3

Basic plate count techniques for the enumeration of microorganisms

3.1

Introduction

Most of the recommendations and guidelines contained in this chapter are taken from American Public Health Association (APHA), as described in the Chapter 6 of the 4th Edition of the Compendium of Methods for the Microbiological Examination of Foods (Swanson et al., 2001). When different from or complementary to those of the Compendium, they were complemented with information and recommendations from ISO 6887-1:1999 and ISO 7218:2007, recommended for performing tests using methods developed by the International Organization for Standardization. Microbiological examination of foods is predominantly based on culturing techniques, to detect and enumerate living microorganisms. In view of the huge variety and multiplicity of groups, genera and species that may be present, a great number of tests are used, and which can be of one of two types: qualitative tests, which are aimed at detecting the presence or absence of the target microorganism(s), without quantifying, and quantitative tests which determine the quantity of the target microorganism(s) in the sample, generally per unit of weight or volume. Each of these tests follow differentiated procedures, which in turn depend on the target microorganism(s), but most of them utilize the same basic microbiological culturing techniques. These techniques are the detection of presence/absence (Chapter 5), the Most-Probable-Number counts (MPN) (Chapter 4) and the standard plate counts, described in this chapter. Standard plate counts are used both for quantification of large microbial groups, such as aerobic mesophilic microorganisms, aerobic psychrotrophiles, yeasts and molds, sulphite-reducing clostridia, enterococci and lactic bacteria, but also particular genera and species, such as Staphylococcus aureus, Bacillus cereus and

Clostridium perfringens. The basic procedure consists in inoculating the homogenized sample (and its dilutions) on a solid culture medium (with agar), contained in Petri dish, and followed by incubation of the plates until visible growth occurs. The versatility of the technique derives from the principle underlying the concept of microbiological counts, and which is based on the premise that, when placed on an appropriate solid medium, each microbial cell present in the sample will form an isolated colony. By varying the type of culture medium (enrichment medium, selective medium, differential selective medium) and the incubation conditions (temperature and atmosphere), it is possible to select the group, genus or species to count. Since microbial cells often tend to aggregate into groupings of different shapes and sizes (pairs, tetrads, chains, grape-like clusters), it is not possible to establish a direct relation between the number of colonies and the number of cells. This correlation is between the number of colonies and the number of “colony forming units” (CFU), which may be either individual cells or clusters of a specific size-shape-number configuration that is characteristic of certain microorganisms. The procedures used to homogenize the samples and prepare sample dilutions are described in Chapter 2. With regard to inoculation in a solid culture medium (called plating) four basic procedures may be utilized: a) pour plate (deep plating), b) spread plate (surface plating), c) drop plate and d) membrane filtration.

3.2

Pour plate technique

The standard procedure for pour plating, described below, has a detection limit of 10 CFU/g for solid products or 1 CFU/ml for liquid products. This procedure may be adapted, if necessary, to achieve a detection limit

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of 1 CFU/g for solid products. The main applications for the pour plating technique are total aerobic mesophilic counts, Enterobacteriaceae counts, enterococci counts and lactic acid bacteria counts. There are some limitations to this technique, the main one being the need to melt the culture medium before use. Some media supplemented with heat-sensitive components after sterilization may not be reheated to melt the agar before use.

3.2.1

Material required for the analyses

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• Material for preparing the sample and serial dilutions, described in Chapter 2. • The culture medium recommended for the test to be carried out, described in specific chapters. • Sterile, empty 20 × 100 mm Petri dishes. • Laboratory incubator set to the temperature specified by the test to be performed, described in specific chapters.

