Tài liệu Practical Food Microbiology 3rd Edition - Part 5 pptx

5.6 Surface spread plate5.7 Multiple tube most probable number methods 5.8 Surface contact methods 5.9 Surface swabs 5.10 Membrane slide cultures 5.11 Rinse method for watercress, other

5.6 Surface spread plate

5.7 Multiple tube (most probable number) methods

5.8 Surface contact methods

5.9 Surface swabs

5.10 Membrane slide cultures

5.11 Rinse method for watercress, other leaf vegetables and acidic berry fruits

5.12 Bottle rinse and plate count

• Characteristics of specific media

• Lower limit of enumeration required

• Purpose of the examination

• Time available

Legislation sometimes prescribes a specific counting method for the meration of microorganisms in a particular product, for example the pour platemethod is specified in European Union (EU) milk legislation For environmentalsamples such as surfaces, utensils and equipment a surface contact techniquemay be the most useful method to choose

Any of a number of methods given in this section may be selected for meration of microorganisms in food Whilst the pour plate method using platecount agar is regarded as the standard international method of enumerationfor a total aerobic colony count, it is common for laboratories to use surfacemethods such as the surface drop and spiral plate Apart from the obvious con-venience of using pre-poured plates, these surface methods have the advantagesthat they eliminate possible heat stress to the organisms from the molten agar,provide fully aerobic conditions of growth and facilitate identification of the organism types present

enu-Pour plate methods require the use of a clear growth medium to allow ing of colonies that have grown below the surface of the medium This also applies to counts performed by automated colony counters using transmittedlight

count-5

In most instances surface methods are preferable when selective media are used for enumeration of specific groups of organisms because they allow full manifestation of colonial properties such as morphology, pigmentation,haemolysis, haloes of precipitation around the colonies or changes in colouraround the surrounding medium However, some organisms with particularatmospheric requirements, such as anaerobes, may be best enumerated by apour plate method where the depth of medium helps maintain an anaerobicenvironment.

The use of a liquid method such as a multiple tube method for enumeration

of organisms that are highly stressed, due to drying or high salt content for example, may allow better recovery and growth of the target organism and thusresult in a more accurate assessment of the level of the target organism in thefood sample Multiple tube methods are also useful for enumeration of lownumbers of organisms (below 100/g) but are less suitable when high numbersare expected

If an enumeration is performed in order to determine compliance with limitsset in microbiological standards, guidelines or specifications the choice of enu-meration method may also be affected by the required lower limit of detec-tion Pour plate methods, membrane filtration and multiple tube methods arecapable of detecting lower counts than surface methods of enumeration because

a larger quantity of the sample can be examined

Where large numbers of similar samples are to be checked for a microbial loadwithin a defined range, such as in production runs within a factory, increasinguse is being made of sophisticated equipment that detects bacterial growth elec-tronically by impedance or conductance within the growth medium For anygiven product it is first necessary to produce a calibration curve for growth in adefined medium under carefully controlled test conditions The advantage ofsuch methods is that batch rejection can be triggered as soon as a predefinedpoint on the calibration curve is reached and means that the samples with thehighest bacterial count will be detected in the minimum period of time, some-times within 6 h These methods are not included in this manual because of the diversity of foods which most non-industrial laboratories are required to examine

Factors affecting the results [1]

The successful performance of the pour plate technique depends heavily on adequate and appropriate tempering of the molten agar Bottles of molten agarshould be placed in a water bath set at 44–47°C The length of time required fortempering to that temperature will depend on the volume of agar in each bottleand should be determined on an individual basis The number of bottles placed

in the water bath will also affect the rate of cooling Extended storage of themolten agar will reduce the gelling properties Molten agar should be used with-

in 8 h of melting and preferably within 3 h, and should not be remelted once ithas set For some particularly sensitive media such as agars containing bile, the

duration of holding in the molten state should not exceed 3 h Even if adequatetempering of the molten agar has been ensured, heat stress of organisms maystill occur, particularly in chilled and frozen foods.

