ANNEX III

BASIC COUNTING PROCEDURES

1. COUNTING TECHNIQUES ON PLATES

The counting techniques on plates allow for the visualization of colony formation at
starting from a "fixed" number of viable cells.
They are therefore used to obtain the count of colony-forming units.
(UFC) present in the sample under analysis.
The culture media used to obtain the number of colonies in a sample.
they can be of general use (media with basic nutritious ingredients), enriched (media with
additional nutrients, such as blood, egg yolk and serum), added or not added to systems
inhibitors and/or indicators.
The application of counting techniques is based on the use of serial dilutions, obtained
starting from the homogenization of solid and semi-solid samples or from direct inoculations
of liquid samples and their dilutions.

The basic counting techniques on plates include:

1.1 Deep sowing: Distribute the aliquots of the chosen dilutions in
sterile petri dishes.
Add 12 to 15 mL of agar at 46-48ºC. Homogenize immediately and properly.
Let them solidify and incubate the plates (inverted or not), according to the requirements of
time, temperature, oxygen tension, etc., required by the microorganism to be listed.

1.2 Surface sowing: Use plates on which the

specific medium for the microorganism to be enumerated.
Promote the drying of the surface by leaving the panels inverted, semi-open with the
base supported on the lid, for about 15 minutes in an oven at 45-50ºC.
Inoculate 0.1 mL, or at most 0.5 mL, of the selected dilutions, remembering that 0.1
mL of a dilution provides a result corresponding to the following dilution, that is, 0.1 mL of 10-1
provides a result for the dilution 10-2.
Observe that the inoculum should be deposited in the center of the agar surface, avoiding
touch the tip of the pipette in the middle, keeping it as close as possible.
Spread the inoculum using a Drigalski loop or a hockey stick type glass rod,
spread over the entire surface of the agar until complete absorption.
Invert the plates after complete absorption of the inoculum and incubate as required.
as fast as possible.

1.3 Overlay Seeding: Proceed as for deep seeding.

After homogenizing the culture medium with the inoculum and solidifying the agar,
add a new layer of 10 to 12 mL of the corresponding agar, melted and maintained at 46-
48ºC.
Let it solidify without mixing and incubate as required.
The overlay technique can be done from a surface sowing,
adding a layer of 10 to 12 mL of molten agar after sowing,
distribution and total absorption of the inoculum by the agar.
Let it solidify without mixing and incubate as quickly as possible.
When aiming for the recovery of cells under stress, the time elapsed between the
the inoculation of the first layer and the addition of the overlay can be extended, leaving the plates
inverted in ambient temperature, according to the indication of the specific methodology.

2. MOST LIKELY NUMBER TECHNIQUE

The Most Probable Number (MPN) technique is a method that allows estimating the
density of viable microorganisms present in a sample under analysis.
This technique does not allow for a 'fixed' count of viable cells or forming units.
of colonies (UFC), as occurs with the plate counting technique.
The analysis by NMP is recommended when:
a) A low number of the target microorganism is expected in the food being analyzed.
(<100/g or mL) or when, due to limitations of the method and the necessary dilutions for
the specific food, the acceptance/rejection pattern cannot be met through evidence
of counting.
b) When, due to the technological process undergone by the food, the cells present
are physiologically impaired (stressed cells), and therefore are not able to form
colonies in selective solid media.