net or in the proximity of the flame of a Bunsen burner. Deposit the inoculum off-centre in the Petri dish, since this will later on facilitate mixing with the culture medium. Position the pipette at an angle of about 45° touching the bottom of the plate. Use a different pipette for each dilution, with a maximum holding capacity of 10 ml. The uncertainty of the volume measurement must not exceed 5% (ISO 6887-1:1999). Observe carefully whether the plate used actually corresponds to the sample and dilution that are being inoculated. Change the position of the plates as they are being inoculated, to avoid the risk of inoculating the same plate more than one time, or to leave a plate un-inoculated.
Note b.1) Select the dilutions as a function of the estimated contamination level of the sample, so as to obtain plates containing 25 to 250 colonies. If the expected contamination level of the inoculum falls in range from 2.500 to 25.000 CFU/g or ml, for example, the recommended dilutions are 10−1, 10−2 and 10−3, which correspond to 0.1, 0.01 and 0.001 g or ml of sample. If the contamination level is expected to exceed this range, higher dilutions must be inoculated. If the contamination is expected to be below this range, it is possible to start inoculating 1 ml of the sample without any dilution for liquid products. In the case of solid products it is not possible to inoculate samples without dilution, but it is possible to inoculate up to 2 ml of the initial dilution in one and the same plate, or a greater volume, distributed or divided over several plates (2 ml/plate). If it is not possible to previously estimate the level of contamination of the sample, it is recommended to inoculate more than three dilutions, made from the initial dilution. Note b.2) For the analysis of certain foods, the recommended initial dilution is greater than 1:10 (see Chapter 2). If the counts in these products are expected to be low, the inoculated volume of the initial dilution should be increased, in the same way as described in Note b.1. When using this technique, the inoculation of 0.1 g of solid product or 1 ml of liquid product should be maintained, if possible. Note b.3) To increase the accuracy of the counts, it is recommended not to use pipettes with a holding capacity greater than 2 ml to dispense volumes of 1 ml. It is also possible to inoculate two plates with the same dilution (duplicate). Note b.4) ISO 7218:2007 does not require the inoculation of three dilutions of the sample, nor that of two plates for each dilution. It establishes two successive dilutions, without duplicate, or with a duplicate if only one dilution is to be used. However, the way to cal-

3.2.2

Procedure

Before starting the procedure, observe the precautions and care described in Chapter 2, to ensure that all activities will be carried out under aseptic conditions. Properly identify the tubes and plates that will be inoculated by labeling them with the sample code, the dilution, and the standard abbreviation of the culture medium. Melt the culture media in a boiling water bath, maintaining the boiling for only the time necessary to soften and liquefy the agar. Cool immediately in cold water and keep at a temperature of 44 to 46°C until the time of use (in a temperature-controlled water bath or incubator). a) Preparation of the samples and serial dilutions: For the preparation of the samples and serial dilutions follow the procedures described in Chapter 2. b) Inoculation: In general, inoculation is done for several tests at the same time. For each test that is being conducted, select three adequate dilutions of the samples (see the notes below) and inoculate 1 ml of each dilution in separate, sterile and empty Petri dish, opening the plates only enough to insert the pipette. Work in a laminar flow cabi-

Basic plate count techniques for the enumeration of microorganisms culate the results is somewhat different (see item 3.7).

25

of medium, add 0.5 ml of a 1% aqueous TTC solution, previously sterilized by filtration.

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c) Addition of the culture medium: For each test that is being conducted, withdraw the culture medium from the water bath or incubator at 44–46°C and, if the flask is wet, dry with a paper towel taking care to avoid spattering onto the plates at the moment of plating. Avoid agitation and abrupt movements to prevent the formation of bubbles. Pour 12 to 15 ml of the medium into the inoculated plates, observing whether the identification of the plates corresponds to the culture medium used. Mix the medium with the inoculum, moving the plates gently on a flat surface, in movements forming the number eight or in circular movements, 8–10 times clockwise and 8–10 times counter-clockwise. The plates should be moved about with utmost care, to avoid droplets of medium from spattering onto the rims or lids of the plates. To facilitate this step of the operation, prefer using high plates (20 × 100 mm). The plates can be stacked one on top of the other during the addition of the growth medium and homogenization with the inoculum, but they must subsequently be evenly distributed over the cold surface of a bench, to accelerate cooling and solidification of the medium.
Note c.1) When several tests are being conducted simultaneously, the activities and teamwork should be organized and programmed so as to satisfy the following conditions, established by the Compendium (Swanson et al., 2001): the time between depositing the inoculum in a plate and the addition of culture medium should not exceed 10 minutes, to prevent the inoculum from drying out and to adhere to the plates. Mixing the culture medium with the inoculum should be done immediately after adding the medium, in order to avoid the risk of solidification of the agar. Furthermore, the Compendium recommends that the complete procedure, from the preparation of the first dilution until finishing the inoculation of all the culture media, should not take longer than 20 minutes. ISO 6887-1:1999 recommends not exceed 45 minutes. Note c.2) For foods containing or consisting of particles (meals and flours, for example) the distinction between colonies and particles may be difficult in the first dilution. To avoid this problem, TTC (2,3,5 triphenyltetrazolium chloride) may be added to the culture medium since most bacteria form red colonies in the presence of TTC. For each 100 ml

