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NTC 4092 General Guide For The Counting of Microorganisms

This document presents guidelines for carrying out microbiological counts of foods in accordance with Colombian standards. Explains how to prepare samples, acceptable count ranges, and how to calculate and express results for different types of microorganisms.
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0% found this document useful (0 votes)
23 views8 pages

NTC 4092 General Guide For The Counting of Microorganisms

This document presents guidelines for carrying out microbiological counts of foods in accordance with Colombian standards. Explains how to prepare samples, acceptable count ranges, and how to calculate and express results for different types of microorganisms.
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
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GENERAL GUIDE FOR COUNTING

MICROORGANISMS
IN FOOD
Cabeza Herrera, EA, Ortiz Lemuz, JF
University of Pamplona, Faculty of Basic Sciences, Department of
Microbiology. University Campus, Km. 1 Via Bucaramanga.

This guide presents in an illustrated manner the different methods for plate counting of
microorganisms in foods. This is a summary adapted from the Colombian technical
standard NTC – 4092 and the recommendations handled by the National Institute of
Health of Colombia and endorsed by the Ministry of Social Protection (Formerly the
Ministry of Health). For the method of counting microorganisms by the most probable
number technique, consult NTC-4092.

COUNTING OF MICROORGANISMS IN FOOD (ICONTEC).

This guide for counting microorganisms in foods is based on the Colombian Technical
Standard NTC-4092. This standard establishes general guidelines for performing
microbiological analyzes in accordance with international standards (ISO 7218 of 1997).

1. GENERAL CONSIDERATIONS:
For the correct application of this standard, the preparation of the sample is
essential, therefore it is required that the seeding be carried out from two
consecutive dilutions and in duplicate in addition to inoculating the correct volumes
(0.1 ml for surface - under certain circumstances, 0.2 ml, 0.5 ml or 1 ml can be
inoculated on the surface for samples that are considered to have a low count and
when the culture medium used has the capacity to imbibe said volume - and 1 ml
for depth).

2. COUNTING RANKS:
After the specified incubation period, the CFU colony-forming units present in the
boxes containing between 15 CFU and 300 CFU are counted. The analyzes that
apply this range are: Mesophilic aerobes; coliforms, count of thermophilic,
proteolytic, lipolytic bacteria, sulfite-reducing Clostridium, S. aureus , B. cereus ,
etc. Except for those bacteria whose norm requires presence or absence.
After the specified incubation period, the CFU colony-forming units present in the
boxes containing between 15 CFU and 150 CFU are counted for Molds and yeasts,
osmophilic yeasts.

3. COUNTING AND EXPRESSION OF RESULTS:

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GENERAL GUIDE FOR THE COUNT OF MICROORGANISMS IN FOODS

TO). GENERAL CASE: The number N of microorganisms present in the sample is


calculated from the count of two successive dilutions. It is necessary that at least
one of the two boxes of each dilution be within the range, using the following
formula:

ΣC
N= , where:
V ( n 1 + (0.1* n 2)) d

N: Total number of CFU per gram or milliliter of sample.


Σ C : It is the sum of the number of colonies counted in each box examined from the
successive dilutions.
V : Volume of sample inoculated in each box.
n1: Number of boxes retained from the first dilution.
N2: Number of boxes retained from the second dilution.
d: Dilution factor corresponding to the first dilution retained.
0.1= correction constant.
The calculated result is rounded to two significant figures. For this, if the last figure
is less than 5, the previous figure is not modified; If the last digit is 5 or greater, the
previous digit is incremented by one. This continues until two significant figures are
obtained.

The results are expressed in CFU per gram or milliliter of product depending on its
nature, expressed as a number between 1.0 and 9.9 (Two significant figures, an
integer and a decimal) multiplied by 10 appropriate out of 10.

Example:
From an analysis of mesophilic aerobes for pasteurized milk, the following results
were obtained:

- in the first selected dilution (10 -2 ): 168 CFU and 215 CFU.
- in the second selected dilution (10 -3 ): 14 CFU and 25 CFU.

N= 168 + 215 + 25
1(2 + (0.1*1))0,01
N= 408 = 19,428.57
0,021
When rounding the figure as specified above, the result is 19,000, expressed with
two significant figures 1.9x10 4 CFU per milliliter.

B). CALCULATION METHOD FOR COUNTS OF MICROORGANISMS THAT


REQUIRE LATER CONFIRMATION: (For example S. aureus coagulase positive, B.
cereus .)
The number N of microorganisms present in the sample is calculated from the count
of two successive dilutions. It is necessary that at least one of the two boxes of
each dilution be within the range. In this case it is necessary to confirm a
representative number of typical colonies from each of the boxes (generally 5).