The NMP technique must be carried out including the following analytical steps:
a) Presumptive test (reading the results obtained from the multiple tubes series).
b) Confirmatory test (subculture of the positive tubes from the presumptive test in broth of
higher impedance or in selective differential agar for the researched microorganism.
c) Complete test (biochemical identification of the microbial species present).
This technique is based on the statistical probability related to the frequency of
occurrence of more likely positive results based on the actual number of microorganisms
presents. It is necessary for the sample to be prepared in such a way that the bacteria do not
find them grouped, that are randomly distributed in the sample; and that the means and
incubation conditions allow for the recovery and detection of at least one viable cell from
target organism.
For the determination of the NMP, the sample must be diluted as many times as
necessary for the last dilution of 3, 5, or 10 tubes to show all results
negatives. The higher the expected number of the researched microorganism, the greater they should be.
the dilutions used.
When one cannot estimate what the dilution of the food that will offer this situation is,
more than 3 series with decimal dilutions are inoculated (for example 100 up to 10-4, or more
dilutions).
Meanwhile, in the final reading of the NMP, only the 3 dilutions should be considered.
more significant.
As in the routine of analytical laboratories, this procedure greatly increases the ...
the volume of work and material to be used, normally only three dilutions are used,
considering the estimated number of the microorganism under study.
So, the number of viable cells present is obtained through 3 decimal dilutions.
successive and transfer of determined aliquots (also decimal, such as 10 and 1mL) of
each dilution in series of tubes.
The number of tubes per series is variable, ranging from 2 to 10. More commonly, they are
used series of 3 or 5 tubes per dilution.
When there is a need to inoculate a large volume (10 mL) of the diluted sample or
No, in the first series of tubes, these should contain medium at double concentration.
The arrangement of the number of positive tubes from the 3 dilutions (or the most significant ones) is
transposed into statistical tables that inform the NMP for the different combinations of
positive tubes that also include the confidence limits of the most likely numbers of
microorganisms researched in relation to the table in question.
The constant 95% confidence intervals from the NMP tables provide the information
that, in at least 95% of the times, there is a chance of the actual concentration of the microorganism
the target should be included in the confidence interval calculated for each arrangement of positive tubes.
For example, the positive tube arrangement 3-1-0 offers an NMP value of 43/g. In that
In this case, the 95% confidence interval corresponds to the values between 9 and 180. This
It means that the chance of the real number present in the sample being included in the interval of values.
between 9 and 180 UFC/g is 95%.
The expression of the NMP is made only through the most likely number that
correspond to the positive tube arrangement in series.
In general, the tables are already corrected considering g or mL (and consequently, the
dilutions), for the obtaining of NMP.
The following examples allow for the selection of the most significant dilutions among the
described possibilities.

TABLE 1
Cases Dilution (g or mL) Combination of pipes
1.0 0,1 0.01 0.001 0.0001
1 3/3 3/3 1/3* 3-3-1
2 3/3 3/3 2/3 1/3 3-2-1
3 3/3 2/3 3-2-0
4 2/3 2/3 2-2-0
5 3/3 3/3 2/3 1/3 1/3 3-2-2
6 3/3 2/3 1/3 3-2-1
Numerador = número de tubos positivos
Denominator = number of inoculated tubes
3 dilutions considered significant for the determination of MPN

2.1 Interpretation of NMP results

For the choice of the table in which the corresponding NMP will be verified for the combination of
positive tubes, pay attention to the amount (g or mL) of sample that was actually inoculated in
each tube from different series and not for the inoculated volume.

Example 1:

Inoculation of Amount of sample inoculated in each

series
10 mL of the sample in the 1st series 10mL
1.0 mL of the sample in the 2nd series 1.0mL
1.0 mL of dilution 10-1in the 3rd grade 0.1mL
The table to be consulted should be the one that provides NMP results for 10, 1.0, and 0.1.
g or mL. For water, whose final result must be expressed in CFU per 100 mL, the number
the constant of the table must be multiplied by 10.
In this Manual, for greater practicality, the table for reading NMP results in
water already contains the corrected numbers (multiplied by 10).

Example 2:
Inoculation of Amount of sample inoculated in each
series
10 mL of dilution 10-1in the 1st grade 1.0 g or mL
1.0 mL of dilution 10-1in the 2nd grade 0.1 g or mL
1.0 mL of dilution 10-2in the 3rd grade 0.01 g or mL
The table to be consulted should be the one that offers NMP results for 1, 0.1, and 0.01.
g or mL. Due to the fact that the amount of sample inoculated in this case is equal to
quantity to which the table refers, the result of the NMP will be obtained directly from the
table.

Example 3:
Inoculation of Amount of sample inoculated in each
series
1.0 mL of the sample in the 1st series 1.0 g or mL
1.0 mL of dilution 10-1in the 2nd grade 0.1 g or mL
1.0 mL of dilution 10-2in the 3rd grade 0.01 g or mL
The table to be consulted should be the one that offers NMP results for 1, 0.1, and 0.01 g or
mL. Due to the fact that the amount of sample inoculated in this case is equal to the amount
for which the table refers, the result of the NMP obtained will be the same as found in the table.