Many of the organisms found in foods are obligate aerobes, for example some

species of Pseudomonas and Bacillus The relatively anaerobic conditions found

in the depths of the agar in a pour plate may result in under-recovery of these ganisms Use of surface methods utilizing pre-poured plates will remove thesevariables and may result in a more accurate determination of the levels of theseorganisms Pre-poured plates usually require some drying before use, so that theinoculum used in the test is absorbed within 15 min of application Over-dryingmust be avoided as this can result in concentration of inhibitory components atthe surface of the plate with subsequent inhibition of growth

or-Inoculated plates should be placed in the incubator as soon as possible afterthe agar has set or the inoculum absorbed International standards recommendthat plates should be stacked no more than three high to ensure good heat penetration This may be difficult to achieve in practice and studies have shownthat plates stacked six high are not subject to significant variation in heat penetration [1]

At the end of the incubation period it is not always possible to perform thecolony counting, for example due to lack of time or work of a higher priority Inmost cases it is acceptable to refrigerate the plates until counting can be per-formed ISO 7218 [2] permits refrigerated storage of plates for up to 24 h after theincubation period unless otherwise specified in the method For media contain-ing pH indicators such as violet red bile agars the plates must be allowed to regain ambient temperature before attempting to count the colonies to ensureaccurate identification of suspect colonies

It is good practice to monitor the microbial contamination of the laboratoryenvironment, and this should be performed at regular intervals determined bythe level of activity in the laboratory Settle plates may be used to monitor thelevel of aerial environmental contamination in areas of sample processing byexposing the agar surface for a defined length of time, e.g 15 min The number

of organisms are then counted after incubation An action level should be lished above which remedial action should be taken, for example thoroughcleaning of the laboratory Surface swabs may also be taken to monitor generallevels of hygiene and to ensure the absence of pathogens

estab-Preparation of dilutions [3]

In order to enumerate fully the number of organisms in a food sample it may benecessary to prepare dilutions of the food homogenate Commonly serial deci-mal dilutions in peptone saline solution (maximum recovery diluent, MRD) areprepared from the sample homogenate by adding 1 mL of sample homogenate

to 9 mL of diluent etc to the required endpoint The accuracy of the volumes ofdiluent used should be ±2% and the accuracy of the sample volume dispensedshould be ±5% The use of automatic pipettors and associated sterile tips is advo-

cated to help ensure accuracy when preparing dilutions Precision of ±1% isachievable with automatic pipettors compared with ±5% with volumetric graduated pipettes All automatic pipettors should be checked regularly to ensure that the desired volume is being delivered For dispensing volumes of 0.1 mL or more, the pipettor should be used in total delivery mode, that is theplunger is depressed only to the first stop when drawing up the liquid, but fullydepressed when discharging the liquid If the volume to be dispensed is less than0.1 mL, the reverse pipetting technique should be used whereby the plunger isfully depressed when aspirating the liquid but only depressed to the first stopwhen discharging In all cases care must be taken to prevent jump back of the liquid inoculum that may result in contamination of the pipettor, as this mayalso result in contamination of the sample inocula; regular sanitizing of thepipettor is recommended.

If total delivery volumetric pipettes are used, correct delivery is ensured

by touching the tip of the pipette on an inside wall of the container when emptying

Quality control of media

Solid and liquid media used for the enumeration of microorganisms in foodsshould be subjected to quality control tests using reference cultures Details ofcultures for use in relation to media specific for particular organisms or groups oforganisms are given in Section 6 The organisms listed in Table 5.1 are recom-mended for testing media used for enumeration of ‘total’ microbial content andother non-selective procedures

Table 5.1 Control organisms for testing enumeration and non-selective media

NCTC 6571 Staphylococcus aureus

} Blood agar base, tryptone soya

Plate count agar, yeast extractNCTC 775 Enterococcus faecalis } agar, milk plate count agarNCTC 10418/9001 Escherichia coli

} Nutrient agarNCTC 10418/9001 Escherichia coli

• The standard uncertainty of a test method is defined as one standard deviation.

• The combined standard uncertainty is the result of the combination of all the

standard uncertainty components associated with that test method

• The expanded uncertainty is obtained by multiplying the combined standard

uncertainty by a coverage factor (see below)

• Type A evaluations of uncertainty are done by calculations from a series of

re-peated observations, using statistical methods

• Type B evaluations of uncertainty are derived from other sources, e.g calibration

data

Likely sources of uncertainty are shown in Table 5.2

In microbiological testing the greatest sources of uncertainty arise from sampling and the non-homogeneous distribution of microorganisms in thesample In order to evaluate uncertainty it has to be assumed that the organismsare distributed randomly When performing a microbiological test, type B un-certainties usually form part of a type A evaluation and so may not need to beconsidered separately In addition, they usually represent such a small contribu-

Table 5.2 Factors contributing to uncertainty of measurement in microbiology

Sample stability

Representative nature of subsampling in the laboratory

Uncertainty associated with weighing balance

Uncertainty associated with diluting equipment (dispensers, pipettors)

Uncertainty associated with inoculum volume (pipettes, pipettors)

Integrity of filtration membrane (quality, pore size)

Uncertainty of temperature measurement (thermometers)

Stability of incubation conditions

Penetration of heat during incubation

Achievement of designated incubation duration

Performance of the isolation medium (yield)

Uncertainty associated with counting:

particle statistical variation

crowding effect

between operator variation

accuracy of colony counter

personal interpretation of the target

Uncertainty associated with confirmatory tests:

number of colonies selected

tion to the combined standard uncertainty that they do not make a significantcontribution Thus for microbiological testing purposes, the type A evaluation isthe dominant component and is not significantly different from the standarduncertainty Generally, the type B components can therefore be ignored for microbiological tests.

