Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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novelty drinks, including those that contain suspen-

sions of fruit or fruit pieces.

0037 Many fruits do not retain their physical integrity on

cooking and turn into a nondescript mash. Where

cooking stability is required, the raw fruit pure

e can

be reformed into a fruit shape with an alginate gel.

Such gels do not melt at normal cooking or baking

temperatures. The technique is widely used, though

for a different reason, to reform paprika in cocktail

olives. Currently its use in reformed meat is being

advocated.

0038 Natural meats and fish show a considerable weight

loss on cooking. To counteract this, mixtures of

salts and hydrocolloids, typically carrageenans, are

injected or pumped into the raw meat and the mixture

gels after cooking. In meat pies and pastries, the

escaping cell fluid can also disfigure the pastry sur-

face. This may be obviated by incorporation of cellu-

lose ethers, usually methylcellulose, into the filling. In

contrast to almost all other polysaccharide food addi-

tives, colloidal solutions of methylcellulose form a

gel when heated and thereby prevent the escape or

boil-out of juices. The technique is also applicable to

fruit-filled bakery products. Cellulose ethers have

also been employed as film-building agents to reduce

the penetration and uptake of fats in deep-fried foods.

See also: Cheeses: Quarg and Fromage Frais; Cocoa:

Production, Products, and Use; Cream: Types of Cream;

Dressings and Mayonnaise: The Products and Their

Manufacture; Emulsifiers: Uses in Processed Foods;

Freezing: Principles; Gums: Properties of Individual

Gums; Ice Cream: Methods of Manufacture; Properties

and Analysis; Margarine: Types and Properties; Methods

of Manufacture; Pasteurization: Principles; Pectin: Food

Use; Starch: Modified Starches; Yogurt: The Product and

its Manufacture

Further Reading

Glicksman M (1969) Gum Technology in the Food

Industry. London: Academic Press.

Tamime AY and Robinson RK (1985) Yoghurt – Science and

Technology. Oxford: Pergamon Press.

Webb BH, Johnson AH and Alford JA (1978) Fundamen-

tals of Dairy Chemistry. Westport, Connecticut: AVI

Publishing.

STAPHYLOCOCCUS

Contents

Properties and Occurrence

Detection

Food Poisoning

Properties and Occurrence

A L Wong and M S Bergdoll

, University of Wisconsin,

Madison, WI, USA

This article is reproduced from Encyclopaedia of Food Science,

Classification

0001 In 1880, Ogston first demonstrated that a cluster-

forming coccus caused many types of purulent

infections in humans. He named the organism

‘staphylococcus’. In 1884, Rosenbach isolated

staphylococci from pus and determined that the

organisms were identical to those described by

Ogston. He proposed the genus Staphylococcus to

contain these organisms. Rosenbach observed yellow

and white colonies formed by the staphylococci and

named the yellow colony-forming organism Staphylo-

coccus aureus, and the white colony-forming organ-

ism S. albus. It was later determined that pigmentation

was a variable characteristic and therefore not a valid

criterion for classifying the staphylococci, and S. albus

was reclassified as S. aureus. Staphylococcus was

placed in the family Micrococcaceae in 1920.

0002In 1908, Winslow and Winslow proposed a second

species, S. epidermidis. It was not until 1974 that a

third species, S. saprophyticus, was added. By 1980,

the number of species had increased to 13 and by

1984 to 20. Currently, at least 32 species have been

described, based on DNA homology and immuno-

chemical and biochemical characteristics.

Deceased.

STAPHYLOCOCCUS

/Properties and Occurrence 5547

0003 Staphylococci are Gram-positive, catalase-positive,

nonmotile cocci that characteristically divide in more

than one plane to form irregular clusters. The cell

wall typically contains teichoic acid, and the peptide

subunits of the peptidoglycan are cross-linked by

pentapeptide bridges containing solely or primarily

glycine. Staphylococci are facultative anaerobes but

grow more rapidly and abundantly under aerobic

conditions. A variety of carbohydrates and/or amino

acids can be utilized as carbon and energy sources.

The staphylococci can be differentiated from

members of the strictly aerobic genus Micrococcus

by their ability to grow and produce acid from glu-

cose anaerobically. In addition, the two genera have

different cell wall structures, cytochrome and fatty

acid profiles, menaquinone content, and aliphatic

hydrocarbons.

