10 Staphylococcus aureus
10.1 Introduction The staphylococci cells are spherical and characteris-
Staphylococcus aureus is a pathogenic bacterium, which
causes a foodborne disease classified by the Interna-
tional Commission on Microbiological Specifications
for Foods (ICMSF, 2002) in Risk Group III: “diseases
of moderate hazard usually not life threatening, nor-
mally of short duration without substantial sequelae,
with symptoms that are self-limiting but can cause
severe discomfort”.
10.1.1 Taxonomy
10.1.1.1 The genus Staphylococcus
In the 9th Edition of Bergey’s Manual of Determinative
Bacteriology (Holt et al., 1994) the genus Staphylococcus
was placed in group 17, which includes the Gram posi-
tive cocci. In the 2nd Edition of Bergey’s Manual of System-
atic Bacteriology the members of group 17 are subdivided
into three phyla: the genus Deinococcus was transferred
to the phylum Deinococcus-Thermus, the genus Micro-
coccus and the genus Stomatococcus were transferred to
the phylum Actinobacteria and the other genera of Gram
positive cocci, including Staphylococcus, were transferred
to the phylum Firmicutes (Garrity and Holt, 2001).
The affiliation of Micrococcus and Staphylococcus to
different phyla indicates a great phylogenetic distance
between these genera. However, they share many phe-
notypic characteristics and in the 1st Edition of Bergey’s
Manual of Systematic Bacteriology (Sneath et al., 1986)
the two genera were located in the same family.
The phylum Firmicutes includes the Gram positive
bacteria with a low DNA mol% G+C content (<50)
(Schleifer, 2009). The genus Staphylococcus is a member
of the family Staphylococacaeae, which also contains the
genera Jeotgalicoccus, Macrococcus and Salinicocus (Sch-
leifer and Bell, 2009a).
7007TS-DASILVA-Book.indb 105
tically divide in more than one plane to form irregular
grape like clusters. The Gram stain is positive, the cells
are nonmotile and nonspore-forming. The catalase reac-
tion is usually positive and oxidase is usually negative.
Chemo-organotrophs, the carbohydrate metabolism
is respiratory and fermentative. Susceptible to lysis by
lysostaphin and resistant to lysis by lysozyme. Predomi-
nantly associated with skin, skin glands and mucous
membranes of warm-blooded animals (Schleifer and
Bell, 2009b).
The Staphylococcus species can be grouped into groups
and the most important groups are (Schleifer and Bell,
2009b):
Group S. epidermidis (e.g., S. epidermidis, S. capitis,
S. caprae, S. haemolyticus, S. hominis, S. saccharolyticus,
S. warneri) and Group S. simulans (e.g., S. simulans,
S. carnosos), which are coagulase negative and novo-
biocin susceptible.
Group S. saprophyticus (e.g., S. saprophyticus, S. cohnii,
S. xylosus) and Group S. sciuri (e.g., S. sciuri, S. lentus,
S. vitulinus), which are coagulase negative and novo-
biocin resistant.
Group S. intermedius (e.g., S. intermedius, S. del-
phini) and Group S. aureus (e.g., S. aureus subsp. aureus,
S. aureus subsp. anaerobius), which are coagulase posi-
tive and novobiocin susceptible.
10.1.1.2 The coagulase positive
staphylococci
The coagulase positive staphylococci are S. aureus,
S. intermedius, S. delphini, and S. schleiferi subsp. coag-
ulans. S. hyicus is coagulase variable. These species are
considered potentially serious pathogens (Schleifer and
Bell, 2009b) and, for this reason, the production of
coagulase is considered as an indication of pathogenic-
ity among the species of Staphylococcus.
11/26/2012 11:15:25 AM
 106 Microbiological examination methods of food and water

According to MacFaddin (2000), coagulase is an
enzyme which converts fibrinogen to fibrin, resulting in
a visible clot. It may be present in two forms, the bound
coagulase or “clumping factor” and the free coagulase
or “clotting factor”. The free coagulase is extracellular
and reacts with the coagulase-reacting factor “CRF”
(a thrombin-like substance in the plasma) to form a
coagulase-CRF complex. This complex indirectly con-
verts fibrinogen to fibrin forming a clot. The detection
is achieved by the tube coagulase test. The clumping
factor is located on the surface of cell walls and forms
clots with no involvement of “CRF”. It is not inhib-
ited by antibodies to free coagulase and the detection is
achieved by the slide coagulase test.
The main characteristics of the coagulase positive
staphylococci are shown in Table 10.1. According to
Schleifer and Bell (2009b) S. aureus subsp. aureus is
the most common pathogen among the coagulase posi-
tive staphylococci. Some strains produce enterotoxins.
S. aureus subsp. anaerobius is found in abcesses of sheep
and is also pathogenic for goats. It produces coagulase
but does not produce enterotoxins. S. intermedius is
opportunistic pathogenic of dogs, S. hyicus is associated
with infections in pigs, skin lesions in cattle and horses,
osteomyelitis in poultry and cattle, and occasionally
with mastitis in cattle. S. delphini is associated with skin
lesions in dolphins. S. schleiferi subsp. coagulans is asso-
ciated with ear otitis in dogs.
Among the coagulase positive strains S. aureus, S. hyi-
cus and S. intermedius are the species associated with
food intoxication outbreaks (Bennett and Hait, 2011).
10.1.1.3 Staphylococcus aureus
S. aureus is subdivided into two subspecies, S. aureus
subsp. aureus and S. aureus subsp. anaerobius. The char-
acteristics differentiating the two subspecies are shown
in Table 10.1.
S. aureus subsp. anaerobius grows microaerobically
and anaerobically, but the aerobic growth is weak. It
is distinguished from S. aureus subsp. aureus by three
characteristics: the lack of pigment and clumping fac-
tor, the inability to ferment mannitol anaerobically, and
the inability to grow at 45°C. The temperature range
for optimal growth is 30–40°C and it does not grow
at 20 or 45°C. All strains tolerate 10% of NaCl; most

