabilities. In the Imaging configuration, the deck features two designated positions for single-well plates for each batch of imaging.
Pattern Description: An object of type or subtype Model[Instrument, ColonyHandler] or Object[Instrument, ColonyHandler]
Programmatic Pattern: ObjectP[{Model[Instrument, ColonyHandler], Object[Instrument, ColonyHandler]}]
PreparedSample
Indicates if the provided samples containing microbial cells that are counted have been previously transferred onto a solid media plate and cultivated to grow colonies. If PreparedSample is set to True, the provided sample container is placed directly into the imaging instrument for imaging and counting, bypassing the cell spreading and incubation steps.
Default Calculation: Set to False if the provided samples is not in the state of Solid, otherwise set to True.
SpreaderInstrument
The colony handler that is used to spread the provided samples on solid media to prepare colony samples.
Figure 3.2: Diagram of the deck layout of the ColonyHandler. The ColonyHandler is equipped with both a pipette and a colony spreading tool. In the spreading configuration, the deck features designated positions for a pipette tip container and a tip chute, 8 positions for multi-well plates, two positions for single-well plates, and wash baths.
Default Calculation: When PreparedSample is set to False, automatically set SpreaderInstrument to Model[Instrument, ColonyHandler, "QPix 420 HT"].
Pattern Description: An object of type or subtype Model[Instrument, ColonyHandler] or Object[Instrument, ColonyHandler] or Null.
Programmatic Pattern: (ObjectP[{Model[Instrument, ColonyHandler], Object[Instrument, ColonyHandler]}] | Automatic) | Null
Incubator
The cell incubator that is used to grow colonies in the desired environment before colonies are imaged. Custom incubation actively selects an incubator in the lab and uses a thread to incubate only the cells from this protocol for the specified Time. Selecting a default IncubationCondition (BacterialIncubation, YeastIncubation) will passively store the cells for the specified time in a shared incubator, potentially with samples from other protocols. However, it will not consume a thread while the cells are inside the incubator.
Default Calculation: When PreparedSample is set to False, automatically set Incubator to an incubator that meets the requirements according to IncubateCellsDevices that provides desired incubation conditions.
Pattern Description: An object of type or subtype Model[Instrument, Incubator] or Object[Instrument, Incubator] or Null.
Programmatic Pattern: (ObjectP[{Model[Instrument, Incubator], Object[Instrument, Incubator]}] | Automatic) | Null
WorkCell
Preparation
Indicates if this unit operation is carried out primarily robotically or manually. Manual unit operations are executed by a laboratory operator and robotic unit operations are executed by a liquid handling work cell.
Imaging
ImagingStrategies
The end goals for capturing images. ImagingStrategies can be either a single end goal of simple visualization or a list of multiple end goals. The options include BrightField, BlueWhiteScreen, and various fluorescence imaging techniques. BrightField imaging provides essential baseline images and is required if any other imaging strategies are employed. BlueWhiteScreen is used to distinguish recombinant colonies with disrupted lacZ (white colonies) from blue colonies, using an absorbance filter. Fluorescence imaging allows for the visualization of colonies with fluorescent dyes or proteins.
Figure 3.3: Diagram of the imaging module of the ColonyHandler with various imaging strategies. For BlueWhiteScreen, an absorbance filter which blocks blue light between the sample and the light source, eliminating blue colonies from the image, even when they are newly formed and appear powder blue. For VioletFluorescence, GreenFluorescence, OrangeFluorescence, RedFluorescence, or DarkRedFluorescence, an LED-based light source with a specific fluorescence excitation wavelength illuminates the sample, and emission is filtered before reaching the CCD camera. The transition to capture multiple types of images is accomplished by the robotic gantry of the QPix colony handler, which moves seamlessly between imaging strategies and samples.
Default Calculation: Automatically set to include BlueWhiteScreen along with BrightField if Populations is set to include BlueWhiteScreen; set to include a specific fluorescence strategy along with BrightField if the Model[Cell] information in the sample matches one of the fluorescent excitation and emission pairs supported by the imaging instrument. Otherwise, set to BrightField, as a BrightField image is always taken.
