Colony Counting User Manual A D I V I S I O N O F S Y N O P T I C S L T D

Transcription

1 ProtoCOL Colony Counting User Manual S Y N B I O S I S A D I V I S I O N O F S Y N O P T I C S L T D All possible care has been taken in the preparation of this publication, but Synoptics Limited accepts no liability for any inaccuracies that may be found. Synoptics Limited reserves the right to make changes without notice both to this publication and to the product that it describes. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior permission of the copyright owner. Version 09-1p.1 Published by Synoptics Limited Synoptics Limited 2009

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3 Contents Getting Started 1 Introduction Structure of the manual Using ProtoCOL to count colonies overview Starting ProtoCOL Logging out and closing ProtoCOL Capturing images 2 Loading plates into the instrument The Image tab Capturing an image Setting the exposure Zooming the image Exporting the image to a file Importing an image from a file Calibrating the image Viewing batch images Creating a new batch 3 Creating a new batch using the Create New Batch Wizard Welcome Batch Name and Measurement Type Derive from existing Batch Plate Type Configure Measurements Spiral Plate Other plate types Setting up batches to count colonies Design mode accepting and rejecting new batches Allowing changes in Measurement mode ProtoCOL Systems User Manual Contents-1

4 Contents Classification Frame Color classification Classification panel Adding sub-classifications Size classification Shape classification Grid classification Changing an existing classification Modifying an existing classification Repeating a color classification Results Measure Plate ID Dilution Putting an upper limit on counts for Pour plate batches Test Measure Plate Accepting the batch design Counting colonies 4 Opening and selecting batches Adjusting batch settings in Measurement mode Entering plate identifiers Exclude regions Counting the colonies on a plate Closing batches Working with results 5 The Results table Editing results Selecting results Deleting results Adding and editing comments Editing the plate identifier and/or dilution Editing a count value Manually editing the detected colonies Viewing the audit trail of changes Contents-2 ProtoCOL Systems User Manual

5 Contents Repeating a measurement for an Ames batch plate Compiling reports Working with reports ProtoCOL Systems User Manual Contents-3

6 Contents Contents-4 ProtoCOL Systems User Manual

7 Getting Started Introduction ProtoCOL is a combined hardware and software system for automatically counting colonies and measuring inhibition zones. This manual describes how to use ProtoCOL to count colonies; see the ProtoCOL Zone Measurement User Manual for how to use ProtoCOL to measure zones. You can use ProtoCOL to distinguish between different types of colony according to their color, size and/or shape, and to distinguish between colonies and debris or other artifacts. Once the system has been set up, all you have to do to count the colonies on a series of plates is to put each plate on the plate holder and press a button. The counting of each plate takes no more than a few seconds and the results are shown instantly in a table. ProtoCOL allows you to enter plate identifiers and dilutions manually, to use auto-incrementing plate identifiers or to enter lists of plate identifiers and dilution series within ProtoCOL, from a CSV file or from a LIMS. If you use auto-incrementing plate identifiers or a plate list, ProtoCOL will prompt you for which plate to insert next. The plate identifiers and dilutions are then automatically assigned to the results and recorded. You can also read plate identifiers and dilutions using a barcode reader. ProtoCOL can apply sophisticated algorithms to distinguish touching colonies, but also allows you to add or remove colonies manually after an automatic count. You can also perform a completely manual count if required. If there are any problem areas on an individual plate where colonies cannot be distinguished for some reason, you can exclude them by defining an exclude region, and the missing area is then automatically allowed for in calculating the total count for the plate. All results are displayed and saved automatically, and any manual changes you make to a result are marked against the individual result with comments and coded flags. By default, an image of the plate is automatically saved with each result, and can be ProtoCOL Systems User Manual 1-1

8 Getting Started reloaded at a later time for auditing. You can also view a full audit history for any changes you have made to a result. Once you have counted the colonies on the plates, you can compile a report showing the results. This introduction has only mentioned a few of the features that make ProtoCOL such a powerful but easy tool to use. The rest of this manual and the on-line Help system give full instructions for using all of its many functions. Structure of the manual The rest of this manual is structured as follows: The next section of this Getting Started chapter provides a brief overview of the way you use ProtoCOL to count colonies. This involves two main phases: using Design mode to set up the system for detecting and counting colonies on a series of plates; using Measurement mode to actually count the colonies on the plates and record the results. This section also introduces you to the idea of a batch, which is used to hold the settings made in Design mode and then to store the results produced in Measurement mode. The final two sections of Chapter 1 show you how to start up and log in to ProtoCOL and then how to log out of it and close it down. Chapter 2, Capturing images, shows you how to load plates into ProtoCOL and capture an image of the plate. Chapter 3, Creating a new batch, shows you how to create a new batch and specify how colonies should be counted. Chapter 4, Counting colonies, shows you how to count the colonies on a series of plates. Chapter 5, Working with results, shows you how to view and edit the results of your measurements and how to create reports using the results. Chapter 3 is concerned largely with procedures used in Design mode, and Chapters 4 and 5 with procedures used in Measurement mode; Chapter 2 covers procedures common to both modes. 1-2 ProtoCOL Systems User Manual

9 Using ProtoCOL 3 to count colonies overview Using ProtoCOL 3 to count colonies overview ProtoCOL uses batches to define the settings used to detect colonies on plates and to store the results of the measurements you make using those settings. This means that before you can use ProtoCOL to take any measurements, you must have an open batch. You can do this by opening an existing batch or by creating a new batch. You can create a completely new batch or base a new batch on an existing one, either to use the same settings, or as a starting point for making new settings. ProtoCOL automatically uses the current batch to save your results, so once you have created a batch, you do not need to take any action to save it again. Each batch contains the settings and results for detecting and counting the colonies on a series of plates that have the same types of colony, use the same medium and are of the same type (Pour Plate, Spiral Plate, Dilution Series, Multi-Sector Plate, Ames or OPKA). Amongst other things, the settings in the batch define the classification of the colonies by color and, optionally, size and shape. The color classification is the primary classification and is used to distinguish between different types of colony according to their color (if required), and to distinguish between colonies and the plate background (medium) and any debris or other artifacts. Hence the first step in using ProtoCOL to count colonies is to create a batch and specify its settings using ProtoCOL in Design mode. Then, when you have completed the design, the second step is to accept the batch and switch ProtoCOL to Measurement mode, which you then use to count the colonies on each plate and record the results. ProtoCOL takes you through the various stages of designing the batch and classifying the colonies using wizards, and for most types of plate and colony, the classification can be performed completely automatically all you need to do is choose how many classifications you want and specify how the results should be interpreted: for example, whether a particular color represents a type of colony or background debris. However, you can also make manual settings to handle difficult cases. While you are working in Design mode, you can perform test measurements and experiment with and change the settings used to detect and classify the colonies. However, once you have accepted the batch, most of the batch settings, including the definitions of the color, size and shape classifications, are fixed and cannot be changed. There are some settings that can be changed in Measurement mode, but if required, they can also be locked when the batch is designed so that they cannot be changed in Measurement mode. ProtoCOL Systems User Manual 1-3

10 Getting Started Once the batch has been accepted for Measurement mode, the process of counting the colonies is very straightforward: put a plate in the plate holder, press a button and move on to the next plate. In most cases this is all you will need to do, but if there are any problems with difficult plates, you can add exclude regions, edit the results or manually add to or remove colonies from the count any changes to a result are recorded in an audit history for that result. Finally, once you have taken all the measurements, all you need to do to compile a report on the results is press a few buttons. Starting ProtoCOL 3 You can control ProtoCOL using the touch-sensitive screen, a mouse or keyboard shortcuts. See the ProtoCOL Quick Guide for how to set up the hardware ready for use. To turn on the ProtoCOL system: 1 Make sure the system is connected to a suitable mains power supply. 2 Switch on the system using the toggle switch on the rear of the unit. The orange LED on the front of the unit will light. 3 Switch on the built-in PC using the push switch on the right-hand side of the unit. The green LED on the front of the unit will light. 1-4 ProtoCOL Systems User Manual

11 Starting ProtoCOL 3 To start the ProtoCOL colony counting program: 1 Double-click on the ProtoCOL icon on the Windows Desktop to display the Please log on to ProtoCOL dialog box: s This dialog box is also displayed when you log out from ProtoCOL or if you are logged out automatically by ProtoCOL because you have not used it for a set period of time (5 minutes by default) see the next section for more details. 2 Enter your Windows Username the name you use to log on to Windows on this PC. 3 Enter your Windows Password the password you use to log on to Windows on this PC. ProtoCOL checks that the username/password is a valid combination to log on to Windows on this PC, but does not check that it is the currently logged on user. This means that you can log on to ProtoCOL while someone else is logged on to Windows on this PC. The default Username is P2Admin and the default Password is the serial number of your instrument, for example, P2C\xxxx. 4 Press OK. The ProtoCOL Application window will open. You can either create a new batch for counting colonies (see Creating a new batch using the Create New Batch Wizard, ProtoCOL Systems User Manual 1-5

12 Getting Started page 3-2) or open an existing batch if there is one (see Opening and selecting batches, page 4-2) and start counting colonies. Logging out and closing ProtoCOL 3 To log out from ProtoCOL : Press the Log out button near the top right-hand corner of the ProtoCOL application window: The Please log on to ProtoCOL dialog box will be displayed. s You will also be logged out automatically and this dialog box displayed if you do not use ProtoCOL for more than a set period of time (3 minutes by default). You (or someone else) can then: Log on again see previous section. Press Close to exit ProtoCOL. 1-6 ProtoCOL Systems User Manual

13 Capturing images This chapter shows you how to capture and work with images. You will find out about: Loading plates into the instrument see next section The Image tab see page 2-2 Capturing an image see page 2-4 Setting the exposure see page 2-4 Zooming the image see page 2-6 Exporting the image to a file see page 2-7 Importing an image from a file see page 2-7 Calibrating the image see page 2-8 Viewing batch images see page The view batch images procedure is used to view images captured during the measurement process, so is only available in Measurement mode. All of the other procedures described in this chapter are used in both Design and Measurement modes. Loading plates into the instrument To load a sample plate into the instrument: 1 Open the curtain doors at the front of the instrument by sliding them into the instrument. 2 If required, change the sample holder fitted in the instrument: Circular plate holders should be fitted with the plate stop pillars to the rear. Rectangular plate holders should be fitted with the plate stop pillars to the left and rear. ProtoCOL Systems User Manual 2-1

14 Capturing images ProtoCOL is supplied with five sample holders: transparent, translucent white and opaque black holders for circular plates; translucent white and opaque black holders for rectangular plates. Apart from the shape of plate, the choice of which type to use depends on the properties of the samples you are using and is best determined by experiment. The Image tab 3 Place the sample plate on the holder and push it up against the plate stop pillars to fix its position. For rectangular plates, push the plate against the two side pillars first, then slide it back along the side pillars until it hits the rear pillar. 4 Close the instrument doors. To display the image controls: Press the Image tab: 2-2 ProtoCOL Systems User Manual

15 The Image tab You use the controls on the Image tab to capture and work with images: When you are working in Measurement mode (see Using ProtoCOL to count colonies overview, page 1-3, for more information about Design and Measurement modes), there is an additional button: For instructions on how to use the image controls, see the following sections: Capturing an image, the next section Setting the exposure, page 2-4 Exporting the image to a file, page 2-7 Importing an image from a file, page 2-7 Viewing batch images, page See also: Zooming the image, page 2-6. ProtoCOL Systems User Manual 2-3

16 Capturing images Capturing an image To capture an image in ProtoCOL : 1 Press the Image tab: to display the image controls. 2 Press The image will be captured and displayed in the ProtoCOL window. In fact, three images will be captured, with red, green and blue illumination, respectively, and the results combined to form a color image this procedure produces more accurate results than can be obtained using a color camera. If the image is too light or too dark, you can adjust the exposure (see the next section) and capture the image again. Setting the exposure In Design mode the image controls have a padlock button allowing you to unlock or lock the controls in Measurement mode the pictures below were taken in Measurement mode, so no padlock appears. The exposure controls are disabled in Measurement mode if they were not unlocked in Design mode. See Using ProtoCOL to count colonies overview, page 1-3, for more information about Design and Measurement modes; see Allowing changes in Measurement mode, page 3-13, for information about locking and unlocking controls. 2-4 ProtoCOL Systems User Manual