Table 10.1 Biochemical and growth characteristics of the species and subspecies of coagulase positive Staphylococcus (Schleifer and Bell,
2009).

S. aureus S. aureus S. schleiferi

Characteristic subsp. aureus subsp. anaerobius S. hyicus S. intermedius S. delphini subsp. coagulans

Catalase + −
+ + + +
Oxidase − − − − − −
Pigment +w − − − − −
Aerobic growth + −
+ + + +
Anaerobic growth + + + (+) + +
(thiglycolate medium)
Growth on 10% of NaCl (w/v) + + + + + ND
Growth on 15% of NaCl (w/v) w d −
w d + ND
Growth at 15°C + ND + + ND ND
Growth at 45°C + − −
w + + ND
Alkaline phosphatase + + + + + +
Tellurite reduction + + ND −
ND ND
Coagulase + + d + + +
Clumping factor + − −
d − −
Protein A + −
ND −
ND −
Heat stable nuclease + + + + −
+
Hemolysisa + + −
d + +
Acid from mannitol + − −
(d) + d

+, 90% or more strains positive; −, 90% or more strains negative; d, 11–89% strains positive; ( ), delayed reaction; w, weak reaction;
–w, negative to weak reaction; +w, positive to weak reaction; ND, not determined.
a
Positive hemolytic reactions include greening of the agar as well as clearing.

