30 Microbiological examination methods of food and water

Note. ISO 7218:2007 considers acceptable plates containing
between 10 and 300 colonies, but plates of two consecutive
dilutions, with a number of colonies within this range, are
used to calculate the results (vide item 3.7).
To calculate the results, two situations are to be con-
sidered. The first is the standard situation and the sec-
ond is the samples prepared by the surface swabbing or
surface washing techniques.
3.6.1.1 Calculating the pour plate results
in the standard situation
The standard situation is that in which the analytical
unit consists of a mass (weight) or volume of the
sample, homogenized with the diluent. The general
rule for calculating the results is: CFU/g or CFU/ml =
c/d.v, where c is the number of colonies on the
counted plate, d the dilution rate of the counted
plate and v the inoculated volume of this dilution.
More detailed rules for calculating the results follow
below.
Note. The general rule for calculating the results of ISO 7218:2007
is different (see item 3.7).
Rule 1 – If the count was performed on a plate inocu-
lated with an undiluted sample, without duplicate, the
number of colony forming units (CFU) is equal to the
number of colonies (Examples 1 and 2). If a duplicate
was made, the number of CFU is equal to the arithme-
tic average of the counts obtained in each of the plates
of the duplicate (Examples 3 and 4).
Rule 2 – If the count was performed on a plate
inoculated with a 10−1 dilution or greater, without
duplicate, calculate the number of CFU/g or ml by
multiplying the number of colonies by the inverse of
the inoculated dilution. The inverse of the 10−1 dilution
is 101, the inverse of the 10−2 dilution is 102 and so forth
(Examples 5 and 6). If a duplicate was made, consider
as the number of colonies the arithmetic average of the
counts obtained in each of the plates of the duplicate
and multiply by the inverse of the dilution (Examples 7
and 8).

Rule 1

N° colonies in the plate(s)

without
Example dilution (100) 10−1 10−2 Count (CFU/ml)

Without duplicate
1 199* 8 0 199 = 2.0 × 102
2 245* 22 2 245 = 2.5 × 102
With duplicate
3 62*–57* 6–5 0–0 (62 + 57)/2 = 59.5 = 60
4 123*–136* 12–10 0–0 (123 + 136)/2 = 129.5 = 1.3 × 102

*Counts effectively used to calculate the result.

Rule 2

N° colonies in the plate(s)

Example 10−1 10−2 10−3 Count (CFU/g or CFU/ml)

Without duplicate
5 199* 18 2 199 × 101 = 1,990 = 2.0 × 103
6 TNTC 245* 22 245 × 102 = 2,450 = 2.5 × 104
With duplicate
7 TNTC–TNTC 62*–57* 6–5 [(62 + 57)/2] × 102 = 59.5 × 102 = 6.0 × 103
8 TNTC–TNTC TNTC–TNTC 239*–242* [(239 + 242)/2] × 103 = 240.5 × 103 = 2.4 × 105

*Counts effectively used to calculate the result. TNTC = Too numerous to count.

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 Basic plate count techniques for the enumeration of microorganisms 31