0004 Some of the characteristics used to differentiate

staphylococcal species are given in Table 1. S. aureus

is the predominant species involved in staphylococcal

food poisoning outbreaks. The illness is caused by the

ingestion of preformed staphylococcal enterotoxins

in foods. Thirteen enterotoxins have been identified:

enterotoxins A (SEA), B (SEB), C1 (SEC1), C2

(SEC2), C3 (SEC3), D (SED), E (SEE), G (SEG), H

(SEH), I (SEI), J (SEJ), K (SEK), and L (SEL). Two of

the newer species, S. intermedius and S. hyicus, were

formerly considered to be S. aureus. Although they

differ in some of their characteristics, they can pro-

duce coagulase and thermonuclease (TNase), the two

characteristics used most frequently to distinguish

S. aureus from other staphylococci. All the other

staphylococcal species are coagulase- and TNase-

negative. The use of the name S. aureus herein

includes both S. intermedius and S. hyicus because,

essentially, all of the information presented was

obtained before these two species were separated

from S. aureus. Occasional strains of a few coagu-

lase-negative species have been reported to produce

enterotoxin. The degree of involvement of these or-

ganisms in food poisoning is unknown.

Ecology

0005Staphylococci are ubiquitous. Their main habitats are

the noses, skin, and throats of humans and warm-

blooded animals. Staphylococci can also be found

in the air, soil, water, sewage, plant surfaces and

products, meats, poultry, and dairy products.

0006Certain staphylococcal species show host prefer-

ences. The primary host for S. aureus is the human;

however, the organism can be found also in a variety

of animals and birds, and can cause many types of

infections and diseases. S. epidermidis is the most

prevalent on human skin, whereas S. hyicus com-

monly inhabits the nares and skin of chickens and

pigs. Dogs are the preferred host for S. intermedius,

which can also be found on pigeons.

0007About 30–50% of healthy individuals are carriers

of S. aureus, with 40–50% of the isolates being enter-

otoxin producers. The incidence of enterotoxigenic

strains among individuals with staphylococcal infec-

tions is higher than among healthy people. In general,

the incidence of enterotoxin-producing staphylococci

in healthy animals is relatively low. S. aureus causes

many types of infections and diseases in animals. The

incidence of enterotoxigenic staphylococci from mas-

titic animals depends on the animal species. A high

percentage (60–80%) of staphylococcal isolates from

mastitic sheep and goats are enterotoxigenic, whereas

less than 15% of those isolated from mastitic cows

produce enterotoxin.

0008Essentially all raw foods, especially raw meats and

poultry, can be contaminated with S. aureus,by

humans or animals, or both. Raw milk often contains

staphylococci, and the organism may be isolated from

30–50% of poultry carcasses. Staphylococci isolated

from goats and sheep usually produce SEC, and those

tbl0001 Table 1 Characteristics of Staphylococcus species

Property S. aureus S. intermedius S. hyicus S. epidermidis

Pigment þ



Coagulase þþ + 

Thermonuclease þþ þ

Hemolysins þþ +

Mannitol

þ 

Acetoin þ þ

Clumping factor þþ + 

Hyaluronidase þ þ

Lysostaphin HS

HS HS SS

þ, over 90% positive; , over, 90% negative; +, 11–89% positive.

Anaerobic conditions.

HS, high sensitivity.

SS, slight sensitivity.

5548

STAPHYLOCOCCUS

/Properties and Occurrence

from cows produce either SEC or SED, whereas

strains isolated from humans produce primarily

SEA, the enterotoxin type most commonly involved

in staphylococcal food poisoning. (See Meat: Preser-

vation; Milk: Processing of Liquid Milk; Poultry:

Ducks and Geese; Turkey.)

Growth and Enterotoxin Production

0009 Growth and enterotoxin production by S. aureus are

influenced by a variety of environmental and nutri-

tional factors including temperature, pH, water activ-

ity (a

), inoculum size, atmospheric composition,

carbon and nitrogen sources, salt levels, and compet-

ing microflora. Generally, growth is necessary for

enterotoxin production, although toxin production

does not always accompany growth, especially in

foods. Experimental nongrowing cell cultures have

been observed to produce enterotoxin. Production

of SEB and SEC is affected more by the culture condi-

tions than is SEA production, which is more closely

related to the growth of S. aureus.

Temperature

0010 S. aureus can grow between 7 and 47.8



C, with an

optimum of 37



C. Enterotoxin is produced at 10–



C, with an optimum temperature range of 37–



C. The temperature for supporting growth of

staphylococci in a variety of foods ranges from 6.7

to 45.6



C, with no growth occurring below 5.6



Vanilla pudding inoculated with S. aureus supported

enterotoxin production over a temperature range

of 10–45



C, whereas pasteurized milk supported

growth and enterotoxin production at 20–35



C, but

not at 10



C. Detectable levels of SEA were produced

in 12 h when incubated at 35



C. Longer incubation

times were necessary at lower temperatures for

enterotoxin production. SEB was produced in cured

hams that were incubated for 2 weeks at 10



0011 The optimum pH for growth is between 6.0 and 7.0,

but the organism can grow over a pH range of 4.0–

9.8. The pH at which a strain will grow depends on

other cultural parameters such as the atmosphere, a

value, the type of medium and the salt concentration.