17 The Image tab To set the exposure for capturing an image: 1 Press the Image tab: to display the image controls, including the exposure controls: 2 The current exposure time is shown between the arrow buttons. To change the exposure time, press: to decrease the exposure by 100 ms to decrease the exposure by 10 ms to increase the exposure by 10 ms to increase the exposure by 100 ms. Or: Drag the handle in the exposure slider bar: ProtoCOL Systems User Manual 2-5

18 Capturing images Zooming the image By default, the image is scaled to fit the image pane in the ProtoCOL window. To zoom the image: Press When it is zoomed, the button shows the scaling factor: To zoom out: Press To return to fitting the image to the image pane Press The zoom controls are also displayed with the image in dialog boxes you use them in exactly the same way as in the main ProtoCOL window. 2-6 ProtoCOL Systems User Manual

19 Zooming the image Exporting the image to a file To export the current image in the ProtoCOL window to a file: 1 Press the Image tab: to display the image controls. 2 Press to display the Export Image dialog box this is a standard Windows Save As dialog box. 3 Use the Export Image dialog box to: select an image format from the Save as type drop-down list select a folder to hold the image enter a file name for the image. 4 Press Save to save the file. Importing an image from a file To import an image from a file into the ProtoCOL window: 1 Press the Image tab: ProtoCOL Systems User Manual 2-7

20 Capturing images to display the image controls. 2 Press Calibrating the image to display the Import Image dialog box this is a standard Windows Open dialog box. 3 Use the Import Image dialog box to locate the folder holding the required image and select the image. 4 Press Open to open the file and load it into the ProtoCOL window. A warning dialog box will be displayed asking you to calibrate the image see the next section, Calibrating the image, for details. When you import an image from a file (see Importing an image from a file, page 2-7), or try to perform a measurement from an uncalibrated imported image, a warning dialog box will be displayed asking if you want to calibrate the image, for example: In order to create an accurate calibration you need to know the actual size of some feature in the image. To calibrate the image: 1 Press Yes in the warning dialog box to display the Calibrate Camera for Measurements dialog box. 2-8 ProtoCOL Systems User Manual

21 Zooming the image 2 Choose some horizontal or vertical feature of known length in the image, such as the plate itself, and click on one end of it: ProtoCOL Systems User Manual 2-9

22 Capturing images 3 Move the pointer to the other end of the feature as you do this, a line will be drawn back to the starting point: 4 Click to mark the other end of the feature. The calibration edit box and units button will be displayed on the page: 2-10 ProtoCOL Systems User Manual

23 Zooming the image 5 If required, to change the units: a Press the units button to display the units menu: b Select the required units from the menu. 6 Type the length of the known feature into the calibration edit box. 7 Press Viewing batch images to set the calibration and return to the main ProtoCOL window. When you create a new batch the image used for the color classification (see Color classification, page 3-22) is saved with the batch. After you have accepted the batch, when you take a measurement, the image is also saved with the batch. To view the color classification image and images used for measurements: 1 Press ProtoCOL Systems User Manual 2-11

24 Capturing images to display a dialog box: The box on the left-hand side lists the batch images, beginning with the color classification image followed by the measurement images listed in order of their plate identifiers and dilutions. 2 To view a measurement image, click on the dilution line within the entry for its plate identifier; to view the classification image, click on the batch identifier (the top line). 3 Press Close to close the dialog box and return to the main ProtoCOL window ProtoCOL Systems User Manual

25 Creating a new batch This chapter show you how to create and set up a new batch for counting colonies. It is divided into two main sections: Creating a new batch using the Create New Batch Wizard see next section. This section guides you through the choices presented by the Create New Batch Wizard. Setting up batches to count colonies see page This section describes: Design mode, including how to accept new batches for Measurement mode see page 3-11 How to allow users to make changes to settings when they are working in Measurement mode see page 3-13 How to use the Classification tab, including: Working with measurement frames see page 3-15 Creating a Color classification see page 3-22 The classification panel see page 3-40 Adding sub-classifications see page 3-42 Creating a Size classification see page 3-43 Creating a Shape classification see page 3-47 Creating a Grid classification see page 3-50 Changing an existing classification see page 3-56 How to use the Results tab to choose what to display in the Results table see page 3-63 ProtoCOL Systems User Manual 3-1

26 Creating a new batch How to use the Measure tab (see page 3-65), including: Specifying plate identifiers see page 3-67 Specifying dilutions see page 3-81 Carrying out a test measurement see page Many of the operations described in the Setting up batches to count colonies section for Design mode may also be carried out in Measurement mode, though in some cases only if they have been unlocked in Design mode when the batch was being set up (see Allowing changes in Measurement mode, page 3-13). Creating a new batch using the Create New Batch Wizard Before you can start counting the colonies on plates you need to set up and create a batch to define how the colonies should be counted, and to hold the results. The Create New Batch Wizard guides you through the process of creating the new batch. Once you have created the new batch, you will still need to set it up and test it before you can start using it to count the colonies on plates see Setting up batches to count colonies, page 3-11, for details. To run the Create New Batch Wizard: 1 Press the batch button: If there is no batch open currently, the batch button is labeled Create or Open Batch: If there is already a batch open in ProtoCOL, the batch button will show the name of the current batch, for example: 3-2 ProtoCOL Systems User Manual

27 Creating a new batch using the Create New Batch Wizard The Start menu will be displayed: 2 Press Welcome to run the Create New Batch Wizard. The Create New Batch Wizard takes you through the process of creating the batch one step at a time by displaying different pages according to the selections you have made. The following sections describe each of these in turn. ProtoCOL Systems User Manual 3-3

28 Creating a new batch By default, the Create New Batch Wizard displays a welcome page when it starts up. If you wish, you can prevent this happening by unchecking Always show this welcome page. The Welcome page cannot be re-enabled from within ProtoCOL. To display the next step in the Wizard: Press Next to display the Batch Name and Measurement Type page see the next section for how to use this page. Batch Name and Measurement Type The Batch Name and Measurement Type page is displayed after you press Next in the welcome page (see previous section) or, if the welcome page has been disabled, immediately after you start up the Create New Batch Wizard: 3-4 ProtoCOL Systems User Manual

29 Creating a new batch using the Create New Batch Wizard If the zone measurement option was purchased with your ProtoCOL system, the dialog box will also include a Zone Measurement radio button see the ProtoCOL Zone Measurement User Manual for details. 1 Enter a Batch Name for the new batch. 2 If you want the new batch to have the same settings as an existing batch: a b Select Base new batch on existing batch. Press Next to display the Derive from existing Batch page see the following section. If you want to create a completely new batch from scratch: a Select Plate Counting. b Press Next to display the Plate Type page see Plate Type, page 3-7. ProtoCOL Systems User Manual 3-5

30 Creating a new batch Derive from existing Batch The Derive from existing Batch page is displayed when you press Next in the Batch Name and Measurement Type page (see previous section) after choosing Base new batch on existing batch: Batches are marked in the Available column if they are currently open in ProtoCOL you cannot base a new batch on an existing batch if it is open. To base a new batch on an existing batch: 1 If there are a large number of existing batches and the batch you want to use as a basis for the new batch is not shown in the list, to filter the list according to the batch names: a Type the name, or a part of the name, of the required batch into the Batch Name box. b Press. 3-6 ProtoCOL Systems User Manual

31 Creating a new batch using the Create New Batch Wizard Plate Type Only batches with names that contain the text you entered will be listed. 2 Select the required batch in the list. 3 Press Create to create the new batch. When you press Create, the new batch will be created and loaded into ProtoCOL in Design mode ready for you to set it up and test it before using it to carry out and record measurements see Setting up batches to count colonies, page 3-11, for details. The Plate Type page is displayed when you press Next in the Batch Name and Measurement Type page (see page 3-4) after choosing Colony Counting: The plate types listed in your system will depend on the options purchased with ProtoCOL. ProtoCOL Systems User Manual 3-7

32 Creating a new batch To create a completely new batch: Configure Measurements 1 Select the type of plate you wish to use in the new batch. 2 Press Next to display the Configure Measurements page see next section. The controls included on the Configure Measurements page depend on whether you selected Spiral Plate or one of the other types on the Plate Type page (see the previous section). Spiral Plate 3-8 ProtoCOL Systems User Manual

33 Creating a new batch using the Create New Batch Wizard 1 To change the units used for the Spiral Plate Diameter: a Press the Spiral Plate Diameter unit button to display a units menu: b Select the required unit from the list. 2 Type the diameter of the plates you will be using in this batch into the Spiral Plate Diameter box. 3 Press the Spiral Volume button corresponding to the volume dispensed by the spiral plater used the selected button is highlighted in orange. 4 Press the Measurement Units unit button to display the units menu and select the units to be used for measuring the colony sizes. 5 Press Create to create the new batch. When you press Create, the new batch will be created and loaded into ProtoCOL 3 in Design mode ready for you to set it up and test it before you use it to carry out and record measurements see the next section, Setting up batches to count colonies, for details. ProtoCOL Systems User Manual 3-9

34 Creating a new batch Other plate types 1 Select the Circular Plate or Rectangular Plate radio button to specify the shape of plates you are going to use. The Rectangular Plate radio button is disabled for Multi-Sector Plate batches. 2 To change the units used for the plate dimensions: a Press the unit button to display a units menu: b Select the required unit from the list ProtoCOL Systems User Manual

35 Setting up batches to count colonies 3 Enter the Diameter (circular plates) or Width & Height (rectangular plates) of the plates you will be using in this batch. 4 Enter the Sample volume you will be using in this batch you can change the units used by pressing the unit button. 5 Press the Measurement Units button to display the units menu and select the units to be used for measuring the colony sizes. 6 Press Create to create the new batch. When you press Create, the new batch will be created and loaded into ProtoCOL 3 in Design mode ready for you to set it up and test it before using it to carry out and record measurements see the next section for details. Setting up batches to count colonies Design mode accepting and rejecting new batches Once you have created a new batch (see previous section), it will be loaded into ProtoCOL 3 in Design mode for final set-up and testing before you use it to carry out and record measurements. The steps you will need to follow before you can start carrying out measurements depend on the type of plate you are using and on whether you based the new batch on an existing batch. At the very least, you will need to carry out a test; this is the case even if the new batch is based on an existing batch and you do not want to change any of the settings. While ProtoCOL 3 is in Design mode: The message area at the bottom of the ProtoCOL 3 window shows, for example: where Test 3 is the name of the new batch. ProtoCOL Systems User Manual 3-11

36 Creating a new batch The Discard New Batch button appears near the top left corner of the ProtoCOL 3 window: Press Discard New Batch to abort the new batch you will be asked to confirm that you want to do this. The Accept New Batch button appears near the top left corner of the ProtoCOL 3 window. Initially, the button will be disabled: When you have completed all the required procedures for setting up the batch, including testing it, the button will become enabled: Press Accept New Batch to confirm that you have completed the set-up procedure and want to proceed to taking measurements. The Accept New Batch button is enabled to show that you can proceed to taking measurements if you wish you can still make further changes to the set-up if required. Once you have pressed Accept New Batch after setting up the batch, there will be restrictions on the changes you can make to the settings while you carry out the measurements. In fact, part of the set-up procedure is to choose what settings can be changed after the batch has been accepted see the next section for details. The sections following that describe all of the other settings, which are grouped together on the following tabs: Classification see page 3-14 Measure see page ProtoCOL Systems User Manual

37 Setting up batches to count colonies Results see page Allowing changes in Measurement mode By default, most of the settings you make when creating a batch are fixed permanently when you press Accept New Batch. However, you can unlock some of these settings so that they can be changed for individual plates after you have begun taking measurements. Settings that can be locked or unlocked have a padlock button. For example, the exposure time on the Image tab: In some cases there are two or more lockable/unlockable settings connected with a control, such as the frame button on the Classification tab: The two padlocks on the button show that there are two or more settings that can be locked/unlocked the button shows two padlocks even if there are more than two lockable settings. To lock or unlock a setting: 1 Press its padlock button to display a menu listing all the settings that can be locked/unlocked. For example: 2 Choose the setting you want to lock or unlock. ProtoCOL Systems User Manual 3-13