Rule 3 – If the inoculated volume of the first dilution
(or of the sample without dilution) is different from
1 ml and the count was performed on the plate inoculated
with this volume, rules 2 and 3 apply, but the number
of colonies must be divided by the inoculated volume in
order to calculate the result (Examples 9, 10, 11 and 12).
Rule 4 – If the initial dilution is not decimal,
(1:20, 1:50, 1:200, or other), rules 2 and 3 apply, but
it is necessary to insert into the calculations the actual
initial dilution used. Considering an analytical unit of
m grams or milliliters, diluted in v milliliters of diluent,
the initial dilution will be equal to m/(m+v), that is, the
analytical unit divided by the total volume (diluent +
analytical unit). The subsequent decimal dilutions will
be the initial dilution multiplied by 10−1 (1st decimal),
the initial dilution multiplied by 10−2 (2nd decimal) and
so forth. For example, for an analytical unit of 50 g pre-
pared with 950 ml of diluent, the initial dilution is 50/
(50 + 950) = 50/1.000 = 1/20 (1:20). The 1st decimal
is 10−1/20, the 2nd decimal is 10−2/20 and so forth. The
results can also be calculated by multiplying the number
of colonies by the inverse of the dilution, but in this
case, the inverse of the dilution is the inverted fraction:
the inverse of a 1/20 dilution = 20/1, the inverse of a
10−1/20 dilution = 20 × 101, the inverse of a 10−2/20
dilution = 20 × 102 and so forth (Examples 13, 14, 15,
16, 17 and 18).
The examples given above are calculations made under
ideal conditions, with the number of colonies falling in
the 25 to 250 range, in plates of the same dilution, with-
out spreading. However, quite frequently the plates do
not present such ideal situations and require the applica-
tion of some basic rules to calculate the results. The rules
are presented below, including examples in Table 3.1.
Rule 5 – One duplicate plate with counts above or
below the range of 25–250 colonies. If the other plate
exhibits counts in the 25 to 250 range, the number of
both plates must be considered when calculating the
result (Example 30 of Table 3.1)
Rule 6 – Two consecutive dilutions with 25–250
colonies. Calculate the number of CFU of each dilu-
tion and compare the results.
6.a) If one of the results is greater than the double of
the other, consider only the lower count (Exam-
ples 19 and 31 of Table 3.1).

Rule 3

N° colonies in the plate(s) (volume inoculated)

Example 10−1 (2 ml) 10−2 (1 ml) 10−3 (1 ml) Count (CFU/g or CFU/ml)

Without duplicate
9 199* 18 2 (199/2) × 101 = 1.0 × 103
10 123* 2 0 (123/2) × 101 = 6.2 × 102
With duplicate
11 62*–57* 6–5 0–0 {[(62 + 57)/2]/2} × 101 = 29.75 × 101 = 3.0 × 102
12 27*–35* 3–3 0–0 {[(27 + 35)/2)]/2} × 101 = 15.5 × 101 = 1.6 × 102

*Counts effectively used to calculate the result.

Rule 4

N° colonies in the plate(s)

Analytical Volume of Initial
Example unity diluent dilution Initial dilution 1st decimal 2nd decimal Count (CFU/g or CFU/ml)

Without duplicate
13 10 g 490 ml 10/500 = 1/50 199* 18 2 199 × 50 = 1.0 × 104
14 25 g 350 ml 25/375 = 1/15 280 30* 2 30 × 15 × 101 = 4.5 × 103
15 25 g 975 ml 25/1,000 = 1/40 TNTC TNTC 133* 133 × 40 × 102 = 5.3 × 105
With duplicate
16 25 g 475 ml 25/500 = 1/20 237*–229* 21–20 2–1 [(237 + 229)/2] × 20 = 4.7 × 103
17 10 g 490 ml 10/500 = 1/50 TNTC-TNTC 62*–57* 6–5 [(62 + 57)/2] × 50 × 101 = 3.0 × 104
18 10 g 290 ml 10/300 = 1/30 TNTC-TNTC TNTC-TNTC 239*–242* [(239 + 242)/2] × 30 × 102 = 7.2 × 105

*Counts effectively used to calculate the result. TNTC = Too numerous to count.

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 32 Microbiological examination methods of food and water

Table 3.1 Examples for calculating the pour plate results in not ideal conditions.

N° colonies in the plate(s)

Example Rules used 10−1 10−2 10−3 Count (CFU/g or CFU/ml)