Example 4:
Inoculation of Amount of sample inoculated in each
series
1.0 mL of dilution 10-1in the 1st grade 0.1 g or mL
1.0 mL of dilution 10-2in the 2nd grade 0.01 g or mL
1.0 mL of dilution 10-3in 3rd grade 0.001 g or mL
The table to be consulted should be the one that provides NMP results for inocula of
0.1, 0.01, and 0.001 g or mL. The NMP table can also be used for inocula of 1, 0.1 and
0.01 mL since the result of the table is to be multiplied by 10, since the amount of
The inoculated sample in this case was 10 times smaller than the amount for which the table is.
referee.

2.2 Specific cases

a) Inoculation of more than 3 serial dilutions
When inoculating more than 3 serial dilutions, select the largest dilution in the
that all inoculated tubes are positive and consider the next 2 larger dilutions following that.
selected (cases 2 and 3 from table 1 above and examples below).

Example 1: Sample of liquid food that was inoculated in the dilutions below and showed the following
resultados:
1003 positive tubes
10-13 positive tubes
10-23 positive tubes
10-32 positive tubes
10-40 positive tubes
10-50 positive tubes

The value of NMP to be read in the table would be that for the positive tube arrangement 3/2/0.
which corresponds to the value of 9.3 NMP/g or mL. As in this case the sample was diluted to 10-2, or
the result obtained in the table must be multiplied by 100 to obtain the real NMP value
per gram or mL of the food being analyzed. Thus, the final result would be 930 NMP/g or mL.

Example 2: Liquid food sample from which only the 10 dilutions were inoculated010-1, 10-2the 10-3, and
the results obtained were the following:
1003 positive tubes
10-13 positive tubes
10-23 positive tubes
10-32 positive tubes
The value of NMP to be read in the table would be that for the arrangement of positive tubes 3/3/2.
which corresponds to the value of 110 NMP/g or mL. As in this case the sample was diluted to 10-1, o
the result must be multiplied by 10 to obtain the value of NMP per gram or mL of
food under analysis. Thus, the final result would be 1100 NMP/g or mL.

Example 3: In the analysis of the same food above, if the inoculated dilutions were as follows:
10-3 2 positive tubes
10-40 positive tubes
10-50 positive pipes
10-60 positive tubes

The value of NMP to be read in the table would be that for the positive tube arrangement 2/0/0,
which corresponds to the value 0.92 NMP/g or mL. As in this case, the sample was diluted to 10.-3,
o
the result must be multiplied by 1000 to obtain the value of NMP/g or mL of the food in
analysis. Thus, the final result would be 920 NMP/g or mL.
Example 4: In the same example above, if the inoculated dilutions were 10.010-1and 10-2considering the
same results:

1003 positive tubes

10-13 positive tubes
10-23 positive tubes
The value of NMP to be read in the table would be that for the combination of positive tubes.
3/3/3, which corresponds to the value >110 NMP/g or mL. As in this case the sample was not
diluted (100), the final result to be issued is obtained directly from the table.
The differences in results observed in the different work examples with the
the same sample and different dilutions demonstrate the importance of making dilutions of the
sample considering the estimated number of the test microorganism, as the same sample
analyzed according to examples 4 (without sufficient dilution) and 1 (with adequate dilutions)
show very different results (> 110 NMP/g or mL and 930 NMP/g or mL).
The result >110 NMP/g or mL is vague and may not be suitable for decision making.
decision regarding the fate of the lot to which the analyzed food refers, especially
when there is a tolerance in numbers larger than this.

Example 5: In the cases of inoculation where 5 dilutions are used in series of 3 tubes and that presents the
results below:

1003 positive tubes

10-13 positive tubes
10-2 1 positive tube
10-30 positive tubes
10-40 positive tubes

Consider the arrangement 3-1-0, since the result closest to the real one is obtained when the
the first considered series contains the 3 positive tubes and the last series contains the 3 negative tubes.
b) Inoculation of more than 3 serial dilutions in which positive tubes occurred in more
of two subsequent dilutions to the chosen one.
In this case, transfer a positive tube from the highest positive dilution to the immediately.
previously, successively, until obtaining an arrangement of tubes that fits the previous situation
(cases 5 and 6 of table 1).