Programmatic Pattern: (DuplicateFreeListableP[BrightField | BlueWhiteScreen | VioletFluorescence | GreenFluorescence | OrangeFluorescence | RedFluorescence | DarkRedFluorescence] | BrightField) | Automatic
ExposureTimes
A single length of time that the camera sensor collects light during image acquisition for each imaging strategy. An increased exposure time leads to brighter images based on a linear scale. When set as Automatic, optimal exposure time is automatically determined during the experiment. This is done by running AnalyzeImageExposure on images taken with suggested initial exposure times. The process adjusts the exposure time for subsequent image acquisitions until the optimal value is found.
Figure 3.4: This table outlines the suggested exposure times for various imaging strategies and lists the corresponding fields where the raw image data is stored for each imaging strategy.
Programmatic Pattern: ({(RangeP[1*Millisecond, 2000*Millisecond] | Automatic)..} | (RangeP[1*Millisecond, 2000*Millisecond] | Automatic)) | Automatic
Spreading
DilutionType
Indicates the type of dilution performed on the sample. Linear dilution represents a single stage dilution of the Analyte in the sample to a specified concentration or by a specified dilution factor. Serial dilution represents a stepwise dilution of the Analyte in the sample resulting in multiple samples and a geometric progression of the concentration. The progression can be described by either a series of target concentrations or a series of dilution factors. In a serial dilution the source of a dilution round is the resulting sample of the previous dilution round. The first source sample is the original sample provided.
DilutionStrategy
Indicates if only the final sample (Endpoint) or all diluted samples (Series) produced by serial dilution are used for spreading on solid media plate.
NumberOfDilutions
Default Calculation: Automatically set to the length of TargetAnalyteConcentration, CumulativeDilutionFactor, or SerialDilutionFactor if provided, otherwise set to 1.
Pattern Description: Greater than or equal to 1 and less than or equal to 100 in increments of 1 or Null.
CumulativeDilutionFactor
For each sample, the factor by which the concentration of the TargetAnalyte in the original sample is reduced during the dilution. The length of this list must match the corresponding value in NumberOfDilutions.
Default Calculation: Automatically set based on the systems of equations specified under the DilutionType option.
Pattern Description: Greater than or equal to 1 and less than or equal to 100000000000000000000000 or Null.
SerialDilutionFactor
For each sample, the factor by which the concentration of the TargetAnalyte in the resulting sample of the previous dilution step is reduced. For example, if the CumulativeDilutionFactor is equal to {10,100,1000}. SerialDilutionFactor will resolve to {10,10,10}. The length of this list must match the corresponding value in NumberOfDilutions.
Default Calculation: Automatically set based on the systems of equations specified under the DilutionType option.
Pattern Description: Greater than or equal to 1 and less than or equal to 100000000000000000000000 or Null.
ColonySpreadingTool
For each sample, the tool used to spread the suspended cells from the input sample onto the destination plate or into a destination well.
Figure 3.6: Various colony spreading tools. In Spreading application, the ColonyHandlerHeadCassette containing either 1/8-pin spreading head is installed into the robot gantry is used to spread suspended cells from the input sample onto the destination plate or into a destination well.
Default Calculation: If DestinationContainer is an 8 well solid media tray, resolves to Model[Part, ColonyHandlerHeadCassette, "8-pin spreading head"]. Otherwise resolves to Model[Part, ColonyHandlerHeadCassette, "1-pin spreading head"].
Pattern Description: An object of type or subtype Model[Part, ColonyHandlerHeadCassette] or Object[Part, ColonyHandlerHeadCassette] or Null.
Programmatic Pattern: (ObjectP[{Model[Part, ColonyHandlerHeadCassette], Object[Part, ColonyHandlerHeadCassette]}] | Automatic) | Null
SpreadVolume
Default Calculation: Automatically set to the median among 30 Microliter, the sample volume divided by the length of dispense coordinates, and 100 Microliter.
Pattern Description: Greater than or equal to 10 microliters and less than or equal to 130 microliters or Null.
DispenseCoordinates
For each sample, the location to dispense the suspended cells to be spread on the destination plate.
Programmatic Pattern: ({{RangeP[-63*Millimeter, 63*Millimeter], RangeP[-43*Millimeter, 43*Millimeter]}..} | Automatic) | Null
SpreadPatternType
For each sample, the pattern the spreading colony handler head will move when spreading the colony on the plate. Can be specified as a pre-determined pattern or Custom to indicate the coordinates in CustomSpreadPattern should be used.
