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OCR for page 104
5
Selection of Indicator
Organisms and Agents
as Components
of Microbiological Criteria
Regulatory agencies and industrial quality assurance personnel regularly
examine foods or ingredients for microorganisms or their metabolic prod-
ucts that may indicate (1) the possible presence of a pathogen or harmful
toxin, (2) the possibility that faulty practices occurred during production,
processing, storage, and distribution, and/or (3) the suitability of a food
or ingredient for a desired purpose.
The indicator organisms and agents discussed in this chapter are divided
into four categories: (1) those that assess numbers of microorganisms and/
or microbial activity, (2) indicators of potential human or fecal contam-
ination or possible presence of pathogens, (3) indicators of post-heat pro-
cessing contamination, and (4) metabolic products of pathogens that indicate
a health hazard.
The section on assessment of numbers of microorganisms and/or mi-
crobial activity includes (1) the aerobic plate count, thermoduric, psy-
chrotrophic, thermophilic, proteolytic, and lipolytic counts; the direct
microscopic count; Howard mold count; rot fragment count; "machinery
mold"; yeast and mold count; heat-resistant molds and thermophilic spore
count; and (2) examination for metabolic products such as by organoleptic
examination, dye or indicator reduction time, pH, trimethylamine (TMA),
total volatile nitrogen (TVN), indole, ethanol, diacetyl, histamine, en-
dotoxins (Limulus amoebocyte lysate test), extract release volume, and
adenosine triphosphate.
Indicators of potential human or fecal contamination or of possible
presence of pathogens include staphylococci, Escherichia coli, fecal col-
iforms, enterococci (fecal streptococci, group D streptococci), and Pseu-
domonas aeruginosa. Indicators of post-heat processing contamination
104
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SELECTION OF INDICATOR ORGANISMS AND AGENTS
105
include coliform bacteria and Enterobacteriaceae. Thermonuclease and
U.V. light examination of grains are discussed in a section on metabolic
products of pathogens that indicate a potential health hazard. The signif-
icance of the presence of phosphatase in certain dairy products is also
discussed. Metabolic products such as tested for by organoleptic evalu-
ation, pH, TMA, TVN, indole, ethanol, and diacetyl are not commonly
used as components of microbiological criteria as are microorganisms.
They are included in this chapter because they result from microbial
activity and are intended as an adjunct or substitute for microbiological
testing for one or more reasons such as convenience, economy, or effec-
tiveness.
Each of these indicators (organisms, group of organisms, or agents)
will be discussed in reference to (1) its relative importance, (2) status and
limitations of method for detection or enumeration, and (3) suitability as
part of a microbiological criterion. Recommended uses of these indicators
as part of microbiological criteria for foods are given in Chapter 9.
ASSESSMENT OF NUMBERS OF
MICROORGANISMS AND/OR MICROBIAL ACTIVITY
Estimating Numbers of Microorganisms
Aerobic Plate Count
The aerobic plate count (APC) is used as part of a microbiological
standard for raw and pasteurized milk and many other dairy products, and
for shellfish at the wholesale level (see Chapters 8 and 91. It is also used
(1) to monitor a large number of other foods for compliance with standards
or guidelines set by various regulatory agencies, (2) to monitor foods for
compliance with purchase specifications, and (3) to monitor adherence to
good manufacturing practices. The test is based on the assumption that
each microbial cell, pair of cells, chain of cells, or clump or cluster of
cells present in an analytical unit (which is mixed with agar containing
appropriate nutrients) forms visible, separate colonies when incubated for
a sufficient period of time in an aerobic atmosphere and at a temperature
suitable for growth.
While the APC is the most popular method for estimating the number
of viable microorganisms, it is often misused. Contrary to some assump-
tions, the APC does not measure the size of the total bacterial population
in a sample of food. Instead it measures only that fraction of the microbial
flora that is able to produce colonies in the medium used under the pre-
vailing conditions of plate incubation. Alterations in medium or environ-
mental conditions change the fraction of the organisms that can grow.
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106 EVALUATION OF THE ROLE OF MICROBIOLOGICAL CRITERIA
Hence, rigid adherence to standardized test conditions is required if the
APC is to be part of a microbiological criterion.
The value of total counts as indexes of sanitary quality, organoleptic
quality, safety, and utility of foods was reviewed by Silliker (19631. The
APC of refrigerated perishable foods such as milk, meat, poultry, and
fish may reflect conditions such as the microbial content of the raw ma-
terials and ingredients, the effectiveness of the processing procedureks),
the sanitary condition of equipment and utensils, and the time-temperature
profile of storage and distribution. Specific causes of unexpected high
counts can be identified by examination of samples at critical control
points and by plant inspection. However, even perishable foods prepared
from wholesome raw materials and properly processed, packaged, and
stored develop high counts, lose quality, and ultimately spoil if held for
long periods. Under these conditions, the APC reflects not sanitary quality
but continued growth of psychrotrophic bacteria that were already present
in the food. Thus, the usefulness of the APC depends to a great extent
on the point in the process or distribution at which the sample was taken.
