Breif discussion about the organism and food through which the outbreaks have occured. It is also added with Bacteriological Analytical Methods (BAM) for the isolation and enumeration of the organism from the food sample.

1. Staphylococcus aureus
Meritta Joseph
M.Phil
Microbiologist

2.  First described by Scottish surgeon Sir. Alexander Ogston
 Causes pyogenic infection in human
 Greek: staphyle, bunch of grapes; coccus, a grain or berry
 Gram positive coccus
 Spherical to ovoid cells of 1 micrometer diameter.
 Catalase positive, Oxidase negative and glucose
fermentation

3.  Mesophile with a growth temperature range between 7
and 48ºC and an optimum at 37ºC
 Enterotoxin is produced at an optimum of 35-40ºC.
 Production of enterotoxin is adversely affected by
anaerobic conditions.
 Growth occurs at pH 6 -7 with minimum and maximum
limits of 4.0 and 9.8 – 10.
 Readily grows in media containing 5 – 7% NaCl and
some capable of growth in 20%.
 It can grow down to an water activity of 0.83

5.  First outbreak of illness through cheese in Michigan.
 Barber (1914) demonstrated staphylococci were able to
cause poisoning in milk from cow with staphylococcal
mastitis.
 Dack (1930) showed staphylococcal food poisoning by
a filterable enterotoxin.
 The infections are relatively mild and short lived type
of infections.
 Enterotoxin toxin producing strains include S.aureus,
S.intermedius and S.hycius

6.  Characterized by short incubation period 2 – 4h.
 Nausea, vomiting, stomach cramps, retching and
prostration are the predominant symptoms, diarrhoea is
also often.
 Severe cases dehydration, marked pallor and collapse
may require treatment by intravenous infusion.
 S.aureus produces 11 enterotoxins (SEA to SEJ). There
are three variants of SEC.
 When illness is of short incubation period, it is due to
ingestion of a pre-formed toxin in the food.
 Frequently implicated in outbreaks of food, toxins A and
D

8.  Naturally occur in poultry and other raw meat as skin
microflora.
 Isolated from raw milk and milk products as
enterotoxin can survive pasteurization
 Contamination by food handlers. Contaminated from
infected skin lesions or by coughing and sneezing.
 Poultry products and cold, cooked meats.
 Salted meats such as ham and corned beef,
 Canned foods, hard cheeses, cold sweets, custards
and cream-filled bakery products

9. Equipments and materials

10. Media and Reagents
 Baird-Parker medium
 Trypticase (tryptic) soy agar (TSA)
 Brain heart infusion (BHI) broth
 Coagulase plasma (rabbit) with EDTA
 Toluidine blue-DNA agar
 Lysostaphin (Schwartz-Mann, Mountain View Ave.,
Orangeburg, NY 10962)
 Tryptone yeast extract agar
 Paraffin oil, sterile
 0.02 M phosphate-saline buffer , containing 1% NaCl
 Catalase test

11.  Serial diution of the sample to be analysed
 Aseptically transfer 1 ml sample suspension to 3 plates of
Baird-Parker agar, distributing 1 ml of inoculum equitably to 3
plates.
 Select plates containing 20-200 colonies, unless only plates at
lower dilutions (>200 colonies) have colonies with typical
appearance of S. aureus.
 Count and record colonies.
 When plates of the lowest dilution contain <20
colonies, these may be used.
 Plates containing >200 colonies have colonies with
the typical appearance of S. aureus and typical
colonies do not appear at higher dilutions, use
these plates for the enumeration of S. aureus, but
do not count nontypical colonies.

12. COLONY MORPHOLOGY
Colonies of S. aureus are circular,
smooth, convex, moist, 2-3 mm in
diameter on uncrowded plates,
gray to jet-black, frequently with
light-colored (off-white) margin,
surrounded by opaque zone and
frequently with an outer clear
zone; colonies have buttery to
gummy consistency when touched
with inoculating needle.

13.  Select > 1 colony of each type counted and
test for coagulase production.
 Add number of colonies on triplicate plates
represented by colonies giving positive
coagulase test and multiply by the sample
dilution factor. Report this number as number
of S. aureus/g of food tested.

14.  Transfer suspect S. aureus colonies into small tubes
containing 0.2-0.3 ml BHI broth and emulsify
thoroughly
 Inoculate agar slant of suitable maintenance medium,
e.g., TSA, with loopful of BHI suspension. Incubate
BHI culture suspension and slants 18-24 h at 35-37°C.
 Add 0.5 ml reconstituted coagulase plasma with EDTA
(B-4, above) to the BHI culture and mix thoroughly.
 Incubate at 35-37°C and examine periodically over 6 h
period for clot formation

15.  Only firm and complete clot that stays in place when
tube is tilted or inverted is considered positive
for S. aureus.
 Test known positive and negative cultures
simultaneously with suspect cultures of unknown
coagulase activity.
 Stain all suspect cultures with Gram reagent and observe
microscopically.
 A latex agglutination test (AUREUS TESTTM, Trisum
Corp., Taipei, Taiwan) may be substituted for the
coagulase test if a more rapid procedure is desired.

18.  Catalase test :

19.  Inoculate tube of carbohydrate fermentation medium
containing glucose (0.5%).
 Immediately inoculate each tube heavily with wire loop.
Make certain inoculum reaches bottom of tube.
 Cover surface of agar with layer of sterile paraffin oil at
least 25 mm thick. Incubate 5 days at 35-37°C.
 Acid is produced anaerobically if indicator changes to
yellow throughout tube, indicating presence of S. aureus.
 Run controls simultaneously (positive and negative
cultures and medium controls).

21.  Repeat 2, above, using mannitol as
carbohydrate in medium. S. aureus is usually
positive but some strains are negative. Run
controls simultaneously.
Anaerobic utilization of mannitol.

22.  Transfer isolated colony from agar plate with
inoculating loop to 0.2 ml phosphate-saline buffer, and
emulsify.
 Transfer half of suspended cells to another tube (13 ×
100 mm) and mix with 0.1 ml phosphate-saline buffer
as control.
 Add 0.1 ml lysostaphin (dissolved in 0.02 M phosphate-
saline buffer containing 1% NaCl) to original tube for
concentration of 25 µg lysostaphin/ml.
 Incubate both tubes at 35-37°C for not more than 2 h. If
turbidity clears in test mixture, test is considered
positive.
 If clearing has not occurred in 2 h, test is
negative. S. aureus is generally positive.
Lysostaphin sensitivity.