38 Creating a new batch The padlock on the button is red and closed when a setting is locked; it is green and open when it is unlocked. The lock status is also shown in the menu, for example: If a control has more than one lockable setting, the padlocks show whether: all the settings are locked both padlocks red and closed; all the settings are unlocked both padlocks green and open; at least one setting is locked and at least one setting is unlocked the front padlock is green and open and the rear padlock is red and closed (to find out which settings are locked/unlocked, press the padlock button to display the menu), for example: Remember that locking/unlocking a setting only affects the ability to change the setting once you have started making measurements; it has no effect on your ability to change the setting while the batch is still in Design mode. When a setting is unlocked, you use the same procedure to adjust it in both Design and Measurement modes. See Locking and unlocking frame adjustment, page 3-18, for how to lock and unlock frame adjustment in Measurement mode once it has been unlocked in Design mode. Classification The classification tab in Design mode allows you to set the following properties: Frame see next section Color classification see page 3-22 Size classification see page 3-43 Grid classification see page ProtoCOL Systems User Manual

39 Setting up batches to count colonies Shape classification see page 3-47 and to Modify an existing classification see page Frame Choosing the type of frame to use The frame defines the area within which colonies will be counted. The choice of frames depends on the type of batch: Pour Plate, Dilution Series and Ames: the frame can be circular, rectangular or a single sector of a circle; Multi-Sector Plate: the frame is a circle divided into the selected number of sectors; OPKA: the frame is a rectangle divided into a grid of rectangular sectors; Spiral Plate: Whole Frame: circular Two Sector: a series of concentric circles, with two opposite highlighted sectors. For example: ProtoCOL Systems User Manual 3-15

40 Creating a new batch To change the type of frame used to define the region for counting colonies: 1 Press the frame button to display the frame menu. 2 Choose the required option the contents of the menu depend on the type of plate and are described in the following subsections. You cannot change the frames for an OPKA batch after you have accepted its design for Measurement mode. When you are creating other batch types, you can choose to unlock or lock the Change Frame command using the padlock button on the frame button. When it is unlocked, you will be able to change the type of frame for an individual plate when you are taking results in Measurement mode see Allowing changes in Measurement mode, page Pour Plate, Dilution Series and Ames batches You can choose Circular Frame or Rectangular Frame, or to use a frame covering a single sector only, 1/2, 1/4, 1/8 or 1/10 from the Partial Plate Frame submenu: Partial plate frames can be useful when you have plates that are heavily loaded with bacteria, or if it there is some problem with a plate and you just want to read results from an unaffected part. This means that you are more likely to want to choose a partial plate frame for an individual plate while you are working in Measurement mode rather than for all the plates in a batch when you are creating it in Design mode. However, remember that in order to do this, you must unlock the Change Frame command see Allowing changes in Measurement mode, page You may also want to be able to adjust the partial plate frame once you have selected it, for example to rotate it, which means that you must also unlock the Adjust Frame command see Locking and unlocking frame adjustment, page ProtoCOL Systems User Manual

41 Setting up batches to count colonies You can also avoid counting in problematic areas of individual plates by placing Exclude regions over them see Exclude regions, page 4-8. Spiral Plate batches You can choose Whole Frame or 2 Sector: As a general rule, you should use Whole Frame if there has only been a small amount of colony growth, otherwise, use 2 Sector. Multi-Sector Plate batches You can choose the number of sectors required from the Multi-Sector Frame submenu: OPKA batches (Design mode only): You can choose the number of columns and rows in the grid of OPKA frame sectors from the OPKA Frame submenu: Adjusting the position and size of frames You should load a plate from the batch into the instrument and capture an image before adjusting the frame see Loading plates into the instrument, page 2-1, and Capturing an image, page 2-4. ProtoCOL Systems User Manual 3-17

42 Creating a new batch Locking and unlocking frame adjustment In Design mode, you can use the padlock button on the frame button to lock or unlock Adjust Frame to prevent or allow adjustments to the frame position and size when you are taking results in Measurement mode see Allowing changes in Measurement mode, page s You cannot unlock Adjust Frame for OPKA batches; you cannot adjust the size of spiral frames. In Measurement mode, as a further safeguard against accidental changes, you can also allow or prevent frame adjustment within Measurement mode itself. To allow or prevent frame adjustment in Measurement mode: Click the Adjust check box: Frame adjustment is allowed when the Adjust check box is checked. The Adjust check box is disabled in Measurement mode if Adjust Frame is locked in Design mode. The Adjust check box is always disabled for OPKA frames since you cannot unlock Adjust Frame for OPKA batches. The Adjust check box also appears in Design mode, but has no function there. When frame adjustment is allowed in Measurement mode, you use the same procedures for adjusting the position and size of frames as in Design mode the instructions in the following sections apply in both cases ProtoCOL Systems User Manual

43 Setting up batches to count colonies Moving frames To move a frame: 1 Move the pointer near to the center of the frame an orange circular drag handle will appear in the center of the frame: s If you are working in Measurement mode and the orange circle does not appear, make sure the frame is unlocked and Adjust is selected see Locking and unlocking frame adjustment, page Drag the frame and drop it in the required position. Resizing frames Generally, frames should be made as large as possible within the area of the plate but avoiding the very edge (as this may produce spurious results) and allowing some margin for variation between plates. You can unlock frame adjustment (see previous section) to allow the frame size/position to be adjusted for individual plates in Measurement mode, but it will save time if you can use a single setting for all the plates in a batch. You cannot resize a spiral frame. ProtoCOL Systems User Manual 3-19

44 Creating a new batch To resize a frame: 1 Move the pointer near to the frame boundary (for Partial Plate frames, the frame boundary is the circle of which the frame forms a sector). The frame boundary will turn orange showing that you can adjust its size: If you are working in Measurement mode and the frame boundary does not turn orange, make sure the frame is unlocked see Locking and unlocking frame adjustment, page Drag the frame boundary to resize/reshape the frame. For rectangular frames, drag the top or bottom side to adjust the height; drag the left or right side to adjust the width; drag a corner if you want the frame to be forced to be a square when you adjust the size ProtoCOL Systems User Manual

45 Setting up batches to count colonies Rotating frames You can rotate 2-Sector Spiral Plate frames, Partial Plate frames (any type) and Multi-Sector frames. To rotate a frame: 1 Move the pointer to a point about half way between the center and edge of the frame. An orange line will appear joining the pointer to the center: s If you are working in Measurement mode and the orange line does not appear, make sure the frame is unlocked see Locking and unlocking frame adjustment, page Drag the line around the center to rotate the frame. ProtoCOL Systems User Manual 3-21

46 Creating a new batch Color classification In order to count the colonies on a plate, ProtoCOL 3 has to distinguish between the different types of colony, the background and any debris on the plate. It does this through the process of color classification. If you base a new batch on an existing batch, it will use the same color classification as the existing batch by default (you can change the color classification if required). If you are creating a completely new batch, you will not be able to accept it for counting colonies until it has been color classified. To carry out a color classification procedure: 1 Insert a typical plate from the batch into ProtoCOL 3 see Loading plates into the instrument, page 2-1. This plate should have the full range of colony, background and debris colors that appear on plates in the batch. 2 Capture the image see Capturing an image, page Press to start the Colony Classification Wizard for color classification ProtoCOL Systems User Manual

47 Setting up batches to count colonies The Configure Color Classification page will be displayed in the wizard: If there is no current image, an image will be automatically captured when you press the Color Classification button. If the image shown in the wizard is too light or too dark, press Cancel to close the wizard and adjust the exposure accordingly (see Setting the exposure, page 2-4), then press the Color Classification button again to restart the wizard. 4 If required, you can move and/or resize the frame shown on the image using the same techniques as in Adjusting the position and size of frames, page This may be useful in restricting the area used for color classification. The frame in the Colony Classification Wizard defines the area used for color classification adjusting the color classification frame has no effect on the size or position of the main colony counting frame. ProtoCOL Systems User Manual 3-23

48 Creating a new batch 5 If required, you can zoom the image in the same way as in the main window see Zooming the image, page By default, ProtoCOL 3 automatically compensates for any irregularities in the illumination of the plate this is called background correction. In most cases, automatic background correction produces excellent results, but you may find that if the plates in a batch have some unusual properties, you can get better results by manually adjusting the background correction parameters, or by disabling background correction completely. You should only use manual background correction after you have tried using automatic background correction and found that it does not produce satisfactory results. To set the background correction manually, or to disable background correction: Press the Automatic background correction check box so that it becomes unchecked (see the next section for how to carry out the manual background correction procedure). To allow ProtoCOL 3 to carry out the background correction automatically: Leave Automatic background correction checked. 7 In this step you choose whether to make ProtoCOL 3 carry out an automatic color classification of the colonies or to carry out a manual color classification yourself: To make ProtoCOL 3 carry out an automatic color classification of the colonies: a Decide on the number of colors to be classified on the plate (excluding the background): this is the number of distinct colony types plus the number of distinct colors of debris ProtoCOL Systems User Manual

49 Setting up batches to count colonies As an example, suppose the plates have colonies with two different colors but no debris. If you want the results to show separate totals for the two different colors of colony, you should set the number of colony types to 2, but if you just want the total number of colonies, irrespective of color, the number of colony types should be set to 1. However, the automatic color classification may have problems if there are more than two colors and you want to distinguish between some of them, but not others: for example, if there are two colony colors that you want to count together, but also some debris that you want to distinguish from the colonies. In this case, you could: Set the number of distinct colony types plus the number of distinct colors of debris to 2, and check whether the automatic color classification successfully distinguishes the colonies from the debris (see Review color classification, page 3-33) if it does not, you can press Back to return to the Configure Color Classification page to try one of the following alternatives. Set the number of colors for automatic color classification to be three and combine the results for the different colony colors later (outside ProtoCOL 3) Use manual color classification. b Select the Automatic Classification radio button corresponding to the number of different colors to be classified. If you want to enter a number greater than 3, select More and press the arrow buttons to set the required number. To set the color classification manually: 8 Press Next. Select the Choose colors in next step radio button. ProtoCOL Systems User Manual 3-25

50 Creating a new batch What happens next depends on the selections you have made: If you have selected manual background correction, the wizard will display the Background Correction page see the next section, Manual background correction, for instructions. Otherwise (ie you have selected automatic manual background correction): If you have selected manual color classification, the wizard will display the Manual Color Classification page see Manual color classification, page 3-29, for instructions. Otherwise (ie you have selected automatic background correction and automatic color classification), the automatic colony color classification will be carried out immediately and the wizard will then display the Review Color Classification page showing the results see Review color classification, page 3-33). Manual background correction In most cases the ProtoCOL 3 automatic background correction produces very good results and should be used in the first instance. In exceptional cases, however, you may find you can get better results by using manual background correction, or no background correction at all. You should only use manual background correction after you have tried using automatic background correction and found that it does not produce satisfactory results ProtoCOL Systems User Manual

51 Setting up batches to count colonies If you chose not to carry out automatic background correction (see Step 6 in the previous section), the Background Correction page is displayed in the Color Classification Wizard: 1 If you do not want to apply any background correction, press the Use Background Correction check box to uncheck it and go to Step 5. Otherwise, leave Use Background Correction checked and go on to the next step. 2 If required, you can zoom the image in the same way as in the main window see Zooming the image, page Adjust the Detect and Reject sliders to optimize the background correction so that the areas shown as background/non-background features in the colormap image correspond as accurately as possible to the actual background/ non-background in the image (see following note). ProtoCOL Systems User Manual 3-27