The APC of shelf-stable foods reflects the same conditions as listed for
perishable foods except that certain attributes of the shelf-stable food (aw,
pH, heat treatment, frozen state) prevent further growth of contaminating
bacteria. In the use or interpretation of the APC, additional factors need
to be considered: (1) the APC measures only living microbial cells. Pro-
cessing procedures, for example a heat treatment, can mask high-count
raw materials or insanitary conditions. In addition, continued storage of
a food in the frozen or dried state or at low pH values results in death
and thus in decreases in count; (2) the APC is of little value in assessing
the existing organoleptic quality of foods. Perceptible changes in quality
characteristics of foods because of microbial activity do not occur until
very high viable counts (106-107 per g or ml) have developed. Bacteri-
ological analysis will then only confirm the results of an organoleptic
examination; (3) the APC does not differentiate types of bacteria. Thus,
even if numbers are approximately the same, biochemical activities leading
to changes in a food may be different because of differences in microbial
types or in composition of the food. In addition, high counts resulting
from microbial growth are more likely to cause defects in foods than are
similar levels resulting from massive contamination.
For some foods, the APC can be used successfully to estimate potential
shelf-life. For example, a standard plate count (SPC) on freshly pasteurized
milk that was subsequently held at 7°C (44.6°F) for 5 days can provide
an estimate of the potential shelf-life of this food (APHA, 19781. This
application of the APC requires an intimate knowledge of the association
between count and shelf-life of a food.
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SELECTION OF INDICATOR ORGANISMS AND AGENTS
107
The APC may or may not be useful as a measure of the utility of an
ingredient for a food. In many cases, it may be necessary to analyze the
ingredient for the presence of specific organisms known to be of impor-
tance for the stability of the final product. APCs have little if any rela-
tionship to safety of foods. Also, APCs of fermented or retorted foods
are of little value.
Thermoduric, Psychrotrophic, Thermophilic, Proteolytic, and
Lipolytic Counts
Minor modifications of the APC can be applied to enumerate specific
groups of microorganisms such as thermoduric, psychrotrophic, thermo-
philic, proteolytic, or lipolytic bacteria. The thermoduric or laboratory
pasteurization count (APHA, 1978) is used in the milk industry to check
individual producer milk in order to identify samples with excessive num-
bers of these organisms. Thermoduric organisms survive pasteurization
of milk and thus may contribute to high SPCs on pasteurized milk. High
thermoduric counts are closely associated with persistent improper clean-
ing and sanitizing of equipment at the producer farm or at the processing
plant.
From a quality control point of view, psychrotrophic microorganisms
are of great significance in refrigerated perishable foods because they
grow, although not at optimum rate, at refrigeration temperatures. Some
of these psychrotrophs can cause serious off-tastes, off-odors, or sliminess
of foods when numbers are allowed to increase to 106-108 cells per g
or ml. Although the initial number of psychrotrophs in a refrigerated
perishable food may be low, it can increase to large numbers during
storage, the time depending upon factors such as the initial level of con-
tamination and the temperature. Shelf-life of such foods will be severely
impaired when initial numbers are high, particularly when foods are held
at marginal refrigeration temperatures. Unless consumed prior to this event,
high microbial counts will definitely occur in perishable foods even when
these foods are prepared from wholesome materials and are properly pro-
cessed and stored. Psychrotrophic bacteria are often sources of heat-re-
sistant proteolytic and lipolytic enzymes that may affect adversely the
quality of foods during long-term storage after heat processing (Griffiths
et al., 19811.
Proteolytic and lipolytic microorganisms can be responsible for a variety
of odor and flavor problems in foods. Some of the common psychrotrophic
bacteria are strongly proteolytic and/or lipolytic and cause serious defects
in dairy, meat, poultry, and seafood products when high counts (106 per g
or ml or above) are reached during refrigerated storage. The proteolytic
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108 EVALUATION OF THE ROLE OF MICROBIOLOGICAL CRITERIA
count (Chapter 8, Table 8-4) is part of a microbiological criterion for
butter and whipped butter. The proteolytic count and lipolytic count of
butter can be helpful in drawing attention to unsatisfactory manufacturing
practices.
In addition to the APC (AOAC, 1980) and SPC (APHA, 1978), there
are alternative procedures for determining viable bacterial counts such as
the surface or spread plate method, plate loop, oval tube or boKle culture,
drop plate, membrane filter, and spiral plate (AOAC, 1980; APHA, 1976,
1978, 19841.
