2. Automated Counting of Microbial Colonies on Rehydratable Film Media Mehrdad Saadat Biology 4400 Spring Semester 1999

3. Automated Counting of Microbial Colonies on Rehydratable Film Media I.Background II.Methods and Results III.Conclusions

4. Background Use of Petri dishes –Every day, biologists use petri dishes filled with media to culture bacteria –One use for culturing is enumeration –A simple formula can be applied to a set of cells grown on media to enumerate the number of cells in the original sample being tested

5. Background BioCount  –Developed by Apogee Systems

6. Background BioCount  –This is a scanner based system used for detecting and enumerating bacterial colonies in petri dishes –The system uses raster imaging to detect curvatures on a given media –The program then accepts a set of curvatures as a colony

7. Background Previous Results –The BioCount system has proven to be reliable in counting colonies on mFC agar –Paula Tennant-Clegg showed in her research that the BioCount system could accurately count coliform colonies on mFC agar when properly cultured

8. Background Rehydratable Film Media –Recently the 3M company has developed the Petrifilm ® –This is a much easier and faster way of plating for several reasons:  no need to spend hours making a selective media  no accidents in “cutting into” the agar while streak plating

11. Background Can the BioCount system be adapted for use with Petrifilms? If so, it provides a possibility for –More rapid analysis –Greater objectivity: Elimination of variability associated with manual counts –Automated documentation

12. Methods and Results Objectives of this Project –Determine the Biocount ® parameters for optimal counts of total aerobic Petrifilms –Compare manual and automated colony counts –Identify possible causes of false positive and false negative errors in the automated counts

13. Methods and Results Procedure –First: Prepare a fresh culture of aerobic bacteria Bacillus subtilis, Enterococcus fecalis, Staphylococcus aureus –Once a turbid broth was obtained, it could be diluted down to give a desired concentration of bacterial cells (10 -14 )

14. Methods and Results Procedure (cont.) –One ml of the dilution was then plated on the Petrifilm aseptically using a pipette –Proper procedure was followed to cover the film and incubate it at the temperature given by 3M™ (approximately 33º C) –After 24 hours the films were removed from the incubator –At this time colonies were manually counted

15. Methods and Results Procedure (cont.) –The films were then scanned by the desktop scanner, four films at a time –Once plated and counted by hand, the colonies were scanned using a typical desktop scanner by HP at 150 dpi

16. Methods and Results Hypothesis: There is not a significant difference in the count of bacterial colonies on a Petrifilm between a manual count and that of one done by an automated system

18. Methods and Results Parameters –Using the parameters tool, I attempted to optimize the parameters so that the program would detect an almost exact number of colonies on a given film — if this were to work I would then test the same parameters on another film.

20. Methods and Results Parameters (cont.) –Optimal parameters should pass two tests: They must only allow for detection of few, if any false positives nor any false negatives. They must accurately count the present colonies

21. Methods and Results Parameters (cont.) –Minimum Peak Size: 6 –Radius Step: 1 –Vector Radius Maximum: 3 –Gray Threshhold: 135 –Radius Power: 1.01 –RGB color settings: 240, 169, 25 –Intensity settings: 2.15, 1.25,.08

23. Methods and Results We already knew that the program was set to utilize a gray scale in order to detect colonies We also know that if we pointed to a given colony with the computer’s mouse, and used the right button of the mouse to click on the colony, we would set the gray-scale parameters to that colony’s colors

24. An area of the film may be enlarged to give more distinct colonies When enlarged the colonies were not all the same color and there was a gradient of colors within a given colony. This appeared to be a major source of error in optimizing the system

28. Methods and Results ManualAutomated Film # CountCount Difference --------- ------------- ----------------------------- 1 745 707 38 2 286 323 17 3 525 509 16 4 498 475 23 5 442 504 62 6 387 433 46 7 240 332 92 8 413 500 87

29. Methods and Results Statistical Analysis: t-test t = 1.582 –At a 95% confidence level, the deviation between the automated and manual count is not significant –Therefore, the data support the hypothesis

30. Conclusions Overall, there is good agreement between manual and automated colony counts on total aerobic Petrifilms If we were to choose a dark colony as our basis for a gray-scale, the program would detect anything below this level of darkness If we were to choose a light colony the program would detect, light colonies, as well as dark colonies, but it would also detect many false positives and background interference.

31. Conclusions Areas for future work –Microbiology research: Find ways to promote colony growth with greater uniformity of color –Computer research: Enhance the colony detection method to handle color gradients within colonies