7007TS-DASILVA-Book.indb 106 11/26/2012 11:15:25 AM


do not tolerate 15%. The primary isolation requires a
medium supplemented with serum, egg yolk or blood
(Schleifer and Bell, 2009b).
S. aureus subsp. aureus is usually called only S. aureus
in the literature, without mention of the subspecies.
According to Schleifer and Bell (2009b) it is faculta-
tive anaerobic but growth is best under aerobic condi-
tions. Protein A is produced and the positive reactions
include alkaline phosphatase, coagulase, clumping fac-
tor, heat stable nuclease (thermonuclease), hemolysin,
and lipase. Acid is produced aerobically from glucose
and mannitol. The temperature range for growth is 10
to 45°C and the optimum is 30 to 37°C.
S. aureus is not heat resistant, and is easily destroyed
by pasteurization or by normal cooking. Enterotoxins,
on the other hand, are highly heat-resistant and survive
heat treatments as severe as those used to sterilize low-
acid foods (ICMSF, 1996).
S. aureus is a salt tolerant microorganism and accord-
ing to Schleifer and Bell (2009b) its growth is good in
medium containing 10% of NaCl and poor at 15%.
According to ICMSF (1996) it grows at a water activity
as low as 0.85 (salt content 25% w/w). From this aspect,
S. aureus is an atypical bacterium among the foodborne
pathogens, which normally do not grow at such a low
water activity.
10.1.2 Pathogenicity
According to Bien et al. (2011) S. aureus can cause a
wide variety of infections, including wound infection,
toxinoses (food poisoning, scalded skin syndrome, toxic
shock syndrome) and systemic conditions (endocardi-
tis, osteomyelitis, pneumonia, brain abscesses, menin-
gitis, bacteremia).
According to Schleifer and Bell (2009) S. aureus was
responsible for considerable morbidity and mortal-
ity among hospitalized patients from 1950 to 1960.
Methicillin-resistant S. aureus (MRSA) strains emerged
in 1980 and become a great epidemiological problem
in hospitals. Enterotoxin-producing S. aureus strains
are the most common coagulase positive staphylococci
associated with food intoxication outbreaks.
10.1.2.1 Staphylococcus aureus
enterotoxins
S. aureus produces various types of toxins. The alpha,
beta, delta and gamma toxins, and the leukocidins are
7007TS-DASILVA-Book.indb 107
Staphylococcus aureus 107
involved with cell lysis and tissue invasion (Ferry et al.,
2005). The exfoliative toxins (ETs) (also known as
“epidermolytic” toxins) are responsible for the staphy-
lococcal scalded skin syndrome (disease characterized
by the loss of superficial skin layers, dehydration, and
secondary infections) (Bukowski et al., 2010). The toxic
shock syndrome toxin (TSST-1) is responsible for the
toxic shock syndrome (acute onset illness characterized
by fever, rash formation and hypotension that can lead
to multiple organ failure and lethal shock (Ferry et al.,
2005). The enterotoxins are involved in staphylococcal
food poisoning, one of the most common food-borne
diseases worldwide.
The enterotoxins responsible for staphylococcal
food poisoning are produced primarily by Staphyloco-
ccus aureus, although S. intermedius and S. hyicus also
have been shown to be enterotoxigenic (Bennett and
Hait, 2011). S. intermedius was isolated from butter
blend and margarine in a food poisoning outbreak in
United States (Khambaty et al., 1994, Bennett, 1996).
A coagulase negative S. epidermidis was reported to have
caused at least one outbreak (Breckinridge and Berg-
doll, 1971).
S. aureus enterotoxins (SEs) and toxic shock syn-
drome toxin (TSST-1) are broadly classified as super-
antigens (SAgs), which have the ability to stimulate
large populations of T cells leading to the production
of a cytokine bolus (Pinchuk et al., 2010). In 1990 the
staphylococcal research community published a stand-
ard nomenclature for the superantigens expressed by
Staphylococcus aureus (Betley et al., 1990). At this time
the classical members of this family included toxic
shock–syndrome toxin-1 (TSST-1) and five antigenic
variants of S. aureus enterotoxins, designated “SEA”,
“SEB”, “SEC”, “SED”, and “SEE” (Lina et al., 2004).
The TSST-1 was initially designated as “SEF” (Bergdoll
et al., 1981) but was later designated as TSST-1 because
did not show in-vivo biological activity characteristic of
true enterotoxins (Fueyo et al., 2005).
Newly SEs described after 1990 received a letter desig-
nation in the order in which they have been discovered.
In 2004 the International Nomenclature Committee for
Staphylococcal Superantigens proposed an international
procedure for the designation of newly described SAgs
and putative SAgs, reported by Lina et al. (2004). The
rules are: a) Toxin genes identified but not confirmed
to be expressed should not be subject to the standard-
ized toxin nomenclature. b) Only toxins that induce
emesis after oral administration in a primate model
should be designated as enterotoxin. c) The current
11/26/2012 11:15:26 AM
 108 Microbiological examination methods of food and water
letter designation should be retained for SEs in which
S = S. aureus and E = enterotoxin. d) The SE should be
followed by a letter assigned sequentially until the 25th
toxin (SEZ) has been assigned (SEF has been retired).
Thereafter, newly described toxins should be numbered
sequentially beginning with SE26. e) Related toxins that
lack emetic properties (or have not been tested) should
be designated “staphylococcal enterotoxin-like” (SEl), to
indicate that their potential role in staphylococcal food
poisoning has not been confirmed. To minimize confu-
sion and significant renaming, SEls should receive a let-
ter designation in the order in which they are described.
If the proteins are later shown to have enterotoxic activi-
ties, the SEl designation can be changed to SE.
In the IAFP 4th European Symposium on Food Safety
a presentation made by Smith (2008) showed the fol-
lowing situation:
• Classic staphylococcal enterotoxins: SEA, SEB,
SEC (including three variants SEC1, SEC2, SEC3
and SEC ovine and SEC bovine variants), SED, and
SEE.
• Newer SEs: SEG, SEH, SEI, SER, SES, and SET.
• Enterotoxin-like proteins (SEls): SElJ, SElK, SElL,
SElM, SElN, SElO, SElP, SElQ, SElU, SElV, SElW.
Only a few of the staphylococcal enterotoxins have
been studied in depth. They are pyrogenic and share
some other important properties that include the super-
antigenicity and the ability to induce emesis and gastro-
enteritis (Pinchuk et al., 2010).
The different SE serotypes are identified serologically
as separate proteins (Bennett and Hait, 2011). They
resist acid and are stable over a wide pH range. They are
highly heat-resistant and are not completely denatured
by cooking. They are resistant to inactivation by gas-
trointestinal and other proteases (pepsin, trypsin, chy-
motrypsin, rennin, papain) (Smith, 2008).
10.1.2.2 Staphylococcal food poisoning
The disease transmitted by S. aureus is an intoxication,
caused by the ingestion of enterotoxins formed in the
food as a result of the multiplication of the bacterial cells.
The intake of a dose smaller than 1μg may cause symp-
toms of intoxication and this quantity is reached when
the population of S. aureus attains values greater than
105 UFC/g of food (Hait, 2012). The serotype A is the
most frequently involved in foodborne staphylococcal
illness (Bennett and Hait, 2011).
7007TS-DASILVA-Book.indb 108
The symptoms become evident between one to seven
hours after ingestion, and include nausea, vomiting,
retching and abdominal cramping. Dehydration, head-
ache, muscle cramping, and changes in blood pressure
and pulse rate may occur in more severe cases. Recov-
ery occurs in few hours to one day and complications
or death are rare (Hait, 2012). It is easily diagnosed,
especially in the cause of outbreaks in which nausea and
vomiting predominate, and with a short interval between
the food ingestion and the onset of symptoms.
S. aureus can be found in the nasal airways, throat,
skin and hair of 50% or more of healthy human indi-
viduals. Food handlers are a common source of con-
tamination, although equipment and food handling
surfaces in processing environments may also contami-
nate the foods (Hait, 2012).
Foods that have already been implicated in outbreaks
include meat and meat products; poultry and egg prod-
ucts; salads, such as egg, tuna, chicken, potato, and
macaroni; bakery products, such as cream-filled pastries,
cream pies, and chocolate éclairs; sandwich fillings; and
milk and dairy products. The foods at greatest risk are
those that are intensely handled during their prepara-
tion and/or those that remain at room temperature after
preparation (Hait, 2012).
10.1.3 Methods of analysis
There are several methods available for counting
S. aureus, the degree of sensitivity of which may vary
both in function of the selective/differential character-
istics used in formulating the culture media, as in func-
tion of the enumeration technique itself (direct plate
count or the Most Probable Number technique). Even-
tually, and depending on the objective of analysis and
the conditions of the injuries inflicted on the cells, it
may be advisable to use a simple presence/absence test,
with a non-selective pre-enrichment step, which will
ensure recovery from the injuries.
The main selective characteristics used to isolate
S. aureus are the ability of this microorganism to grow
in the presence of NaCl (5.5 to 10%), potassium tellur-
ite (0.0025 a 0.05%), lithium chloride (0.01 to 0.05%),
glycine (0.12 to 1.26%) and polymyxin (40 mg/l).The
differential characteristics are the capacity to reduce
potassium tellurite (producing black colonies in solid
media), the capacity to hydrolyze egg yolk, the capacity
to use mannitol and grow at 42–43°C under selective
conditions, the activity of coagulase and the activity of
11/26/2012 11:15:26 AM
 Staphylococcus aureus 109