Reliable interpretation of the APC of a food depends upon an intimate
knowledge of the expected microbial population at the point in a process
or distribution at which the sample was collected. Higher than expected
counts can serve as an alerting mechanism to investigate the potential
causers) of this event. The two most widely used APC methods are the
AOAC method (AOAC, 1980) and the standard plate count (SPC) method
as described in Standard Methods for the Examination of Dairy Products
(APHA, 19781. These reliable methods are used routinely to determine
APC as part of many microbiological criteria. With due consideration of
the precautions and limitations discussed in this section, the APC can be
a useful component of microbiological criteria of many foods. Specific
applications of the APC in microbiological criteria for foods are given in
Chapter 9.
Direct Microscopic Count
The direct microscopic count (DMC) is used as a component of mi-
crobiological criteria (standards) of raw (non-Grade A) milk, dried milks,
liquid and frozen eggs, and dried eggs. The DMC gives an estimate of
the total number of microorganisms, viable and nonviable, in a sample.
The DMC is rapid, requires a minimum amount of equipment, and pro-
vides information about the morphological characteristics of the cells. The
DMC is also used to count the number of leucocytes in raw milk and
thereby detect mastitic milk. Disadvantages of the method are that it is
suitable only for foods containing relatively large numbers of microor-
ganisms (105-106 per ml) and that the small quantity of sample (0.01 ml)
examined limits precision. The lower limit of precision for the DMC is
dictated by the Microscopic Factor and the number of fields examined.
Separation of viable and nonviable cells by this method is not possible,
and consequently counts may exceed corresponding agar plate counts many
times. For these reasons the use of the DMC as part of microbiological
criteria is very limited and applicable only when fairly high microbial
populations (viable, nonviable, or both) are expected and information on
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SELECTION OF INDICATOR ORGANISMS AND AGENTS
109
the microbial history of the product (such as dried milk) can be useful in
quality control programs. Rapid standardized methods (AOAC, 1980;
APHA, 1976, 1978, 1984) are available and are satisfactory for use in
microbiological criteria.
Other applications of the DMC use counting chambers such as the
Petroff-Hauser slide or the haemocytometer. These counting chambers are
used to determine populations of yeasts and other microorganisms in foods
such as fruit juice concentrates.
Microscopic MoldF Counts
Microscopic mold counts are used to assess the soundness of raw hor-
ticultural products and the sanitary conditions of processing lines. The
Howard mold count and rot fragment count are used for regulatory pur-
poses (see Chapter 9~.
Howard Mold Count. The Howard mold count, one of the oldest
microbiological criteria, is widely used to detect introduction of moldy,
decomposed fruits and vegetables into processed foods. It is used to mon-
itor both raw products and sorting and trimming operations. The test was
originally developed for tomato products. It is also applied to foods such
as apple butter, drupelet berries, citrus and pineapple juices, cranberry
sauce, strawberries, pureed infant food, and garlic powder and other spices.
Microbiological criteria (see APHA, 1976, 1984) vary with the type of
food being evaluated.
Rot Fragment Count. The rot fragment count is applied mainly to
tomato products such as catsup and sauce. This test, a count of tomato
cellular materials that exhibit mold filaments, is used mainly to evaluate
whether or not a food was prepared from sound raw produce. Practical
methods for these mold counts are applicable for microbiological criteria
(AOAC, 1980; APHA, 1976, 19841. There is a sufficiently close rela-
tionship between the Howard mold and rot fragment counts and the amount
of decayed tissue present for the counts to serve as useful microbiological
criteria for certain fruits and vegetable products and to be an index of
manufacturing practices. However, processing conditions such as milling
and homogenization can affect microscopic mold counts.
"Machinery Mold." Geotrichum candidum is the predominant mold
on tomato processing equipment and can be responsible for the slime that
accumulates on this equipment. It has been referred to as "machinery
mold." Geotrichum filaments have also been recovered from a variety of
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110 EVALUATION OF THE ROLE OF MlCROBlOLOGICAL CRITERIA
fruits and vegetables at different stages of processing. Careful cleaning
of processing equipment results in a marked reduction in the Geotrichum
count. Methods to determine G. candidum for purposes of microbiological
criteria are adequate (AOAC, 1980; APHA, 1976, 1984~. G. candidum
is used as a microbiological criterion to check sanitation of equipment in
fruit and vegetable processing plants and in bottling works.
Yeast and Mold Counts
Yeast and mold counts are used as part of microbiological standards of
various dairy products, for example cottage cheese (Chapter 8, Table 8-
4) and sugar (National Soft Drink Association, 19751.
