Computer assisted biological assay system

• US 4,637,053 A
• Filed: 05/03/1984
• Issued: 01/13/1987
• Est. Priority Date: 05/03/1984
• Status: Expired due to Fees

• Current Assignee: Spiral System Instruments Incorporated
• Original Assignee: Spiral System Instruments Incorporated
• Inventors: Schalkowsky, Samuel
• Primary Examiner(s): Kreitman, Stephen A.
• Application Number:
  US06/606,886
• Time in Patent Office: 985 Days
• Field of Search: 382/6, 382/66, 382/68, 364/555, 364/557, 364/582, 364/416, 364/413, 356/39, 377/7, 377/10, 435/808, 435/291, 435/39, 435/32, 435/30
• US Class Current: 382/133
• CPC Class Codes: C12M 41/36 of biomass, e.g. colony counters or by turbidity measurements electrooptical investigation of individual particles G01N15/14, flow cytometers G01N15/1404
• CPC Class Codes: G06M 11/00 Counting of objects distributed at random, e.g. on a surface

Abstract

The present invention is an improved system for counting of bacterial colonies on a plate. The system permits the counting of colonies under adverse colony counting conditions where there is overlap of colonies and analyzes the plate in a plurality of segments. The colony counts for each segment are subjected to count correction, size qualification and distribution qualification to correct for missed counts and to eliminate data from the system data base when the count for a segment will not produce reliable data. The entire plate is also analyzed for subsample qualification which provides the best estimate of the bacteria containing liquid which was used to produce the colonies as represented by a coefficient of variation of the bacterial density in the liquid.

Claims

1. In a system for counting the number of colonies in a segment of an area by scanning the area with a scanning spot, a method for determining the number of counts missed because of colony overlap to produce a corrected count indicative of the actual number of colonies in the segment comprising:

(a) scanning the segment with the scanning spot to determine a number of distinct colonies within the segment;

(b) determining the average diameter of the scanned colonies; and

(c) calculating the corrected count of colonies within the segment as a function of the sensed number of distinct colonies, the computed average diameter and segment geometry.

2. A method in accordance with claim 1 wherein the data of the segment is discarded when the calculation of a corrected count is mathematically impossible.

3. A method in accordance with claim 1 wherein the data of the segment is discarded when a predetermined condition is determined to exist.

4. In a system for counting the number of colonies in a plurality of segments of an area by scanning the area with a scanning spot scanner, a method for comparing average colony size in each individual segment to the average of all of the segments and for qualifying individual segments in accordance with the average size of the colonies contained in relationship to the average size of all of the colonies contained in the plurality of segments comprising:

(a) scanning the plurality of segments with the scanning spot to detect the first intercepts of the scanning spot with each of the visible colonies of each segment during the scanning of the segments to permit determination of the number of distinct colonies in each segment; and

(b) determining average colony size in each segment from said number of distinct colonies in each segment and a total number of intercepts in each segment.

5. A method in accordance with claim 4 wherein the average size of the colonies in all of the segments is computed from the average size of the colonies in each segment and the average size of the colonies in each segment is compared to the aerage colony size of all of the segments.

6. A method in accordance with claim 4 wherein the determining of average colony size also utilizes parameters derived from scan geometry of the scanning of the plurality of segments with the scanning spot.

7. In a system for counting the number of colonies in a plurality of segments of an area by scanning the area with a scanning spot scanner, a method for comparing average colony size in each individual segment to the average of all of the segments and for qualifying individual segments in accordance with the average size of the colonies contained in relationship to the average size of all of the colonies contained in the plurality of segments comprising:

(a) scanning the plurality of segments with the scanning spot to the first intercepts of the scanning spot with the visibile colonies of each segment during the scanning of the segments to permit determination of the of distinct colonies in each segment; and

determining average colony size in each segment from said number distinct colonies and a total number clock pulses occurring during interception of the within each segment.

8. A method in accordance with claim 7 wherein the average size of colonies in all of the segments is computed from the size of the colonies in each segment and the size of the colonies in each segment is compared to the average colony size of all of the segments.

9. A method in accordance with claim 7 wherein the determining of average colony size also utilizes parameters derived from scan geometry of the scanning of the plurality of segments with the scanning spot.