CustomSpreadPattern
For each sample, the user defined pattern used to spread the suspended cells across the plate. Specify a series of Spread[{{xCoordinate,yCoordinate}}..] to specify the pattern. In order to draw separate lines, specify series of coordinates in different Spread[...]'s. Between each Spread[...], the pin on colony spreading tool will be lifted off of the agar and then repositioned at the first coordinate of the next Spread[...].
Programmatic Pattern: ((Null | (Verbatim[Spread][{{RangeP[-63*Millimeter, 63*Millimeter], RangeP[-43*Millimeter, 43*Millimeter]}..}] | {Verbatim[Spread][{{RangeP[-63*Millimeter, 63*Millimeter], RangeP[-43*Millimeter, 43*Millimeter]}..}]..})) | Automatic) | Null
DestinationContainer
For each Sample, the desired type of container to have suspended cells spread in, with indices indicating grouping of samples in the same plate, if desired.
Pattern Description: An object of type or subtype Model[Container] or Object[Container] or a prepared sample or {Index, Container} or Null.
Programmatic Pattern: (((ObjectP[{Model[Container], Object[Container]}] | _String) | {GreaterEqualP[1, 1] | Automatic, (ObjectP[{Model[Container]}] | _String) | Automatic}) | Automatic) | Null
DestinationWell
DestinationMedia
Default Calculation: Automatically set to the PreferredSolidMedia field of the first Model[Cell] of the composition of the Sample.
Pattern Description: An object of type or subtype Object[Sample] or Model[Sample] or a prepared sample or Null.
Incubation
IncubationCondition
The type of incubation that defines the Temperature, Carbon Dioxide Percentage, Relative Humidity, Shaking Rate and Shaking Radius, under which the input cells are incubated. Custom incubation actively selects an incubator in the lab and uses a thread to incubate only the cells from this protocol for the specified Time. Selecting an IncubationCondition, through a symbol or an object, will passively store the cells for the specified time in a shared incubator, potentially with samples from other protocols. However, it will not consume a thread while the cells are inside the incubator. Currently, MammalianIncubation, BacterialIncubation, BacterialShakingIncubation, YeastIncubation, and YeastShakingIncubation are supported cell culture incubation conditions with shared incubators.
Figure 3.8: This table indicates how the CellType impact the resolution of IncubationCondition when no other options are specified.
Default Calculation: Automatically set to a storage condition matching specified CellType, CultureAdhesion, Temperature, CarbonDioxide, RelativeHumidity, ShakingRate and ShakingRadius options, or set to Custom if no existing storage conditions can provide specified incubation condition options. If no Temperature, RelativeHumidity, CarbonDioxide, ShakingRate, or ShakingRadius are provided, automatically set based on the CellType and CultureAdhesion as described in the below table.
Programmatic Pattern: (((BacterialIncubation | YeastIncubation | Custom) | ObjectP[Model[StorageCondition]]) | Automatic) | Null
Temperature
Temperature at which the input cells are incubated. 30 Degrees Celsius and 37 Degrees Celsius are supported by default cell culture incubation conditions. Alternatively, a customized temperature can be requested with a dedicated custom incubator between 28 degrees Celsius and 80 degrees Celsius until the protocol is completed. See the IncubationCondition option for more information.
Default Calculation: Automatically set to match the Temperature field of specified IncubationCondition. If IncubationCondition is set to Custom, automatically set to 30 Celsius.
Programmatic Pattern: ((CellIncubationTemperatureP | RangeP[$MinCellIncubationTemperature, $MaxCellIncubationTemperature]) | Automatic) | Null
ColonyIncubationTime
Default Calculation: If PreparedSample is set to False, automatically set to the shorter time of 10 Hour or ten times the DoublingTime of the cells in the samples, with a minimum of 1 hour.
Analysis
MinReliableColonyCount
The smallest number of colonies that can be counted on a solid media plate to provide a statistically reliable estimate of the concentration of microorganisms in a sample. Below this number, the count is considered statistically unreliable and the result is typically referred to as "Too Few To Count" (TFTC).
Figure 3.9: The reliable counting range, between MinReliableColonyCount and MaxReliableColonyCount colonies per plate, is chosen to ensure reliability and accuracy in statistical analysis. Counts below MinReliableColonyCount are often not statistically significant, leading to potential inaccuracies, while counts above MaxReliableColonyCount can result in overlapping colonies and nutrient depletion, making accurate counting and colony growth assessment difficult.
Default Calculation: If PreparedSample is set to False, automatically set to 30, otherwise set to 1.