Generally, the less optimal the other parameters, the

narrower the pH range tolerated by S. aureus. For

example, the lowest pH at which S. aureus grew and

produced enterotoxin in aerobic cultures was 4.0,

whereas the lowest pH values that supported growth

and enterotoxin formation anaerobically were 4.6

and 5.3, respectively. (See pH – Principles and Meas-

urement.)

0012Enterotoxin can be produced over a pH range

of 4.0–9.0, the optimum being 6.5–7.5. As with

staphylococcal growth, whether enterotoxin will be

produced at a certain pH depends on other cultural

parameters. The acid used for pH adjustment is also

an influencing factor. When milk was acidified with

hydrochloric acid, SEA was produced at pH levels of

4.5, 5.0, 6.0, and 6.4. However, when lactic acid was

used, growth and enterotoxin production were

observed at the higher pH levels, but not at pH 4.5.

Foods having pH values below 5.0 or above 9.0 do

not support enterotoxin production.

Water Activity

0013S. aureus can grow over a much wider a

range than

many other food-associated pathogens, with growth

of some strains occurring at an a

of 0.86; the

optimum is > 0.99. The minimum a

for anaerobic

growth is 0.90. The minimum a

for enterotoxin

production is 0.86, and the optimum is > 0.99. (See

Water Activity: Effect on Food Stability.)

0014The humectant used for a

adjustment has a sig-

nificant effect. For example, when sodium chloride

was used, the minimum a

for SEB production was

0.90–0.92. In a mixture of sodium chloride, potas-

sium chloride and sodium sulfate, SEB production

occurred at an a

< 0.90. When glycerol was used,

the minimum a

was 0.98–0.99.

0015Temperature and pH also affect the a

at which

S. aureus will grow and produce enterotoxin. When

these parameters deviate from their optimum levels,

the minimum a

tolerated by S. aureus is elevated.

Atmospheric Conditions

0016Staphylococci are facultative anaerobes, but the

amount and rate of growth and enterotoxin produc-

tion are considerably less under anaerobic conditions

than under aerobic conditions. Excessive aeration or

oxygen levels can decrease the amount of SEB pro-

duced. At a dissolved oxygen (DO) level of 100%

growth of S. aureus at 37



C was maximal, but no

SEB was produced. The optimal DO level for SEB

production was 10%. In contrast, SEA production

was related more to growth and not affected by the

level of DO.

0017Staphylococcal growth and enterotoxin production

have been observed in Canadian bacon and ham, and

sausage, turkey, and hamburger sandwiches stored

anaerobically. As with culture media, enterotoxin

yields were higher under aerobic conditions.

Nutritional Factors

0018Most species of staphylococci require an organic

nitrogen source and one or more B vitamins for

STAPHYLOCOCCUS

/Properties and Occurrence 5549

aerobic growth. Media containing protein digests

generally enhance growth and enterotoxin produc-

tion. For anaerobic growth, a fermentable carbon

source and uracil are required.

0019 Addition of readily fermentable carbon sources such

as glucose or pyruvate to aerobic cultures repressed

enterotoxin synthesis by S. aureus. The repression is

partially attributed to decreased pH due to acid

production, and partly to catabolic repression.

Sodium Chloride

0020 One characteristic of S. aureus is its ability to survive

and grow in relatively high salt concentrations. The

organism can grow in up to 20% sodium chloride,

whereas enterotoxin production occurs with up to

10% sodium chloride. Growth and enterotoxin

production are generally retarded at increased salt

concentrations.

Competing Microorganisms

0021 Staphylococci are not good competitors, especially

when the inoculum is small compared with that of

the other organisms present. Many common food

bacteria have been shown to inhibit growth of

S. aureus and/or its ability to produce enterotoxin.

In raw foods, S. aureus will not grow appreciably

because of the presence of other organisms. An ex-

ception would be in the case of raw milk from a cow

with staphylococcal mastitis. The high numbers of

staphylococci present would be able to overcome

other microorganisms. In heat-processed foods,

contaminating S. aureus would have a competitive

edge, especially if the food contains salt and has a

reduced a

Survival in Foods

0022 Staphylococcal food poisoning results from the inges-

tion of enterotoxin produced by S. aureus growing in

the food. Raw foods such as meat and milk are fre-

quently contaminated with staphylococci. However,

they are seldom involved in food poisoning because

other contaminating microorganisms inhibit their

growth and enterotoxin production. In addition, not

all staphylococcal isolates are enterotoxigenic.

0023 Heat is the most effective way to inactivate

S. aureus in food. Heating meat to an internal tem-

perature of 73.9–76.7



C will kill any staphylococci

present. Temperatures normally used for cooking

meats should be sufficient to inactivate S. aureus.