Yeasts and molds are widely distributed in the environment and can
enter foods through inadequately sanitized equipment or as airborne con-
taminants. Yeasts and molds frequently become predominant on foods
when conditions for bacterial growth are less favorable, e.g., foods with
low aw, low pH, high salt, or high sugar content. Therefore, they become
a problem in fermented dairy products, fruits, fruit beverages and soft
drinks. They manifest themselves by producing undesirable odors, flavors,
appearance (discoloration), gas, and sediment. Satisfactory methods are
available for yeast and mold counts that are applicable for purposes of
microbiological criteria. These methods use either acidified agars or agar
media with added antibiotics to inhibit bacterial growth (APHA, 1976,
1978, 19841. Methods for osmophilic yeasts are available (APHA, 1984~.
Heat-resistant MoldEs
A few molds such as Byssochlamys fulva and Aspergillus fisher) produce
ascospores that are sufficiently heat resistant to survive the thermal pro-
cesses applied to fruit and fruit products. Food processors sometimes set
limits for these molds in purchase specifications for ingredients such as
fruit concentrates and tapioca starch. Satisfactory methods for isolation
and enumeration of these molds applicable for microbiological criteria are
available (APHA, 1976, 19841.
Thermophilic Spore Count
Various types of sporeformers are used as part of the microbiological
criteria, primarily specifications, by the canning industry to monitor the
quality of ingredients such as sugar, starch, flour, spices, mushrooms,
nonfat dry milk, and cereals that are intended for low-acid heat processed
foods. National Food Processors Association criteria (NCA, 1968) for
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SELECTION OF INDICATOR ORGANISMS AND AGENTS
111
sugar and starch to be used for this purpose include limits for total ther-
mophilic spore count, flat sour spores, thermophilic anaerobic spores, and
sulfide spoilage spores.
Concern for thermophilic spores in these ingredients is related to their
high heat resistance and their ability to cause defects in foods held at
elevated temperatures because of inadequate cooling and/or storage at too
high temperatures. Examination of equipment surfaces, product in process,
or finished product is in some cases useful for location of foci of spore
buildup. Methods to determine these spores (AOAC, 1980; APHA, 1976,
1984; NCA, 1968) are adequate for application in microbiological criteria.
Microbiological criteria involving thermophilic spore counts have been
useful as purchase specifications to specify quality of or check ingredients
intended for use in low-acid heat processed foods (see sections of Chapter 9
that apply).
Measuring Metabolic Products
Metabolic products produced during growth of microorganisms have
been used to estimate bacterial populations and to express quantitatively
the effect of microbial activity on the quality of foods.
Organoleptic Examination
Organoleptic examinations of foods are used extensively for evaluation
of quality attributes such as taste, odor, body and texture, color, and
appearance. The food industry uses these examinations to classify certain
foods into quality grades. For basic information about organoleptic eval-
uations of foods, see Amerine et al. (1965) and Larmond (19774.
Use of organoleptic examinations of foods to evaluate microbial activity
is limited, as the effect of microbes on organoleptic attributes varies.
Certain microorganisms act upon the constituents of a food and produce
marked changes in perceptible characteristics of the food; others are rel-
atively inert biochemically and produce little change. The effect of certain
levels and/or types of microorganisms on perceptible characteristics in
foods varies with differences in food composition and physical-chemical
characteristics. For example, objectionable odors in a perishable food may
result from extensive microbial activity; on the other hand, large microbial
populations do not necessarily cause perceptible odor problems. In ad-
dition, alterations in packaging and distribution methods can interfere with
normal expected changes in quality characteristics of a perishable food
(see Chapter 11. Microbial types on meat, poultry, and fish stored under
refrigeration in vacuum packages or in modified gaseous atmospheres
OCR for page 112
112 EVALUATION OF TlIE ROLE OF MICROBIOLOGICAL CRITERIA
differ considerably from those on products exposed to air.
Nevertheless, organoleptic examinations of certain foods can be useful
in evaluation of microbial activity. For example, organoleptic examina-
tions are used to evaluate the amount of acid and/or aroma compounds
produced by various lactic acid bacteria in cultured dairy products. The
organoleptic evaluation of cheese for typical flavor and body and texture,
to a large extent, reflects the activities of desirable bacteria. Organoleptic
examination is also used with some foods to evaluate the activity of
spoilage bacteria that may lead to quality deterioration and subsequent
spoilage of the food. In these cases, an attempt is made to relate the
organoleptic trait, usually odor, to the level of activity of spoilage bacteria.
Although organoleptic examinations for this purpose are used for many
commodities such as raw milk, meat, and poultry, they are used exten-
sively for fish and other seafoods (see Chapter 91. In general, seafoods
are more perishable than are other high-protein meat foods, because many
seafoods contain relatively large amounts of nonprotein nitrogen com-
pounds that are readily available to support microbial growth. Also, sea-
foods harvested from cold waters contain microbes that are not as effectively
inhibited by refrigeration. The information in Table 5-1 is an example of
organoleptic changes in relation to chemical changes and bacterial counts
of fish.