MaxReliableColonyCount
The largest number of colonies that can be counted on a solid media plate beyond which accurate counting becomes impractical and unreliable. This threshold is typically set at 300 colonies. When the number of colonies exceeds this limit, it is difficult to distinguish and count individual colonies accurately, and the result is typically referred as "Too Numerous To Count" (TNTC).
Figure 3.10: The reliable counting range, between MinReliableColonyCount and MaxReliableColonyCount colonies per plate, is chosen to ensure reliability and accuracy in statistical analysis. Counts below MinReliableColonyCount are often not statistically significant, leading to potential inaccuracies, while counts above MaxReliableColonyCount can result in overlapping colonies and nutrient depletion, making accurate counting and colony growth assessment difficult.
MinDiameter
The smallest diameter value from which colonies will be included in TotalColonyCounts in the data and analysis. The diameter is defined as the diameter of a circle with the same area as the colony.
Figure 3.11: Diameter is the distance of the longest line connecting 2 points on the edge of the colony that goes through the centerpoint.
Pattern Description: Greater than or equal to 0.2 millimeters and less than or equal to 10 millimeters or Null.
MaxDiameter
The largest diameter value from which colonies will be included in TotalColonyCounts in the data and analysis. The diameter is defined as the diameter of a circle with the same area as the colony.
Figure 3.12: Diameter is the distance of the longest line connecting 2 points on the edge of the colony that goes through the centerpoint.
Pattern Description: Greater than or equal to 0.2 millimeters and less than or equal to 10 millimeters or Null.
MinColonySeparation
The closest distance included colonies can be from each other from which colonies will be included in the data and analysis. The separation of a colony is the shortest path between the perimeter of the colony and the perimeter of any other colony.
MinRegularityRatio
The smallest regularity ratio from which colonies will be included in the data and analysis. The regularity ratio is the ratio of the area of the colony to the area of a circle with the colony's perimeter. For example, jagged edged shapes will have a longer perimeter than smoother ones and therefore a smaller regularity ratio.
Figure 3.14: Regularity is the ratio of the area of the colony to the area of a circle with the colony's perimeter.
MaxRegularityRatio
The largest regularity ratio from which colonies will be included in the data and analysis. The regularity ratio is the ratio of the area of the colony to the area of a circle with the colony's perimeter. For example, jagged edged shapes will have a longer perimeter than smoother ones and therefore a smaller regularity ratio.
Figure 3.15: Regularity is the ratio of the area of the colony to the area of a circle with the colony's perimeter.
MinCircularityRatio
The smallest circularity ratio from which colonies will be included in the data and analysis. The circularity ratio is defined as the ratio of the minor axis to the major axis of the best fit ellipse. For example, a very oblong colony will have a much larger major axis compared to its minor axis and therefore a low circularity ratio.
Figure 3.16: Circularity is a measure of the ratio of the minor axis to the major axis of the best fit ellipse.
MaxCircularityRatio
The largest circularity ratio from which colonies will be included in the data and analysis. The circularity ratio is defined as the ratio of the minor axis to the major axis of the best fit ellipse. For example, a very oblong colony will have a much larger major axis compared to its minor axis and therefore a low circularity ratio.
Figure 3.17: Circularity is a measure of the ratio of the minor axis to the major axis of the best fit ellipse.
Populations
The criteria used to group colonies together into a population to count. Criteria are based on the ordering of colonies by the desired feature(s): Diameter, Regularity, Circularity, Isolation, Fluorescence, and BlueWhiteScreen. For more information see documentation on colony population Unit Operations: Diameter, Isolation, Regularity, Circularity, Fluorescence, BlueWhiteScreen, MultiFeatured, and AllColonies under Experiment Principles section.
Default Calculation: If the Model[Cell] information in the sample object matches one of the fluorescent excitation and emission pairs of the colony picking instrument, Populations is set to Fluorescence. If BlueWhiteScreen is specified in ImagingStrategies, Populations is set to BlueWhiteScreen. Otherwise, Populations is set to All.