The time–temperature treatments used to pasteurize

milk are adequate to destroy the organisms. The D

values of S. aureus in skim milk at 60.0 and 65.5



are 3.44 and 0.28 min, respectively. The enterotox-

ins, however, are very heat-resistant, and are not

inactivated by pasteurization. An outbreak occurred

in 1985 from chocolate milk served to schoolchil-

dren. The milk had been held inadvertently for several

hours at a warm temperature before pasteurization.

S. aureus present in the milk was able to grow and

produce SEA. No viable staphylococci were present

in the pasteurized milk, but SEA was detectable. (See

Heat Treatment: Chemical and Microbiological

Changes.)

0024Temperatures used in commercial canning are

sufficient to destroy staphylococci and the amount

of enterotoxin usually present in foods involved in

food poisoning outbreaks (1 ng to > 50 ng per gram

of food). There have been outbreaks associated with

the consumption of canned corned beef, but these

were traced to recontamination of improperly sealed

cans after processing. Two people were reported to

become ill after eating lobster bisque that had been

heated at 118



C for 86 min, but this was due to

inadequate processing of some of the cans. In general,

the heat stability of the enterotoxins is greater in

foods than in buffer. The degree of inactivation

depends on a variety of factors, including the nature

of the food, pH, and concentration and type of enter-

otoxin. (See Canning: Principles.)

0025S. aureus inoculated on to frankfurters was inacti-

vated when they were heated to an internal tempera-

ture of 71.1



C in the smoking procedure. S. aureus in

the interior of ham that survived the curing process

was slowly inactivated during heating in a smoke-

house at 48.9



C for 48 h. (See Curing.)

0026Freezing and thawing have no significant effects on

the viability of S. aureus. However, prolonged storage

at subfreezing temperatures reduces the number of

S. aureus in meats. The population of S. aureus in

raw minced beef was reduced by 91% after storage at

22



C for 4 months.

0027S. aureus is relatively resistant to drying. Nonfat

dry milk and foods containing nonfat dry milk have

been implicated in several staphylococcal food

poisoning outbreaks. Staphylococci present in the

milk may survive spray drying, depending on the

temperature, the moisture content of the product,

and the strain of S. aureus.

See also: Canning: Principles; Curing; Heat Treatment:

Chemical and Microbiological Changes; Meat:

Preservation; Milk: Processing of Liquid Milk; pH –

Principles and Measurement; Poultry: Ducks and

Geese; Turkey; Water Activity: Effect on Food Stability

Further Reading

Bergdoll MS (1989) Staphylococcus aureus. In: Doyle MP

(ed.) Foodborne Bacterial Pathogens, pp. 463–523.

New York: Marcel Dekker.

5550

STAPHYLOCOCCUS

/Properties and Occurrence

Jablonski LM and Bohach GA (2001) Staphylococcus

aureus. In: Doyle MP, Beuchat LR and Montville TJ

(eds) Food Microbiology, pp. 411–434. Washington,

DC: American Society for Microbiology.

Martin SE and Myers ER (1994) Staphylococcus aureus. In:

Hui YH, Gorham JR, Murrell KD and Cliver DO (eds)

Foodborne Disease Handbook, vol. 1, pp. 345–394.

New York: Marcel Dekker.

Detection

M S Bergdoll

and A L Wong, University of Wisconsin,

Madison, WI, USA

This article is reproduced from Encyclopaedia of Food Science,

Introduction

0001 The fact that staphylococcal food poisoning is

caused by the ingestion of enterotoxins produced by

staphylococci requires that methods be available for

detection of both the organisms and the enterotoxins.

Although it is essential that staphylococci are present

and grow in the food to produce enterotoxin at some

point in the preparation or processing of the food,

their presence in the food is not proof that entero-

toxin is present. Not all staphylococci are capable of

producing enterotoxin. On the other hand, the ab-

sence of staphylococci in food does not prove that

enterotoxins are absent because these organisms are

easily destroyed by heat, whereas the enterotoxins are

not. Detection of enterotoxins is most important be-

cause, if they are present at detectable levels in a food,

ingestion of the food most likely will result in food

poisoning.

Detection of Staphylococci in Foods

0002 The isolation of coagulase-positive staphylococci

from foods is of greatest concern because these are

the types most often involved in food poisoning, al-

though occasionally coagulase-negative staphylococci

have been implicated. Coagulase-positive staphylo-

cocci include not only Staphylococcus aureus but

also S. intermedius and S. hyicus; some strains from

each of these species are known to produce entero-

toxin. Hence, coagulase production by suspected

food-poisoning isolates is one of the most important

properties tested for. Thermonuclease (TNase)

production is also commonly associated with food-

poisoning staphylococci and is another important

characteristic of these species.

Detection in Raw Foods

0003Normally, a 50-g sample of the food is used for detec-

tion of the presence of staphylococci. The sample

should be as representative of the food from which

it is taken as possible. Frozen samples should be

thawed under refrigeration just before testing. All

sampling should be done under sterile conditions and

samples should remain under sterile conditions

and kept refrigerated until tested.