In recent years, FDA has used organoleptic examination to determine
the degree of decomposition of imported shrimp. The shrimp are placed
in one of three classes:
Class 1, passable: This class includes fishery products that range from
very fresh to those that contain fishy or other odors characteristic of the
product, but not definitely identifiable as odors of decomposition.
Class 2, decomposed (slight but definite): This is the first stage of
definitely identifiable decomposition. An odor is present that is not really
intense, but is persistent and readily perceptible to th experienced ex
aminer as that of decomposition.
Class 3, decomposed (advanced): This class of products possesses a
strong odor of decomposition that is persistent, distinct, and unmistakable.
Limits of acceptability of a lot are based on the number of shrimp in
a sample that are placed in the three classes (Anonymous, 1979~. In
general, trained personnel could consistently place shrimp correctly in the
appropriate class.
The presence of objectionable odors in raw seafood usually indicates
that extensive microbial activity has taken place. When such changes are
perceptible, bacterial numbers exceed 106 per gram.
Successful application of organoleptic examination of fish and other
foods to determine the degree of decomposition by microbial and tissue
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SELECTION OF INDICATOR ORGANISMS AND AGENTS
113
enzyme activity requires well-trained personnel. The organoleptic ex-
amination of specific foods including milk, meat, and poultry, but par-
ticularly raw fish and other seafoods, can be a most valuable tool to
determine the degree of quality deterioration by microbial activity. If off-
odors of microbial origin are perceptible, then counts are high and the
product should be rejected.
Dye or Indicator Reduction Time
Attempts have been made to use oxidation-reduction indicators (dyes)
for estimating the microbial quality of various foods such as milk, meat,
poultry, seafoods, and vegetables (Bush, 19701. Present use of these dyes
is restricted to the milk industry where they are used for classifying certain
manufacturing milks into grades (APHA, 1978) and in rapid tests to
estimate the potential shelf-life of freshly pasteurized milk (Broitman et
al., 1958; Parmelee, 19741. The application of dye reduction tests to foods
other than milk usually has not been successful because naturally present
reducing enzymes and agents interfere with the dye reduction pattern.
Comparisons of reduction times in milk with estimates of bacterial pop-
ulations by the agar plate method and other tests have demonstrated that
reduction times are, in general, inversely related to the initial bacterial
content of the sample. Dye reduction tests provide only estimates of
bacterial activity, as reduction times are influenced not only by bacterial
numbers but also by the growth phase of the bacteria at time of testing
and by the types present. With improvements in milk quality resulting in
lower bacterial counts, reduction tests have become less applicable for
quality evaluation of raw milk. Results of oxidation-reduction tests on
pasteurized milk can be used by the processor as guidelines for estimation
of the potential shelf-life of freshly pasteurized milk. Simple, practical
dye reduction methods are available (APHA, 19784.
pN
The pH of some foods changes as a result of microbial activity; con-
sequently, pH measurements have been applied to determine the degree
of quality deterioration. The pH of fish and shrimp increases during storage
because of the production of ammonia and amines by microbial and tissue
enzyme activity. The pH of molluscs, on the other hand, decreases during
spoilage. Microbial activity results in the production of basic substances
in refrigerated ground meat stored under aerobic conditions (Shelef and
Jay, 19701. However, changes in pH because of microbial activity vary
with conditions such as nature of the food (available substrate for microbial
OCR for page 114
114
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OCR for page 121
SELECTION OF INDICATOR ORGANISMS AND AGENTS
121
Staphylococci are usually killed during heat processing. Their reap-
pearance on heat-processed foods is due to handling by workers or, less
commonly, by contact with contaminated equipment or air. Small numbers
of S. aureus are to be expected in foods that have been exposed to or
handled by food workers. Large numbers (the number depending on the
type of product) usually result from growth and almost invariably occur
in foods that have been heat processed to eliminate competing organisms.
Temperature abuse then sets the stage for growth. The presence of S. aureus
can indicate a potential health hazard (see Chapter 41.
Large numbers of staphylococci may be indicative of the presence of
toxins, although small numbers do not mean absence of toxin since large
populations may have been reduced to smaller ones by a processing step,
e.g., heating or fermentation. Methods for the detection and enumeration
of S. aureus are adequate for their use in microbiological criteria (AOAC,
1980; APHA, 19841. The AOAC procedure was originally designed to
increase the sensitivity of the plating procedure but unfortunately is in-
hibitory to injured cells. The direct plating method employing Baird-Parker
agar effectively recovers both injured and noninjured S. aureus (Rayman
et al., 19784. S. aureus counts are useful as part of microbiological criteria
for foods that are handled after heat processing.