d) Incubation: Wait until solidification of the culture medium is completed, invert the plates (if required by the method being used) and incubate at the conditions of temperature, time and atmosphere specified for each test. The culture medium should reach the incubation temperature within a maximum time interval of two hours. Avoid excessive stacking of the plates and do not place an excessive number of plates in each incubator, to ensure even distribution of the temperature. Within the first 48 h of incubation, the plates may not lose more than 15% of their weight caused by drying out. Excessive moisture is also undesirable, since it increases the risk of spreading. Depending on the temperature, humidity control of the incubator may be necessary. e) Counting the colonies and calculating the results: Follow the guidelines and instructions described in item 3.6.1.

3.3 Spread plate technique The main difference between surface plate and pour plate is that the sample and/or its dilutions are inoculated directly onto the surface of a solid medium, previously distributed over a certain number of Petri dishes. Surface inoculation is considered advantageous in some aspects, since it does not expose the microorganisms to the high temperature of the melted medium, allows visualization of the morphological and differential characteristics of the colonies, facilitates the transferring of colonies, allows to use media that may not be re-heated to melt the agar, and does not require that the culture media be transparent or translucent. Its main disadvantage is the volume to be inoculated, which is limited to the maximum liquid absorption capacity of the culture medium (0.5 ml per plate). The standard procedure is the inoculation of 0.1 ml/plate of each dilution, with a detection limit of 100 CFU/g for solid products or 10 CFU/ml for liquid products. This procedure can be adapted, if necessary, to a detection limit of 10 CFU/g for solid products or 1 CFU/ml for liquid products. Its main applications are total aerobic psychrotrophic counts, yeast and mold counts, S. aureus counts and B. cereus counts.

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Microbiological examination methods of food and water

3.3.1

Material required for the analyses

• Materials for preparing the sample and serial dilutions, described in Chapter 2. • Petri dishes containing the medium recommended for the test, described in specific chapters. • Glass or plastic spreaders (Drigalski) immersed in ethanol 70%. • Laboratory incubator with the temperature set at the temperature specified by the test to be performed, described in specific chapters.
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surface of the medium as fast as possible, using glass or plastic spreader (Drigalski), and continue until all excess liquid is absorbed. Utilize a different spreader for each plate or, alternatively, flamesterilize the spreader after each plate, starting with the greatest dilution plate and going to the smallest dilution plates.
Note b.1) When several tests are being performed simultaneously, the activities and teamwork should be organized and programmed so as to satisfy the following conditions, established by Compendium (Swanson et al., 2001): the complete procedure, from the preparation of the first dilution until finishing the inoculation of all the culture media, should not take longer than 20 minutes and the inoculum spreading should be started immediately after depositing the three dilutions onto the medium surface. ISO 6887-1:1999 recommends not exceed 45 minutes. Note b.2) As described for pour plating, select the dilutions as a function of the estimated contamination level of the sample, so as to obtain plates containing 25 to 250 colonies. However, it should be taken into account that the inoculated volume is ten times smaller. In the case of samples with a low level of contamination, a greater volume of the first dilution may be inoculated, distributing this volume over several plates. A distribution commonly used is inoculating three plates with 0.3 ml and one plate with 0.1 ml. The spreading of 0.3 ml onto the plates requires a longer time of absorption of the liquid, thus care and precautions must be taken to avoid that moisture films remain on the surface, with the consequent formation of spreading zones. Note b.3) For the analysis of certain foods, the recommended initial dilution is greater than 1:10 (see Chapter 2). If the expected counts in these products are low, the inoculated volume of the initial dilution should be increased, in the same way as described in Note b.1. When using this technique the inoculation of 0.01 g of solid products or 0.1 ml of liquid products should be maintained, if possible. Note b.4) To increase the accuracy of the counts, it is recommended not to utilize pipettes with a capacity greater than 1 ml to dispense volumes of 0.1 ml. Note b.5) ISO 7218:2007 does not require the inoculation of three dilutions of the sample, establishing only two successive dilutions, without duplicate, or with a duplicate if only one dilution is to be used. However, the way to calculate the results is somewhat different (see item 3.7).