0004The food samples are suspended in or mixed with a

suitable diluent, plated on Baird–Parker agar and

incubated. One or more colonies of each colony type

are selected from a plate containing 20–200 colonies

for coagulase determination. Any colonies testing

negative for coagulase should be tested for TNase.

Any colonies positive for either coagulase or TNase

can be considered candidates for enterotoxin pro-

duction.

Detection in Processed Foods

0005Collection of processed foods and preparation of the

dilutions are the same as for the raw foods. The

diluted samples are incubated in double-strength

trypticase soya broth before single-strength trypticase

soya broth containing 20% sodium chloride is added

and the incubation continued. The cultures are plated

on Baird–Parker agar and the same procedures as

described above are used for testing colonies. This

procedure can be used for raw or nonprocessed

foods suspected of containing < 100 S. aureus organ-

isms per gram in the presence of large numbers of

competing organisms.

Detection in Cases of Food Poisoning

0006In most cases of food poisoning the food will have

been contaminated after it was cooked or heated. In

these cases the same procedures used for isolation of

staphylococci from raw foods are employed. In those

cases where the staphylococci have grown in the

foods and produced enterotoxin before the food was

processed and in those cases where the food history is

not known, the procedure for processed foods is used.

In some cases the staphylococci have either been

killed or else they died during storage. Because

TNase is heat-stable and survives in food for long

periods of storage, testing for it can be undertaken

to indicate whether sufficient staphylococcal growth

had occurred to produce enterotoxin.

0007If staphylococci are isolated from the food they

should be tested for enterotoxin production. If they

Deceased.

STAPHYLOCOCCUS

/Detection 5551

produce enterotoxin, the food can be tested for the

type of enterotoxin produced by them. In the case

where staphylococci cannot be isolated but the

TNase test is positive, the foods must be tested for

enterotoxin because it is not known whether the

staphylococci that grew in the food were capable of

producing enterotoxin.

0008 Phage typing of the staphylococci isolated from the

food is done in order to aid in the identification of the

source of contamination. Any staphylococci isolated

from an individual who handled food that was

implicated in a food-poisoning outbreak should be

phage-typed. If the isolates from the food and the

individual have the same phage pattern, it can be

concluded that the individual was the source of the

contamination. (See Food Poisoning: Tracing Origins

and Testing.)

Detection of the Enterotoxins

0009 All of the methods for the detection of the enterotox-

ins are based on the use of specific antibodies to the

enterotoxins. Detection of the enterotoxins is compli-

cated by the fact that seven enterotoxins have been

identified – enterotoxins A (SEA), B (SEB), C

(SEC

), C

(SEC

), D (SED), and E (SEE). Five

specific antibodies are needed for their analysis; the

SECs can be detected by one antibody. Unidentified

enterotoxins do exist, but their involvement in food

poisoning is minor. Antibodies prepared in animals

such as rabbits are polyclonal and react with entero-

toxins in gels to give precipitin reactions. Monoclonal

antibodies cannot be used in gels because their reac-

tions with the enterotoxins do not result in the forma-

tion of precipitates.

0010 The detection of enterotoxin in foods requires

methods that are much more sensitive than those

required for determination of the enterotoxigenicity

of strains. The quantity of enterotoxin present in

foods involved in food-poisoning outbreaks may

vary considerably, from less than 1 ng g

1

to greater

than 50 ng g

1

. Usually, little difficulty is encountered

in detecting the enterotoxin in foods involved in

staphylococcal food-poisoning outbreaks. However,

outbreaks do occur in which the amount of entero-

toxin is less than 1 ng per gram of food. In such cases,

the enterotoxin can only be detected by the most

sensitive methods. Another situation in which it is

essential to use a very sensitive method is in determin-

ing the safety of a food for consumption.

Gel Diffusion Methods

0011 Many types of gel reactions have been used in the

detection of the enterotoxins; the most common ones

are some form of either the Ouchterlony gel plate or

the microslide (Figure 1). These methods have been

used widely in the determination of the enterotoxi-

genicity of staphylococcal strains. The modification

of the Ouchterlony gel plate test that is used in the

Food Research Institute, University of Wisconsin, and

recommended to others is the optimum sensitivity

plate (OSP) method (Figure 2). It is easy to use and,

in conjunction with production of the enterotoxins by

the membrane-over-agar method (Figure 3) or the sac

culture method, is of adequate sensitivity to detect

most enterotoxigenic staphylococci. In the sac culture

method, the medium is inside a dialysis sac that is

placed in an Erlenmeyer flask with the inoculum in

buffer outside the bag; incubation is with shaking.