Example 6: Sample of liquid food that was inoculated at the dilutions below, presenting the following
resultados:

1003 positive tubes

10-13 positive tubes
10-22 positive tubes
10-31 positive tube
10-41 positive tube

The final result, in this case, will be the value of the NMP table corresponding to the arrangement 3-2-
2 positive tubes.
This arrangement is obtained by transposing the positive tube of dilution 10.-4for the dilution
previous

Example 7: Liquid food sample that was inoculated in the dilutions below, presenting the following
resultados:

1003 positive tubes

10-12 positive tubes
10-20 positive tubes
10-31 positive tube
10-40 positive tubes

The final result, in this case, will be the value of the NMP table corresponding to the arrangement 3-2-
1 of positive tubes.
This arrangement of positive tubes is obtained by transposing the positive tube from the dilution.
10-3for the dilution 10-2.
c) In cases of inoculation where 5 dilutions are used in series of 3 tubes, whose
results indicate two possibilities of all tubes in the first series being positive and
all of the last series being negative, consider the highest value of NMP presented
by the corresponding table.

100 3 positive tubes

10-13 positive tubes
10-20 positive tubes
10-30 positive tubes
10-40 positive tubes
3-3-0 - 24 NMP/g or mL (100, 10-1the 10-2)
3-0-0 - 23 NMP/g or mL (10-1, 10-2and 10-3)
The final result, in this case, will be the NMP value corresponding to the arrangement 3-3-0 (24
NMP/g or mL.
d) Inoculation of only 3 serial dilutions In cases of inoculation of only 3
serial dilutions, read directly from the table (cases 1 and 4 and example 4).
e) Nonexistent arrangements in the tables
Repeat the analysis whenever the results of the controls applied in the analytical process
pointing to this need, and/or the positive pipe arrangements, in the pipe series
multiples, revealing inconsistent behavior such as 0-1-3 or other arrangements nonexistent in
NMP tables.
f) Complying with the legislation
Use the inoculation of 3 series of 10, 5, or 3 tubes when the legislation establishes
standard for NMP less than 1.0, 2.0, or 3.0, respectively.

2.3 NMP Tables

Table 1. Most Probable Number per gram or mL, for series of 3 tubes with
0.1, 0.01 and 0.001 g or mL inoculums and their respective 95% confidence intervals.

Number of Positive Tubes NMP/g or mL Confidence Interval (95%)

0,1 0.01 0.001 Inferior Superior

0 0 0 <3,0 -.- 9.5
0 0 1 3.0 0.15 9,6
0 1 0 3.0 0.15 11
0 1 1 6.1 1,2 18
0 2 0 6.2 1,2 18
0 3 0 9.4 3.6 38
1 0 0 3.6 0.17 18
1 0 1 7.2 1,3 18
1 0 2 11 3.6 38
1 1 0 7.4 1.3 20
1 1 1 11 3.6 38
1 2 0 11 3.6 42
1 2 1 15 4.5 42
1 3 0 16 4.5 42
2 0 0 9.2 1.4 38
2 0 1 14 3.6 42
2 0 2 20 4.5 42
2 1 0 15 3.7 42
2 1 1 20 4.5 42
2 1 2 27 8.7 94
2 2 0 21 4.5 42
2 2 1 28 8.7 94
2 2 2 35 8.7 94
2 3 0 29 8.7 94
2 3 1 36 8.7 94
3 0 0 23 4.6 94
3 0 1 38 8.7 110
3 0 2 64 17 180
3 1 0 43 9 180
3 1 1 75 17 200
3 1 2 120 37 420
3 1 3 160 40 420
3 2 0 93 18 420
3 2 1 150 37 420
3 2 2 210 40 430
3 2 3 290 90 1000
3 3 0 240 42 1000
3 3 1 460 90 2000
3 3 2 1100 180 4100
3 3 3 >1100 420 -.-

Source: Bacteriological Analytical Manual Online, 2001.

Note: To obtain the NMP/g or mL, for series of 3 tubes, with inocula of 1.0, 0.1, and 0.01 g or mL, and
respective 95% confidence intervals, divide the values in Table 1 corresponding to the arrangement by 10
positive tubes obtained in the analysis.

Table 2. Most Probable Number per 100mL, for series of 3 tubes with 10 mL inocula,
1.0 mL and 0.1 mL, and respective 95% confidence intervals.

