Multiple tube methods, also known as most probable number (MPN) methods, are suitable for liquid food products or food homogenates. The methods are based on the probability of finding bacterial growth after culture of successive dilutions of the food sample in a liquid medium. They are used for enumeration of specific organisms or groups of organisms such as Escherichia coli and col-iforms, and not for ‘total’ microbial counts on a product. The most commonly used procedures are based on the use of three serial decimal dilutions of the sam-ple (methods 3 and 4) using three or five aliquots of each dilution; further dilu-tions can be made if necessary. The degree of dilution necessary and the number of tubes at each dilution used depends on the initial bacterial load. For a sample that is unlikely to contain many organisms to be counted, culture of 6 ¥ 18 mL aliquots of a single dilution (method 1) would give a counting range of 1–10 organisms/100 mL of the dilution used. For a wider count range, 10 ¥ 1 mL aliquots of a single dilution will allow counting of 10–230 organisms/100 mL of the dilution used (method 2). Higher number of organisms can be counted and greater accuracy achieved with the methods utilizing three serial dilutions.
5.7
(d) Incubate all plates as appropriate for the organisms sought (see Section 6 for guid-ance). For a total mesophilic aerobic colony count, incubation is normally per-formed at 30 ± 1°C for 48 ± 2 h or 72 ± 3 h.
After incubation
Count all colonies on plates containing 300 or fewer colonies per plate or, if the medium is selective, 150 colonies or fewer per plate.
Calculation
Use the plates containing not more than 300 colonies (or 150 for selective media) at two consecutive dilutions to calculate the results. Calculate the number (N) of cfu/g or mL of test sample as follows:
N = C/v (n1+ 0.1n2) d.
For the key to symbols and an example of calculation see Section 5.3. In this instance, the volume added per plate, v, is 0.1 mL (or 0.5 mL).
Method 1 Six tube (six by 18 mL or 18 g) test
Procedure
(a) If the sample is liquid, add 18 mL volumes to each of six 180 mL volumes of the chosen liquid medium (e.g. buffered peptone water (BPW) or selective enrich-ment medium). If the sample is solid, prepare six homogenates using 18 g of sample with 180 mL of chosen liquid medium.
continued
(b) Incubate overnight at the temperature appropriate for the organism sought (see Section 6 for guidance).
After incubation
Test all six tubes for the characteristic reactions of the organisms sought by subculture from each tube to a suitable selective agar or liquid medium. If the medium used has no characteristic reactions, subculture all the tubes. Incubate the plates or broths and record the number of tubes that contain the target bacteria. Obtain the MPN of bacte-ria/g or mL of the food sample from Table 5.5, taking into account any dilution factor of the original sample.
Method 2 Ten tube (10 by 1 mL or 1 g) test
Procedure
(a) For liquid samples add 1 mL volumes to 10 tubes of chosen liquid medium (e.g. buffered peptone water or selective enrichment medium). For solid samples add 10 mL of the 10-1homogenate (equivalent to 1 g) to either 10 mL of double strength medium or 90 mL of single strength medium.
(b) Incubate overnight at the temperature appropriate for the organism sought (see Section 6 for guidance).
After incubation
Test all 10 tubes for the characteristic reactions of the organisms sought by subculture from each tube to a suitable selective agar or liquid medium. If the medium has no characteristic reactions subculture all tubes. Incubate the plates or broths and record the number of original tubes that contain the target bacteria. Obtain the MPN of bacteria/g or mL of the food sample from Table 5.6.
Table 5.5 Most probable number (MPN)/100 mL or 100 g using six 18 mL or 18 g aliquots.
Number positive MPN/100 mL or 100 g
1 1
2 2
3 4
4 6
5 10
6 >10
Method 3 Nine tube (3,3,3 tube) test
[8,9]Procedure
(a) Make serial decimal dilutions of the liquid food sample, or 10-1food homogenate prepared as described in Section 4.3, using MRD as diluent.
(b) Prepare nine tubes, each containing 10 mL of single strength growth medium appropriate for the organisms sought (e.g. BPW or a selective enrichment broth).
(c) Add 1 mL of the liquid food sample or food homogenate to each of three tubes containing growth medium.
(d) Repeat step (c) for each of two subsequent decimal dilutions.
(e) Incubate all nine tubes as appropriate for the organisms sought (see Section 6 for guidance).