10. In a system for counting the number of colonies in a plurality of segments in a subsample scanned by a scanning spot, a method for detecting deviation of the distribution of colonies in a subsample with respect to a reference distribution comprising:

(a) detecting the number of discrete colonies in each of the plurality of segments within the subsample;

(b) normalizing the segments as a function of liquid sample volume deposited in each segment;

(c) computing a coefficient of variation for the normalized segments of the subsample;

(d) computing a coefficient of variation for the reference distribution; and

(e) comparing the coefficient of variation for the normalized segments of the subsample with the coefficient of variation of the reference distribution to determine if the colonies in the subsample conform to the reference distribution.

11. A method in accordance with claim 10 wherein if the coefficient of variation of the subsample does not conform to that of the reference distribution, the data of individual segments is removed from the computation of the coefficient of variation of the subsample to produce a newly computed coefficient of variation of the subsample and the newly computed coefficient of variation is compared with the coefficient of variation of the reference distribution to determine whether better conformance to the reference distribution is achieved by the absence of the excluded data.

12. A method in accordance with claim 11 wherein the effect of each segment within the subsample in computation of the coefficient of variation of the subsample is determined and the removal of those segments which produce better conformance is performed until either acceptable conformance to the reference distribution is obtained or the effect of the removal of all but a predetermined remaining number of segments.

13. In a system for counting colonies in a plurality of subsamples which each contain a plurality of segments by scanning the subsamples with a scanning spot, a method for estimating the bacterial concentration in the sample used for producing the scanned colonies and providing an assessment of how well the estimated concentration for the sample represents the true concentration from which the sample was derived comprising:

(a) performing count correction, size qualification and distribution qualification for each segment within each subsample to produce a total corrected count for all of the segments of the subsamples, a measure of the quality of the subsample as represented by the degree of conformance to a reference distribution and a determination of the proportion of the counts added by the correction procedure to the corrected count;

(b) determining the estimated bacterial concentration of the sample from the total count and the total volume contained in the segments which remain after count correction, size qualification and distribution qualification; and

(c) determining the measure of the conformance of the estimated concentration of the bacteria in the subsample to the bacterial concentration in a substance from which the sample was derived by calculation of an estimated coefficient of variation comprising a component due to statistical variation assuming that the corrected count of the subsample follows the distribution of the bacteria in the source, modifying the component due to statistical variation by the quality measure of the subsample obtained from the distribution qualification and a component associated with the fraction of the total corrected count derived from the count correction procedure.

14. In a system for counting the number of colonies in a plurality of segments of an area by scanning the area with a scanning spot, a method for comparing average colony size in each individual segment to the average of all of the segments and for qualifying individual segments in accordance with the average size of the colonies contained in relationship to the average size of all of the colonies contained in the plurality of segments comprising:

(a) scanning the segment with the scanning spot to determine a number of distinct colonies within the segment; and

(b) determining average colony size in each segment from said number of distinct colonies in each segment and a total number of intercepts in each segment.

15. In a system for counting colonies in a plurality of subsamples which each contain a plurality of segments by scanning the subsamples with a scanning spot, a method for estimating the bacerial concentration in a deposited sample used for producing the scanned colonies and providing an assessment of how well the estimated concentration for the sample represents the true concentration from which the sample was derived comprising:

(a) performing count correction, size qualification and distribution qualification for each segment within each subsample to produce a total corrected count for all of the segments of the subsamples, a measure of the quality of the subsample as represented by the degree of conformance to a reference distribution and a determination of the proportion of the counts added by the correction procedure to the corrected count;

(b) determining the estimated bacterial concentration of the sample from the total count and the total volume contained in the segments which remain after count correction, size qualification and distribution qualification; and

(c) determining the measure of the conformance of the estimated concentration of the bacteria in the subsample to the bacterial concentration in a substance from which the sample was derived by calculation of an estimated coefficient of variation comprising a component due to statistical variation based upon the number of colonies used in estimating the bacterial concentration, a component associated with the quality measure of the subsample obtained from the distribution qualification and a component associated with the total corrected count derived from the count correction procedure.

16. The method of claim 15 wherein the estimated coefficient of variation is used for the purpose of qualifying subsamples and selecting among the subsamples those subsamples with data which is preferable in estimating the bacterial concentration of the deposited sample.