Number of Positive Tubes NMP/g or mL Confidence Interval (95%)

10 1.0 0,1 Inferior Superior

0 0 0 <3,0 -.- 9.5
0 0 1 3.0 0.15 9.6
0 1 0 3.0 0.15 11
0 1 1 6.1 1,2 18
0 2 0 6.2 1,2 18
0 3 0 9.4 3.6 38
1 0 0 3.6 0.17 18
1 0 1 7,2 1.3 18
1 0 2 11 3.6 38
1 1 0 7.4 1.3 20
1 1 1 11 3.6 38
1 2 0 11 3.6 42
1 2 1 15 4.5 42
1 3 0 16 4.5 42
2 0 0 9.2 1.4 38
2 0 1 14 3.6 42
2 0 2 20 4.5 42
2 1 0 15 3.7 42
2 1 1 20 4.5 42
2 1 2 27 8.7 94
2 2 0 21 4.5 42
2 2 1 28 8.7 94
2 2 2 35 8,7 94
2 3 0 29 8.7 94
2 3 1 36 8.7 94
3 0 0 23 4.6 94
3 0 1 38 8.7 110
3 0 2 64 17 180
3 1 0 43 9 180
3 1 1 75 17 200
3 1 2 120 37 420
3 1 3 160 40 420
3 2 0 93 18 420
3 2 1 150 37 420

3 2 2 210 40 430
3 2 3 290 90 1000
3 3 0 240 42 1000
3 3 1 460 90 2000
3 3 2 1100 180 4100
3 3 3 >1100 420 -.-
Source: Bacteriological Analytical Manual Online, 2001.

Table 3. NMP per gram or mL for series of 10 tubes with inoculums of 10 mL, 1.0 mL and
0.1mL and respective 95% confidence intervals.

Number of Positive Tubes NMP/g or mL Confidence Interval (95%)

10 1,0 0,1 Inferior Superior

0 0 0 <0,9 -.- 3.1
0 0 1 0.9 0.04 3.1
0 0 2 1.8 0,35 5.1
0 1 0 0.9 0.04 3.6
0 1 1 1.8 0.33 5.1
0 2 0 1.8 0.33 5.1
0 2 1 2.7 0.8 7.2
0 3 0 2.7 0.8 7.2
1 0 0 0.94 0.05 5.1
1 0 1 1.9 0.33 5.1
1 0 2 2.8 0.8 7.2
1 1 0 1.9 0.33 5.7
1 1 1 2.9 0.8 7.2
1 1 2 3.8 1.4 9
1 2 0 2.9 0.8 7.2
1 2 1 3.8 1.4 9
1 3 0 3.8 1.4 9
1 3 1 4.8 2.1 11
1 4 0 4.8 2.1 11
2 0 0 2 0.37 7.2
2 0 1 3 0.81 7.3
2 0 2 4 1.4 4.9
2 1 0 3 0.82 7.8
2 1 1 4 1 4.9
2 1 2 5 2.1 11
2 2 0 4 1,4 9.1
2 2 1 5 2.1 11
2 2 2 6.1 3 14
2 3 0 5.1 2.1 11
2 3 1 6.1 3 14
2 4 0 6.1 3 14
2 4 1 7.2 3.1 15
2 5 0 7.2 3.1 15
3 0 0 3.2 0.9 9
3 0 1 4.2 1.4 9.1
3 0 2 5.3 2.1 11
3 1 0 4.2 1.4 10
3 1 1 5.3 2.1 11
3 1 2 6.4 3 14