3.3.2

Procedure

As recommended for pour plate, observe the precautions and care described in Chapter 2 before starting the procedure, to ensure that all activities be carried out under aseptic conditions. Identify all the tubes and plates that will be inoculated with the sample code, the dilution and the standard abbreviation of the culture medium. Prepare the plates on beforehand and dry them in a laminar flow cabinet for 30–60 min with the lids partially open or in an incubator at 50°C/1.5–2 h with the lids partially open or in an incubator at 25–30°C/18–24 h with the lids closed. a) Preparation of the samples and serial dilutions: For the preparation of the samples and serial dilutions follow the procedures described in Chapter 2. b) Inoculation: In general, inoculation is done for several tests at the same time. For each test that is being conducted, select three adequate dilutions of the sample to be inoculated (see note b.1). Using a pipette with a maximum holding capacity of 1 ml (and 0.1 ml graduation markings), inoculate 0.1 ml of each dilution onto the surface of previously prepared plates. Verify whether the identification of the plate actually corresponds to the sample and dilution that are being inoculated and whether the plate contains the correct culture medium. Change the position of the plates as they are being inoculated, to avoid the risk of inoculating the same plate more than one time, or to leave a plate un-inoculated. Work in a laminar flow cabinet or in the proximity of the flame of a Bunsen burner. Spread the inoculum onto the entire

c) Incubation: Follow the same instructions and guidelines as those described for pour plating.

Basic plate count techniques for the enumeration of microorganisms

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d) Counting the colonies and calculating the results: Follow the guidelines and instructions described in item 3.6.2. 3.4 Drop plate technique

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The drop plate method is a surface inoculation technique that has the same advantages as the spread plating technique. The main difference is that the inoculum is not spread, but deposited onto the surface of the culture medium in the form of 0.01 ml-droplets. Since the droplets occupy a minimum amount of space, it is possible, on one and the same plate, to inoculate three dilutions in triplicate, three drops per dilution. This makes this technique extremely cost-friendly, with a detection limit 1,000 CFU/g for solid products or 100 CFU/ml for liquid products. Although the drop plate technique is not routinely used for the microbiological examination of foods, it can be very useful in situations that require the inoculation of a large number of dilutions. 3.4.1 Material required for the analyses

a) Preparation of the samples and serial dilutions: For the preparation of the samples and serial dilutions follow the procedures described in Chapter 2, but prepare the diluent supplemented with 0.1% agar, to make it easier to fix the droplets later on onto the surface of the culture medium. b) Inoculation: Divide the plate into nine sectors, marking the bottom with a glass-marking pen (tracing three horizontal lines and three vertical lines). For each test that is being conducted, select three adequate dilutions of the sample to be inoculated (see note b.1 below). Before collecting the volume of each dilution to be inoculated onto the plates, vigorously agitate or shake the dilution tubes, by inverting them 25 times in an 30 cm-arc or with the aid of a vortex shaker. Using 0.1 ml (and 0.01 ml graduated) pipettes or pipettes with disposable tips, deposit three 0.01 ml drops of each dilution in three adjacent quadrates of the plate (triplicate). This procedure must be performed with care, to avoid any droplets from running out of their respective quadrate. Do not spread the drops. Keep the plates placed on a flat surface, wait until all the liquid is absorbed by the culture medium, which will require approximately 30 minutes.
Note b.1) Select the dilutions as a function of the estimated contamination level of the sample, so as to obtain drops containing 30 colonies, at most. Take into account that drop plating cannot be used with samples with a contamination level lower than 103 CFU/g or 102 CFU/ml, except when the purpose of the test is not to quantify, but rather to show or prove that the count is below this limit.

• Materials for preparing the sample and serial dilutions, described in Chapter 2. • Sterile 0.1 ml graduated pipettes or pipettes with disposable tips to dispense the droplets. • Petri plates containing the medium recommended for the test, described in specific chapters. • A laboratory incubator with the temperature set at the temperature specified by the test to be performed, described in specific chapters. 3.4.2 Procedure

c) Incubation: Wait until the liquid of the drops is completely absorbed by the culture medium and incubate under the same conditions recommended for pour plating. d) Counting the colonies and calculating the results: Follow the guidelines and instructions described in item 3.6.3.