52 Creating a new batch You can drag the sliders, press the arrow buttons to adjust the sliders by a single step or click anywhere on a slider bar to set the slider to that position. You can view the effect of the background correction by pressing the display button (labeled Colormap Image in the example picture). You can view: Colormap Image: this uses contrasting colors to show which areas of the image have been detected as the background and which as foreground features Original image: the uncorrected image Corrected image: the image after the background correction has been applied to the image. 4 If you wish to revert to the automatic background correction settings (Detect and Reject both set to 5.0), press 5 When you are satisfied with the background correction, press Next. What happens next depends on the selections you made earlier (see the previous section): If you selected manual color classification, the wizard will display the Manual Color Classification page see Manual color classification, page 3-28, for instructions. Otherwise (ie you selected automatic color classification) the automatic color classification will be carried out immediately and the wizard will then display the Review Color Classification page showing the results see Review color classification, page 3-33) ProtoCOL Systems User Manual

53 Setting up batches to count colonies Manual color classification If you selected Choose colors in next step in the first page of the wizard see color classification, 3-22 (Step 7), the Manual Color Classification page is displayed in the Colony Classification Wizard : You use the Manual Color Classification page to enable ProtoCOL 3 to distinguish the colonies you want to count from the background and any debris you want ignored. You do this by identifying the distinct colony types, and debris and background colors appearing in the image and creating a color channel for each of them. You then define the color of each color channel by taking samples of the corresponding color from the image. However, with the exception of the default background channel, you do not specify at this stage whether each channel corresponds to a colony type, debris or background you will do this in the Classification Details page later in the wizard (see Classification Details, page 3-37). ProtoCOL Systems User Manual 3-29

54 Creating a new batch You must define at least one background color channel, but you can add further background channels if required, for example, if the background has some patches of different colors. You can then add as many debris and colony channels as required. However, you should bear in mind that when you perform a count, the results will show a separate count for each colony channel you define. This means that if the plates have colonies with different colors, but you do not want to distinguish between them in the results (you just want the total number of colonies, irrespective of color), you should define a single color channel for these colonies, and sample all of the different colony colors to define that channel. To carry out a manual color classification: 1 Click on the Click here to select background item at the bottom of the channel list to select it. The selected item will be highlighted in orange and the wording changed: 2 In the image, click on the background to sample its color. When you click, a circle showing a sample of the color you clicked on will appear in the color channel: 3-30 ProtoCOL Systems User Manual

55 Setting up batches to count colonies 3 If you click at the wrong point and want to remove the last color sample added: Press This removes the last color sample from the currently selected color channel if the required channel is not currently selected, click on it first to select it. 4 Repeat Steps 2 3 as required to select a representative sample covering the full range of the background color as it appears in the image. 5 Identify the next color in the image for which you want to define a color channel: it may be a colony color, a debris color or another background color you will specify which it is in the Classification Details page later in the wizard (see page 3-37). 6 Either, if an undefined color channel already exists:: Click on the Click here to select this colony item for the channel to select it. Or, if there are currently no undefined color channels: Press to create a new channel the new channel will be selected. The selected channel will be highlighted in orange and labeled Click on image to sample colony color (see pictures in Step 1). 7 If required, you can zoom the image in the same way as in the main window see Zooming the image, page 2-6. Zooming the image may help you make sure you click accurately on colonies or debris when you are selecting color samples in Step 8. 8 In the image, click on areas of the image (colony, debris or background) that have the required color you should select a representative sample covering the full range of the color as it appears for these objects in the image. ProtoCOL Systems User Manual 3-31

56 Creating a new batch Remember that when you perform a count, the results will show a separate count for each colony channel you define if you do not want the results to distinguish between different colored colonies, you should sample all of the colony colors for a single colony channel. When you click, a circle showing a sample of the color you clicked on will appear in the color channel: See Step 3 if you want to remove the last sample added. 9 When you have added a representative number of samples to the channel: If you want to add another color channel, go back to Step 5. If you do not want to add another color channel, go to Step If after adding samples for all the colors appearing in the image there are any unused channels, or if you want to remove a channel you have added: a b Click on the Click here to select this colony item for the first channel you want to remove to select it (you cannot remove the default background item). Press c Repeat Steps a and b for any other channels you want to remove. 11 Press Next to display the Review Color Classification page see next section. (Next is disabled if there are any unused channels see previous step.) 3-32 ProtoCOL Systems User Manual

57 Setting up batches to count colonies Review Color Classification The Review Color Classification page is displayed in the Colony Classification Wizard after you have carried out an automatic (see page 3-22) or manual color classification (see previous section): You can press the display button (labeled Colormap Image All Colony Markers in the example picture) and choose to display the image as the: Colormap Image: this uses contrasting colors to show which areas of the image have been detected as the background and which as foreground features Original Image: the original uncorrected image Corrected Image: the image after the background correction has been applied to the image. ProtoCOL Systems User Manual 3-33

58 Creating a new batch You can also press the display button to choose how to mark the detected colonies on the image. Here colonies means non-background parts of the image that have one of the classified colors (see previous sections), which may include different types of colony, different colors of the same type of colony, or debris. You will be able to specify whether each of these colony types should be counted as a colony in the Classification Details page later see next section. You can choose to display: No Colony Markers All Colony Markers: each detected colony is identified with a symbol showing the type of colony (color classification) a key to the colony symbols is shown to the right of the image All Colony Outlines: the boundary of each detected colony is marked in the color of the symbol representing the type of colony All Colony Regions: the area of each detected colony is marked in the color of the symbol representing the type of colony. If two or more colonies overlap, they will be shown as a single colony. You can choose to use the colony splitter to separate overlapping colonies see later in this section. ProtoCOL 3 applies a size filter to remove the effect of small colored regions of the image that may be wrongly interpreted as colonies. You can use the Reject Small Particles slider bar to set the level of size filtering used. To adjust the Reject Small Particles slider: Drag the slider, click the arrow buttons at the ends of the slider to adjust the slider by a single step or press anywhere on the slider bar to set the slider to that position. To choose whether or not to use the colony splitter for a colony type to separate overlapping colonies: Click on the Split check button next to the colony type: 3-34 ProtoCOL Systems User Manual

59 Setting up batches to count colonies For example: Split unchecked Split checked The settings for the Reject Small Particles slider and colony splitter you make here will be used to perform the initial counts for plates when you are working in Measurement mode. However, you will be able to vary these settings for individual plates by editing the measurement after you have performed the count see Manually editing the detected colonies, page 5-11, for details. To review the color classification: 1 Examine the detected colonies and image carefully to check that: the colonies in the image are being detected successfully different colors of colony are being distinguished successfully the colonies are distinguished from debris with a different color. It may help to change the way the image is displayed and/or the way the colonies are marked. Also, it may be easier to see the results if you zoom in on areas of the image: you can zoom the image in the same way as in the main window see Zooming the image, page If there are any problems with the colony detection/discrimination: If small debris particles are wrongly detected as colonies, try increasing the Reject Small Particles setting. If small colonies are being wrongly rejected, try decreasing the Reject Small Particles setting. If colors are being wrongly classified (for example, if colonies are not being detected because they are being treated as background, or if colonies and debris with different colors are not being distinguished accurately): ProtoCOL Systems User Manual 3-35

60 Creating a new batch If you performed an automatic color classification, try pressing Back until you get to the Configure Color Classification page and try increasing or decreasing the number of colors you selected, or try using a manual color classification instead see Manual color classification, page If you performed an automatic background correction, try pressing Back until you get to the Configure Color Classification page and use a manual background correction instead see Manual background correction, page When you are happy that the color classification is as good as possible, press Next to display the Classification Details page see the next section ProtoCOL Systems User Manual

61 Setting up batches to count colonies Classification Details You use the Classification Details page to specify whether the detected colors correspond to colonies, debris or background, and, if there is more than one type of colony color, what organisms they correspond to. The left-hand pane of the Classification Details page shows the image with the colony markers superimposed. The right-hand pane lists the classified colors in the image and shows whether they represent colonies, debris or the background, with a button allowing you to change the classification: the button for a colony type is labeled with the colony name and marker, otherwise it is labeled Debris or Background. ProtoCOL Systems User Manual 3-37

62 Creating a new batch To change the classification for a color: 1 Press the classification button to display the classification menu: When the current classification is Colony, the classification details panel is shown alongside the classification menu see example in next instructions. 2 Select Colony, Debris or Background, as required. If necessary, you can assign more than one color to Debris or Background. The button will show the selected classification type: Debris, Background or the name of the colony type. The following instructions show you how to change the classification details for a colony type produced by the color classification. However, if you intend to use one of the other classifications (size, shape or grid see Adding sub-classifications, page 3-42) as a sub-classification for this colony type, you can omit this step, as any settings you make here will be overridden by the sub-classification settings. To change the classification details for a colony type: 1 Press the colony classification button to display the classification details panel next to the classification menu: 3-38 ProtoCOL Systems User Manual

63 Setting up batches to count colonies 2 Edit the Colony Name as required for example, to show the name of the organism forming the colonies. 3 If you do not want to change the colony marker or color, press the Return key or press 4 Select the shape of colony marker you want to use from the Colony Marker panel. The classification menu and panel will close after you have selected the colony marker, so you will need to go back to Step 1 if you want to carry out any more changes. 5 To change the color used for the colony marker, press the Color button to display the Standard Colors control: 6 Click on the required color in the Standard Colors control. When you have completed the classification details, press Finish to complete the color classification and return to the main ProtoCOL 3 window. ProtoCOL Systems User Manual 3-39

64 Creating a new batch Classification panel The Classification panel appears below the frame button in the classification tab and shows the current classification: In this example, the only classification is a color classification with a single colony type A and the Background. If you add sub-classifications (see next section), the classification will appear as a tree see the following sections for examples. As well as showing you the current classification, the Classification panel allows you to change the colony marker and color (but not the name), and control whether changes can be made to the colony markers and colors when the batch is in Measurement mode. To change the colony marker or color: 1 Move the pointer over the colony item to display the colony button: 3-40 ProtoCOL Systems User Manual

65 Setting up batches to count colonies 2 Press the colony button to display the marker controls: 3 Either: Or: Select the shape of colony marker you want to use from the marker controls. a Press Color to display the Standard Colors control: b Click on the required color in the Standard Colors control. To allow/prevent changes to the colony markers or colors in Measurement mode: 1 Click on the padlock icon at the top left corner of the classification panel to display the lock menu: 2 Select Change Marker. The padlock icon and lock menu show the current status of the lock see Allowing changes in Measurement mode, page 3-13, for more details. ProtoCOL Systems User Manual 3-41

66 Creating a new batch Adding sub-classifications Once you have added a color classification to the batch, the other classification buttons become enabled. You cannot add a color sub-classification if you press the Color Classification button, you will be asked if you want to discard the current color classification and all of its sub-classifications, if there are any, and replace it with a new color classification. You can add a size or shape sub-classifications to any existing colony type. You cannot add a grid sub-classification to a colony type that has any other sub-classifications a grid sub-classification must be the highest level sub-classification. Choosing where to add the sub-classification When you press one of the sub-classification buttons, the appropriate wizard opens. However, if there is already more than one colony type, the first step in all of these wizards is the same, for example: The left-hand panel shows the image with colony markers; the right-hand panel shows the current colony classification as a list. The first step in adding a new classification is to specify which of the existing colony types you want to divide with the new classification ProtoCOL Systems User Manual

67 Setting up batches to count colonies The wizards will skip this step if there is currently only one colony type, since in that case there is no choice to be made. To choose where to add the new classification: Size classification Click in the classification list on the existing colony that you want to divide. The Add Size Classification button becomes enabled once you have performed a color classification (see Color Classification, page 3-22). You can use a size classification to distinguish between different sizes of a particular colony type, or to set maximum and/or minimum size limits for colonies of that type to be counted. You can use the Reject Small Particles slider in the Review color classification page of the Color classification wizard (see page 3-33) to set a minimum size for colonies of all colony types. To add a size classification to the batch so that you can distinguish between colonies or between colonies and debris according to their sizes: 1 If the current image does not have the full range of sizes appearing on plates in the batch, you can replace it by loading a plate having the full distribution (see Loading plates into the instrument, page 2-1) and capturing a new image (see Capturing an image, page 2-4). 2 Press to start up the Size Classification Wizard. If there is already more than one colony type in the colony classification, the Add New Size Sub-Classification page will be displayed go to Step 3; otherwise (if there is currently only one type of colony in the colony classification), the wizard will skip straight to the Configure Size Classification page go to Step 5. ProtoCOL Systems User Manual 3-43