The normal sensitivity of the OSP method is 0.5 mg

1

, but can be increased to 0.1 mgml

1

by a fivefold

concentration of the staphylococcal culture super-

natant fluids. The microslide is used by some investi-

gators, but care is needed in preparing the slides; even

so, the results are often difficult to interpret. Experi-

ence is important in achieving its maximum sensitiv-

ity (50–100 ng ml

1

), but even with experience many

individuals are unable to achieve this sensitivity.

0012The original methods for the detection of entero-

toxin in foods employed the microslide as the test

method for detection of the enterotoxins. These

methods required the use of 100 g of food and extrac-

tion and concentration procedures to reduce the ex-

tract to 0.1–0.2 ml. These were cumbersome and

time-consuming procedures which have been out-

dated by more sensitive methods for the detection of

the enterotoxin in the extracts. The US Food and

Drug Administration personnel still use the original

method developed in 1965 and consider it the official

method for the detection of enterotoxins in foods.

Sensitive Detection Methods

0013Development of sensitive methods for the detection of

proteins at less than 1 ng per milliliter of fluid greatly

simplified the detection of enterotoxins in foods.

Thus, it was possible to use a simple procedure for

extracting the enterotoxin from foods. The one used

in the Food Research Institute, University of Wiscon-

sin, is an example: (1) grind the food to a homoge-

neous slurry with 1–1.5 ml of fluid per gram of food;

(2) adjust the pH to 4.5 and centrifuge; (3) adjust the

supernatant fluid to pH 7.5 and centrifuge if neces-

sary; (4) extract with chloroform if fat interferes,

centrifuge, and filter. Use of simple extraction proced-

ures not only greatly shortens the time required to

prepare the sample for enterotoxin detection (from

2–3daysto1–2 h), but also improves the recovery of

the enterotoxin from the food. This is particularly

important in cases where very low amounts of enter-

otoxin (1ngg

1

) are present. In these cases one can

5552

STAPHYLOCOCCUS

/Detection

expect recovery of at least 50% of the enterotoxin

but, in the long extraction and concentration proced-

ures, less than 10% will be recovered.

Enzyme-Linked Immunosorbent Assay (ELISA)

0014 The ELISA methods were applied to the detection of

the enterotoxins in foods soon after they were origin-

ally developed for the detection of other proteins.

Essentially, all of the ELISA methods used are of the

sandwich type; in this type the enzyme is coupled to

the antibody instead of the enterotoxin. The amount

of enzyme and, thus, the color developed from the

enzyme–substrate reaction, is directly proportional to

the amount of enterotoxin present in the unknown

sample. (See Immunoassays: Radioimmunoassay and

Enzyme Immunoassay.)

0015Most users of ELISA methods employ microtiter

plates to which the antibodies are attached. The large

number of wells in a microtiter plate allow examin-

ation of several samples at one time, although there

may not be uniformity in all of the wells, particularly

those around the edge of the plate. A plate reader is

necessary for recording the results, which adds to the

expense of the method.

0016An alternative method is to use polystyrene balls to

which the antibodies are attached. The ball method is

more cumbersome because each ball must be handled

separately. A relatively large volume of the unknown

sample can be used, thus increasing the amount of

enterotoxin adsorbed, and hence the sensitivity of

the assay. The use of 1-ml volumes of substrate allows

the color developed to be read in a simple colorimeter,

0.357 cm

0.238 cm

0.45 cm

0.1587 cm

0.3175 cm

2.54 cm

Template

Two layers of

waterproof tape

Clean agar-coated

microscope slide

Melted agar

2.54 cm

7.62 cm

fig0001 Figure 1 Microslide gel diffusion assembly showing typical results with the microslide test. The antibody is placed in the center well

and the control enterotoxin is placed in wells 1 and 3. Slide 1, positive reactions with control toxin; slide 2, positive reactions with

unknowns in wells 2 and 4; slide 3, positive reaction with unknown in well 2; reaction when unknown in well 4 contained a larger

amount of toxin and unknown in well 2 contained less toxin than the unknown in slide 3. Reproduced from Staphylococcus: Detection,

Encyclopaedia of Food Science, Food Technology and Nutrition, Macrae R, Robinson RK and Sadler MJ (eds), 1993, Academic Press.

STAPHYLOCOCCUS

/Detection 5553

an instrument that most laboratories would have

available. The sensitivity of the ELISA methods is

between 0.1 and 1.0 ng per gram of food (Table 1).

0017Although polyclonal antibodies are used in most of

the enterotoxin detection methods, the development

of monoclonal antibodies to the enterotoxins has

made possible their use in enterotoxin analysis. Two

monoclonal antibodies are required for each entero-

toxin in the sandwich ELISA as only one site exists on

the molecule for each monoclonal antibody. Not all

monoclonal antibodies are usable, particularly as the

coating antibody, because the reacting site may be

inactivated in the coating reaction.

