The PhenePlate software

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

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

3. 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

4. Reading 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’ Go to beginning Go to PhP menu

5. Click here to se a detailed list on files available 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 Either select a PhP data file to be analysed Data analysis Or select PhP data from another application(e.g. Excel) file to be analysed

6. The main menu 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 Clear memory and load new data Calculate similarities for clustering and dendrogram presentation Comparison to reference data Pairwise comparisons of isolates Population statistics (Diversity, Sp etc) Evaluation of tests

7. View and edit data Wait until this message appears 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

8. Paste!

9. Creating a reference data file 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. Selected data can be copied to a database containing data from reference strains, e.g. of known species.

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

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

12. 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 Remove tests Go to beginning Go to main menu

13. Sort and remove samples 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

14. Normalise data 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’ Normalization towards negative controltest: 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). Convert data: Data may be converted to other numerical values, or to dichotomous (+/-) values Go to beginning Go to main menu

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

16. Analyse data - clustering

17. Analyse data - clustering

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

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

20. 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 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

21. Adding marks and text to the dendrogram

22. Use this option to be able to edit the dendrogram in e.g. an imaging software or in Excel... 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

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

24. Presenting several dendrograms on the same page

25. Presenting several dendrograms on the same page

26. Presenting several dendrograms on the same page Up to 10 dendrograms per page can be presented Go to beginning Go to main menu

27. Comparisons to reference data 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)

28. 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 Selected reference samples 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)

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

30. 1 (*) P1:1 Select two samples Similariy between sample 1 and sample 2 Results for sample 1 Results for sample 2 Differences between sample 1 and sample 2 Pairwise comparisons of samples Go to beginning Go to main menu

31. 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

32. 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’. The first isolate in each population will be marked with an X

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

34. 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 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

35. Use scroll barto move list Mean and median similariesmeasure the similarities between all isolates in two different compared populations. Population similarity coefficients, mean and median similaritiesmay be clustered to yield a dendrogram showing the relations beween different populations (samples) 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 Go to beginning Go to main menu

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

37. 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

38. Click here to obtain informationon the files Select a plate image and click ’Open’ 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.

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

40. Before proceeding with the first plate……. Remember to select the color to measure! 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

41. When all plates from one reading occasion have been converted, click ’Exit’. You must exit after the last plate has been converted! 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’ The PhP values are shown. On the first reading occasions names of samples can be added to the text box Sample 1

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

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

44. 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

45. 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

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

47. Go to beginning Go to main menu

48. Some useful hints Go to beginning Go to main menu

49. How to transfer data between Microsoft Excel and PhP

50. Other data PhP data Excel file with PhP data