68 Creating a new batch 3 In the Add New Size Sub-Classification page, click on the existing colony type that you want to subdivide with the new size classification (see Choosing where to add the sub-classification, page 3-42, for more details). 4 Press Next to display the Configure Size Classification page: 5 In the Configure Size Classification page (see picture in previous step), click on the radio button for the number of size sub-classifications you want to add or, if you want to add more than four, click on More and use the arrow buttons to set the required number ProtoCOL Systems User Manual

69 Setting up batches to count colonies 6 Press Next to display the Review Size Classification page: The Review Size Classification page allows you to review and adjust the automatic size classification results. 7 Examine the marked colonies on the image to check whether the size classification is as required. It may be easier to see the results if you zoom in on areas of the image (as in the example above): you can zoom the image in the same way as in the main window see Zooming the image, page 2-6. It may also help to change the way the colonies are marked by pressing the display button (labeled All Colony Markers in the picture). 8 If required, adjust the boundaries between the different size classifications: a If necessary, scroll to the classification you want to adjust. ProtoCOL Systems User Manual 3-45

70 Creating a new batch If the number of size ranges is larger than the space available, a scroll bar will appear so that you can display the required controls, for example: b Adjust the upper or lower size boundary for the classification by typing in the edit box or clicking on the up or down arrow buttons attached to the right-hand side of the edit box. The lower/upper boundary of the adjacent classification will be adjusted accordingly and the results of the new classification shown. 9 When you are satisfied with the classification, press Next to display the Classification Details page showing the new classification tree: 3-46 ProtoCOL Systems User Manual

71 Setting up batches to count colonies 10 Use the classification tree controls in the Classification Details page in the same way as in the Color Classification Wizard (see Classification Details, page 3-37) to specify for each sub-classification: whether it corresponds to colonies, debris or background and for colony sub-classifications (if you are not going to sub-classify them any further): the name to be used to identify it the marker used to identify it on the image the color used for the marker. 11 When you have completed the classification details, press Finish to return to the main ProtoCOL 3 window, which will show the updated classification tree. Shape classification The Add Shape Classification button becomes enabled once you have performed a color classification (see Color Classification, page 3-22). You can use a shape classification to distinguish differently shaped colonies as different types or classes of colony, or to distinguish between colonies and unevenly shaped debris. To add a shape classification to the batch so that you can distinguish between colonies and/or debris according to their shape (how close they are to being circular): 1 If the current image does not have the full range of shapes appearing on plates in the batch, you can replace it by loading a plate having the full distribution (see Loading plates into the instrument, page 2-1) and capturing a new image (see Capturing an image, page 2-4). 2 Press to start up the Shape Classification Wizard. ProtoCOL Systems User Manual 3-47

72 Creating a new batch If there is already more than one colony type in the colony classification, the Add New Shape Sub-Classification page will be displayed go to Step 3; otherwise (if there is currently only one type of colony in the colony classification), the wizard will skip straight to the Configure Shape Classification page go to Step 5. 3 In the Add New Shape Sub-Classification page, click on the existing colony type that you want to subdivide with the new shape classification (see Choosing where to add the sub-classification, page 3-42, for more details). 4 Press Next to display the Configure Shape Classification page: 5 In the Configure Shape Classification page (see picture in previous step), click on the radio button for the number of shape sub-classifications you want to add or, if you want to add more than four, click on More and use the arrow buttons to set the required number ProtoCOL Systems User Manual

73 Setting up batches to count colonies 6 Press Next to display the Review Shape Classification page: The Review Shape Classification page allows you to review the automatic shape classification results. 7 Examine the marked colonies on the image to check whether the shape classification is as required. It may be easier to see the results if you zoom in on areas of the image (as in the example above): you can zoom the image in the same way as in the main window see Zooming the image, page 2-6. It may also help to change the way the colonies are marked by pressing the display button (labeled All Colony Markers in the picture). 8 If the automatic shape classification has not produced the required results, press Back to return to the Configure Shape Classification page and try selecting a different number of sub-classifications see Step 5. ProtoCOL Systems User Manual 3-49

74 Creating a new batch 9 When you are satisfied with the new classification, press Next to display the Classification Details page showing the new classification tree: 10 Use the classification tree controls in the Classification Details page in the same way as in the Color Classification Wizard (see Classification Details page 3-37) to specify for each sub-classification: whether it corresponds to colonies, debris or background and for colony sub-classifications (if you are not going to sub-classify them any further): the name to be used to identify it the marker used to identify it on the image the color used for the marker. 11 When you have completed the classification details, press Finish to return to the main ProtoCOL 3 window, which will show the updated classification tree. Grid classification The Add Grid Classification button becomes enabled once you have performed a color classification (see Color Classification, page 3-22), but is disabled again if all the colony types already have a sub-classification. When you are using plates that have a grid pattern, the grid lines may interfere with the detection of colonies that have a similar color to the grid. The grid classification allows you to remove the effects of the grid on the count ProtoCOL Systems User Manual

75 Setting up batches to count colonies To add a grid classification to the batch: 1 If the current image does not show a typical grid pattern for the batch, you can replace it by loading a plate with the typical pattern (see Loading plates into the instrument, page 2-1) and capturing a new image (see Capturing an image, page 2-4). 2 Press to start up the Grid Classification Wizard. If there is more than one colony type in the colony classification, the Add New Grid Sub-Classification page will be displayed go to Step 3; otherwise (if there is currently only one type of colony in the colony classification), the wizard will skip straight to the Configure Grid Classification page go to Step 5. 3 In the Add New Grid Sub-Classification page, click on the existing colony type that you want to subdivide with the new shape classification (see Choosing where to add the sub-classification, page 3-42, for more details). You cannot select a colony type from another sub-classification a grid sub-classification must be the highest level sub-classification. ProtoCOL Systems User Manual 3-51

76 Creating a new batch 4 Press Next to display the Configure Grid Classification page: 5 In the Configure Grid Classification page (see picture in previous step), either: Click on the Detect grid automatically radio button if you want ProtoCOL to detect the grid without any assistance. Or, if you want to narrow ProtoCOL s search for the grid to a specific range: a Click on the Detect grid between limits radio button. The Line Spacing controls will become enabled: 3-52 ProtoCOL Systems User Manual

77 Setting up batches to count colonies b Set Minimum and Maximum limits for the grid Line Spacing in ProtoCOL s search for the grid by typing directly into the edit boxes or by clicking on the up or down arrow buttons attached to the right-hand side of the edit boxes. In most cases ProtoCOL will be able to detect the grid without any assistance, so you should try Detect grid automatically on a first pass you should only need to set spacing limits if the automatic detection procedure has a problem. 6 Press Next to display the Review Grid Classification page: The Review Grid Classification page allows you to review the automatic grid classification results, and change the colony splitter setting if required. 7 Examine the image carefully to check whether the marker lines showing where ProtoCOL has detected the grid correspond to the actual grid lines on the image. It may be easier to see the results if you zoom in on areas of the image: you can zoom the image in the same way as in the main window see Zooming the image, page 2-6. ProtoCOL Systems User Manual 3-53

78 Creating a new batch 8 If the grid classification has not produced the required results: a b c d e the Line spacing value of the wrongly detected grid. Press Back to return to the Configure Grid Classification page. Click Detect grid between limits and enter Minimum and Maximum values to set a range including your estimate for the spacing of the actual grid on the image but excluding the Line spacing value noted in Step a. Press Next to make ProtoCOL redetect the grid and redisplay the Review Shape Classification page. Go back to Step 7 to check whether the new grid that ProtoCOL has detected matches the actual grid on the plate. 9 When you are satisfied with the grid classification, examine the image to see if you need to use the colony splitter to separate overlapping colonies. The initial setting of the Split check button is determined by the choice you made in the Review color classification page (see page 3-34). However, you made that choice before you could see the effect of removing the grid, which may make separate colonies appear to be joined, so you may find after examination of the image that you want to change your mind about whether to use the colony splitter. The choice you make here overrides your earlier choice the splitter is only applied once, and then only after any grid sub-classifications have been performed. The colony splitter setting you make here will be used to perform the initial counts for plates when you are working in Measurement mode. However, you will be able to change the setting for individual plates by editing the measurement after you have performed the count see Manually editing the detected colonies, page 5-11, for details. To change the colony splitter setting: Click on the Split check button next to the colony type: 3-54 ProtoCOL Systems User Manual

79 Setting up batches to count colonies For example: Split unchecked Split checked 10 Press Next to display the Classification Details page showing the new classification tree: 11 If you are not going to make any further sub-classifications of the grid classification, use the classification tree controls in the Classification Details page in the same way as in the Color Classification Wizard (see Classification Details page 3-37) to specify: the name to be used to identify it the marker used to identify it on the image the color used for the marker. 12 When you have completed the classification details, press Finish to return to the main ProtoCOL window, which will show the updated classification tree. ProtoCOL Systems User Manual 3-55

80 Creating a new batch Changing an existing classification Once you have accepted a batch for use in Measurement mode (see Design mode accepting and rejecting new batches, page 3-11), you cannot make any changes to the way colonies are detected and classified you can change the colony markers and colors, but only if you have unlocked these changes in Design mode (see Classification panel, page 3-40). You will also be able to edit measurements to change the settings for the Reject Small Particles slider and colony splitter, but only after you have performed the initial counts using the settings selected when designing the batch see Manually editing the detected colonies, page 5-11, for details. However, you can change the way colonies are detected and classified while you are still working on the batch in Design mode. You cannot do this by editing the definition of an existing classification (for example, the color samples defining the background, the boundary between two size classes or the number of shape classes), but you can remove an existing (sub-)classification and replace it with a new one with the required properties. The next section describes how to change classifications using the Modify Existing Classification button, but you can also: Add new size, shape and grid sub-classifications using the procedures described in the previous sections (you can also do this using the Modify Existing Classification button see Adding a new sub-classification, page 3-59). Create a new color classification replacing the existing color classification and any sub-classifications it may have by pressing the Color Classification button (see Removing a (sub-)classification, page 3-60, for how to remove sub-classifications individually). Modifying an existing classification To modify an existing classification: 1 If required, you can replace the current image by loading a different plate (see Loading plates into the instrument, page 2-1) and capturing a new image (see Capturing an image, page 2-4) ProtoCOL Systems User Manual

81 Setting up batches to count colonies 2 Press to display the Modify Previous Classification page of the Colony Classification Wizard: If there are no sub-classifications, the final item in the list is Repeat Color Classification instead of Remove Classification. 3 Press: Review Existing Classification to change the classification details (you can also adjust the Reject Small Particles and colony splitter settings for a color classification provided it has no sub-classifications) Add New Sub-Classification to add a new sub-classification Remove Classification to remove a (sub-)classification Repeat Color Classification to replace the existing color classification with a new one. ProtoCOL Systems User Manual 3-57

82 Creating a new batch 4 Press Next to go on to the next step, which depends on the selection you made in Step 3. For details, see: Reviewing an existing classification next section Adding a new sub-classification page 3-59 Removing a (sub-)classification page 3-60 Repeating a color classification page Reviewing an existing classification The next step after you select Review Existing Classification and press Next in the Modify Previous Classification page of the Colony Classification Wizard (see previous section) depends on whether the existing classification has any sub-classifications: If there are no sub-classifications, go to Step 1 If there are any sub-classifications, go straight to Step 3. 1 If the existing classification has no sub-classifications, the Review Color Classification page of the Colony Classification Wizard is displayed again. This shows the results of the color classification on the current image and allows you to adjust the Reject Small Particles and colony splitter settings see Review color classification, page 3-33, for details. 2 Press Next to display the Classification Details page. 3 The Classification Details page shows the current classification tree: 3-58 ProtoCOL Systems User Manual