Without duplicate
19 6.a TNTC 140* 32 140 × 102 = 1.4 × 104
20 6.b TNTC 243* 34* [(243 × 102) + (34 × 103)]/2 = 2.9 × 104
21 7 18* 2 0 18 × 101 = 1.8 × 102 (est)
22 8 0 0 0 <1 × 101 = <10 (est)
23 9a TNTC TNTC 370* 370 × 103 = 3.7 × 105 (est)
24 9b TNTC TNTC 8/cm2* 8 × 65 × 103 = 520 × 103 = 5.2 × 105 (est)
25 9c TNTC TNTC 21/cm2* 21 × 65 × 103 = 1,365 × 103 = 1.4 × 106 (est)
26 9d TNTC TNTC >100/cm2* >100 × 65 × 103 = >6.5 × 106 (est)
27 10 TNTC 325* 20 325 × 102 = 3.3 × 104 (est)
28 11 TNTC 243* Spr Spr 243 × 102 = 2.4 × 104
29 12 27 215 20 Unacceptable result, repeat the analysis
With duplicate
30 5 TNTC-TNTC TNTC-TNTC 239*–328* [(239 + 328)/2] × 103 = 283.5 × 103 = 2.8 × 105
31 6a 138*–162* 42–30 1–2 [(138 + 162)/2] × 101 = 150 × 101 = 1.5 × 103
32 6b 228*–240* 28*–26* 2–2 {[(228 + 240)/2] × 101 + [(28 + 26)/2]×102}/2 = 2,520 = 2.5 × 103
33 7 18*–16* 2–0 0–0 [(18 + 16)/2] × 101 = 17 × 101 = 1.7 × 102
34 8 0–0 0–0 0–0 <10 (est)
35 9a TNTC-TNTC TNTC-TNTC 320*–295* [(320 + 295)/2] × 103 = 307.5 × 103 = 3.1 × 105
36 10 287*–263* 23–19 2–2 [(287 + 263)/2] × 101 = 275 × 101 = 2.8 × 103
37 11 TNTC-TNTC 224*–180* 28*–Spr {[(224 + 180)/2] × 102 + (28 × 103)}/2 = 24,100 = 2.4 × 104

*Counts effectively used to calculate the result. TNTC = too numerous to count, Spr = spreader and adjoining area of repressed growth
covering more than one-half of the plate, est = estimated count.

6.b) If one of the results does not exceed the double of
the other, then both results must be considered,
and the mean value should be presented as the
final result (Examples 20 and 32 of Table 3.1).
Rule 7 – None of the plates reached 25 colonies.
Count the plates exhibiting a number of colonies
closest to 25, calculate CFU number (Examples 21
and 33 of Table 3.1) and report the result as estimated
count (est).
Rule 8 – No plate showing growth. Consider the
number of colonies of the 1st inoculated dilution as
being one and calculate the result in accordance with
rules 1, 2, 3 or 4 (Examples 22 and 34 of Table 3.1).
Report the final result as being smaller than the value
obtained by the calculation, estimated value.
Rule 9 – All plates containing more than 250 colo-
nies. In these cases, there are four alternatives for esti-
mating the number of CFU/g or ml. In all cases, the
result must be reported as estimated count (est).
9.a) If it is possible to count all the colonies on the
plate, count and calculate the number of CFU
from the counts obtained (Examples 23 and 35
of Table 3.1).
9.b) If it is not possible to count all colonies on the
plate, but the number of colonies per cm2 is lower
than 10, count the colonies in 12 of the 1 cm2
squares, six consecutive squares in a row and six
consecutive squares in a column, using the squares
traced on the grid background of the colony
counter as counting guide. Calculate the average
number of colonies/cm2 and use this average value
to determine the total number of colonies on the
plate by multiplying the average value by the total
surface area of the plate. Remember that the total
surface area of the plate is equal to πd2/4, where d
is the inner diameter. For example, 100 mm-plates
have an inner diameter of about 9 cm and a total
surface area of 65 cm2. Use the total number of
colonies thus calculated to determine the number
of CFU (Example 24 of Table 3.1).
9.c) If the number of colonies per cm2 is greater than
10, count the colonies in four squares representa-
tive of the distribution of the colonies on the plates
and calculate the number of CFU in the same way
as described for the case of 12 squares (Example 25
of Table 3.1).