Duplicate results from tests performed by different operators as part of nal or external quality control samples can be used to calculate uncertainty ofmeasurement using the analysis of variance to obtain the repeatability standarddeviation This is equivalent to the standard uncertainty In order to obtain alevel of confidence of approximately 95% the standard uncertainty (standarddeviation) is multiplied by a coverage factor of two The value obtained is known

inter-as the expanded uncertainty of the test

This analysis should be repeated on a regular basis to maintain an estimatethat is relevant to the laboratory in its current situation Results from all staffshould be included, to provide a result for the laboratory as a whole

Interpretation of counts [4]

If a numerical limit is specified in a standard, guideline or specification and astatement of compliance is required but no reference is made to taking uncer-tainty into account, the following approach is recommended [4]

• Expand the count obtained in the test by the uncertainty interval at a level

of confidence of 95% before comparison with the numerical standard For microbiological tests, maximum values are usually specified

• Compliance is achieved if the standard lies above the upper limit of the tainty interval

uncer-• If the standard is exceeded even when the measured count is decreased by halfthe uncertainty interval, a statement of non-compliance can be made

• If the lower limit of the uncertainty interval does not exceed the standard it isnot possible to confirm compliance or non-compliance The test result andexpanded uncertainty should be reported together with a statement thatcompliance was not demonstrated

E X A M P L E

The uncertainty for a test at a 95% confidence level is ±0.21 (expressed as a logarithmicvalue)

The standard to be met is 1.0 ¥ 105/g (or log105.0000)

The measured count for the test is 1.3 ¥ 105/g (or log105.1139)

The measured count expanded by the uncertainty is:

Log104.9039 - log105.3239 or 8.0 ¥ 104- 2.1 ¥ 105

Because the measured count lowered by half the uncertainty interval (8.0 ¥ 104) is less thanthe standard it is not possible to confirm compliance or non-compliance

E N U M E R AT I O N M E T H O D S

Dip slide culture

Dip slides may be used for estimating numbers of bacteria in liquid food ucts and in food homogenates prepared as described in Section 4.2 The use ofdip slides for surface contact methods is described in method 3 of Section 5.8.There is a wide choice of dip slides available and the selection of a particular typewill depend on the following:

prod-• The organism or group of organisms sought (and therefore the agar mediumused)

• The potential use of the dip slide (the same medium or different media can beused to coat the two sides of the slide)

• The surface area of the slide

• The availability and storage life

(b) Remove the dip slide and drain

(c) Replace the dip slide in its container and incubate as appropriate for the isms sought (see Section 6 for guidance)

organ-After incubation

Estimate the number of microorganisms/mL of sample from diagrams supplied bythe manufacturer of the slide or count the number of colonies on each side of theslide

Calculation

For watery liquids only:

Calibration is necessary for other types of liquids, e.g oil–water emulsions, milk ormilk products

Total colonies on slide

Agar surface area cm( 2)¥1000=colony forming units cfu mL.( )

Membrane filtration [5]

This method is suitable for water, beverages and liquid food products Any ured volume of sample that is compatible with the equipment available may beused, so this method is particularly useful for examining larger sample sizes such

meas-as 100 mL or 1 L If the sample is likely to contain high numbers of organisms,

5.2

the use of a small volume or preparation of serial decimal dilutions is recommended.

(b) After filtration, remove the filter membrane with sterile forceps and place it on aculture pad previously soaked in appropriate culture medium or on the surface of

a suitable agar medium (see Section 6 for guidance)

(c) Incubate the culture pad or agar plus filter membrane as appropriate for the organisms sought (see Section 6 for guidance)

5.3

Procedure

(a) Place 1 mL of the dilution into each of two sterile Petri dishes

(b) Add about 15 mL of molten clear agar, tempered to 44–47°C, to each plate (e.g.plate count agar for a total colony count)

(c) Mix each plate well by moving it five times in a vertical, clockwise, horizontal andanticlockwise direction as shown, then allow the plates to set

(d) Incubate all plates as appropriate for the organisms sought (see Section 6 for guidance) For a total mesophilic aerobic colony count using plate count agar, incubate for 72 ± 3 h at 30°C

Use the plates containing fewer than 300 colonies at two consecutive dilutions to

cal-culate the results from a weighted mean The number (N) of cfu/g or mL of test sample

is calculated as follows:

N = C/v (n 1 + 0.1 n 2 ) d

where: C is the sum of colonies on all plates counted

v is the volume applied to each plate

n 1is the number of plates counted at the first dilution

n 2is the number of plates counted at the second dilution

d is the dilution from which the first count was obtained.

