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

Enumeration of microorganisms 105

Enumeration of microorganisms

5.1 Dip slide culture

5.2 Membrane filtration

5.3 Pour plate

5.4 Spiral plate

5.5 Surface drop

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

Choice of method

A range of methods is available for the enumeration of microorganisms in food.

The choice of method will depend on a number of factors.

• Type of sample.

• Characteristics, including the physiological state, of specific organisms

sought.

• Characteristics of specific media.

• Lower limit of enumeration required.

• Purpose of the examination.

• Time available.

Legislation sometimes prescribes a specific counting method for the enu￾meration of microorganisms in a particular product, for example the pour plate

method is specified in European Union (EU) milk legislation. For environmental

samples such as surfaces, utensils and equipment a surface contact technique

may be the most useful method to choose.

Any of a number of methods given in this section may be selected for enu￾meration of microorganisms in food. Whilst the pour plate method using plate

count agar is regarded as the standard international method of enumeration

for a total aerobic colony count, it is common for laboratories to use surface

methods such as the surface drop and spiral plate. Apart from the obvious con￾venience of using pre-poured plates, these surface methods have the advantages

that 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.

Pour plate methods require the use of a clear growth medium to allow count￾ing of colonies that have grown below the surface of the medium. This also

applies to counts performed by automated colony counters using transmitted

light.

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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 colour

around the surrounding medium. However, some organisms with particular

atmospheric requirements, such as anaerobes, may be best enumerated by a

pour plate method where the depth of medium helps maintain an anaerobic

environment.

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 thus

result in a more accurate assessment of the level of the target organism in the

food sample. Multiple tube methods are also useful for enumeration of low

numbers of organisms (below 100/g) but are less suitable when high numbers

are expected.

If an enumeration is performed in order to determine compliance with limits

set 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 are

capable 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 load

within a defined range, such as in production runs within a factory, increasing

use is being made of sophisticated equipment that detects bacterial growth elec￾tronically by impedance or conductance within the growth medium. For any

given product it is first necessary to produce a calibration curve for growth in a

defined medium under carefully controlled test conditions. The advantage of

such methods is that batch rejection can be triggered as soon as a predefined

point on the calibration curve is reached and means that the samples with the

highest bacterial count will be detected in the minimum period of time, some￾times within 6h. 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 agar

should be placed in a water bath set at 44–47°C. The length of time required for

tempering to that temperature will depend on the volume of agar in each bottle

and 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 the

molten agar will reduce the gelling properties. Molten agar should be used with￾in 8h of melting and preferably within 3h, and should not be remelted once it

has set. For some particularly sensitive media such as agars containing bile, the

106 Section five