Table 10.2 Analytical kits adopted as AOAC Official Methods for Staphylococcus aureus in foods (Horwitz and Latimer, 2010, AOAC Inter-
national, 2010).

Method Kit Name and Manufacturer Principle Matrices

993.06 TECRA™ Staph Enterotoxin
VIA, 3M Microbiology Products
995.12 Aureus Test™, Trisum Corp.
2001.05 Petrifilm™ Rapid S. aureus
Count Plate, 3M Microbiology
Products
2003.07 Petrifilm™ Staph Express Count Plate,
3M Microbiology Products
2003.08 Petrifilm™ Staph Express Count Plate,
3M Microbiology Products
2003.11 Petrifilm™ Staph Express Count Plate,
3M Microbiology Products
EIA (colorimetric visual or
photometer) in microtiter plate
Polystyrene latex particles coated
with anti-protein A immunoglobulin
and fibrinogen that bind protein A
and coagulase.
Cultural, plate count in dry
rehydratable film
Cultural, plate count in dry
rehydratable film
Cultural, plate count in dry
rehydratable film
Cultural, plate count in dry
rehydratable film
Extracts prepared from beef, pasta,
chicken, lobster bisque, mushrooms,
nonfat milk.
Pure culture isolated from foods.
Pasta filled with beef and cheese,
frozen hash browns, cooked chicken
patty, egg custard, frozen ground raw
pork, and instant nonfat dried milk.
Frozen lasagna, custard, frozen mixed
vegetables, frozen hash browns, and
frozen batter-coated mushrooms.
Ice cream, raw milk, yogurt, whey
powder and cheese.
Cooked and diced chicken, ham,
salmon, and pepperoni.

EIA = Enzyme immunoassay.

