The Most Suitable Number of Colonies on Plates for Counting

Abstract

Major events that led to acceptance of 30 to 300 as the most suitable number of colonies on plates for counting were reviewed. Three new sets of data were collected, involving triplicate plates of fifteen 1: 1.4 serial dilutions of 65 samples of raw milk. Statistical methods were developed to analyze bias (variability introduced primarily by crowding and analyst counting errors) and variance (sampling and dilution errors), Bias and variance were combined as mean-squared error, which was expresed as a function of the number of colonies per plate, The counting range that minimized the mean squared error could then be determined for selected dilution series. For twofold , five-fold and tenfold dilution series, respectively, the most suitable limits on plates for counting were 70 to 140, 40 to 200 and 25 to 250 colonies/plate. A range of 2S to 250 colonies/plate was suggested for the analysis of dairy prodUcts. Limitations in application of the data to other systems are discussed. Almost every textbook, laboratory manual and methods volume in microbiology contains the statement that plates for counting bacteria should contain, when possible, 30-300 colonies. The "30-300" concept has been so ingrained in our thinking that the limits are rarely questioned. We report here the historical background of the "30-300" concept and results of a study wherein modern plating methods were analyzed statistically to determine the counting range that would result in minimal errors when making plate counts, The "30-300" concept originated with two publications by Breed and Dotterrer (7,8); the text and tables presented in both publications are identical. The authors summarized results of a few early studies and then proceeded to more clearly define the problem and provide a solution. Three dilutions (10-2 , 10-3 and 10-4) of each of a large number of raw milk samples were plated in triplicate on standard agar. Colonies were counted after incubation for 5 days at 20 C and again after incubation for 2 additional days at 37 C. Colonies on all plates were counted, when possible. Individual plate counts were tabulated as "satisfactory" if the count could be used in making an average without deviating by more than 20 % from the average. Thus, each average was determined by a variable number of plates in the serial dilution series for a sample; occasionally, all nine of the plates made from a sample could be included in the final average. An individual plate count was tabulated as a "discrepancy" if the count varied by more than 20% from the average. The 21 C, S-day data of Breed and Dotterrer (7.8) are reproduced in Table 1 because these data have, through the years, served as the major basis for specifying the most suitable number of colonies on plates for counting. Their description of the data (slightly paraphased) follows: "Table 1 gives the number of plate counts made after 5 days at 21 C, arranged in groups according to the number of colonies which appeared on the plates. Of the 1435 plates, 439 had less than 10 colonies/ plate. Only 22 % of these checked within the 200/0 limit. Of 180 plates in the group having more than 10 and less than 20 colonies/plate, 54% checked within the 20 % Iimit. Percentages calculated for the other groups of plates were more or less variable, showing that from 66 to 93%ofthe total number of plates agreed within the 20% limit. The best percentage of agreement is shown by the group having more than 100 and less than 200 colonies/plate, and the next highest by the group having between SO and 100 colonies/plate. There were decidedly fewer plates giving satisfactory results among those which had more than 400 colonies/plate, the percentage of plates which checked within 20 % being 44. "The results given in the lower part of Table 1 were calculated from the same counts. the groups of plates having been arranged differently. From this part of the table it will be seen that the percentages of discrepant plates is practically the same for groups of plates having 20 to 400, 30 to 400. 20 to 200, 30 to 200, or 40 to 200 colonies/plate, the best showing being made by the group of plates having more than 40 and less than 200 colonies/plate. Plates having less than 30 or more than 400 colonies show very large percentages of discrepancies ." Breed and Dotterrer (7,8) concluded that.