Escher~chia cold
E. cold conforms to the definitions of Enterobacteriaceae, coliforms,
and fecal coliforms, but is further identified by an IMViC (indole, methyl
red, Voges-Proskauer, citrate utilization) pattern of + + - - or
-. Its natural habitat is the intestines of vertebrate animals.
Thus, the presence of E. cold in a food indicates the possibility that fecal
contamination has occurred and that other microorganisms of fecal origin,
including pathogens, may be present. At present, E. cold is the best in-
dicator of fecal contamination among the commonly used fecal-indicator
organisms. In evaluating foods for safety, the presence of E. cold signifies
a more positive assumption of hazard than the presence of other coliform
bacteria. The failure to detect E. cold in a food, however, does not assure
the absence of enteric pathogens (Mossel, 1967; Silliker and Gabis, 19761.
In many raw foods of animal origin, small numbers of E. cold can be
expected because of the close association of these foods with the animal
environment and the likelihood of contamination of carcasses from fecal
material, hides, or feathers during slaughter-dressing procedures. These
organisms are easily destroyed by heat processing of foods. Thus, the
presence of E. cold in a heat-processed food means either process failure
or, more commonly, postprocessing contamination from equipment or
OCR for page 122
122 EVALUATION OF THE ROLE OF MICROBIOLOGICAL CRITERIA
employees or from contact with contaminated raw foods. If the objective
is to check for postprocessing contamination, then coliforms, rather than
E. colt, should be the target organisms. Large numbers of E. cold in some
foods (soft cheese, for example) may be the result of growth in the product
or buildup on the equipment.
Routine methods for the detection and enumeration of E. cold (AOAC,
1980; APHA, 1984) are applicable for use in microbiological criteria.
However, the MEN procedures for the enumeration of E. cold in foods
are time-consuming, costly, inhibitory to injured cells, and lacking in
precision. A direct plating procedure is now available (Anderson and
Baird-Parker, 1975; Holbrook et al., 1980~. A comparative study showed
that the direct plating procedure was preferable to the MEN method for
enumerating E. cold in meats, because of its lower variability, better re-
covery of E. cold from frozen samples, rapidity (24 hours vs. 8 days),
decreased requirements for media, and decreased cost of analysts' time
(Rayman et al., 19791. In addition, Anderson and Baird-Parker (1975)
considered indole production, the index in the direct plating method a
more reliable characteristic of E. cold from foods than gas production from
lactose.
Microbiological criteria involving E. cold are useful in those cases where
it is desirable to determine if fecal contamination may have occurred.
Contamination of a food with E. cold implies a risk that one or more of
a wide diversity of enteric pathogens may have gained access to the food
and introduced a health hazard.
Fecal Coliforms
Fecal coliform bacteria!are used as a component of microbiological
standards to monitor the Wholesomeness of shellfish and the quality of
shellfish growing waters (USDHEW, 19651. The term fecal coliform has
arisen from attempts to find rapid, dependable methods to establish the
presence of E. cold without isolation and purification of cultures and ap-
plication of the lengthy and costly IMViC tests. The fecal coliforms are
a group of organisms selected by incubating an inoculum derived from a
coliform enrichment broth at higher temperatures (44° to 45.5°C/111° to
114°F) than used for incubating coliforms. Such preparations usually con-
tain a high proportion of E. cold and are thus useful for indication of a
probable fecal source. The fecal coliforms have a higher probability of
containing organisms of fecal origin and hence of indicating fecal con-
tamination than do coliforms that have received no further differential
tests.
The fecal coliforms comprise a population presumed to contain a high
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SELECTION OF INDICATOR ORGANISMS AND AGENTS
123
proportion of E. cold but without the actual proportion of E. cold being
positively established. In many foods, most fecal coliforms are E. colt,
but some strains of Enterobacter and Klebsiella are included. In some
foods, frozen vegetables for example, the composition may be different.
Thus, if fecal coliforms in a food are used as an index of E. cold and
therefore as an index of fecal contamination, this population's proportions
must be established first. Fecal coliforms can become established on equip-
ment and utensils in the food processing and handling environment and
contaminate processed foods. They are easily destroyed by heat and are
injured sublethally or may die during freezing and frozen storage of foods.
Routine MPN procedures applicable for use in microbiological criteria
are well established (AOAC, 1980; APHA, 1976, 1981, 19841. These
MPN procedures are subject to limitations similar to those mentioned for
E. cold MPN methods.
The fecal coliform test is a useful part of the microbiological standard
to evaluate the quality of shellfish growing waters. Its purpose is to reduce
the risk of harvesting shellfish from waters polluted with fecal material.