As recommended for pour plate, observe the precautions and care described in Chapter 2 before starting the procedure, to ensure that all activities be carried out under aseptic conditions. Identify by labeling all the tubes and plates that will be inoculated with the sample code, the dilution and the standard abbreviation of the culture medium. Prepare the plates with the culture medium on beforehand and leave to dry in an incubator for 24 hours at 25–30°C with the lids closed.

3.5

Membrane filtration

The procedure of membrane filtration is limited to the examination of limpid or crystal-clear liquid samples, without solids in suspension, and which may be filtered through a membrane with a pore size of 0.45 μm. The main advantage of this technique is that it makes it possible to inoculate larger volumes

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Microbiological examination methods of food and water

of the sample, concentrating in the membrane the microorganisms present in the inoculated quantity. The detection limit is 1 CFU per inoculated volume, which makes it the technique of choice for examining samples containing counts lower than the detection limit of the other procedures. Its main applications are total aerobic mesophilic counts, yeast and mold counts, lactic acid bacteria counts, enterococci counts and counts of total coliforms, fecal coliforms and E. coli in water, carbonated soft drinks and other liquid products, in addition to solid products, provided they can be transformed into a limpid solution, such as salt and sugar.
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3.5.1

Material required for the analyses

• Material for preparing the sample and serial dilutions, described in Chapter 2. • A previously sterilized filtration set. • A vacuum pump. • Membrane-filters, 47 mm in diameter, porosity of 0.45 μm. • Petri dishes containing the culture medium recommended for the test, described in specific chapters. • Sterile empty Petri dishes and sterile pads, optional for use with broth media. • A laboratory incubator with the temperature set at the temperature specified by the test to be performed, described in specific chapters.

3.5.2

Procedure

As recommended for pour plating, observe the precautions and care described in Chapter 2 before starting the procedure, to ensure that all activities be carried out under aseptic conditions. Identify by labeling all the tubes and plates that will be inoculated with the sample code, the dilution and the standard abbreviation of the culture medium. a) Preparation of the filtration set: The membrane filtration set is composed of a membrane filter holder, a kitasato flask and a filtration cup. The filter holder is a kind of funnel the upper part of which is plane, to accommodate the filtration membrane and onto the top of which the filtration

cup is held in place by a clamp. The lower part of the filter holder is coupled to the kitasato flask, which, connected to a vacuum pump, collects and retains the filtered liquid. Before beginning the analyses, the filter holder must be coupled to the kitasato, wrapped in kraft paper and sterilized in an autoclave (121°C/30 min). The filtration cups must be wrapped separately in kraft paper and also sterilized in an autoclave (121°C/30 min). Alternatively, disposable sterile cups may be used, which are especially useful when a large number of samples are to be filtered. At the time of use, the two parts must be unwrapped in a laminar flow cabinet or, when such equipment is not available, in the close proximity of the flame of a Bunsen burner. The membrane filtration set is prepared by adjusting the sterile membrane onto the filter holder (graph side up) and the filtration cup on the membrane. Next, the kitasato flask is connected to the vacuum pump to start filtration. Another possibility is to use manifolds fitted with various filter holders and which allow to filter several samples simultaneously. b) Preparation of the plates: The most commonly used plates to perform the membrane filtration technique are 50 mm in diameter and 9 mm in height. If a solid culture medium is used, the plates must be previously prepared, in the same way as recommended for spread plating. It is also common practice to utilize broth medium, a situation in which no previous preparation of the plates is necessary. At the time of analysis, a sterile absorbent pad is placed inside the sterile plates and soaked with 2 ml-portions of the culture medium in liquid form. c) Homogenization of the sample and withdrawing the analytical unit: Homogenize the sample following the procedures described in Chapter 2. Measure 100 ml in a sterile measuring cylinder and pour the content into the cup of the filtration set, avoiding spattering. If the cup of the filtration set is graduated and the graduation scale has a marking that matches the required volume, the volume of the sample may be measured directly without using the measuring cylinder. d) Serial dilution of the sample: Since the filtration method is a technique that concentrates the microorganisms in samples with low counts, serial dilutions of the samples are generally not made. The