thermonuclease. There are several growth media avail-
able for direct plate counts, which combine one or more
selective/differential characteristics, such as Mannitol
Salt Agar, Vogel-Johnson Agar, Egg Yolk Azide Agar,
Phenolphthalein Polymyxin Phosphate Agar, Milk-Salt
Agar, Tellurite Glycine Agar, Tellurite Polymyxin Egg
Yolk and Staphylococcus Medium number 110. In gen-
eral, these media should not be used for foods in which
the presence of injured cells is expected, since they are
considered restrictive to the recovery of injuries.
The most widely used medium is Baird-Parker Agar
(BP), which combines potassium tellurite (0.01%), gly-
cine (1.2%) and lithium chloride (0.5%), as selective
agents and the reduction of tellurite and the hydrolysis
of egg yolk as differential characteristics. In addition,
the medium contains 1% of sodium pyruvate, which is
considered an excellent means to recover injured cells,
since it avoids accumulation of hydrogen peroxide (toxic
for cells). It may be used for direct plating of processed
or fresh (“in natura”) foods, both for enumeration for
indicative purposes as for enumeration for public health
purposes. On the other hand, BP Agar, as well as all
the other media cited above, is not able to completely
suppress the growth of competitors of S. aureus. Other
non-pathogenic species of the Staphylococcus genus may
grow, producing similar colonies, thereby creating the
need to subject typical colonies to the coagulase test for
confirmation.
Other methods that already have been officially rec-
ognized by the AOAC International are microbiological
test kits described in Table 10.2.
10.2 Plate count method APHA
2001 for coagulase positive
staphylococci and S. aureus
in foods
Method of the American Public Health Association
(APHA), as described in the Chapter 39 of the 4th Edi-
tion of the Compendium of Methods for the Microbiologi-
cal Examination of Foods (Lancette & Bennett, 2001)
and Chapter 5 of the 17th Edition of the Standard
Methods for the Examination of Dairy Products (Henning
et al., 2004).
This method is suitable for the analysis of foods
in which more than 100 S. aureus cells/g may be
expected.
Before starting activities, read the guidelines in
Chapter 3, which deal with all details and measures

7007TS-DASILVA-Book.indb 109 11/26/2012 11:15:26 AM

 110 Microbiological examination methods of food and water
required for performing plate counts of microorgan-
isms, from dilution selection to calculating the results.
The procedure described below does not present
these details, as they are supposed to be known to the
analyst.
10.2.1 Material required for analysis
Preparation of the sample and
serial dilutions
• Diluent: 0.1% Peptone Water (PW) or Butterfield’s
Phosphate Buffer
• Dilution tubes containing 9 ml of 0.1% Peptone
Water (PW) or Butterfield’s Phosphate Buffer
• Observation: consult Annex 2.2 of Chapter 2 to
check on special cases in which either the type or
volume of diluent vary as a function of the sample
to be examined.
Direct plate count method
• Baird-Parker (BP) Agar plates
• Laboratory incubator set to 35–37°C (35 ± 1°C for
dairy products)
Confirmation
• Brain Hearth Infusion Broth (BHI) tubes
• Trypticase Soy Agar (TSA) slants
• Coagulase plasma (rabbit) with EDTA
• Toluidine Blue-DNA agar plates or slides
• 3% Hydrogen Peroxide (for catalase test)
• 0.02M Phosphate-Saline Buffer
• Lysostaphin
• Purple Broth with 0.5% Glucose
• Purple Broth with 0.5% Mannitol
• Paraffin oil, sterile
• Gram Stain Reagents
• Laboratory incubator set to 35–37°C
10.2.2 Procedure
A general flowchart for the enumeration of coagulase
positive staphylococci and Staphylococus aureus in foods
using the plate count method APHA 2001 is shown in
Figure 10.1.
a) Preparation of the samples and serial dilutions:
Follow the procedures described in Chapter 2. For
dairy product analysis the Standard Methods for the
7007TS-DASILVA-Book.indb 110
Examination of Dairy Products recommends an ana-
lytical unit of 50 g.
b) Inoculation: Select three appropriate dilutions of
the sample and inoculate 0.1 ml each on Baird Parker
Agar, using the spread plate technique. Spread inoc-
ulum over surface of agar plate, using sterile bent
glass streaking rod. Retain plates in upright position
until inoculum is absorbed by agar.
Note b.1) For solid samples with low count, inoculate 1 ml of
the first dilution and 0.1 ml of the two subsequent
dilutions (distribute 1 ml of first dilution on four
plates: 0.3 ml, 0.3 ml, 0.3 ml, and 0.1 ml). For
liquid samples with low count, start with 1 ml of
the sample without dilution.
c) Incubation and colony counting: Incubate plates
(inverted) at 35–37°C/45–48 h.
Note c.1) The incubation temperature is 35 ± 1°C in the Stand-
ard Methods for the Examination of Dairy Products
(Henning et al., 2004).
Examine plates for typical S. aureus colonies:
black or gray, small (maximum 2–3 mm in diam-
eter), surrounded by an opaque halo and frequently
with an outer clear halo. Non-lipolytic strains form
similar colonies but without the opaque and clear
halos. Select plates containing 20–200 colonies for
counting and if more than one type of presump-
tive S. aureus colonies are present count each type
separately.
d) Confirmation: Select five typical colonies for coag-
ulase test. If there are fewer than five colonies, select
all. If several types of presumptive S.aureus colo-
nies are present, select one or more colonies of each
type.
Transfer suspect S. aureus colonies to Brain Heart
Infusion (BHI) Broth tubes and emulsify. From
the BHI tubes transfer a loopful to Trypticase Soy
Agar (TSA) slants. Incubate BHI and TSA tubes at
35–37°C/18–24 h. Use the BHI culture for coagu-
lase test. Keep TSA cultures at ambient tempera-
ture for other tests, if necessary.
d.1) Coagulase test: From the BHI culture trans-
fer 0.2 ml to an empty sterile tube. Add
0.5 ml of reconstituted coagulase plasma
with EDTA and mix. Use a tube with 0.2 ml
of BHI and 0.5 ml of reconstituted coagu-
lase plasma with EDTA as a negative control.
Incubate the tubes at 35–37°C in a water
bath and examine periodically over a six-hour
period for clot formation. Classify the result
as described below.
11/26/2012 11:15:26 AM
 -3
10 -1 10 -2 10
Homogenization 1ml 1ml

