2. The PhenePlate software Instructions: Step forward with Page Down or by mouse click If you encounter a you can click on that and jump to other items Otherwise just step forward

3. To start the PhenePlate software: Click on the PhP icon

4. The PhP menu Read PhP data with a microplate reader - DOS reading version Installation of reader parameters for the DOS reading version Windows reading version (only for certain readers) Create PhP data from absorbance data and from scanned images Create PhP data after the last reading with a microplate reader - OBS! Only nessecary if you used the DOS reading software Analysis of PhP data

5. Reading menu Go to beginning Go to PhP menu Go to beginning Go to PhP menu Read PhP data with a microplate reader - DOS reading version Installation of reader parameters for the DOS reading version Windows reading version (only for certain readers) The PhP software only works for certain readers. See the manual for the PhP software on how to install and run a microplate reader. If you do not succeed to install your particular reader for the PhP software, go back to PhP menu and select ’Create PhP data from absorbance data and from scanned images’ If you have used a flatbed scanner for reading plates, go back to PhP menu and select ’Create PhP data from absorbance data and from scanned images’

6. Either select a PhP data file to be analysed Or select PhP data from another application (e.g. Excel) file to be analysed Click ’Select all data from file’ if you want all data in the file Click ’Select certain samples’ if you want certain data only When ready, click ’Ok’ to continue Data analysis Click here to se a detailed list on files available

7. View and edit data Input more data Save data in a new file Sort and remove samples Normalise data Normalisation towards negative control sample Normalisation towards negative control test Convert data Convert to -/+ values Calculate similarities for clustering and dendrogram presentation Comparison to reference data Pairwise comparisons of isolates Population statistics (Diversity, Sp etc) Evaluation of tests Clear memory and load new data The main menu

8. It is more easy to handle the PhP data in Microsoft Excel. This can be done by copying the data to clipboard and then pasting into Excel Open an Excel sheet Wait until this message appears View and edit data

9. Paste!

10. Selected data can be copied to a database containing data from reference strains, e.g. of known species. Select an existing reference file to add new samples to, or type a new file name to create a new reference file. All these files are stored in the folder ’References’ in the PhPWIN folder. Creating a reference data file

11. Click ’Save’ to save selected samples in the data base Creating a reference data file Compare to reference data Back to main menu Compare to reference data Back to main menu

12. XXX You can add text strings to sample names, e.g to simplify search for certain samples Add text to sample names

13. Remove tests You can create a file containing information on which tests to remove. Use e.g. Notepad, and type information as in the example file EXDEL.TST Then save the file with a name ending with.tst in the PHPWIN folder Go to beginning Go to main menu Go to beginning Go to main menu

14. The samples will be sorted in the same order as they are selected. Note that samples that are not selected will be removed from the computer memory (you can still load them again from the data file) Go to beginning Go to main menu Go to beginning Go to main menu Sort and remove samples

15. Go to beginning Go to main menu Go to beginning Go to main menu Normalization towards negative control test: Useful for the PhP-48 plates. Data from each isolate will be normalized according to the result on the first negative control test (test no. 24). Normalization to negative control sample: Normally negative wells should give OD values of 20 - 23. If this was not the case, the values can be transformed here, provided that one of the samples in the actual file was a negative control, containing only substrate. If you want substrate normalization, give the number of the sample containing the negative control. The computer will suggest a value for the negative control in the actual file (e.g. 25 if absorbances are slightly high) and if this value is accepted, all data in the file will be multiplied by a factor (20/25 in this case). Note that the normalisation procedures will not affect the raw data file. By selecting this item when you are using PhP-RE plates, you will also obtain information on which isolates are not E.coli. The names of such isolates will be printed with * or ** if you look at them using ’View and edit data’ Convert data: Data may be converted to other numerical values, or to dichotomous (+/- ) values Normalise data