83 Setting up batches to count colonies You can use the classification tree in the same way as in the Color Classification Wizard (see Classification Details, page 3-37) to specify for each sub-classification: whether it corresponds to debris, background or a type of colony, and for colony types: the name to be used to identify it the marker used to identify it on the image the color used for the marker. 4 Press Finish to close the Colony Classification Wizard and return to the main ProtoCOL window, which will show the updated classification details. Adding a new sub-classification The procedure described in this section is an alternative to the procedures described earlier in the Size classification (see page 3-43), Shape classification (see page 3-47) and Grid classification (see page 3-50) sections. The next step after you select Add New Sub-classification and press Next in the Modify Previous Classification page of the Colony Classification Wizard (see Modify Existing Classification, page 3-56) is to select where to create the new sub-classification using the classification tree in the Add New Sub-Classification page: 1 Click on the existing colony type that you want to subdivide with the new classification (see Adding sub-classifications, page 3-42, for more details). ProtoCOL Systems User Manual 3-59

84 Creating a new batch 2 Press Next to display the Modify Previous Classification page, which has buttons for selecting the type of classification to add: Grid classification is disabled if the colony type selected in Step 1 is already a sub-classification. 3 Click on the radio button for the classification you want to add. 4 Press Next to create the new sub-classification. 5 The remaining steps depend on which type of classification you selected in Step 3, and are exactly the same as the corresponding step (Step 5) in the procedures for adding sub-classifications using the classification buttons in the Classification tab for details see: Size classification (see page 3-43) Shape classification (see page 3-47) Grid classification (see page 3-50). Removing a (sub-)classification Remove Classification is only available if the color classification has one or more sub-classifications ProtoCOL Systems User Manual

85 Setting up batches to count colonies The next step after you select Remove Classification and press Next in the Modify Previous Classification page of the Colony Classification Wizard (see Modify Existing Classification, page 3-56) is to select which classification to remove using the classification tree in the Remove Sub-Classification page: 1 Click in the tree to select which classification to remove. 2 Press Next. 3 What happens next depends on whether in Step 1 you selected: A sub-classification: The Classification Details page will be displayed showing the new classification tree with the selected sub-classification removed (you cannot change any of the details in the tree): Press Finish to confirm the change and return to the main ProtoCOL window. ProtoCOL Systems User Manual 3-61

86 Creating a new batch The color classification: A dialog box will be displayed: Press Yes if you want to replace the existing classification and its sub-classifications with a new color classification. You will be taken to the Configure Color Classification page so that you can create the new color classification see Color classification, page 3-22, for details. Press No if you want to remove the existing classification and its sub-classifications without replacing it with a new color classification. The Classification Details page will be displayed with an empty classification tree. If you then press Finish in the Classification Details page, another warning dialog box will be displayed: Press Yes if you want to continue with an empty classification. Press No if you have changed your mind about removing the color classification. You will be returned to the Classification Details 3-62 ProtoCOL Systems User Manual

87 Setting up batches to count colonies Repeating a color classification page, where you can press Cancel to stop the classification removal procedure altogether or press Back to return to Step 1. Repeat Color Classification is only available if the color classification has no sub-classifications. The next step after you select Repeat Color Classification and press Next in the Modify Previous Classification page of the Colony Classification Wizard (see Modify Existing Classification, page 3-56) is to create the new color classification using the Configure Color Classification page see Color classification, page 3-22, for details. The new color classification will replace the existing one. Results To display the results controls: Press the Results tab: ProtoCOL Systems User Manual 3-63

88 Creating a new batch The Results tab in Design mode allows you to specify what results should be included in the Results table and the order of the columns: The results included in the list depend on the type of plate used in the batch. Results with a selected check box will be included in the Results table; unselected results will not be included. The order of the results in the list is the order they will appear in the Results table. To change whether a result will be included in the Results table: 1 Click on the result to select it. 2 Click on the result again to select/deselect the check box ProtoCOL Systems User Manual

89 Setting up batches to count colonies To move a result up or down the list to change the order of results in the Results table: 1 Click on the result to select it. 2 To move the result up the list, press To move the result down the list, press Measure To display the measure controls: Press the Measure tab: ProtoCOL Systems User Manual 3-65

90 Creating a new batch The Measure tab in Design mode allows you to specify identifiers for the individual plates in the batch and the dilution ratio, and to carry out a test count of the current plate so that you can check the batch settings (you must carry out a test measurement before you can accept the batch for counting colonies in Measurement mode): The Limit count per frame controls only appear for Pour plate batches see Putting an upper limit on counts for Pour plate batches, page ProtoCOL Systems User Manual

91 Setting up batches to count colonies Plate ID The Plate ID controls allow you to specify identifiers for the individual plates in the batch: s The Use Barcode button is disabled if there is no barcode reader connected to the PC. You can use a barcode reader in combination with any of the external sources. You can: set a base name, which will be used for the first plate and then auto-incremented for each following plate see the following section, Auto-incrementing plate identifiers enter an identifier manually for each of the plates as you measure it see Manually entered plate identifiers, page 3-69 use plate identifiers and dilutions from one of the following external sources: a LIMS system see Linking to a LIMS system, page 3-70 a CSV file see Using a CSV list of identifiers, page 3-73 a plate list created in ProtoCOL see Creating a plate list, page You can also use: a barcode reader to read the identifier/dilution from the plate in combination with an external source or manual identifiers see Using a barcode reader for plate identifiers, page Auto-incrementing plate identifiers This option is selected by default for all batch types apart from Dilution Series batches, for which manually entered plate identifiers (see next section) is the default. ProtoCOL Systems User Manual 3-67

92 Creating a new batch To use auto-incrementing plate identifiers: 1 Press the Measure tab: to display the measure controls. 2 If it is not already selected (the default), press so that it is selected: 3 In the Template box, type in text to act as a basis for the identifier. If the text includes a sequence of digits, these will be used as the starting point for the auto-incrementing; if it includes more than one sequence, the rightmost sequence will be used as the starting point; if there are no digits in the text, auto-incrementing digits will be added to the end of the text. For example: Template First identifier Second identifier Third identifier abc abc1 abc2 abc3 abc18 abc19 abc20 abc21 abc18def abc19def abc20def abc21def 66abc18def 66abc19def 66abc20def 66abc21def 3-68 ProtoCOL Systems User Manual

93 Setting up batches to count colonies Manually entered plate identifiers If you want to add an identifier manually for each of the plates as you measure them: Make sure you leave both the Use External Source and Auto-increment buttons deselected when you accept the batch (if one is selected, press it to deselect it). s If you leave Auto-increment selected and you have not entered any text in the Template box when you accept the batch, a dialog box will be displayed telling you to enter a name. If you want to use auto-incrementing plate identifiers, enter the base name; if you want to use manually entered plate identifiers, press Auto-increment to deselect it. When you are working in Measurement mode you can use a barcode reader to read the identifier and dilution from the plate see Using a barcode reader for plate identifiers, page Using an external source for plate identifiers You can use the following external sources for plate identifiers: a LIMS see Linking to a LIMS, page 3-70 a CSV file see Using a CSV list of identifiers, page 3-73 a list created in ProtoCOL see Creating a plate list, page To specify an external source for plate identifiers: 1 Press the Measure tab: to display the measure controls. 2 Press ProtoCOL Systems User Manual 3-69

94 Creating a new batch to display the Use External Source for Plate ID dialog box: The following sections describe each of the options in turn. Linking to a LIMS To make a link between the batch and the LIMS generated plate identifiers: 1 Press 3-70 ProtoCOL Systems User Manual

95 Setting up batches to count colonies to display the LIMS link controls: 2 Make a note of the ProtoCOL GUID shown you can press to copy it to the clipboard. When the LIMS generated identifiers are added to the ProtoCOL database they should be labeled with the ProtoCOL GUID for the batch to link them to the batch see the paragraphs following these instructions for more information about adding LIMS generated identifiers to the ProtoCOL database. 3 If you wish to link the ProtoCOL plate table back to the LIMS system, you can add a LIMS Identifier. This is not essential for the operation of ProtoCOL, but can be used to form a link between the table in ProtoCOL and a table in your LIMS database. The details of how to form the link and what identifier to use will depend on the LIMS you are using. 4 Press OK to close the Use External Source for Plate ID dialog box. ProtoCOL Systems User Manual 3-71

96 Creating a new batch The Template box will show that the plate ids will be linked to the LIMS system: The precise details of how you add the LIMS generated plate identifiers and dilutions to the ProtoCOL database will depend on your LIMS. As a simple example, the following SQL code could be used to add the plate identifier Plate1 with a dilution of 1:10 to the ProtoCOL database for a batch that has been given the ProtoCOL GUID a94d6efd-1fe a14f-0cb77e : INSERT INTO Lims_plates (Dilution, PlateID, GUID) VALUES ('1:10','Plate1','a94d6efd-1fe a14f-0cb77e680363') If required (see Step 3), you can add a LIMS identifier as well: INSERT INTO Lims_plates (Dilution, PlateID, GUID, LimsID) VALUES ('1:10','Plate1','a94d6efd-1fe a14f-0cb77e680363','xfs') s ProtoCOL does not require you to use unique identifiers/dilutions. For Dilution Series batches, if an identifier in the LIMS list has a dilution, that dilution will be used and any of the dilution(s) set in ProtoCOL (see Dilution Series batches, page 3-83) will be ignored. On the other hand, if the LIMS list does not include a dilution, each of the dilutions in the ProtoCOL dilutions list will be used in turn for that identifier. When you are working in Measurement mode, you can use a barcode reader in conjunction with a LIMS list to match each plate you are measuring to its identifier/dilution in the LIMS list see Using a barcode reader for plate identifiers, page ProtoCOL Systems User Manual

97 Setting up batches to count colonies Using a CSV list of identifiers To use a CSV file containing a list of plate identifiers and dilutions (see the text following the instructions for the format to use for CSV files): 1 Press to display controls for opening the CSV file: 2 Press to open a standard Windows Open dialog box. 3 Use the Open dialog box to locate and select the required CSV file. ProtoCOL Systems User Manual 3-73

98 Creating a new batch 4 Press Open to open the selected file. If you are creating a Dilution Series batch and the CSV file does not contain any dilution information, you will be asked if you want to import a dilution series (otherwise, go straight to Step 5): a If you want to import a csv file containing a dilution series, press Yes and go to Step b. If you do not want to import a dilution series, press No the default dilution will be used for each of the imported plate identifiers. Go to Step 5. If you want to abort the procedure for importing plate identifiers completely, press Cancel ProtoCOL Systems User Manual

99 Setting up batches to count colonies b If you pressed Yes in the previous step, controls will be displayed for you to import a second csv file containing the dilution series: c Press to open a standard Windows Open dialog box. d e Use the Open dialog box to locate and select the required CSV file. Press Open to open the selected file. ProtoCOL Systems User Manual 3-75

100 Creating a new batch The contents of the file will be shown in the preview box: 3-76 ProtoCOL Systems User Manual

101 Setting up batches to count colonies f Check the preview box to make sure the contents are correct. If the contents are correct, press OK to show the combined plate identifiers and dilutions: 5 The contents of the file will be shown in the preview box (see example above) check the preview box to make sure the contents are correct (see the subsection following these instructions for the format to use for the CSV file). 6 If the contents are correct, press OK to close the Use External Source for Plate ID dialog box and load the plate list into the batch. The Template box will show that the plate identifiers have been taken from an external source: ProtoCOL Systems User Manual 3-77

102 Creating a new batch When you are working in Measurement mode, you can use a barcode reader in conjunction with a CSV list to match each plate you are measuring to its identifier/dilution in the CSV list see Using a barcode reader for plate identifiers, page CSV file format Each line of the file should consist of two strings separated by a comma: where: Identifier,Dilution Identifier is the plate identifier Dilution is text describing the dilution (if the string is not recognized or no dilution is given, No Dilution is used). The order of the lines in the file defines the order of the plates in the list. For example: Test1,No Dilution Test2,1:5 Test3,1 in 1000 Test4,1 in 10^6 ProtoCOL does not require you to use unique identifiers/dilutions. Creating a plate list To create a plate list containing a list of plate identifiers and dilutions: 1 Press 3-78 ProtoCOL Systems User Manual