3 2 0 5.3 2.1 12
3 2 1 6.4 3 14
3 2 2 7.5 3.1 15
3 3 0 6.5 3 14
3 3 1 7.6 3.1 15
3 3 2 8.7 3.6 17
3 4 0 7.6 3.1 15
3 4 1 8.7 3.6 17
3 5 0 8.8 3.6 17
4 0 0 4,5 1.6 11
4 0 1 5.6 2.2 12
4 0 2 6.8 3 14
4 1 0 5.6 2.2 12
4 1 1 6.8 3 14
4 1 2 8 3.6 17
4 2 0 6.8 3 15
4 2 1 8 3.6 17
4 2 2 9.2 3.7 17
4 3 0 8.1 3.6 17
4 3 1 9.3 4.5 18
4 3 2 10 5 20
4 4 0 9.3 4.5 18
4 4 1 11 5 20
4 5 0 11 5 20
4 5 1 12 5.6 22
4 6 0 12 5.6 22
4 6 0 12 5,6 22
5 0 0 6 2.5 14
5 0 1 7.2 3.1 15
5 0 2 8.5 3.6 17
5 0 3 9.8 4.5 18
5 1 0 7.3 3.1 15
5 1 1 8.5 3.6 17
5 1 2 9.8 4.5 18
5 1 3 11 5 21
5 2 0 8.6 3.6 17
5 2 1 9.9 4.5 18
5 2 2 11 5 21
5 3 0 10 4.5 18
5 3 1 11 5 21
5 3 2 13 5.6 23
5 4 0 11 5 21
5 4 1 13 5.6 23
5 4 2 14 7 26
5 5 0 13 6.3 25
5 5 1 14 7 26
5 6 0 14 7 26
6 0 0 7.8 3.1 17
6 0 1 9.2 3.6 17
6 0 2 11 5 20
6 0 3 12 5.6 22
6 1 0 9.2 3.7 18
6 1 1 11 5 21
6 1 2 12 5,6 22

6 1 3 14 7 26
6 2 0 11 5 21
6 2 1 12 5,6 22
6 2 2 14 7 26
6 2 3 15 7.4 30
6 3 0 15 5.6 23
6 3 1 14 7 26
6 3 2 15 7.4 30
6 4 0 14 7 26
6 4 1 15 7.4 30
6 4 2 17 9 34
6 5 10 16 7.4 30
6 5 1 17 9 34
6 5 2 19 9 34
6 6 0 17 9 34
6 6 1 19 9 34
6 7 0 19 9 34
7 0 0 10 4.5 20
7 0 1 12 5 21
7 0 2 13 6.3 25
7 0 3 15 7.2 28
7 1 0 12 5 22
7 1 1 13 6.3 25
7 1 2 15 7.2 28
7 1 3 17 7.7 31
7 2 0 13 6.4 26
7 2 1 15 7.2 28
7 2 2 17 7.7 31
7 2 3 19 9 34
7 0 3 15 72 30
7 3 0 15 7.2 30
7 3 1 17 9 34
7 3 2 19 9 34
7 3 3 21 10 39
7 4 0 17 9 34
7 4 1 19 9 34
7 4 2 21 10 39
7 4 3 23 11 44
7 5 0 19 9 34
7 5 1 21 10 39
7 5 2 23 11 44
7 6 0 21 10 39
7 6 1 23 11 44
7 6 2 25 12 46
7 7 0 23 11 44
7 7 1 26 12 50
8 0 0 13 5,6 25
8 0 1 15 7 26
8 0 2 17 7.5 30
8 0 3 19 9 34
8 1 0 15 7.1 28
8 1 1 17 7.7 31
8 1 2 19 9 34

8 1 3 21 10 39
8 2 0 17 7.7 34
8 2 1 19 9 34
8 2 2 21 10 39
8 2 3 23 22 44
8 3 0 19 9 34
8 3 1 21 10 39
8 3 2 24 11 44
8 3 3 26 12 50
8 4 0 22 10 39
8 4 1 24 11 44
8 4 2 26 12 50
8 4 3 29 14 58
8 5 0 24 11 44
8 5 1 27 12 50
8 5 2 29 14 58
8 5 3 32 15 62
8 6 0 27 12 50
8 6 1 30 14 58
8 6 2 33 15 62
8 7 0 30 14 58
8 7 1 33 17 73
8 7 2 36 17 74
8 8 0 34 17 73
8 8 1 37 17 74
9 0 0 17 7.5 31
9 0 1 19 9 34
9 0 2 22 10 39
9 0 3 24 11 44
9 1 0 19 9 39
9 1 1 22 10 40
9 1 2 25 11 44
9 1 3 28 14 58
9 1 4 31 14 58
9 2 0 22 10 44
9 2 1 25 11 46
9 2 2 28 14 58
9 2 3 32 14 58
9 2 4 35 17 73
9 3 0 25 12 50
9 3 1 29 14 58
9 3 2 32 15 62
9 3 3 36 17 74
9 3 4 40 20 91
9 4 0 29 14 58
9 4 1 33 15 62
9 4 2 37 17 74
9 4 3 41 20 91
9 4 4 45 20 91
9 5 0 33 17 73
9 5 1 37 17 74
9 5 2 42 20 91
9 5 3 46 20 91