Because rapid, direct plating methods for E. cold (Anderson and Baird-
Parker, 1975; Holbrook et al., 1980), including provisions for resusci-
tation of injured cells, are now available it may be advantageous to use
E. cold rather than fecal coliforms as a component of microbiological
criteria for foods.
Enterococci
Enterococci have certain features that make them unique as indicator
organisms. Most are quite salt-tolerant (grow in the presence of 6.5% NaCl).
All are facultatively anaerobic and grow well at 45°C (1 13°F). Except for
Streptococcus bovis and Streptococcus equines, they can grow at 7-10°C
(44.6°-50°F). Unlike E. colt, they are relatively resistant to freezing.
Streptococcus faecalis and Streptococcus faecium, the most common en-
terococci of foods, are relatively heat-resistant and may survive usual milk
pasteurization procedures. S. faecium may survive and cause defects in
marginally pasteurized canned cured meats.
Enterococci may be of fecal origin from both warm-blooded and cold-
blooded animals; they also can establish an epiphytic relationship with
growing plants (Mundt, 1970~. They are often associated with insects and
are thus a part of the natural microflora of many foods. In addition, they
can establish themselves and persist in the food processing establishment
long after their introduction.
Many foods normally contain small to large numbers of enterococci,
especially S. faecalis and S. faecium. For instance, certain cheeses and
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124 EVALUATION OF THE ROLE OF MICROBIOLOGICAL CRITERIA
some fermented sausages may contain more than 106 enterococci per gram.
A wide variety of other foods such as raw meats, poultry, and raw veg-
etables may have relatively low levels (10~-103 per gram). It is well es-
tablished that enterococci counts of foods are not a reliable index of fecal
contamination. A thorough understanding of the role and significance of
enterococci in a food is required before any meaning can be attached to
their presence and population numbers.
Many media have been proposed (APHA, 1976, 1984; ICMSF, 1978)
for the selective isolation and/or enumeration of enterococci. Present meth-
ods have definite shortcomings relative to the degree of selectivity, quan-
titative recovery or differential ability (APHA, 19841. Enterococci counts
have little useful application in microbiological criteria for foods. If they
are used in specific cases to identify poor manufacturing practices, it is
necessary to first establish the normal population levels at different stages
of processing and handling with a standardized test procedure.
Pseudomonas aeruginosa
P. aeruginosa is a frequent contaminant of the environment (including
water) and enters the environment with fecal wastes of human or of animals
associated with humans (Hoadley, 19761. It is an opportunistic pathogen
and, in some countries, is of particular concern for infants who may
become infected when contaminated water or equipment is used to make
infant formulas. This organism has been used in Europe as an indicator
of contamination of bottled mineral water. It is oligocarbo-tolerant; there-
fore, after a period of adaptation, it can multiply in water of low nutrient
level. Although there is not an acceptable standard method of detection
of P. aeruginosa, methods are given in APHA (1981) and by Mossel
et al. (1976~. Most other test procedures, except for incubation at 42°C
(107.6°F), are of little value in detecting P. aeruginosa. At the present
time, P. aeruginosa has no application in microbiological criteria for water
in the United States.
Indicators of Post-Heat Processing Contamination
Coliform Bacteria
Coliform bacteria are members of the Enterobacteriaceae that are ca-
pable of fermenting lactose with the production of acid and gas within
48 hours at 35°C (95°F); for dairy products some workers specify 32°C
(89.6°F). Some coliforms (E. coli) are common in feces of man and other
animals, but others (Enterobacter, Klebsiella, Serratia, Erwinia, and
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SELECTION OF INDICATOR ORGANISMS AND AGENTS
Aeromonas) are commonly found in soil, water, and grains.
125
Small numbers of coliform bacteria are usually present in raw milk,
vegetables, meat, poultry, and many other raw foods. Therefore, they are
of little, if any, value in monitoring raw foods. These organisms are easily
killed by heat; hence, their presence in heat-processed foods suggests post-
heat contamination or, possibly, process failure. To locate the source and/
or mode of entry of coliform bacteria into a food may require the ex-
amination of line samples. When large numbers are present in a heat-
processed food, growth most likely occurred because of a lack of or
improper refrigeration. Coliform bacteria are easily sublethally stressed
by freezing, so coliform counts of frozen foods should be interpreted
cautiously. Special resuscitation procedures are recommended (APHA,
1976, 1984) for their detection and enumeration from frozen foods.