25g of sample +
9ml 9ml
225ml of Peptone Water (PW) PW PW

0.1ml 0.1ml
0.3ml 0.3ml 0.3ml 0.1ml

Baird-Parker (BP) Agar

(spread plate)

BP BP BP BP BP

35-37oC/45-48h
Typical colonies (5)

1 loopful
Trypticase Soy Agar (TSA) Brain Heart Infusion (BHI) Broth

35-37oC/18-24h 35-37oC/18-24h

COAGULASE TEST
Save for additional tests
(if required)

Culture 0.2ml Control (-)

Coagulase Plasma EDTA 0.5ml BHI Broth 0.2ml
Coagulase Plasma EDTA 0.5ml

Water bath

35-37oC/6h

(-) (1+) (2+) (3+) (4+)

Additional Confirmed
tests required tubes

S. aureus
CFU/g

Figure 10.1 Scheme of analysis for the enumeration of coagulase positive staphylococci and Staphylococus aureus in foods using the plate
count method APHA 2001 (Lancette & Bennett, 2001).

7007TS-DASILVA-Book.indb 111 11/26/2012 11:15:26 AM

 112 Microbiological examination methods of food and water
4+ positive: The entire content of tube coagulates
and is not displaced when tube is inverted.
3+ positive: Large and organized clot.
2+ positive: Small organized clot.
1+ positive: Small unorganized clot.
Negative: No evidence of fibrin formation
A 3+ or 4+ clot formation is considered a positive
reaction for S. aureus.
Note d.1.1) The Standard Methods for the Examination
of Dairy Products (Henning et al., 2004)
and the Bacteriological Analytical Manual
(Bennett & Lancette, 2001) consider
confirmed only cultures showing level 4+
positive coagulase test.
Note d.1.2.) A clumping factor latex agglutination test
may be used if a more rapid procedure
than coagulase test is desired. In this case,
follow the manufacturers’ instructions.
Cultures showing levels 1+ or 2+ should be
confirmed by performing the additional tests
listed below. Observe the cultures for Gram
stain and morphology. S. aureus are Gram
positive cocci occurring typically in irregular
clusters (resembling clusters of grapes).
d.2) Catalase test: From the TSA culture emul-
sify a loopful in one drop of 3% Hydrogen
Peroxide on a glass slide. Immediate bubbling
is a positive reaction. S. aureus is catalase
positive.
d.3) Thermostable nuclease test (thermonu-
clease test): Boil a portion of the culture
grown in BHI for 15 min and use for ther-
monuclease test. Prepare microslides by
spreading 3 ml Toluidine Blue DNA Agar
on the surface of each microscope slide.
When agar has solidified, cut 2 mm or larger
wells in agar (10–12 per slide) and fill with
the boiled culture (about 0.01 ml). Also
include S. aureus ATCC 12600 as a positive
control and S. epidermidis ATCC 14990 as
a negative control in the assay Incubate at
35–37°C/4 h or 50°C/2 h. Development of
bright pink halo extending at least 1 mm
from periphery of well indicates a positive
reaction. Lack of the pink halo indicates a
negative reaction. S. aureus is thermonucle-
ase positive.
d.4) Lysostaphin sensitivity test: Mix 0.1 ml of
the BHI culture with 0.1 ml of Lysostaphin
(dissolved in 0.02 M Phosphate Buffer con-
taining 2% NaCl) to give a final concentration
7007TS-DASILVA-Book.indb 112
of 25 μg lysostaphin/ml. To another 0.1 ml
portion of the BHI culture, add 0.1 ml of
0.02 M Phosphate Buffer containing 2%
NaCl (negative control). Also include S. aureus
ATCC 12600 as a positive control and Kocu-
ria varians ATCC 15306 (formerly Micrococ-
cus varians) as a negative control in the assay.
Incubate the tubes at 35°C for not more than
2 h. If turbidity clears in the test mixture, the
test is considered positive (susceptible). No
clearing after 2 h is considered a negative result
(resistant). S. aureus is generally susceptible to
lysostaphin, showing positive result in this
test.
Note d.4.1) The Standard Methods for the Examination
of Dairy Products (Henning et al., 2004)
uses Lysostaphin dissolved in 0.02M
Phosphate Buffer containing 1% NaCl.
d.5) Anaerobic utilization of glucose and man-
nitol. From the TSA slant, inoculate a tube
of Purple Broth with 0.5% glucose and a
tube of Purple Broth with 0.5% mannitol
(exhaust oxygen from tubes before inocula-
tion). Also include S. aureus ATCC 12600
as a positive control and Kocuria varians
ATCC 15306 (formerly Micrococcus varians)
as a negative control in the assay. Cover the
surface of agar with a 25 mm layer of sterile
paraffin oil. Incubate the tubes at 37°C for
five days. The acid production anaerobically
(color change to yellow throughout tube)
indicates a positive test. Lack of change in
color indicates a negative test. S aureus is
positive for anaerobic utilization of glucose.
Most strains are positive for mannitol, but
some strains are negative. K. varians is nega-
tive for both.
e) Interpretation and calculation of results: Con-
sider as S. aureus the cultures showing coagulase
test level 3+ and 4+ or cultures level 1+ and 2+ with
typical characteristics presented in Table 10.3.
Calculate number of S. aureus cells/g of sample, based
on percentage of colonies tested that are confirmed as
S. aureus.
Example 1: The presumptive count obtained with 10−4
dilution of sample was 65 and four of five colonies
tested were confirmed (80%). The number of S. aureus
cells/g of food is 65 × 0.8 × 104 × 10 = 5.2 × 106 CFU/g
(dilution factor is tenfold higher than sample dilution
because only 0.1 ml was tested).
11/26/2012 11:15:26 AM