0018In some instances it may not be necessary to deter-

mine the type of enterotoxin, only if enterotoxin is

present; for example, in examining the marketability

of suspect foods. The food would not be marketable

if any enterotoxin were present. For this purpose,

including all of the enterotoxins in one test saves

time as only one test is needed. This would not save

time in examining foods implicated in food-poisoning

outbreaks, because identification of the type of entero-

toxin is valuable in tracing the source of the contamin-

ation. However, if more than one enterotoxin was

present, the amount of each could be below detectable

levels for the individual enterotoxins. In this case it

would not be possible to detect them by the methods

used to identify individual enterotoxins.

Reversed Passive Latex Agglutination (RPLA)

0019In the RPLA method the specific antibodies are at-

tached to latex particles. When these particles are

added to a solution containing enterotoxin, the latex

particles agglutinate. The sensitivity is less than 1 ng

per gram of food. The method is adequately sensitive

for the detection of enterotoxin in most foods impli-

cated in food-poisoning outbreaks; however, it may

be inadequate for detection of the small amounts

fig0002 Figure 2 Optimum sensitivity plate (OSP) with typical results.

The wells containing the control toxins are one-half the cross-

sectional area of the antibody wells (middle) and the wells con-

taining the unknowns (U1–U4). This results in a doubling of the

sensitivity. Unknowns in wells U1 and U3 in the first place, U1 in

plate 2, and U4 in plate 3 are enterotoxin-positive. The hook in U1

in plate 1 is confirmed by fivefold concentration of the unknown

sample. The unknown in well 2 in plate 2 is not positive; the

observed result is affected by the large amount of toxin in the

unknown in well 1. Reproduced from Staphylococcus: Detection,

Encyclopaedia of Food Science, Food Technology and Nutrition,

Macrae R, Robinson RK and Sadler MJ (eds), 1993, Academic

Press.

Spread inoculum

Dialysis membrane

Glass petri dish

10 ml 1.2% agar layer

3.7% BHI, 1% YE

fig0003 Figure 3 Membrane-over-agar enterotoxin production method.

The organisms grow on the surface of the dialysis membrane as

the nutrients in the medium (brain–heart infusion (BHI) plus yeast

extract (YE)) in the agar can diffuse through the membrane, but

the enterotoxin cannot pass through the membrane. The culture

is removed from the membrane with buffer and centrifuged

before analysis. Reproduced from Staphylococcus: Detection, En-

cyclopaedia of Food Science, Food Technology and Nutrition,

Macrae R, Robinson RK and Sadler MJ (eds), 1993, Academic

Press.

tbl0001Table 1 Detection of staphylococcal enterotoxins (SEs) in

foods by enzyme-linked immunosorbent assay

Food SE Amount of SE

added (ng g

1

)

Amount of SE

detected (ng g

1

)

Milk A 0.63 0.63

Ham A 0.63 0.34

1.25 0.55

Genoa sausage A 0.63 0.36

Cheese A 0.63 0.59

D 0.63 0.15

1.25 0.64

E 0.63 0.38

Cheese food A 0.63 0.36

Potato salad B 0.63 0.18

1.25 0.44

Spaghetti A 0.63 0.54

Reproduced from Freed RC, Evenson ML, Reiser RF and Bergdoll MS

(1982) Enzyme-linked immunosorbent assay for the detection of

staphylococcal enterotoxins in foods. Applied and Environmental

Microbiology 44: 1349–1355.

5554

STAPHYLOCOCCUS

/Detection

of enterotoxin that are sometimes present (Table 2).

One problem with the RPLA method is that the food

extract from an occasional food may give a non-

specific agglutination.

Use of Sensitive Methods for Strain Testing

0020 More recently, a question has arisen regarding the

sensitivity of the gel diffusion methods for determin-

ing the enterotoxigenicity of staphylococcal strains. It

has been reported that enterotoxin production by

strains was observed by the RPLA method that was

not detectable by the OSP method. The amounts

produced were approximately 10–20 ng ml

1

. This

was confirmed by concentrating the culture super-

natant fluid from five strains that tested positive for

SEA by ELISA about 100-fold for testing by the OSP

method. The importance of this low production may

be questioned; however, we have found strains that

were implicated in food-poisoning outbreaks to be

negative by OSP but positive by ELISA. This resulted

from examination of strains by ELISA that were nega-

tive by OSP but positive by the monkey feeding test. A

number of these strains were positive for one or more

of the identified enterotoxins by the ELISA method,

particularly for SED (23 strains). Some of these

strains had been isolated from food-poisoning out-

breaks, which is significant because SED has been

implicated as the second most important enterotoxin

in food poisoning. It should be pointed out that, of all

of the enterotoxins, SED is produced in the smallest

amounts. Only three of the strains produced low

amounts of SEA, the enterotoxin implicated in 75%

of staphylococcal food-poisoning outbreaks. The

production of 10–20 ng of enterotoxin per milliliter

is probably of significance because only 100–200 ng

of SEA has been shown to be necessary to produce

food poisoning. The amount present in the vehicle,

2% chocolate milk, was 0.50–0.75 ng ml

1

. Admit-

tedly, the amounts of enterotoxin produced by the

membrane-over-agar or sac culture methods are

5–10 times those produced in shake flasks or even

possibly in food, yet, if growth is sufficient, 10

colony-forming units (CFU) and 1–2 ng of entero-

toxin per gram of food can be produced. This would

be enough to result in food poisoning in sensitive

individuals.