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CABEZA HERRERA, EA, ORTIZ LEMUZ J. F.

Before calculating the final report, it is necessary to apply a correction factor to


each of the retained boxes using the following formula:

a= b * C , where:
TO

b : Number of colonies identified as positive according to the microbiological criteria.


A : Number of colonies examined
C : Total number of typical colonies counted in each of the retained boxes.

To express the final result, the following formula is applied:


Σa
N= , where:
V ( n 1 + (0.1* n 2)) d
N: Total number of CFU per gram or milliliter of sample.
Σ a : Sum of the number of corrected typical colonies in each box examined.
V: Volume of sample inoculated in each box.
n1: Number of boxes retained from the first dilution.
N2: Number of boxes retained from the second dilution.
d: Dilution factor of the first dilution retained.
0.1= correction constant.

The calculated result is rounded to two significant figures. For this, if the last figure is
less than 5, the previous figure is not modified; If the last digit is 5 or greater, the
previous digit is incremented by one. This continues until two significant figures are
obtained.

The results are expressed in CFU per gram or milliliter of product depending on its
nature, expressed as a number between 1.0 and 9.9 multiplied by 10 X where x is the
appropriate power of 10.

Example:
From an analysis by S. aureus coagulase positive in a cheese, the following results
were obtained:

- in the first selected dilution (10 -3 ): 66 CFU and 80 CFU.


- in the second selected dilution (10 -4 ): 4 CFU and 17 CFU.

Correction factor:
Colonies counted in each box 66 and 80 CFU. 4 an 17 CFU.
Colonies examined (A): 6 and 8 4an 5
Positive colonies (b): 4 and 5 4an 3
d
4 4 a = *4 3 a = *17
a = *66 a = 5 *80 8 4 5
6
a= 44 col. a= 50 col. a= 4 col. a= 28 col

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GENERAL GUIDE FOR THE COUNT OF MICROORGANISMS IN FOODS

Report:
Σ
N= a
V ( n 1 + 0.1* n 2) d

N= 44+50+28
0,1(2 + (0.1*1))0,001
122
N= = 580,952.38
0,00021

When rounding the figure as specified above, the result is 580,000, expressed with
two significant figures 5.8x10 5 CFU per gram.

C). SPECIAL CASES:

- CASE 1: When the number of CFU is below the minimum range in both boxes of
one or two consecutive dilutions, it will be reported as follows:

The count obtained in the highest dilution is discarded and the arithmetic mean
of the colonies present in the two boxes of the lowest dilution is calculated by
multiplying by the inverse of the dilution and the result is reported as follows:

Estimated CFU count per gram or milliliter (Taking into account the way
of reporting the final result: two significant figures per....................)

- CASE 2: Absence of growth, if the two boxes corresponding to the first dilution,
the result is reported as follows:

- 1x10 X CFU per gram or milliliter as appropriate.

NOTE: when the colony count exceeds the maximum range, it is recommended to
repeat the analysis using higher dilutions to obtain a more reliable result, or report
it as TNTC (too numerous to count).

COUNT OF MICROORGANISMS IN FOOD (Recommendations of the National


Institute of Health and Ministry of Social Protection of Colombia).

This guide for counting microorganisms in foods is based on the recommendations of the
National Institute of Health and endorsed by the Ministry of Social Protection of Colombia
and the National Institute for Food and Drug Surveillance –INVIMA-. This guide
establishes general guidelines for performing microbiological analysis counts in
accordance with national standards.

1. GENERAL CONSIDERATIONS:
For the correct application of this standard, the preparation of the sample is
essential, therefore it is required that the seeding be carried out from two
consecutive dilutions and in duplicate in addition to inoculating the correct volumes

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CABEZA HERRERA, EA, ORTIZ LEMUZ J. F.

(0.1 ml for surface and under certain circumstances, 1 ml can be inoculated on the
surface for samples that are considered to have a low count and 1 ml for depth).

2. COUNTING RANKS:
After the specified incubation period, the colony-forming units CFU present in the
boxes containing between 30 CFU and 300 CFU are counted, the analyzes that
apply said range1 They are: Mesophilic aerobes; coliforms, count of thermophilic
bacteria, and in general for analysis of microorganisms where no enzymatic
reactions or growth are observed on the surface of the Agar.
To count these latter microorganisms, the range of 20 CFU – 200 CFU is used, for
example for: S. aureus , B. cereus , etc.
For the count of Molds and Yeasts, and osmophilic yeasts, the range of 10 CFU –
100 CFU is taken into account.