Programmatic Pattern: (((Fluorescence | BlueWhiteScreen | Diameter | Isolation | Circularity | Regularity | All) | FluorescencePrimitiveP | BlueWhiteScreenPrimitiveP | DiameterPrimitiveP | IsolationPrimitiveP | CircularityPrimitiveP | RegularityPrimitiveP | AllColoniesPrimitiveP | MultiFeaturedPrimitiveP) | {((Fluorescence | BlueWhiteScreen | Diameter | Isolation | Circularity | Regularity | All) | FluorescencePrimitiveP | BlueWhiteScreenPrimitiveP | DiameterPrimitiveP | IsolationPrimitiveP | CircularityPrimitiveP | RegularityPrimitiveP | AllColoniesPrimitiveP | MultiFeaturedPrimitiveP)..}) | Automatic
PopulationCellTypes
The cell type thought to represent the physiological characteristics defined in each of the Populations.
Default Calculation: Automatically set to the cell type of the specified PopulationIdentities if it is a Model[Cell]. Otherwise, PopulationCellTypes is set to the cell type of the analyte or the cell model with the highest concentration from the input sample.
Pattern Description: CellType for single Colony Population or CellTypes for Multiple Colony Populations.
PopulationIdentities
The cell constituent thought to represent the physiological characteristics defined in each of the Populations. If given as a string, a new Model[Cell] with the given name will be created with the type specified in option PopulationCellTypes and the current model of the given sample will be removed after the quantification step.
Default Calculation: Automatically set to the cell model of the analyte or the cell model if there is only one cell model in the Composition of the input sample, otherwise set to "Characterized Colony from <SampleID> #", and the protocol ID is always appended to the end of name string automatically at the time of model creation.
Pattern Description: CellIdentities for Multiple Colony Populations or CellIdentity for single Colony Population.
Programmatic Pattern: ((_String | ObjectP[{Model[Cell, Bacteria], Model[Cell, Yeast]}]) | {(_String | ObjectP[{Model[Cell, Bacteria], Model[Cell, Yeast]}])..}) | Automatic
Incubation Repeat
IncubateUntilCountable
Indicates whether colony samples should undergo repeated cycles of incubation, imaging, and analysis until one of the following conditions is met: 1) The TotalColonyCounts from analyses are no longer increasing after surpassing the MinReliableColonyCount. 2) The TotalColonyCounts from analyses exceed the MaxReliableColonyCount. 3) A predetermined maximum incubation time is reached. The criteria for identifying colonies large enough to be counted can be set using the following options: MinDiameter, MaxColonySeparation, MinRegularityRatio, MaxRegularityRatio, MinCircularityRatio, and MaxCircularityRatio. Refer to Figure 3.2 for more information on colony identification criteria.
NumberOfStableIntervals
The number of consecutive intervals during which the TotalColonyCounts remain stable (do not increase). For example, if the TotalColonyCounts (e.g. {200, 240, 240}) do not increase for the last two consecutive incubation intervals, the stable interval number is 1. This metric is used to determine whether all viable colonies have been counted when calculating Colony Forming Units (CFU). This stability indicates that the growth phase of the colonies has plateaued, ensuring an accurate count of all viable colonies present.
MaxColonyIncubationTime
The maximum duration during which the colony samples are allowed to be incubated inside of cell incubator.
Default Calculation: If PreparedSample is set to False and IncubateUntilCountable is set to True, automatically set to the shorter time of 20 Hour or twenty times the DoublingTime of the cells in the samples, with a minimum of 2 hour. Otherwise set to the same value as IncubationTime if IncubateUntilCountable is set to False.
IncubationInterval
The duration during which colony samples are placed inside a cell incubator as part of repeated cycles of incubation, imaging, and analysis. Following this interval, the samples are moved out of the cell incubator and undergo imaging and analysis until all analyses return at least one value of TotalColonyCounts above the MinReliableColonyCount or until the MaxIncubationTime is reached.
Default Calculation: If IncubateUntilCountable is set to True, automatically set to the shorter time of 5 hours or five times the DoublingTime of the cells in the samples, with a minimum of 1 hour. Any time less than 1 hour is not sufficient to complete the cycle of setting up incubator and imaging instrument.
Post Experiment
SamplesInStorageCondition
The non-default conditions under which the SamplesIn of this experiment should be stored after the protocol is completed. If left unset, SamplesIn will be stored according to their current StorageCondition.
Pattern Description: {AmbientStorage, EnclosedAmbientStorage, Refrigerator, Freezer, DeepFreezer, CryogenicStorage, YeastIncubation, YeastShakingIncubation, BacterialIncubation, BacterialShakingIncubation, MammalianIncubation, ViralIncubation, CrystalIncubation, AcceleratedTesting, IntermediateTesting, LongTermTesting, UVVisLightTesting} or Disposal or Null.