Kits Available for Enterotoxin Detection

0021Several commercial kits are available for enterotoxin

detection:

0022An ELISA ball kit, available from Labor Dr W.

Bommeli, La

nggassstrasse 7, CH-3012 Bern,

Switzerland. In this kit the specific antibodies to

SEA, SEB, SEC, and SED are absorbed on to sep-

arate polystyrene beads and the enzyme alkaline

phosphatase is coupled to the specific antibodies.

The substrate, p-nitrophenyl phosphate, gives a

yellow color with the enzyme. Although the test

can be completed in 1 day, recommendations are

that the antibody-coated balls are shaken with

20 ml of extract overnight to obtain the highest

sensitivity (0.1–1.0 ng ml

1

). The color can be

measured in a colorimeter because 1 ml of sub-

strate is used. The method can be used for quanti-

tative measurements.

0023An ELISA dipstick kit, available from Transia,

8 Rue Saint Jean de Dieu, 69007 Lyon, France.

Monoclonal antibodies to SEA, SEB, SEC, SED,

and SEE are coated on nitrocellulose paper in

wells in the dipstick. The conjugate consists of

different monoclonal antibodies coupled to horse-

radish peroxidase, which reacts with the substrate

tbl0002 Table 2 Detection of staphylococcal enterotoxin (SE) in foods from outbreaks

Food S. aureus count g

1

SEdetectedin food

SEstrain ELISA plate ELISA kit RPLA kit

Ham 1.5  10

AA AA

Lasagne, dried 2.0  10

AA AA

Salmon, canned 3.5  10

AA AA

Sheep’s milk cheese ND A A NSA

Corned beef 4.0  10

A, B A, B A, B A, B

Smoked bacon spread 1.0  10

A, D A

A, D A, D

Beef rolls 4.0  10

A, D A

AND

Pork 6.0  10

BB BB

Chicken 1.0  10

CCND

Meat pies 1.0  10

AND NDND

Reproduced from Wieneke AA and Gilbert RJ (1987) Comparison of four methods for the detection of staphylococcal enterotoxin in foods from outbreaks

of food poisoning. International Journal of Food Microbiology 4: 135–143.

ND, not detectable; NSA, nonspecific agglutination; ELISA, enzyme-linked immunosorbent assay; RPLA, reversed passive latex agglutination.

Extract not tested for SED.

STAPHYLOCOCCUS

/Detection 5555

to give a blue color. The method is sensitive to 0.5–

1.0 ng per gram of food; the test can be completed

in 6–7 h. The results are read visually; only one test

is needed per sample.

0024 An RPLA kit, available from Oxoid Ltd, Wade

Road, Basingstoke, Hampshire RG24 0PW, UK.

Latex particles are coated with specific antibodies

to SEA, SEB, SEC, and SED. More than 24 h is

required for completion of the test.

0025 An ELISA tube screening kit, available from Tran-

sia. Monoclonal antibodies to SEA, SEB, SEC,

SED, and SEE are coated on to the bottom of a

small flanged tube. The conjugate is a mixture of

monoclonal antibodies coupled to horseradish

peroxidase. The enzyme reacts with the substrate

to give a blue color. The sensitivity of the test is less

than 0.2 ng ml

1

. The time required for an assay

is 1 h.

0026 An ELISA microtiter plate screening kit, available

from Biotechnology Australia Pty Ltd, PO Box 20,

Roseville, NSW 2069, Australia. A mixture of

specific antibodies to SEA, SEB, SEC, SED, and

SEE is adsorbed to the wells of microtiter plates.

The enzyme used is horseradish peroxidase and

the substrate is ABTS (a sulfonic acid), which

gives a green color. The sensitivity of the method

is less than 1 ng ml

1

and the time required is 4 h.

An additional step of treating the food extracts

with urea followed by concentrating 20-fold is

included in the procedures in this kit for recovery

of enterotoxin from heated foods. Supposedly, the

purpose of the urea is to renature any denatured

enterotoxin in the heated food, but it is doubtful

that this is possible. In any case, the denatured

enterotoxin would be digestible by pepsin in the

stomach and would not cause food poisoning.

However, the concentration procedure does in-

crease the sensitivity of the method, but whether

this additional sensitivity is necessary is ques-

tionable.

See also: Food Poisoning: Tracing Origins and Testing;

Immunoassays: Radioimmunoassay and Enzyme

Immunoassay