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 Basic plate count techniques for the enumeration of microorganisms
9.d) If the number of colonies per cm2 is greater than
100, report the result as being greater than the
total surface area of the plate × inverse of the dilu-
tion (Example 26 of Table 3.1).
Rule 10 – Number of colonies greater than 250
in one dilution and lower than 25 in the next. If
in a dilution the number of colonies was higher than
250 and in the next dilution the number of colonies
was below this number, select the plates with the
counts closest to 250 and calculate the number of
CFU from the count obtained (Examples 27 and 36
of Table 3.1).
Rule 11 – Plates with spreading. There are two
types of spreading. The first type results from the disin-
tegration of cell clusters or groupings which may occur
when mixing the inoculum with the culture medium.
The second type is the result of inadequate mixing of
the inoculum with the medium, leading to the forma-
tion of thin films of moisture either onto the surface of
the medium or between the medium and the bottom
of the plate. The difference between the two types is
visually distinguishable, since in the case of spreading
of the first type the growth of individual colonies can be
observed, whereas in the other case, the growth of the
cell mass is continuous, without individual colonies.
Plates displaying spreading can be counted under the
following conditions: if none of the individual spread-
ing zones is of a size exceeding 25% of the surface area
of the plate, and, also, if the total surface area covered
with spreading zones does not surpass 50% of the plate.
If these two conditions are not met, report the result
as a “laboratory accident” and repeat the test. If the
laboratory observes the occurrence of spreading of the
second type, with spreading zones consistently greater
than 25% of the total plate surface, in more than 5%
of the plates prepared within a certain period of work
time, preventive measures should be taken to minimize
this problem. To count plates with spreading zones of
the first type, each zone should be counted as one sin-
gle CFU, and the individual colonies within each of
these zones should not be counted. To count plates dis-
playing spreading zones of the second type, select one
region of the plate, free of spreading and count the col-
onies within several of the 1 cm2 squares. Calculate the
average of the colonies per cm2, multiply by the total
surface area of the plate (65 cm2 in the case of plates
with an external diameter of 100 mm) and use this esti-
mated value to calculate the number of CFU. Report
the result as estimated count (est) (Examples 28 and 37
of Table 3.1).
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33
Rule 12 – Plates in which microbial growth is
proportionally greater in the greatest dilutions. This
situation may occur as a result of accidental contami-
nation of the sample during plating, incorrect identi-
fication of the sample dilution rate on the plates or be
caused by the presence of inhibitory substances in the
sample. Consider the result as a “laboratory accident”
and repeat the test. If the suspicion of the presence of
inhibitory substances in the sample is high, repeat the
test using an adequate procedure to eliminate or reduce
the influence of these components on the result (Exam-
ple 29 of Table 3.1).
3.6.1.2 Calculating the pour plates results
for samples prepared by the surface
swabbing technique (swabs or
sponges)
The results should be expressed in CFU/cm2 of sample.
Initially it is necessary to calculate the number of CFU
per milliliter of the diluent in which the swabs were
placed prior to analysis. For that purpose, consider this
suspension as a non-diluted sample and, as a function
of the dilutions inoculated of this suspension, calculate
the result in exactly the same way as described for the
standard situation (item 3.6.1.1 above)
Next, the CFU/ml count of the suspension should
be converted to CFU/cm2 of the sample. To that pur-
pose, calculate to how many cm2‘s each milliliter of
the suspension corresponds. In the standard procedure
described in Chapter 2 for swab sampling, a surface area
of 50 cm2 is sampled and the swabs placed in 10 ml
diluent, with each milliliter of diluent corresponding to
5 cm2 of the sampled surface. This ratio, however, may
be changed at the discretion of the laboratory, depend-
ing on the type of sample and the objective of sampling.
It is recommendable to work always with diluent vol-
umes that are a multiple of the sampled areas to facilitate
calculations. In the case above, the CFU/cm2 count will
be equal to the value obtained per ml of the suspension,
divided by five. In the procedure described in Chapter 2
for sponge sampling, a surface area of 100 cm2 is sam-
pled and the sponges placed in 25 ml diluent, with each
milliliter of the diluent corresponding to 4 cm2 of the
sampled surface. In this case, the CFU/cm2 count will
be equal to the value obtained per ml of the suspension,
divided by four. In another situation, in which a swab-
bing suspension yielded by swabbing a surface area of
100 cm2 were to be suspended in 10 ml of diluent, for
example, each ml of the suspension would correspond to
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