Automated Counting of Microbial Colonies on

Rehydratable Film Media

Mehrdad Saadat

Biology 4400

Spring Semester 1999

Automated Counting of Microbial Colonies on

Rehydratable Film Media

I. Background

II. Methods and Results

III. Conclusions

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

Background

• BioCount

– Developed by Apogee Systems

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

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

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

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

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

Methods and Results

• Procedure– First: Prepare a fresh culture of aerobic

bacteriaBacillus 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)

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

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

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

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.

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

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

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

• 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

Methods and ResultsManual Automated

Film # Count Count 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

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

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.

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