Table 10.3 Typical characteristics used to differentiate Staphylococcus aureus from Staphylococcus epidermidis and Micrococcus (Bennett &
Lancette, 2001).
Characteristica
Catalase activity
Coagulase production
Thermonuclease production
Lysostaphin sensitivity
Anaerobic utilization of glucose
Anaerobic utilization of mannitol
a
+ = 90% or more strains are positive; − = 90% or more strains are negative.
b
The 4th Edition of the Compendium (Bennett & Lancette, 2001) does not differentiate Micrococcus and Kocuria.
Example 2: The count obtained with 10−2 dilution of
sample was 30 (20 colonies of one type and 10 colonies
of another type). Five colonies of the first type tested
were confirmed (100%). Two of five colonies of the
second type tested were confirmed (40%). The number
of S. aureus cells/g of food is (20 × 1 × 102 × 10) +
(10 × 0.4 × 102 × 10) = 2.4 × 104 CFU/g
10.3 Most probable number (MPN)
method APHA 2001 for
coagulase positive staphylococci
and S. aureus in foods
Method of the American Public Health Association
(APHA), as described in Chapter 39 of the 4th Edition
of the Compendium of Methods for the Microbiological
Examination of Foods (Lancette & Bennett, 2001).
This method is recommended for products in which
small numbers of S. aureus and a large population of
competing species are expected.
Before starting activities, carefully read the guidelines
in Chapter 4, which deals with all the details and meas-
ures required for MPN counts of microorganisms, from
dilution selection to calculating the results. The proce-
dure described below does not present these details, as
they are supposed to be known to the analyst.
10.3.1 Material required for analysis
Preparation of the sample and
serial dilutions
• Diluent: 0.1% Peptone Water (PW) or Butterfield’s
Phosphate Buffer
7007TS-DASILVA-Book.indb 113
Staphylococcus aureus 113
S. aureus S. epidermidis Micrococcusb
+ + +
+ − −
+ − −
Susceptible Susceptible Resistant
+ + −
+ − −
• Dilution tubes containing 9 ml of 0.1% Peptone
Water (PW) or Butterfield’s Phosphate Buffer
• Observation: consult Annex 2.2 of Chapter 2 to
check on special cases in which either the type or
volume of diluent vary as a function of the sample
to be examined.
MPN Counting
• Trypticase Soy Broth (TSB) with 10% NaCl and
1% Sodium Pyruvate (10 ml tubes)
• Baird Parker (BP) Agar plates
• Laboratory incubator set to 35–37°C
Confirmation
• The same material required as for the plate count
method APHA 2001 (item 10.2.1)
10.3.2 Procedure
A general flowchart for the enumeration of coagulase
positive staphylococci and Staphylococus aureus in foods
using the Most Probable Number (MPN) method
APHA 2001 is shown in Figure 10.2.
a) Preparation of the samples and inoculation.
For preparation of the samples and serial dilu-
tions (10−1, 10−2 and 10−3) follow the procedures
described in Chapter 2. Inoculate three 1 ml por-
tions of each dilution into three tubes containing
10 ml of Trypticase Soy Broth (TSB) with 10%
NaCl and 1% Sodium Pyruvate. Incubate tubes at
35–37°C/48 ± 2 h and examine for visible growth.
b) Confirmation. Streak a loopful from each tube
showing growth (turbidity) on the surface of
11/26/2012 11:15:26 AM
 -1 -3
10 10-2 10
Homogenization 1ml 1ml

9ml 9ml
PW PW
25g of sample +
225ml of Peptone Water (PW)