9 5 4 51 25 120
9 6 0 38 17 74
9 6 1 43 20 91
9 6 2 47 21 100
9 6 3 53 25 120
9 7 0 44 20 91
9 7 1 49 21 100
9 7 2 54 25 120
9 7 3 60 26 120
9 8 0 50 25 120
9 8 1 55 25 120
9 8 2 61 26 120
9 8 3 68 30 140
9 9 0 57 25 120
9 9 1 63 30 140
9 9 2 70 30 140
10 0 0 23 11 44
10 0 1 27 12 50
10 0 2 31 14 58
10 0 3 37 17 73
10 1 0 27 12 57
10 1 1 32 14 61
10 1 2 38 17 74
10 1 3 44 20 91
10 1 4 52 25 120
10 2 0 33 15 73
10 2 1 39 17 79
10 2 2 46 20 91
10 2 3 54 25 120
10 2 4 63 30 140
10 3 0 40 17 91
10 3 1 47 20 100
10 3 2 56 25 120
10 3 3 66 30 140
10 3 4 77 34 150
10 3 5 89 39 180
10 4 0 49 21 120
10 4 1 59 25 120
10 4 2 70 30 150
10 4 3 82 38 180
10 4 4 94 44 180
10 4 5 110 50 210
10 5 0 62 26 140
10 5 1 74 30 150
10 5 2 87 38 180
10 5 3 100 44 180
10 5 4 110 50 210
10 5 5 130 57 220
10 5 6 140 70 280
10 6 0 79 34 180
10 6 1 94 39 180
10 6 2 110 50 210
10 6 3 120 57 220

10 6 4 140 70 280
10 6 5 160 74 208
10 6 6 180 91 350
10 7 0 100 44 210
10 7 1 120 50 220
10 7 2 140 61 280
10 7 3 150 73 280
10 7 4 170 91 350
10 7 5 190 91 350
10 7 6 220 100 380
10 7 7 240 110 480
10 8 0 130 60 250
10 8 1 150 70 280
10 8 2 170 80 350
10 8 3 200 90 350
10 8 4 220 100 380
10 8 5 250 120 480
10 8 5 250 120 480
10 8 6 280 120 480
10 8 7 310 150 620
10 8 8 350 150 620
10 9 0 170 74 310
10 9 1 200 91 380
10 9 2 230 100 480
10 9 3 260 120 480
10 9 4 300 140 620
10 9 5 350 150 630
10 9 6 400 180 820
10 9 7 460 210 970
10 9 8 530 210 970
10 9 9 610 280 1300
10 10 0 240 110 480
10 10 1 290 120 620
10 10 2 350 150 820
10 10 3 430 180 970
10 10 4 540 210 1300
10 10 6 700 280 1500
10 10 6 920 350 1900
10 10 7 1200 480 2400
10 10 8 1600 620 3400
10 10 9 2300 810 5300
10 10 10 2300 1300 -.-
Source: Adapted Bacteriological Analytical Manual Online, 2001.

3. BIBLIOGRAPHY CONSULTED
BRAZIL. Ministry of Agriculture, Supply and Agrarian Reform. Defense Secretariat
Agriculture. Department of Animal Defense. Manual of microbiological methods for food.
General Coordination of Animal Laboratory. 1991/1992 2nd
revision. 136p.
MATURIN, L.J.; PEELER, J.T. Aerobic Plate Count. In: Bacteriological Analytical Manual. 8
ed. Arlington, AOAC International, 1995. p. 3.01 - 3.10.
SWANSON, K.M.J.; PETRAN, R.L.; HANLIN, J.H. Culture Methods for Enumeration of
Microorganisms. In: Compendium of Methods for the Microbiological Examination of Foods. 4 ed.
Frances Pouch Downes & Keith Ito (Eds.), Washington: American Public Health Association,
2001. p. 53-62.
FDA. Bacteriological Analytical Manual Online. 2001. Available at: [Link]