16. Select ’All samples’ (or select only the samples to be included in the analysis) Selected samples are marked with x Analyse data - clustering

19. Analyse data – clustering options Click ’Help’ if you want information on the options

20. The dendrogram A short or extended list of PhP types can be shown

21. The extended list of types The list is sorted according to the order of samples. Click on it to view the list sorted according to types Use this option to paste the list into e.g. an Excel file Move the cursor over the header row in order to see information on the items in the list

22. Adding marks and text to the dendrogram

23. Number of samples that have been clustered The Co-phenetic correlation describes how well the dendrogram reflects the data it was generated from. More than 0.90 is good, 0.80 – 0.90 not so good, and lower than 0.80 is bad Di (diversity index) describes the distribution of samples into types. A high value (maximum 1.0) indicates even distribution into many different types. A low value (minimum 0) indicates dominance of one or a few types The first Di value is calculated from the clustered data (i.e. the types shown in the dendrogram). The second Di value (the true Di) is calculated from the original data. It is thus a better value to present The dotted line shows the identity (ID) level. Isolates that are connected together above this line are assigned to the same PhP type. The ID level is determined by the reproducibility of the assay, and can be selected in the clustering options Use this option to be able to edit the dendrogram in e.g. an imaging software or in Excel...

24. With Excel, the dendrogram can be combined with other information on the isolates (e.g. PFGE patterns, MIC values etc.)

25. Presenting several dendrograms on the same page

27. Up to 10 dendrograms per page can be presented Go to beginning Go to main menu Go to beginning Go to main menu

28. If a reference (database) file has already been created How? If a reference file has not been created yet, first make sure that the reference data has been loaded (if not - select ’Back to main menu - Data manager’ and ’Input more data’ - load the file containing reference data) Comparisons to reference data

29. Selected reference samples Comparisons to reference data Prints a list of all similarities above the selected value Prints only the reference sample to which the highest similarity is obtained. Use this option if you are searching a large data base for one or a few types The lowest similarity level depends on the purpose of the comparison - for species identification, use a lower value (e.g. 0.80) - for detecting clonal groups, use a higher value (e.g. 0.95)

30. Comparison to reference data All reference isolates showing similarity of the selected level (0.80) or higher to the unknown samples Unknown samples to be compared to reference samples Number and % of unknown samples that were identified to the selected reference samples Go to beginning Go to main menu Go to beginning Go to main menu

31. Pairwise comparisons of samples 1 (*) P1:1 Select two samples Results for sample 1 Results for sample 2 Differences between sample 1 and sample 2 Similariy between sample 1 and sample 2 Go to beginning Go to main menu Go to beginning Go to main menu

32. Population statistics A population (of bacteria) in this case may consist of a sample (e.g. a fecal sample or a water sample) which has been cultivated on selective agar media. From these media several pure colonies are picked and typed with the appropriate PhP plates. The aim of the population statistics is to be able to determine the diversity of the bacterial populations in the samples, and to determine the similarities between bacterial populations in different samples

33. The first isolate in each population will be marked with an X Select the first isolate in each bacterial population by clicking on it. Alternatively, add the digits (*) to the name the first isolate in each population (e.g. by using Data manager in PhPWIN), and click ’Use pre-defined’.

34. The identity level within each population is normally determined by the intra assay reproducibility True diversities and homogeneities (as mean and median similarity coefficients between all isolates) for each sample (population)

35. The identity level between populations may be set lower if isolates from several assays are compared, and the inter assay reproducibility has been found to be lower than the intra assay reproducibility In addition to the population similarity coefficients (Sp values), also the mean and median similarities between all isolates in two compared populations may be presented

36. Use scroll bar to move list Population similarity coefficients (Sp) measure the proportion of identical strains in two different compared populations. It thus reflects the possible spread of bacterial clones between the samples Mean and median similaries measure the similarities between all isolates in two different compared populations. Population similarity coefficients, mean and median similarities may be clustered to yield a dendrogram showing the relations beween different populations (samples) Go to beginning Go to main menu Go to beginning Go to main menu