103 Setting up batches to count colonies to display controls for creating a plate list: 2 Type the identifier for the first plate into the edit box at the top of the dialog box. 3 Use the dilution boxes to enter the dilution: you can type directly into the boxes or use the + and buttons to increase or decrease the values. The left-hand box is the sample proportion; the right-hand box is the total. 1:10 means one part sample in ten parts total volume. This means that the left-hand number must be smaller than the right-hand number. 4 Press to add the identifier/dilution to the plate list. 5 Repeat Steps 2 4 for the remaining plates in the batch. ProtoCOL Systems User Manual 3-79

104 Creating a new batch The identifiers must be unique. 6 If you change your mind about one of the plates in the list, click on it in the list to select it and press 7 When you are satisfied that the list is complete and correct, press OK to close the Use External Source for Plate ID dialog box and load the plate list into the batch. The Template box will show that the plate ids have been taken from a custom plate list: s When you are working in Measurement mode, you can use a barcode reader in conjunction with a custom plate list to match each plate you are measuring to its identifier/dilution in the plate list see the following section, Using a barcode reader for plate identifiers. Using a barcode reader for plate identifiers Depending on how the barcode reader is programmed, the barcode reader may or may not be able to read dilution information. When you are working in Measurement mode, you can use a barcode reader to read the identifier/dilution from the plate in combination with an external source of plate identifiers (see Using an external source for plate identifiers, page 3-69) or to input manual identifiers (see Manually entered plate identifiers, page 3-69) ProtoCOL Systems User Manual

105 Setting up batches to count colonies If you intend to use a barcode reader to read plate identifiers (and dilutions where relevant see the note at the beginning of this section): 1 Press the Measure tab: to display the measure controls. 2 Press See Using a barcode reader for identifiers and dilutions, page 4-8, for more on using the barcode reader to input plate identifiers/dilutions. Dilution If you have chosen to use a LIMS, CSV file or a custom plate list as the source of the plate identifiers for use in Measurement mode (see Using an external source for plate identifiers, page 3-69), you can include individual dilutions with each plate identifier see previous section. If no dilution is supplied in the lists, No dilution is assumed. To specify the dilution to use in calculating results: Press the Measure tab: to display the dilution controls. ProtoCOL Systems User Manual 3-81

106 Creating a new batch The dilution controls used for a Dilution Series batch are different from those for other types of batch. Non-Dilution Series batches To set the dilution for a non-dilution Series batch: 1 Press (the button shows the current dilution) to open the dilution editor: 2 Use the dilution boxes to enter the dilution: you can type directly into the boxes or use the + and buttons to increase or decrease the values. The left-hand box is the sample proportion; the right-hand box is the total. 1:10 means one part sample in ten parts total volume. This means that the left-hand number must be smaller than the right-hand number. 3 Press Update to confirm the dilution you have set and return to the main ProtoCOL window ProtoCOL Systems User Manual

107 Setting up batches to count colonies To choose whether to allow the dilution to be changed in Measurement mode for an individual plate (see Allowing changes in Measurement mode, page 3-13): 1 Click on the padlock button on the dilution button to display the dilution lock menu: 2 Choose Edit Dilution Item to lock or unlock the setting. Dilution Series batches To set the dilutions for a Dilution Series batch: 1 Press to display the dilutions menu (the button shows the current dilution): Selecting a dilution by choosing it from the menu or pressing the arrow buttons below the dilution button has no effect in Design mode; however, you can use these procedures in Measurement mode to change the dilution for a plate, provided Select Dilution is unlocked see the next set of instructions. ProtoCOL Systems User Manual 3-83

108 Creating a new batch 2 Select Edit Dilution List from the menu to open the Dilution List Editor: 3 To add a new dilution to the list: a b Click on the dilution at the point you want to add the new dilution. Press Add to open the Dilution Editor: 3-84 ProtoCOL Systems User Manual

109 Setting up batches to count colonies c Use the dilution boxes to enter the dilution: you can type directly into the boxes or use the + and buttons to increase or decrease the values. The left-hand box is the sample proportion; the right-hand box is the total. 1:10 means one part sample in ten parts total volume. This means that the left-hand number must be smaller than the right-hand number. d Press Add to close the Dilution Editor and add the dilution to the list. 4 To change a dilution in the list: a b c d Click on the dilution in the list to select it. Press Edit to open the Dilution Editor. Use the dilution boxes to edit the dilution: you can type directly into the boxes or use the + and buttons to increase or decrease the values. Press Update to close the Dilution Editor and confirm the change to the dilution. 5 To remove a dilution from the list: a b c Click on the dilution in the list to select it. Press Remove. A confirmation dialog box will be displayed. Press Yes to remove the dilution. 6 To move a dilution up or down in the list to change the order of dilutions: a b Click on the dilution in the list to select it. Press the up or down arrow button. 7 When you have finished editing the dilutions list, press Close to return to the main ProtoCOL window. ProtoCOL Systems User Manual 3-85

110 Creating a new batch To choose whether to allow the dilutions to be changed in Measurement mode: 1 Click on the padlock button on the dilution button to display the dilution lock menu: 2 Choose: Select Dilution to allow/prevent the selection of a different dilution from the list for the current plate If Select Dilution is locked, you will not be able to select a dilution from the list when you are in Measurement mode. Edit Dilution Item to allow/prevent manual changes to the dilution for the current plate Edit Dilution List to allow/prevent changes to the dilution list. Putting an upper limit on counts for Pour plate batches To place an upper limit on the count result for Pour plate batches: 1 Press the Measure tab: 3-86 ProtoCOL Systems User Manual

111 Setting up batches to count colonies to display the measure controls, including the Limit count per frame controls: The Limit count per frame controls only appear for Pour plate batches. 2 Check the Limit count per frame check box to enable the edit box and arrow controls: 3 Either type the required limit directly into the edit box or press the arrow buttons to increase or decrease the value. When you perform a measurement when a count limit has been set, the Last measurement results box will show whether the limit has been breached, for example: As this example shows, if there is more than one colony type, the count limit (100 in this case) is applied to each of them separately. Unlike the Last measurement results box, which only shows that the limit has been breached, the Results table records the actual count result. ProtoCOL Systems User Manual 3-87

112 Creating a new batch Test Measure Plate The final step in setting up a batch is to carry out a test measurement to check the batch settings you will not be allowed to accept a new batch for Measurement mode (see page 3-11) until you have carried out a test measurement. To carry out a test measurement: 1 Press the Measure tab: to display the measure controls. 2 Press The results of the measurement will be shown in the Measurement test results panel the detected colonies will also be marked on the image using the colors and markers selected for each colony type. If you are using an uncalibrated imported image, a warning dialog box will be displayed asking you to calibrate the image see Calibrating the image, page 2-8, for details. 3 Check the image to make sure that the colonies have been correctly detected and distinguished from the background and any debris on the plate. 4 If there are any problems with the colony detection, you will need to go back to change the batch settings using one of the procedures described earlier in this chapter. If you are happy with the measurement results, you can proceed to accept the batch for Measurement mode see next section ProtoCOL Systems User Manual

113 Accepting the batch design Accepting the batch design To confirm that you have completed the set-up procedure and want to proceed to taking measurements: Press The next chapter shows you how to use Measurement mode to count the colonies on a batch of plates; the chapter following that (Chapter 5, Working with results) shows you how to view and edit the results, and then compile a report. ProtoCOL Systems User Manual 3-89

114 Creating a new batch 3-90 ProtoCOL Systems User Manual

115 Counting colonies Once you have created a batch and accepted it (see previous chapter), the general procedure for counting the colonies on plates is: 1 Open or select the batch see next section. 2 Load the first plate into ProtoCOL see page Capture the image see page Adjust the batch settings, if required see page Enter a plate identifier, if required see page Add exclude regions, if required see page Carry out the measurement see page Repeat Steps 2 7 for all the other plates in the batch. 9 Check the results, edit them if necessary, and create a report for the batch see the following chapter, Working with results. 10 Close the batch see Closing batches, page Steps 2 and 3 are also required when creating and setting up the batch see the references to Chapter 2, Capturing images. Step 9 is described in Chapter 5, Working with results. For Ames batches, you may want to count the plates twice: once following the above procedure and a second time using the Remeasure edit command see Repeating a measurement for an Ames batch plate, page 5-15, for details. Instructions for the remaining steps are described in this chapter (though much of Step 4 is common to creating and setting up the batch and is covered by references to Chapter 3, Creating a new batch). ProtoCOL Systems User Manual 4-1

116 Counting colonies Opening and selecting batches If you have just created and accepted a new batch (see previous chapter), it will be automatically open and selected in Measurement mode. However, you can also open a previously created batch if there is no batch open currently, or if you want to open another batch in addition to any currently open batches (you can have several batches open at the same time). To open a batch in Measurement mode: 1 Press the batch button: If there is no batch open currently, the batch button is labeled Create or Open Batch: If there is already a batch open, the batch button shows the name of the currently selected batch, for example: The Start menu will be displayed: 2 Press 4-2 ProtoCOL Systems User Manual

117 Opening and selecting batches to display the Open Batch dialog box: Batches are marked in the Available column if they are already open in ProtoCOL. 3 If the required batch is not in the list of the 10 most recent batches: a b Type the name, or a part of the name, of the required batch into the Search criteria box. Press Only batches with names that contain the text you entered will be listed. 4 Select the required batch in the list. ProtoCOL Systems User Manual 4-3

118 Counting colonies 5 Press Open to open the batch. The batch will be opened and the batch button will be labeled with the batch name. If there is more than one batch currently open in ProtoCOL, you can select which one to work on at any time (unless you are in the process of creating a new batch). To select an open batch: Either: a Press the batch button, which shows the name of the currently selected batch, for example: to display the Start menu: b Select the required batch from the Batch list in the menu. Or: To move on to the next batch in the Batch list, press To move back to the previous batch in the Batch list, press 4-4 ProtoCOL Systems User Manual

119 Adjusting batch settings in Measurement mode Adjusting batch settings in Measurement mode Depending on whether they were unlocked when the batch was created (see Allowing changes in Measurement mode, page 3-13), you may be able to change the following settings: Exposure see Setting the exposure, page 2-4 Frame shape see Choosing the type of frame to use, page 3-15 Frame size, position and orientation see Adjusting the position and size of frames, page 3-17 Dilution see Dilution, page 3-81 (note that the procedure depends on whether you are using a Dilution Series batch or not) Colony marker symbol and color you use the same techniques for changing the colony marker symbol and color as on the Classification Details page of the Colony Classification Wizard (see Classification Details, page 3-37). The procedures for changing these settings in Measurement mode are the same as for Design mode (with a few obvious minor differences) see the above references for instructions. Entering plate identifiers Each plate in the batch must be given a plate identifier. The procedure depends on the options selected when the batch was created. If several plates are given the same identifier (for example, with a Dilution Series), they will be grouped together under the same item in the Results table see the following chapter, Working with results, for details. Auto-incrementing plate identifiers When the batch is first opened, the template identifier set when the batch was created (see Auto-incrementing plate identifiers, page 3-67) will be set automatically in the Next Plate Id box as the first identifier for the batch. Also, if the batch is a Dilution Series batch, the dilution will be set to the first dilution in the dilution list see (Dilution Series batches, page 3-83). ProtoCOL Systems User Manual 4-5

120 Counting colonies After each measurement: For non-dilution Series batches, a new plate identifier will be set ready for the next plate by auto-incrementing the previous identifier. For Dilution Series batches, if there are still some dilutions on the next dilution list, the plate identifier will be unchanged and next dilution in the dilution list will be set. When there are no more dilutions in the list, the plate identifier will be auto-incremented and the first dilution in the dilution list will be set. See Auto-incrementing plate identifiers, page 3-67, for more details, including the rules used to generate the new identifier. Manually entered plate identifiers To enter a manual plate identifier: Type the identifier into the Next Plate ID box: You can also use a barcode reader to enter a manual plate identifier (and dilution if the barcode reader is programed to read dilutions). The Next Plate ID box is not cleared after you have made a measurement it continues to show the previously entered manual plate identifier. This is useful if you want to use the same identifier for a number of plates, for example, with a Dilution Series batch, but may cause problems if you forget to change it when you come to a new set of plates. External source LIMS, CSV file or Custom plate list When the batch is set to use an external source of plate identifiers (see Using an external source for plate identifiers, page 3-69), ProtoCOL creates a list of plate identifiers and dilutions from the external source. 4-6 ProtoCOL Systems User Manual