Round the result to two significant figures and express it as a number between 1.0 and9.9 multiplied by 10x where x is the appropriate power of 10.

If a differential or selective medium (such as violet red bile glucose agar [VRBGA]) isused for the pour plate method, plates containing no more than 150 colonies should

be selected for counting

If plates at only one dilution contain countable colonies, calculate the count using

continued

E X A M P L E

Number of colonies at first dilution (10-3) = 171 and 194

Number of colonies at second dilution (10-4) = 14 and 20

Volume added to each plate = 1 mL

N = (171 + 194 + 14 + 20)/1 ¥ (2 + [0.1 ¥ 2]) ¥ 10-3

= 399/0.0022 = 181 363

When rounded to two significant figures this becomes 180 000 or 1.8 ¥ 105cfu/g

or mL

Note: all counts from plates of the selected dilutions should be used, including any

plate with no colonies if the corresponding plate at that dilution contains colonies,

unless the count exceeds 300 or is overgrown

Table 5.3 Enumeration of total colony count (e.g aerobic plate count) using selective medium.

non-Count

First dilution (d1 ) Second dilution (d2 ) Expression

d, dilution (10-1, 10-2, etc.); n, total number of colonies.

Table 5.4 Enumeration of characteristic colonies on selective media

Count

First dilution (d1 ) Second dilution (d2 ) Expression

n ≥ 15 and £ 150 with cc Any with cc Weighted mean

n ≥ 15 and £ 150 with cc No cc Arithmetic mean d1

n ≥ 150 with cc n £ 150 no cc Less than 1/d2and more than 1/d1

n ≥ 150 no cc n £ 150 no cc Less than 1/d1

n > 150 and £ 167 with cc n < 15 with cc Weighted mean

n > 167 with cc n < 15 with cc Arithmetic mean d2

n > 150 with cc n > 150 with cc More than 150 ¥ 1/d2

n > 150 with cc n £ 150 with cc Arithmetic mean d2

cc, characteristic colonies; d, dilution (10-1, 10-2, etc.); n, total number of colonies.

For a 95% probability, the CI can be calculated from the following equation:

Spiral plate

This method is suitable for liquid food products or food homogenates, but it isnecessary to allow all food particles to settle before proceeding with the test Thespiral plater is a dispenser which distributes a set volume of liquid on to the sur-face of a rotating agar plate The dispensing arm moves from near the centre ofthe plate towards the outside edge, depositing the sample in an Archimedes’ spiral A cam-activated syringe dispenses a continually decreasing volume ofsample, resulting in a concentration range of up to 10 000 : 1 on a single plate.The volume of sample on any particular segment of a plate is known and is constant

5.4

E X A M P L E

No colonies counted

Dilution d Dilution d + 1 Weighted mean CI

Full details of rules for counts by the pour plate method outlined above can be found

Surface drop [7]

This method is suitable for liquid food products or food homogenates Serialdecimal dilutions should be made using MRD as diluent As a guide, with cleanproducts dilutions to 10-3 may be sufficient whereas heavily contaminatedproducts may require dilution to 10-6or higher

(f) Repeat step (c) after each sample or between each dilution if higher than the vious dilution

pre-After incubation

Count the colonies on the agar plate This can be done manually with a viewing grid or with a laser colony counter or other automated counter/image analyser Formanual counting, select any segment and count the colonies from the outer edge intothe centre until 20 colonies have been counted; continue to count the remainingcolonies in the subdivision of the segment containing the 20th colony Record thiscount together with the number assigned to the subdivision of the segment Countthe colonies in the same area on the opposite side of the plate and record the count.Add the counts together to obtain the number of colonies in that designated subdivi-sion If the whole plate contains fewer than 160 colonies count the colonies on thewhole plate

Calculation

To calculate the count, divide the total count obtained by the volume constant for thesubdivision counted, then multiply by the appropriate dilution factor Alternatively,use the tables supplied by the manufacturer If a 0.05-mL volume has been used thecountable range of cfu/mL of test dilution is 20–105

Procedure

(a) Start with the highest dilution of the sample (e.g 10-6)

(b) Mix well, preferably using a vortex mixer

(c) Using the reverse pipetting technique, draw up a known volume of the liquid, e.g

20 µL using an automatic pipettor and sterile tip

(d) Dispense the aspirated volume as a drop onto one sector of at least two agar plates(e.g plate count agar)

continued