Basic plate count techniques for the enumeration of microorganisms

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usual procedure calls for filtering total quantities of 100 ml, which may divided into two portions of 50 ml, 4 portions of 25 ml or 3 portions of 70, 25 and 5 ml, respectively. Selecting the volume to be filtered, however, will depend on the estimated level of contamination of the sample, so as to obtain plates containing a number of colonies within the 20 to 200 range. e) Filtration: Turn on the vacuum pump and start the filtration process. After passing the sample through the filter membrane, and with the vacuum pump still running, rinse off the sides of the cup with 20 to 30 ml of the diluents recommended in Chapter 2, to collect contaminants that may have adhered to the sides. Repeat this procedure one more time. Turn off the pump before the membrane begins to dry out. When the volume to be filtered is smaller than 20 ml, add about 20–30 ml of the diluent to the cup of the filtration set, before adding the sample itself. Accurate measuring of the volume of diluent is not necessary, since its function is limited to merely increasing the volume to be filtered and facilitate obtaining a more even distribution of the microorganisms on the membrane.
Note e.1) Between one sample and the next, and prior to positioning a new membrane, the filter holder should be flame-sterilized with alcohol and the filtration cup replaced. After every 10 samples it is recommended to use the filtration set to filter 100 ml (sterile) of one of the diluents recommended in Chapter 2, and subsequently incubate this membrane to check for possible cross-contamination between the samples. After filtering 30 samples, the filtration set should be re-sterilized in autoclave, prior to starting a new filtration series. If the time interval between one filtration operation and the next is greater than 30 min and the set is being used outside of the laminar flow chamber, it is recommended to autoclave the entire filtration set again, even when the limit of 30 samples has not yet been reached.

contained in the medium. In case of bubble formation, the edge of the membrane that is closest to the bubble(s) should be gently lifted up and replaced in a way so as to eliminate the bubble(s). Incubate the plates under the conditions recommended for the test (described in specific chapters) in an inverted position (if indicated by the method) and, preferably, placed inside bags or trays covered with moistened paper towels or filter paper, to avoid dehydration. g) Counting the colonies and calculating the results: Follow the guidelines and instructions described in item 3.6.4.

3.6

Counting colonies and calculating results

The instructions contained in this item are applicable to the tests in which all the colonies that developed on the plates, after the incubation period, are counted and considered in any further calculation(s). In the case of tests that use differential media – performed to distinguish the target microorganism(s) from accompanying microbiota (i.e. other microorganisms that may grow under the same conditions) – only the typical colonies are counted and considered in any further calculation(s). This is the case of enterococci and Enterobacteriaceae counts, which should be calculated in accordance with the guidelines provided in specific chapters. In the same way, in the case of tests that require confirmation of the colonies, only the percentage of confirmed colonies is considered in the calculation (s). The latter is the case of lactic acid bacteria, C. perfringens, S. aureus and B. cereus counts, all of which should be calculated following the instructions and guidelines provided in specific chapters.

f ) Transferring and incubating the membrane: Remove the cup and, using a pair of tweezers, transfer the membrane to the plate containing the culture medium, with the graph side facing up (flame-sterilize and then cool the pair of tweezers before using). When placing the membrane onto the culture medium it is important that its entire surface stays completely adhered to the medium, to ensure that the microorganisms in the membrane come into contact with the nutrients

3.6.1

Pour plate calculations

Select for counting plates without spreading and with a number of colonies in the range between 25 and 250. Count the colonies with the help of the magnifying glass of a colony counter to facilitate visualization. Use a colony counter with a 1 cm2 square grid background to serve as a counting guide. If no such plates in ideal conditions are available, follow the instructions described in rules 5 through 12 for counting.

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Microbiological examination methods of food and water
Note. The general rule for calculating the results of ISO 7218:2007 is different (see item 3.7).

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 considered. The first is the standard situation and the second 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.

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Rule 1 – If the count was performed on a plate inoculated 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 arithmetic 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) Example without dilution (100) 10−1 8 22 6–5 12–10 10−2 0 2 0–0 0–0 Count (CFU/ml) 199 = 2.0 × 102 245 = 2.5 × 102 (62 + 57)/2 = 59.5 = 60 (123 + 136)/2 = 129.5 = 1.3 × 102

Without duplicate 1 199* 2 245* With duplicate 3 62*–57* 4 123*–136*

*Counts effectively used to calculate the result.