3. COUNTING AND EXPRESSION OF RESULTS:

TO). GENERAL CASE: The average number of colonies is calculated for each of
the dilutions to be examined by multiplying the result by the inverse factor of the
respective dilution, based on the count of two successive dilutions.
Divide the result obtained from the highest dilution (less concentrated) by the result
from the lowest dilution (more concentrated).
If the quotient of this division is greater than 2, report the result obtained in the most
concentrated dilution. However , if the quotient is less than 2, report the arithmetic
mean of the counts of the two consecutive dilutions.

1 Except for those bacteria whose standard requires presence or absence

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GENERAL GUIDE FOR THE COUNT OF MICROORGANISMS IN FOODS

The calculated result is rounded to two significant figures. For this, if the last figure
is less than 5, the previous figure is not modified; If the last digit is 5 or greater, the
previous digit is incremented by one. This continues until two significant figures are
obtained.

The results are expressed in CFU per gram or milliliter of product depending on the
nature of the product, expressed as a number between 10 and 99 (two significant
integer figures) multiplied by 10 X where x is the appropriate power of 10.

Example:
From an analysis of mesophilic aerobes for pasteurized milk, the following results
were obtained:

- in the first selected dilution (10 -2 ): 168 CFU and 215 CFU.
- in the second selected dilution (10 -3 ): 35 CFU and 45 CFU.

10-2= 168 + x 100 =


215
19,150
2
10 -3 = 35 + 45 x 1000 = 40000 2
40000
= 2,087
19150
Since the quotient is greater than two, the result of the lowest dilution is reported:

10 -2 = 19150 CFU/ml.
When rounding the figure as specified above, the result is 19,000, expressed with two
significant figures 19x10 3 CFU/ml.

B). SPECIAL CASES:

- CASE 1: When the number of CFU is below the minimum range in both boxes of one
or two consecutive dilutions, it will be reported as follows:

The count obtained in the highest dilution is discarded and the arithmetic mean of the
colonies present in the two boxes of the lowest dilution is calculated by multiplying by
the inverse of the dilution and the result is reported as follows:

Estimated CFU count per gram or milliliter (Taking into account the way of
reporting the final result: two significant figures per........................)

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CABEZA HERRERA, EA, ORTIZ LEMUZ J. F.

- CASE 2: Absence of growth, if the two boxes corresponding to the first dilution do not
show growth, it will be reported as negative and the result is reported as follows:
Estimated CFU count per gram or milliliter
< 1x10 X CFU per gram or milliliter as appropriate. EC

- CASE 3: Abundant growth, if the two boxes corresponding to the first dilution show
growth greater than 300 colonies, divide the box into 2, 4, 6, 8 quadrants, count the
number of colonies present in one quadrant and multiply by the total number of
quadrants and the inverse of the dilution. The counting is continued as in the general
case but the result is reported as Estimated CFU count per gram or milliliter

Example:
From an analysis of mesophilic aerobes for pasteurized milk, the following results were
obtained:
- in the first selected dilution (10 -2 ): >300 CFU in the two boxes.
- in the second selected dilution (10 -3 ): >300 CFU in the two boxes.
By dividing the boxes of 10 -2 into 4, 180 colonies and 150 colonies were counted in a
quadrant respectively.
By dividing the boxes of 10 -3 into 4, 100 colonies and 120 colonies were counted in a
quadrant respectively.

10 -2 (box 1) = ( (180 x 4) x 100 ) 10 -2 (box 2) = ( (150 x 4) x 100 )

10 -3 (box 1) = ( (100 x 4) x 1000 ) 10 -2 (box 2) = ( (120 x 4) x 1000 )

10-2= 720 + 600 x 100 =


2 66000

10-3= 400 + 480 x 1000 =


400000 440000
= 6,66 66000

Since the quotient is greater than two, the result of the lowest dilution is reported:

10 -2 = 66,000 CFU/ml.
When rounding the figure as specified above, the result is 66,000, expressed with
two significant figures 66x10 3 CFU/ml. EC

NOTE: when the colony count exceeds 200 in 1/8 of the box it will be reported as >
1600 CFU/gr or ml multiplied by the inverse of the lowest dilution and estimated
count.
Example:
From an analysis of mesophilic aerobes for pasteurized milk, the following results
were obtained:

-
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GENERAL GUIDE FOR THE COUNT OF MICROORGANISMS IN FOODS

- in the first selected dilution (10 -2 ): >300 CFU in the two boxes.
- in the second selected dilution (10 -3 ): >300 CFU in the two boxes.
By dividing the boxes of 10 -2 into 4, 240 colonies and 230 colonies were counted in
a quadrant respectively.

It is reported as >1600x10 2 CFU/ml. EC

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