SamplesOutStorageCondition
The non-default conditions under which any new samples generated by this experiment should be stored after the protocol is completed. If left unset, the new samples will be stored according to their Models' DefaultStorageCondition.
Pattern Description: {AmbientStorage, EnclosedAmbientStorage, Refrigerator, Freezer, DeepFreezer, CryogenicStorage, YeastIncubation, YeastShakingIncubation, BacterialIncubation, BacterialShakingIncubation, MammalianIncubation, ViralIncubation, CrystalIncubation, AcceleratedTesting, IntermediateTesting, LongTermTesting, UVVisLightTesting} or Disposal or Null.
Protocol Options
Organizational Information
Template
A template protocol whose methodology should be reproduced in running this experiment. Option values will be inherited from the template protocol, but can be individually overridden by directly specifying values for those options to this Experiment function.
Pattern Description: An object of type or subtype Object[Protocol] or an object of type or subtype of Object[Protocol] with UnresolvedOptions, ResolvedOptions specified or Null.
Programmatic Pattern: (ObjectP[Object[Protocol]] | FieldReferenceP[Object[Protocol], {UnresolvedOptions, ResolvedOptions}]) | Null
Name
Example Calls
Data Processing
Warnings and Errors
Messages (67)
ConflictingDestinationMedia (1)
ConflictingIncubationRepeatOptions (2)
ConflictingPopulationCellTypesWithIdentities (2)
ConflictingPopulationCellTypesWithPopulations (2)
ConflictingPopulationIdentitiesWithPopulations (1)
ConflictingPreparedSampleWithIncubation (2)
ConflictingPreparedSampleWithSpreading (2)
ConflictingUnitOperationMethodRequirement (2)
ConflictingWorkCellWithPreparation (1)
ContainerOutLabelMismatch (3)
If DilutionStrategy is Endpoint and the length of ContainerOutLabel is not 1, an error is thrown:
If DilutionStrategy is Null and the length of ContainerOutLabel is not NumberOfDilutions, an error is thrown:
If DilutionStrategy is Series and the length of ContainerOutLabel is not NumberOfDilutions + 1, an error is thrown:
DestinationMediaNotSolid (1)
DilutionMismatch (1)
DuplicatedPopulationIdentities (2)
ImagingOptionMismatch (2)
IncubationMaxTemperature (1)
IncubatorIsIncompatibleForColonies (2)
InstrumentPrecision (2)
InvalidContainerModels (2)
InvalidDestinationContainer (1)
InvalidDestinationContainerType (1)
InvalidDestinationWell (1)
InvalidIncubationConditions (3)
MissingImagingStrategies (1)
NewColonyNameInUse (1)
NonLiquidSamples (2)
NonSolidSamples (1)
ObjectDoesNotExist (6)
Do NOT throw a message if we have a simulated container but a simulation is specified that indicates that it is simulated:
Do NOT throw a message if we have a simulated sample but a simulation is specified that indicates that it is simulated:
Throw a message if we have a container that does not exist (ID form):
Throw a message if we have a container that does not exist (name form):
Throw a message if we have a sample that does not exist (ID form):
Throw a message if we have a sample that does not exist (name form):
OverlappingPopulations (2)
QuantifyColoniesPreparationNotSupported (1)
SampleOutLabelMismatch (5)
If DilutionStrategy is Endpoint and the length of SampleOutLabel is not 1, an error is thrown:
If DilutionStrategy is Null and the length of SampleOutLabel is not NumberOfDilutions, an error is thrown:
If DilutionStrategy is Series and the length of SampleOutLabel is not NumberOfDilutions + 1, an error is thrown:
If DilutionType is Linear and the length of SampleOutLabel is not NumberOfDilutions, an error is thrown:
If there is no dilution and the length of SampleOutLabel is not 1, an error is thrown:
SpreadingMultipleSamplesOnSamePlate (1)
SpreadPatternMismatch (2)
TooManyIncubationConditions (1)
TooManyIncubationSamples (1)
UnsuitableIncubationInterval (2)
Possible Issues
Overlapping Colonies Counted Toward the First Fitting Population
When multiple populations are specified within the same sample, some colonies may meet the criteria for more than one population. In such cases, colonies are assigned to the first matching population in the list. This may result in an artificial division of colony counts and potentially lead to misinterpretation of the true population composition.
Last modified on Sat 27 Sep 2025 02:15:25