APPENDIX 14

ADDITIONAL EXPLANATIONS TO THE CALCULATION OF THE MOST PROBABLE

NUMBER (MPN)

Example:

Determine the number of B. japonicum cells contained in 1 g of a

100 g bag of inoculant made from nonsterile peat.

1) Dilute the 100 g of inoculant in 900 ml water.

2) Make a tenfold dilution series (Table A14.3)

3) Set up plants in quadruplicates as described in Chapter

6 and inoculate each plant with 1 ml of the dilutions.

4) Record nodulation (+ or -).

5) Beside each dilution, write the number of nodulated (+)

units.

6) Add the total of the nodulated units assuming the

results shown in Table A.6.

7) Note that number of replications, n = 4; dilution

steps, s = 10; number of nodulated units, (+) = 21;

lowest dilution in the series, d = 10-1.

8) Use Table A.10 which is calculated for tenfold

dilutions and locate 21 (for 21+ units) in column n =

4.

9) Find the most likely number (m) in column s = 10

corresponding to 21 in the n = 4 column. The most

Table A.6. Evaluation of soybean inoculant prepared from

nonsterile peat.

NODULATION
---------Replications-------- NUMBER OF
NODULATED
UNITS
DILUTION I II III IV
10-1 + + + + 4
10-2 + + + + 4
10-3 + + + + 4
10-4 + - + + 3
10-5 - - + + 2
10-6 + - + - 2
10-7 - - + - 1
10-8 + - - - 1
10-9 - - - - 0
10-10 - - - - 0
Total 21

likely number is m = 3.1 X 104.

10) Multiply most likely number with the reciprocal of

lowest dilution used in the series (d = 101).

(3.1 X 104) X (101) = 3.1 X 105

The peat inoculant contained 3.1 X 105 rhizobia per

gram. Since the original sample was diluted 1:10 (100

g peat in 900 ml sterile water) and aliquot (v) used

for inoculation was 1 ml, the actual calculation should

be:

X = m X d = 3.1 X 104 X 101 = 3.1 X 105 rhizobia g-1 inoculant

 V 1

Determing the most probable number in soil

The MPN count is often used to determine the number of rhizobia

present in soil. Whereas a tenfold dilution series with two or

four replicates is sufficient for most peat inoculants, which

usually have a relatively high number of rhizobia (>108 cells

g-1), a fourfold or even twofold dilution series with

replications in quadruplicate is usually chosen for soil. The

smaller dilution steps provide a more precise estimate when less

than 10,000 cells of rhizobia per gram soil are expected. The

first sample of the series, however, is frequently diluted

tenfold or a 100-fold.

Example: 100 grams of field soil were diluted in 900 ml of

sterile water. A quadruplicate dilution series was prepared

ranging from 4-1 to 4-9. Aliquots of 2 ml were used for the

inoculations.

Use Table A.9 for four fold dilutions in this appendix. In

column (n = 4), find 25 for 25 + units.

Table A.7. Determination of the population of native cowpea-

rhizobia in field soil using sirato as the trap host.

NODULATION

---------Replications--------- Number of
Nodulated
units
Dilution I II III IV
10-1 + + + + 4
4-1 + + + + 4
4-2 + + + + 4
4-3 + + + + 4
4-4 + + + - 3
4-5 ─ + + + 3
4-6 + + ─ ─ 2
4-7 + ─ ─ ─ 1
4-8 ─ ─ ─ ─ 0
4-9 - - - - 0
Total 25

n = 4; s = 10; d = 10-1; + units = 25

m = 1.6 X 103

d (lowest dilution) = 10-1

v = 2 ml

X = 1.6 X 103 X 101 = 8000

2

The most probable number of rhizobia in the field soil was 8000

cells per gram.

Table A.8. Number (M) of rhizobia estimated by the plant infection count (After
Vincent 1970): A. Two-fold dilutions: (A=2)
Positive tubes Dilution steps (s)

n=4 n=2 s=10

40 20 >520
39
38 19 520
37 370
36 18 290
35 220
34 17 180
33 140 s=8
32 16 120 >130
31 95
30 15 78 130
29 65 93
28 14 54 72
27 45 55
26 13 37 45
25 31 35 s=6
24 12 26 29 >33
23 21 24
22 11 18 19 33
21 15 16 23
20 10 13 13 18
19 11 11 14
18 9 8.9 9.3 11
17 7.4 7.7 8.9 s=4
16 8 6.3 6.4 7.4 >8.3
15 5.2 5.4 6.0
14 7 4.4 4.6 4.9 8.3
13 3.7 3.8 4.1 5.9
12 6 3.2 3.2 3.4 4.6
11 2.6 2.6 2.7 3.4
10 5 2.2 2.2 2.3 2.8
9 1.8 1.9 1.9 2.2
8 4 1.5 1.5 1.6 1.8
7 1.2 1.3 1.3 1.4
6 3 1.0 1.0 1.0 1.1
5 0.79 0.79 0.81 0.97
4 2 0.60 0.60 0.62 0.66
3 0.42 0.43 0.43 0.46
2 1 0.27 0.27 0.27 0.29
1 <0.2 <0.2 <0.2 <0.2
0 0
_____
Approx range 2000 500 120 30
Factor, 95%
fiducial limits n=2 2.7
(x,) n=4 2.0

Calculated from Table VIII2 of Fisher and Yates (1963).