After incubation
Test those tubes showing the characteristic reactions of the organisms sought by subculture from each tube to a suitable confirmatory agar or liquid medium. If the medium has no characteristic reactions subculture all the tubes. Incubate the plates or broths and record the number of original tubes at each dilution that contain the target bacteria.
Selection of dilutions [2,8,9]
Select three consecutive dilutions to obtain the MPN of bacteria/g or mL of the food sample from Table 5.7. Select the highest dilution (i.e. that having the lowest sample continued on p. 122 If greater sensitivity is required, also prepare three tubes containing 10 mL of double strength medium and add 10 mL of liquid sample or 10-1food homogenate to each tube. If high numbers of target organisms are expected, prepare three tubes of single strength growth medium for each additional decimal dilution to be used and inocu-late each of them with 1 mL of the additional dilution.
Table 5.6 Most probable number (MPN)/100 mL or 100 g using 10 ¥ 1 mL or 1 g aliquots.
Number positive MPN/100 mL or 100 g 95% confidence limits
0 0 <0–37
1 10 0.25–59
2 22 3–81
3 36 7–106
4 51 13–134
5 69 21–168
6 92 30–211
7 120 43–270
8 160 59–368
9 230 81–600
10 >230 118–>600
Table 5.7 Most probable number (MPN): three tubes at each dilution.
Adapted from ISO 7218 [2]. 95%CI, 95% confidence interval.
E X A M P L E S
(a) If the reading is 0,3,1 tubes positive, it is reasonable to assume that the first set of tubes should have been positive.
Adjusted reading = 3,3,1 From Table 5.7, MPN/g = 460
(b) If the reading is 0,3,1 tubes positive, the dilution series might be theoretically extended so that the tubes may be read 0,3,1,0. Use the last three figures to obtain the MPN value, then multiply by 10 to compensate for the further decimal dilution.
Reading = 3,1,0
From Table 5.7, MPN value = 43 MPN/g (MPN value ¥ 10) = 430.
Note: as with other counts, MPN counts should be reported to two significant figures with one figure before and one figure after the decimal point multiplied by the appropriate power of 10.
concentration) yielding three positive tubes together with the next two dilutions. If this is not possible because insufficient dilutions were made beyond the highest dilu-tion yielding three positive tubes, select instead the three highest diludilu-tions. If no dilution contains three positive tubes, select the three highest dilutions in the series amongst which at least one positive result was obtained.
Table 5.7 shows the MPN counts/g or mL obtained when the sample homogenate and two further decimal dilutions are used to inoculate the three sets of tubes. If a liquid sample and its 10-1and 10-2dilutions have been used the MPN value must be multi-plied by 10 to obtain the MPN count/mL. This must also be done to obtain the MPN count/g if 10 mL volumes of the sample homogenate are inoculated into double strength medium in the first set of tubes. The MPN/g or mL can be obtained with any set of three dilutions by using the formula:
MPN/g = MPN from Table 5.7 ¥ j/100
where j is the dilution of the middle set of tubes.
The presence of inhibitory substances in the sample may prevent typical reactions taking place in tubes containing the lowest dilution of food or the greatest volume of liquid sample. If this is anticipated the dilution series should be extended. If, how-ever, this is not done, the following examples indicate how to derive the MPN.
Interpretation of the probability tables
Wide variations in the results may occur with the MPN technique. Readings at each dilution may vary by one or two tubes, even in tests made separately on the same well-mixed sample. In addition to the MPN value, Table 5.7 shows the category of result and the range defined by 95% confidence limits (95%CI).
The category indicates the acceptability of the combination of positive results.
Category 1 results are those that have the highest probability of being correct. If
combinations belonging to category 1 are obtained they should be used in pref-erence to category 2, and so on. If more than one combination of the same cate-gory is obtained the combination with the highest number of positive tubes should be used. When the decision to be taken on the basis of the result is of great importance, only category 1 or at most category 1 and 2 results should be accepted. Category 0 results should be viewed with great suspicion as there is only a 0.1% chance of obtaining a result in this category without anything being wrong. For further information, consult the appropriate ISO standards.
Method 4 Fifteen tube (5,5,5 tube) test
[8]For greater accuracy, a 3 ¥ 5 tube method can be used, giving a total of 15 tubes incu-bated. The procedure is the same as described for method 3. The MPN/100 g can be derived from Table 9.2–9.4, pp. 233–7, which gives category 1 and category 2 values for a 15 tube test.