Читать книгу Analytical Food Microbiology - Ahmed E. Yousef - Страница 70
Scoring the Tubes and Calculating MPN
ОглавлениеFollowing incubation, each tube is scored as positive or negative, depending on the target microorganism’s characteristic of interest to the analyst. The positive tubes per each dilution are tallied and the number of positives from consecutive dilutions are reported. The outcome is reported as a series of numbers separated with commas from least dilute to most dilute. Using the results presented in Figure 3.6, the MPN score would be 2, 2, 0.
TABLE 3.2 Most Probable Number (MPN) estimatesa based on the number of positive tubes resulting from three consecutive dilutions (three tubes each) representing 0.1 g, 0.01 g, and 0.001 g of food.
| Number of positive tubes | MPN/g | Number of positive tubes | MPN/g | Number of positive tubes | MPN/g | Number of positive tubes | MPN/g | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 0.1 g | 0.01 g | 0.001 g | 0.1 g | 0.01 g | 0.001 g | 0.1 g | 0.01 g | 0.001 g | 0.1 g | 0.01 g | 0.001 g | ||||
| 0 | 0 | 0 | <3 | 1 | 0 | 0 | 3.6 | 2 | 0 | 0 | 9.2 | 3 | 0 | 0 | 23 |
| 0 | 0 | 1 | 3 | 1 | 0 | 1 | 7.2 | 2 | 0 | 1 | 14 | 3 | 0 | 1 | 38 |
| 0 | 0 | 2 | 6 | 1 | 0 | 2 | 11 | 2 | 0 | 2 | 20 | 3 | 0 | 2 | 64 |
| 0 | 0 | 3 | 9 | 1 | 0 | 3 | 15 | 2 | 0 | 3 | 26 | 3 | 0 | 3 | 95 |
| 0 | 1 | 0 | 3 | 1 | 1 | 0 | 7.4 | 2 | 1 | 0 | 15 | 3 | 1 | 0 | 43 |
| 0 | 1 | 1 | 6.1 | 1 | 1 | 1 | 11 | 2 | 1 | 1 | 20 | 3 | 1 | 1 | 75 |
| 0 | 1 | 2 | 9.2 | 1 | 1 | 2 | 15 | 2 | 1 | 2 | 27 | 3 | 1 | 2 | 120 |
| 0 | 1 | 3 | 12 | 1 | 1 | 3 | 19 | 2 | 1 | 3 | 34 | 3 | 1 | 3 | 160 |
| 0 | 2 | 0 | 6.2 | 1 | 2 | 0 | 11 | 2 | 2 | 0 | 21 | 3 | 2 | 0 | 93 |
| 0 | 2 | 1 | 9.3 | 1 | 2 | 1 | 15 | 2 | 2 | 1 | 28 | 3 | 2 | 1 | 150 |
| 0 | 2 | 2 | 12 | 1 | 2 | 2 | 20 | 2 | 2 | 2 | 35 | 3 | 2 | 2 | 210 |
| 0 | 2 | 3 | 16 | 1 | 2 | 3 | 24 | 2 | 2 | 3 | 42 | 3 | 2 | 3 | 290 |
| 0 | 3 | 0 | 9.4 | 1 | 3 | 0 | 16 | 2 | 3 | 0 | 29 | 3 | 3 | 0 | 240 |
| 0 | 3 | 1 | 13 | 1 | 3 | 1 | 20 | 2 | 3 | 1 | 36 | 3 | 3 | 1 | 460 |
| 0 | 3 | 2 | 16 | 1 | 3 | 2 | 24 | 2 | 3 | 2 | 44 | 3 | 3 | 2 | 1100 |
| 0 | 3 | 3 | 19 | 1 | 3 | 3 | 29 | 2 | 3 | 3 | 53 | 3 | 3 | 3 | > 1100 |
a Confidence intervals are not included to simplify the table (FDA Bacteriological Analytical Manual and other sources).
Figure 3.6 Dilution and inoculation scheme for most probable number technique.
After scoring MPN tubes, the score is converted into an MPN/g food sample using standard MPN tables (Table 3.2). There are equations and computer programs available for calculating MPN; online MPN calculators may also be useful. The MPN table is based on the amount of sample (i.e., food) that is present in the MPN tubes; therefore, column headings of MPN tables are usually expressed in grams. MPN tables are usually constructed for sample sizes of 0.1, 0.01, and 0.001 g, for three consecutive dilutions. In practice, these values would correspond to 1 ml transferred to each MPN tube from 10–1, 10–2, and 10–3 dilutions of the master dilution set. In the example shown in Figure 3.6, 1 ml of the 10–2, 10–3, and 10–4 dilutions were used to inoculate the MPN tubes. This inoculation would correspond to 0.01, 0.001, and 0.0001 g of sample in successive MPN tube sets. Therefore, the value from the MPN table, corresponding to the number of positive tubes, must be adjusted to accommodate the difference in amounts of the sample analyzed. Because the sample is ten times more dilute, the table values should be multiplied by ten. Alternatively, the following equation can be used:
(3.8)
This pattern continues if higher dilutions are used to inoculate MPN tubes. MPN results should be reported in scientific notation with the units of MPN/g. For the example in Figure 3.6, the result after correction would be reported as 2.1×102 MPN/g.
In a situation where all incubated tubes were negative (MPN score: 0, 0, 0), the result would be reported as < 3.0×101 MPN/g (assuming no adjustment for dilution scheme). Analysts should not report 0 MPN/g. Similarly, if all incubated tubes were positive (MPN score: 3, 3, 3) the result would be > 1.1×104 MPN/g. These values do not require the “est.” designation due to the presence of the greater than (>) or the less than (<) signs.