121 Entering plate identifiers When you first open the batch, the Next Plate ID box shows the first plate identifier on the list and the dilution control shows the dilution: Before pressing the Measure plate button to count the colonies, you can do one of the following: accept this plate identifier by doing nothing press to display the plate list and then select the required plate identifier/dilution from the list use a barcode reader to read the plate identifier (and dilution if the barcode reader is programed to read dilutions) from the plate ProtoCOL will give an error if the identifier/dilution are not on its plate list for the batch. After you have performed the measurement, the next identifier and dilution will be shown, and you can repeat this procedure until the list is exhausted. When the list is exhausted, no more measurements can be made and an error message will be displayed if you try to perform a count, but see the following note. Once you have accepted a batch for use in Measurement mode you cannot add plate identifiers to the batch s identifier list if it has been created from a CSV file (Using a CSV list of identifiers, page 3-73) or a custom plate list (Creating a plate list, page 3-78). However, you can add LIMS generated plate identifiers to the ProtoCOL database at any time. So, for example, if you have measured the plates for all the identifiers currently loaded into the ProtoCOL database from the LIMS, you can generate a new list from the LIMS and add those to the ProtoCOL database using the same Protocol GUID as you used for the first set (see Linking to a LIMS, page 3-70), and then count the colonies on those plates. ProtoCOL Systems User Manual 4-7

122 Counting colonies Using a barcode reader for identifiers and dilutions You can use a barcode reader to enter a manual plate identifier (and dilution if the barcode reader is programed to read dilutions) or pick a plate identifier and dilution from the plate list created from an external source (see Using an external source for plate identifiers, page 3-69). Exclude regions On occasions, you may want to avoid counting colonies on part of a plate because of some problem with it. One way of doing this is to create one or more exclude regions, which will be ignored when ProtoCOL counts the colonies the area of the exclude regions will be deducted from the area of the frame to ensure that results such as count/ml are correct. If the batch allows you to change and/or adjust the frame, an alternative for some batch types might be to choose a Partial Plate frame and/or to rotate the frame so that the problem area is not counted. To define an exclude region on an image: 1 Press to select it: You may find it helpful to zoom the image before placing the exclude region on the image see Zooming the image, page ProtoCOL Systems User Manual

123 Exclude regions 2 Move the pointer to the position you want to place one corner of the exclude region: 3 Click to place the first corner of the exclude region. 4 Move the pointer to the position you want to place the opposite corner of the exclude region the exclude region will be dragged out: ProtoCOL Systems User Manual 4-9

124 Counting colonies 5 Click to create the exclude region. 6 Repeat Steps 1 5 to place any other exclude regions required. To remove all exclude regions from the image: Press Counting the colonies on a plate To count the colonies on a plate: Press The results will be added to the Results table see the following chapter, Working with results, for details. Closing batches To close a batch: 1 If the batch you want to close is not selected, select it see Opening and selecting batches, page ProtoCOL Systems User Manual

125 Closing batches 2 Either: Press Or: a Press the batch button, which shows the name of the selected batch, for example: to display the Start menu: b Press ProtoCOL Systems User Manual 4-11

126 Counting colonies 4-12 ProtoCOL Systems User Manual

127 Working with results This chapter shows you how to view and process the results produced by ProtoCOL, and then compile a report based on them (see the previous chapter, Counting colonies, for how to carry out the measurements). The chapter is divided into three main sections: The Results table see the next section Editing results see page 5-3 Compiling reports see page The Results table When ProtoCOL has counted the colonies on a plate, it adds the results to the top of the Results table at the bottom of the window, for example: The choice of which columns are included in the Results table is made when the batch is set up see Results, page In the example, there are results for three plates: Test 3, Test 2 and Test 1 the bottom two have been closed so they only show summary information; the top one is open and shows the detailed results. ProtoCOL Systems User Manual 5-1

128 Working with results To open or close a result: Press the or button in front of the plate name. If required, you can resize the Results table by dragging the handle at the top of the table: You can also resize the table columns by dragging the dividing line between the headings: If there is more than one colony type, the count will be shown in a separate row for each type. In the example at the beginning of this chapter there are two colony types, A and B. If there is more than one result with the same plate identifier (for example, the results for a Dilution Series), they will appear as separate results within the same plate item: 5-2 ProtoCOL Systems User Manual

129 Editing results For Multi-Sector Plate and OPKA batches, the results for each sector are shown as a separate item within each plate item: This example shows that you can open and close a result using and buttons when there is more than one result for a batch with more than one colony type appearing within the same plate item. Editing results ProtoCOL allows you to: Delete results see page 5-6 Add and edit comments see page 5-7 Edit the plate identifier and/or dilution see page 5-8 Edit a count value see page 5-10 Manually edit the detected colonies see page 5-11 View the audit trail of changes see page 5-14 Repeat a measurement for Ames batches see page 5-15 To select any of the edit operations: 1 Select the result you want to edit see next section. ProtoCOL Systems User Manual 5-3

130 Working with results 2 Press on the right-hand edge of the Results table to display the edit menu: The Remeasure command only appears for Ames batches. Selecting results 3 Select the required command. In order to carry out these operations, you first need to select the item in the Results table that you want to edit. Depending on the type of batch and operation, when you make a measurement, ProtoCOL may produce: a single result (for example, a Pour Plate batch with only one type of colony) a collection of results for different types of colony 5-4 ProtoCOL Systems User Manual

131 Editing results a collection of results for different sectors of the plate, where for each sector, the result may be a single result or a collection of results for different types of colony for example: Depending on the edit operation required, you may need to select: the complete set of results from an individual plate (which in the simplest cases will be a single result, but could be a collection of results for different types of colony, or the collection of results for all sectors, such as Test 1a in the example) the results from an individual sector (which could be a single result or a collection of results for different types of colony, such as Sector 2 in the example) an individual result for a single colony type (such as the A in Sector 2 in the example). To select an item in the Results table: Click on it. To select the next or previous plate in the Results table: Press or on the right-hand edge of the Results table. To select the next or previous item in the Results table: Press the down or up arrow key on the keyboard. ProtoCOL Systems User Manual 5-5

132 Working with results Deleting results To delete the result(s) for a plate: 1 Select the plate in the Results table see previous section. 2 Press 3 Press 4 Press Yes in the confirmation dialog box. You will be asked to enter a reason for deleting the result: 5 Enter a reason for deleting the plate. 6 Press OK. The result will be deleted from the Results table. Although the result is deleted from the Results table and can no longer be viewed in ProtoCOL, it is not removed from the ProtoCOL database and can be viewed there if required for audit purposes. 5-6 ProtoCOL Systems User Manual

133 Editing results Adding and editing comments To add a comment to the results for a plate or edit an existing comment: 1 Select the plate in the Results table see Selecting results, page Press 3 Press to display the Edit Comments dialog box: 4 Enter or edit a comment. 5 Press OK. ProtoCOL Systems User Manual 5-7

134 Working with results You will be asked to enter a reason for adding the comment: 6 Enter a reason for adding the comment. 7 Press OK. The comment and reason will appear in the Comments/s column for the result. Editing the plate identifier and/or dilution To edit the plate identifier and/or dilution for a result: 1 Select the plate in the Results table see Selecting results, page Press 3 Press 5-8 ProtoCOL Systems User Manual

135 Editing results to display the Edit results dialog box: 4 Edit the Plate ID as required. 5 Edit the Dilution as required. 6 Press OK. You will be asked to enter a reason for editing the plate identifier and/or dilution: 7 Enter a reason for editing the plate identifier and/or dilution. 8 Press OK. The identifier and/or dilution will be updated for the result and if you have changed the dilution, values depending on the dilution will also be updated. Your reason will appear in the Comments/s column for the result and an E flag will appear in the Flags column. ProtoCOL Systems User Manual 5-9

136 Working with results Editing a count value To edit the result of a count: 1 Select the individual result see Selecting results, page 5-4. If the measurement produces more than one result (for example, if there is more than one colony type or several sectors), select an individual result, not a group of results. 2 Press 3 Press to display the Edit Colony Count dialog box: 4 Edit the Count as required. 5 Press OK ProtoCOL Systems User Manual

137 Editing results You will be asked to enter a reason for editing the count: 6 Enter a reason for editing the count. 7 Press OK. The count will be updated for the result and any values depending on the count will also be updated. Your reason will appear in the Comments/s column and an M flag will appear in the Flags column. Manually editing the detected colonies Immediately after you have completed a result, you can add colonies to or remove colonies from the detected colonies you cannot manually edit the detected colonies for previous results. To add colonies to or remove colonies from the detected colonies for a result, or manually adjust the Reject Small Particles filter and colony splitter settings for an automatic count: 1 Select the required result see Selecting results, page 5-4. For Multi-Sector Plate and OPKA batches, select the sector you want to edit; for other batch types, select the plate. This means that if there is more than one type of colony, you will be editing the results for all the colony types in the sector/plate. 2 Press ProtoCOL Systems User Manual 5-11

138 Working with results 3 Press to display the Manual counting dialog box: You can use the Manual counting dialog box to: count colonies manually see Step 4. manually adjust the Reject Small Particles filter and colony splitter settings for an automatic count see Step 5. 4 To add colonies: a Select the colony type that you want to add by clicking on it in the classification tree ProtoCOL Systems User Manual

139 Editing results b If it is not already selected (as in the picture above), press c Click on the image where you want to add the colonies (you can only add colonies within the frame). 5 To remove colonies: a b Select the colony type that you want to remove by clicking on it in the classification tree. If it is not already selected, press to select it: c Click in the image on the colonies you want to remove. 6 Press OK. You will be asked to enter a reason for editing the count: 7 Enter a reason for editing the count. ProtoCOL Systems User Manual 5-13

140 Working with results 8 Press OK. The count(s) will be updated together with any values depending on the count(s). Your reason will appear in the Comments/s column and an M flag will appear in the Flags column. Viewing the audit trail of changes When you make any changes to a result, ProtoCOL makes an entry in the audit history for that plate. To view the audit history for a plate in the Results table: 1 Select the plate in the Results table see Selecting results, page Press 3 Press 5-14 ProtoCOL Systems User Manual

141 Editing results to display the Audit History dialog box: 4 When you have finished viewing the audit history, press Cancel to close the dialog box. Repeating a measurement for an Ames batch plate Depending on the procedure you are following, you may need to take a second measurement for an Ames batch plate after an interval of time has elapsed. To repeat a measurement for a plate in an Ames batch: 1 Insert the plate to be remeasured into ProtoCOL see Loading plates into the instrument, page Capture the image see Capturing an image, page Select the previous measurement for the plate in the Results table see Selecting results, page Press ProtoCOL Systems User Manual 5-15

142 Working with results 5 Press The new measurement for the plate will be added to the Results table above the previous result in the item for the plate the date and time will allow you to identify the measurements. Compiling reports To compile a results report: 1 Press 5-16 ProtoCOL Systems User Manual

143 Compiling reports to display the ProtoCOL Reports dialog box: 2 Click on the template for the current batch to create a detailed report for the current batch or click on Summary of all Batches to create a summary report covering all the batches in the ProtoCOL database. 3 To choose to include the data for each result for the columns shown in the Results table only (the default): Leave Restrict report by batch headings checked. To include all the data for each result, including columns not selected for the Results table when the batch was designed (see Results, page 3-63): Press Restrict report by batch headings so that it is not checked. The Restrict report by batch headings has no effect on summary reports. ProtoCOL Systems User Manual 5-17

144 Working with results 4 Press to compile the report, for example: 5 When you have finished working with the report (see below for how to work with the report), press: to return to ProtoCOL ProtoCOL Systems User Manual