Rule 2 N° colonies in the plate(s) Example 10−1 10−2 18 245* 62*–57* TNTC–TNTC 10−3 2 22 6–5 239*–242* Count (CFU/g or CFU/ml) 199 × 101 = 1,990 = 2.0 × 103 245 × 102 = 2,450 = 2.5 × 104 [(62 + 57)/2] × 102 = 59.5 × 102 = 6.0 × 103 [(239 + 242)/2] × 103 = 240.5 × 103 = 2.4 × 105

Without duplicate 5 199* 6 TNTC With duplicate 7 TNTC–TNTC 8 TNTC–TNTC

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

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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 prepared 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:
Rule 3

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, without spreading. However, quite frequently the plates do not present such ideal situations and require the application 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 dilution and compare the results. 6.a) If one of the results is greater than the double of the other, consider only the lower count (Examples 19 and 31 of Table 3.1).

N° colonies in the plate(s) (volume inoculated) Example 10−1 (2 ml) 10−2 (1 ml) 18 2 6–5 3–3 10−3 (1 ml) 2 0 0–0 0–0 Count (CFU/g or CFU/ml) (199/2) × 101 = 1.0 × 103 (123/2) × 101 = 6.2 × 102 {[(62 + 57)/2]/2} × 101 = 29.75 × 101 = 3.0 × 102 {[(27 + 35)/2)]/2} × 101 = 15.5 × 101 = 1.6 × 102

Without duplicate 9 199* 10 123* With duplicate 11 62*–57* 12 27*–35*

*Counts effectively used to calculate the result. Rule 4 Analytical Volume of Initial Example unity diluent dilution Without duplicate 13 10 g 14 25 g 15 25 g With duplicate 16 25 g 17 10 g 18 10 g 490 ml 350 ml 975 ml 475 ml 490 ml 290 ml N° colonies in the plate(s) Initial dilution 1st decimal 18 30* TNTC 2nd decimal 2 2 133* Count (CFU/g or CFU/ml) 199 × 50 = 1.0 × 104 30 × 15 × 101 = 4.5 × 103 133 × 40 × 102 = 5.3 × 105 [(237 + 229)/2] × 20 = 4.7 × 103 [(62 + 57)/2] × 50 × 101 = 3.0 × 104 [(239 + 242)/2] × 30 × 102 = 7.2 × 105

10/500 = 1/50 199* 25/375 = 1/15 280 25/1,000 = 1/40 TNTC 25/500 = 1/20 10/500 = 1/50 10/300 = 1/30

237*–229* 21–20 2–1 TNTC-TNTC 62*–57* 6–5 TNTC-TNTC TNTC-TNTC 239*–242*

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

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Microbiological examination methods of food and water
Examples for calculating the pour plate results in not ideal conditions. N° colonies in the plate(s)

Table 3.1

Example

Rules used

10−1 TNTC TNTC 18* 0 TNTC TNTC TNTC TNTC TNTC TNTC 27 TNTC-TNTC 138*–162* 228*–240* 18*–16* 0–0 TNTC-TNTC 287*–263* TNTC-TNTC

10−2 140* 243* 2 0 TNTC TNTC TNTC TNTC 325* 243* Spr 215 TNTC-TNTC 42–30 28*–26* 2–0 0–0 TNTC-TNTC 23–19 224*–180*

10−3 32 34* 0 0 370* 8/cm2* 21/cm2* >100/cm2* 20 Spr 20 239*–328* 1–2 2–2 0–0 0–0 320*–295* 2–2 28*–Spr

Count (CFU/g or CFU/ml) 140 × 102 = 1.4 × 104 [(243 × 102) + (34 × 103)]/2 = 2.9 × 104 18 × 101 = 1.8 × 102 (est) 6.5 × 106 (est) 325 × 102 = 3.3 × 104 (est) 243 × 102 = 2.4 × 104 Unacceptable result, repeat the analysis [(239 + 328)/2] × 103 = 283.5 × 103 = 2.8 × 105 [(138 + 162)/2] × 101 = 150 × 101 = 1.5 × 103 {[(228 + 240)/2] × 101 + [(28 + 26)/2]×102}/2 = 2,520 = 2.5 × 103 [(18 + 16)/2] × 101 = 17 × 101 = 1.7 × 102