37. This option only needs to be used if you have measured PhP plates using the old DOS reading software !

38. Transformation of scanned plate images to PhP data Set scanner to read transmissible originals. Set scanner resolution to 75 - 100 dpi. Use RGB color. Save images as.bmp (windows bitmap) files (or copy scanned images to clipboard and transform directly to PhP data – see later in this presentation) Scan all PhP plates using the same options, and position all plates at the same location on the scanner. Note that it is very useful to write a clear plate identification on the top corner of the plate - the identification will then be visible in the scanned image

39. Select a plate image and click ’Open’ Click here to obtain information on the files Either the image can be loaded from a.BMP file that was created by the scanner. Or load the image from clipboard. The image must then first be copied to clipboard using the scanner or another application software.

40. Always click on well A1 in the plate first (no matter its position in the plate image) !!

41. Select reading no Type a new file name if it was the first reading occasion Select an existing.dta file if it was second and other reading occasions Remember to select the color to measure! Before proceeding with the first plate…….

42. Sample 1 The PhP values are shown. On the first reading occasions names of samples can be added to the text box If the data are not ok, click ’use new co-ordinates’ and reload the same plate (or another plate) If data are ok, click ’Save data’ When all plates from one reading occasion have been converted, click ’Exit’. You must exit after the last plate has been converted!

43. If you want to transform absorbance data..... Go to beginning Go to main menu Go to beginning Go to main menu

44. Select option Select a file that contains absorbance data from PhP plates.

45. Select reading no Type a new file name if it was the first reading occasion Select an existing.dta file if it was second and other reading occasions

46. Look at the converted data – Does it seem to correspond to data from one plate? No – click on the first line in the unconverted data that displays absorbance values Unconverted data Converted to PhP data

47. Look at the converted data – Does it seem to correspond to data from one plate? Isolate 1 Yes – Type the names of the isolates in the text box (optional) The conversion settings may be permanented by selecting ’Change file layout’

48. Go to beginning Go to main menu Go to beginning Go to main menu

49. How to transfer data between Microsoft Excel and PhP How to select the most representative isolates from common PhP types for saving and further analysis How to select the most representative data from common PhP types for creating a reference data base Some useful hints Go to beginning Go to main menu Go to beginning Go to main menu

50. How to transfer data between Microsoft Excel and PhP

51. Excel file with PhP data Other data PhP data

52. Start the PhP software Select ’Load data from clipboard’

53. Select data in the Excel file to be used. The last columns must contain the PhP data. Copy to clipboard

54. Now the data has been loaded and may be used like any other PhP data Go back to PhP software and proceed as usual Go to beginning Other useful hints Go to beginning Other useful hints

55. Analyse the data, create a dendrogram Select ’PhP types’ How to select the most representative isolates from common PhP types for saving and further analysis

56. The list is sorted according to the order of samples. Click on the list to sort it according to the order in the dendrogram The ’X’ in the left column indicate the isolate(s) that is the most representative for each type (showing the highest minimum similarity to other isolates belonging to the same type) Click again to sort it in original order

57. Selected isolates

58. Selected isolates are picked directly from the wells in the incubated PhP plates and saved for further studies Go to beginning Other useful hints Go to beginning Other useful hints

59. Select ’PhP types’ Analyse the data, create a dendrogram How to select the most representative data from common PhP types for creating a reference data base

60. The ’X’ in the left column indicate the isolate(s) that is the most representative for each type (showing the highest minimum similarity to other isolates belonging to the same type)

61. Click on the yellow PhP icon on the bottom menu bar Select Data manager – View and edit data

62. Select a new or existing reference file to save the selected data in Click on samples to save in the reference file Click ’Save’

63. THE END Go to beginning Other useful hints Go to beginning Other useful hints