Table A.9. Number (M) of rhizobia estimated by the plant infection count (After
Vincent 1970): B. four-fold dilutions; (A=4)
Positive tubes Dilution steps (s)
n=4 n=2 s=10
40 20
39 >2.0x105
38 19 2.0x105
37 1.2
36 18 8.1x104
35 5.5
34 17 3.8
33 2.6 s=8
32 16 1.8 >1.3x104
31 1.3
30 15 9.1x103 1.3x104
29 6.3 7.9x103
28 14 4.5 5.1
27 3.5 3.5
26 13 2.2 2.4
25 1.6 1.7 s=6
24 12 1.1 1.1 >7.9x102
23 8.0x102 8.0x102
22 11 5.6 5.6 7.9x102
21 4.0 4.0 5.0
20 10 2.8 2.8 3.2
19 2.0 2.0 2.2
18 9 1.4 1.4 1.5
1.0 1.0 1.0 s=4
16 8 7.1x101 7.1x101 7.2x101 >5.0x101
15 5.0 5.0 5.1
14 7 3.5 3.5 3.5 5.0x101
2.5 2.5 2.5 3.2
12 6 1.8 1.8 1.8 2.0
1.3 1.3 1.3 1.4
10 5 8.9x100 8.9x100 8.9x100 9.6x100
6.3 6.3 6.3 6.6
8 4 4.5 4.5 4.5 4.6
3.2 3.2 3.2 3.2
6 3 2.2 2.2 2.2 2.2
1.6 1.6 1.6 1.6
4 2 1.1 1.1 1.1 1.1
3 7.2x10-1 7.2x10-1 7.2x10-1 7.2x10-1
2 1 4.4 4.4 4.4 4.4
1 0
0 <4.4x10-1 <4.4x10-1 <4.4x10-1 <4.4x10-1

Approx. range 5x105 3x104 2x103 1x102

Factor for 95%
fiducial limits n=2 4.0
(x,) n=4 2.7

Calculated from Table VIII2 of Fisher and Yates (1963).

Table A.10. Number (M) of rhizobia estimated by the plant infection count (After
Vincent 1970): C. Ten-fold dilutions; (A=10)
 Positive tubes Dilution steps (s)
n=4 n=2 s=10
40 20 >7x108
39
38 19 6.9
37 3.4
36 18 1.8
35 1.0
34 17 5.9x107
33 3.1 s=8
32 16 1.7 >7x106
31 1.0
30 15 5.8x106 6.9
29 3.1 3.4
28 14 1.7 1.8
27 1.0 1.0
26 13 5.8x105 5.9x105
25 3.1 3.1 s=6
24 12 1.7 1.7 >7x104
23 1.0 1.0
22 11 5.8x104 5.8x104 6.9
21 3.1 3.1 3.4
20 10 1.7 1.7 1.8
19 1.0 1.0 1.0
18 9 5.8x103 5.8x103 5.9x103
17 3.1 3.1 3.1 s=4
16 8 1.7 1.7 1.7 >7x102
1.0 1.0 1.0
14 7 5.8x102 5.8x102 5.8x102 6.9
3.1 3.1 3.1 3.4
12 6 1.7 1.7 1.7 1.8
1.0 1.0 1.0 1.0
10 5 5.8x101 5.8x101 5.8x101 5.9x101
3.1 3.1 3.1 3.1
8 4 1.7 1.7 1.7 1.7
7 1.0 1.0 1.0 1.0
6 3 5.8x1 5.8x1 5.8x1 5.8x1
3.1 3.1 3.1 3.1
4 2 1.7 1.7 1.7 1.7
1.0 1.0 1.0 1.0
2 1 0.6 0.6 0.6 0.6
1 <0.6 <0.6 <0.6 <0.6
0 0

Approx. range 109 107 105 103

Factor, 95%
fiducial limits* n=2 6.6
(x,): n=4 3.8

Calculated from Table VIII2 of Fisher and Yates (1963).

Table A.10. Number (M) of rhizobia estimated by the plant infection count (After
Vincent 1970): C. Ten-fold dilutions; (A=10)
Positive tubes Dilution steps (s)
n=4 n=2 s=10
40 20 >7x108
39
38 19 6.9
37 3.4
36 18 1.8
35 1.0
34 17 5.9x107
33 3.1 s=8
32 16 1.7 >7x106
31 1.0
30 15 5.8x106 6.9
29 3.1 3.4
28 14 1.7 1.8
27 1.0 1.0
26 13 5.8x105 5.9x105
25 3.1 3.1 s=6
24 12 1.7 1.7 >7x104
23 1.0 1.0
22 11 5.8x104 5.8x104 6.9
21 3.1 3.1 3.4
20 10 1.7 1.7 1.8
19 1.0 1.0 1.0
18 9 5.8x103 5.8x103 5.9x103
17 3.1 3.1 3.1 s=4
16 8 1.7 1.7 1.7 >7x102
1.0 1.0 1.0
14 7 5.8x102 5.8x102 5.8x102 6.9
3.1 3.1 3.1 3.4
12 6 1.7 1.7 1.7 1.8
1.0 1.0 1.0 1.0
10 5 5.8x101 5.8x101 5.8x101 5.9x101
3.1 3.1 3.1 3.1
8 4 1.7 1.7 1.7 1.7
7 1.0 1.0 1.0 1.0
6 3 5.8x1 5.8x1 5.8x1 5.8x1
3.1 3.1 3.1 3.1
4 2 1.7 1.7 1.7 1.7
1.0 1.0 1.0 1.0
2 1 0.6 0.6 0.6 0.6
1 <0.6 <0.6 <0.6 <0.6
0 0

Approx. range 109 107 105 103

Factor, 95%
fiducial limits* n=2 6.6
(x,): n=4 3.8

Calculated from Table VIII2 of Fisher and Yates (1963).

*Cochran; Biometrics, 1950, 6, 105.