From their original fecal, soil, water, or plant environment, coliform
bacteria can reach the food processing plant, food service establishment,
or home environment, where they may be spread via equipment and utensil
surfaces or by employees. The presence of coliforms in food does not
necessarily mean that there was fecal contamination or that pathogens are
present. Their significance in foods depends upon the circumstances to
which the food has been exposed. The fecal connotation of E. colt, a
member of the coliform group, is often inappropriately linked to foods in
which coliform bacteria are found. Further examination (see sections on
fecal coliform bacteria and E. colt, above) is required to establish potential
fecal association. The use of coliform bacteria as an index of contamination
after heat processing of foods requires a thorough understanding of the
production, processing, and distribution practices to which a food is sub-
jected and the effect of these practices on coliform bacteria. Methods for
the enumeration of coliforms are adequate for purpose of use in micro-
biological criteria (AOAC, 1980; APHA, 1978, 1984; ICMSF, 19781.
Coliform bacteria are particularly useful as part of microbiological cri-
teria to indicate postprocessing contamination of foods that have been
processed (heating, irradiation, or chlorination) for safety. They are also
useful indicators in guidelines at critical control points, particularly after
heat processing.
Enterobactertaceae
Enterobacteriaceae counts are not a component of microbiological cri-
teria for foods in the United States; they are, however, used in Europe
for this purpose. The enrichment-plating procedure, which employs violet-
red bile agar with 1% glucose, allows colony formation by a greater
spectrum of members of the Enterobacteriaceae than the usual procedures,
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126 EVALUATION OF THE ROLE OF MICROBIOLOGICAL CRITERIA
which select for the lactose-positive members only (Mossel, 1967; Mossel
et al., 19631. The Enterobacteriaceae test, like the coliform test, is useful
to indicate postprocessing contamination in foods, but the presence of
either group does not imply fecal contamination. Whether or not the
Enterobacteriaceae count should supplant the coliform count in micro-
biological criteria is a debatable question.
TESTS FOR OTHER COMPONENTS
Tests for Metabolic Products of Pathogens
That Indicate a Health Hazard
In a number of circumstances, tests for metabolic products of pathogens
are preferred to tests for pathogens (see Chapter 4) to indicate the presence
of pathogens or their toxins.
Thermonuclease Test for Evidence of Growth of
Staphylococci and Presence of Enterotoxins
When large numbers ('106/g or ml) of S. aureus are present or sus-
pected in processed foods, actual testing for enterotoxins is desirable, but
the procedure is time-consuming and expensive. Furthermore, not all food
laboratories have the capability to perform the test (see Chapter 9, Part A).
Low counts of S. aureus in reheated or fermented foods may be misleading
because the number of viable cells of 5. aureus may be the remainder of
larger populations, whereas enterotoxins may be present. S. aureus pro-
duces thermostable deoxyribonuclease (TNase), which has been used as
a rapid and inexpensive procedure for screening foods for indication of
extensive staphylococcal growth and possible presence of enterotoxin
(APHA, 19841. The method is adequate for application in microbiological
criteria. The TNase test has been recommended for testing foods such as
cheeses and sausages when conditions such as improper lactic starter
performance may have been responsible for the presence of significant
levels of coagulase-positive S. aureus (Emswiler-Rose et al., 1980; Todd
et al., 1981. See also Chapter 9, Part A). TNase can be a useful indicator
because it can almost always be detected in foods whenever enterotoxins
can be detected (Tatini, 19811. TNase-positive samples, whenever pos-
sible, should be tested for enterotoxin.
Afiatoxin Detection by Ultraviolet Light
Long-wave ultraviolet (black) light (U.V.) has been used to detect the
presence of Aspergillus flavus and Aspergillus parasiticus in corn (Shot
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SELECTION OF INDICATOR ORGANISMS AND AGENTS
127
well and Hesseltine, 19811. When corn viewed under U.V. light displays
a bright greenish-yellow fluorescence (BGYF), it is probable that aflatoxin
may be present. Detection procedures for aflatoxins are given in AOAC
(1980~. Because of the association of BGYF with the potential occurrence
of aflatoxins, the examination of corn and other grains with U.V. light
as a rapid screening procedure has been adopted by industry. This ex-
amination is utilized only as a presumptive test; the occurrence of BGYF
is indicative that more sophisticated testing for the presence of aflatoxin,
e.g., by high-pressure liquid chromatography, is needed. The use of U.V.
examination as a component of microbiological criteria is limited to pur-
chase specifications or guidelines.
Test for Phosphatase
The phosphatase test is part of a criterion for certain milk and milk
products to determine whether the product in question was pasteurized
properly and also to detect the possible addition of raw milk to pasteurized
milk (APHA, 1978; USPHS/FDA 19781. Methods, limitations, and inter-
pretation of test results are presented in detail in Standard Methods for
the Examination of Dairy Products (APHA, 19781. Routine methods for
the detection of phosphatase are well established and applicable to evaluate
the acceptability of pasteurized milk and milk products.
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Representative terms from entire chapter:
microbial activity