1ml 1ml 1ml 1ml 1ml 1ml 1ml 1ml 1ml

TSB (10ml) Trypticase Soy Broth (TSB) TSB (10ml)

with 10% NaCl and 1% Sodium
Pyruvate (10ml)

35-37oC/48±2h
Tubes with growth

Streak a loopful on
Baird-Parker (BP) Agar
35-37oC/48±2h

Typical colonies

1 loopful
Trypticase Soy Agar (TSA) Brain Heart Infusion (BHI) Broth

35-37oC/18-24h
35-37oC/18-24h
COAGULASE TEST

Save for additional tests

(if required) Control (-)
Culture 0.2ml
Coagulase Plasma EDTA 0.5ml BHI Broth 0.2ml
Coagulase Plasma EDTA 0.5ml

Water Bath

35-37oC/6h

(-) (1+) (2+) (3+) (4+)

Additional Confirmed
tests required tubes

S. aureus
MPN/g

Figure 10.2 Scheme of analysis for the enumeration of coagulase positive staphylococci and Staphylococus aureus in foods using the Most
Probable Number (MPN) method APHA 2001 (Lancette & Bennett, 2001).

7007TS-DASILVA-Book.indb 114 11/26/2012 11:15:27 AM


Baird-Parker (BP) Agar plates. Incubate the plates
at 35–37°C/48 ± 2 h and examine for typical
S. aureus colonies, as described in the plate count
method APHA 2001 above (10.2.2.c). From each
plate showing growth, select at least one suspect
colony and confirm, as described in the plate count
method APHA 2001 (10.2.2.d). In the absence of
suspected colonies, consider the tube negative for
S. aureus.
c) Calculation of results. Record the number of con-
firmed tubes and determine the MPN/g or MPN/
ml as detailed in Chapter 4, using one of the MPN
tables.
10.4 Presence/absence method
APHA 2001 for coagulase
positive staphylococci
and S. aureus in foods
Method of the American Public Health Association
(APHA), as described in Chapter 39 of the 4th Edition
of the Compendium of Methods for the Microbiological
Examination of Foods (Lancette & Bennett, 2001).
This method is recommended for processed foods
likely to contain a small population of injured cells.
10.4.1 Material required for analysis
Preparation of the sample and
serial dilutions
• Diluent: 0.1% Peptone Water (PW) or Butterfield’s
Phosphate Buffer
• Observation: consult Annex 2.2 of Chapter 2 to
check on special cases in which either the type or
volume of diluent vary as a function of the sample
to be examined.
Presence/absence test
• Double strength Trypticase Soy Broth (TSB) (50 ml
flasks)
• Single strength Trypticase Soy Broth (TSB) with
20% NaCl (100 ml flasks)
• Baird Parker (BP) Agar plates
• Laboratory incubator set to 35–37°C
Confirmation
• The same material required as for the plate count
method APHA 2001 (item 10.2.1)
7007TS-DASILVA-Book.indb 115
Staphylococcus aureus 115
10.4.2 Procedure
A general flowchart for the determination of S. aureus
by the APHA presence/absence method is shown in
Figure 10.3.
a) Pre-enrichment. Following the procedures described
in Chapter 2 homogenize 50 ml of the sample with
450 ml of Peptone Water (PW) (10−1 dilution).
Transfer 50 ml of the 10−1 dilution (5 g of sample)
into 50 ml of double strength Trypticase Soy Broth
(TSB). Incubate at 35–37°C/3 h.
Note a.1) To increase the detection limits an analytical unit
of 100 g (in 900 ml of diluent) may be used. For
liquid samples it is not necessary to dilute.
b) Selective enrichment. After the 3 h incubation
period, add 100 ml of single strength Trypticase
Soy Broth (TSB) with 20% NaCl to the pre-en-
riched sample. Incubate at 35–37°C/24 ± 2 h.
c) Confirmation. Inoculate two 0.1 ml portions
of the selective enrichment broth onto duplicate
plates of Baird-Parker (BP) Agar (spread plate).
Incubate plates at 35–37°C/46 ± 2 h and exam-
ine for typical colonies, as described in the plate
count method APHA 2001 above (10.2.2.c).
From each plate showing growth, select at least one
presumptive colony and confirm, as described in
the plate count method APHA 2001 (10.2.2.d).
Report results as S. aureus presence or absence in
5 g of food.
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Trypticase Soy Agar (TSA)
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35-37oC/18-24h
35-37oC/18-24h
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Save for additional tests

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Coagulase Plasma EDTA 0.5ml

Water Bath

35-37oC/6h

(-) (1+) (2+) (3+) (4+)

Additional Confirmed
tests required tubes

S. aureus
CFU/g

Figure 10.3 Scheme of analysis for the detection of coagulase positive staphylococci and Staphylococus aureus in foods using the presence/
absence method APHA 2001 (Lancette & Bennett, 2001).

7007TS-DASILVA-Book.indb 116 11/26/2012 11:15:27 AM


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