Examination of Foods for Staphylococcus aureus - DB Server Test ...

Experiment 12: Examination of Foods for
Staphylococcus aureus
Based on U.S. FDA Bacteriological Analytical Manual, 8th Edition, Current through Revision A,
1998, Chapter 12: Staphylococcus aureus, Reginald W. Bennett and Gayle A. Lancette
Objective:
1. To learn how to isolate, enumerate and confirm Staphylococcus aureus in foods.
Background:
Staphylococcus aureus is highly vulnerable to destruction by heat treatment and nearly all
sanitizing agents. Thus, the presence of this bacterium or its enterotoxins in processed
foods or on food processing equipment is generally an indication of poor sanitation. S.
aureus can cause severe food poisoning. It has been identified as the causative agent in
many food poisoning outbreaks and is probably responsible for even more cases in
individuals and family groups than the records show. Foods are examined for the
presence of S. aureus and/or its enterotoxins to confirm that S. aureus is the causative
agent of foodborne illness, to determine whether a food is a potential source of "staph"
food poisoning, and to demonstrate post-processing contamination, which is generally
due to human contact or contaminated food-contact surfaces. Conclusions regarding the
significance of S. aureus in foods should be made with circumspection. The presence of a
large number of S. aureus organisms in a food may indicate poor handling or sanitation;
however, it is not sufficient evidence to incriminate a food as the cause of food
poisoning. The isolated S. aureus must be shown to produce enterotoxins. Conversely,
small staphylococcal populations at the time of testing may be remnants of large
populations that produced enterotoxins in sufficient quantity to cause food poisoning.
Therefore, the analyst should consider all possibilities when analyzing a food for S.
aureus.
Methods used to detect and enumerate S. aureus depend on the reasons for testing the
food and on the past history of the test material. Processed foods may contain relatively
small numbers of debilitated viable cells, whose presence must be demonstrated by
appropriate means. Analysis of food for S. aureus may lead to legal action against the
party or parties responsible for a contaminated food. The methods of analysis for S.
aureus that have been studied collaboratively and found suitable for use in providing the
type of information necessary for FDA requirements are presented in this chapter.
There has been considerable controversy about the significance and correct method of
reading the coagulase test. Research results have indicated that the weak coagulase
activity represented by 1+, 2+, and 3+ reactions seldom corresponds with other criteria
associated with S. aureus (4). A consensus of peers has established that a 4+ coagulase
reaction is necessary for unquestioned identification of S. aureus. Those strains suspected
of being S. aureus on the basis of coagulase reactions of less than 4+ should be confirmed
by other tests, such as anaerobic glucose fermentation, lysostaphin sensitivity, and
thermonuclease production. Studies of colonial morphology on Baird-Parker agar,
1

lysostaphin sensitivity, coagulase and thermonuclease production, and glucose and
mannitol fermentation were conducted on 100 enterotoxigenic and 51 nonenterotoxigenic
strains of S. aureus (3). In all cases, the reactions of enterotoxigenic and
nonenterotoxigenic strains varied by 12% or less. This research indicates that none of
these tests can be relied upon to differentiate toxic and nontoxic staphylococci.
Experiment:
Direct Plate Count Method
This method is suitable for the analysis of foods in which more than 100 S. aureus cells/g
may be expected. It conforms to the method in ref. 1.
A. Materials
Tuesday, October 15: Plating on Baird Parker Medium plates, incubating plates at 35 o C
for 48 hours, plating on 3M Petrifilm Rapid S. aureus Count Plates, incubating
plates at 35 o C for 24 hours
1. Food sample
2. Sterile spatulas: 1 per team
3. Scale
4. Sterile stomacher bags: 1 per team
5. Butterfield's phosphate-buffered dilution water: One 225-ml flask per team
6. Butterfield's phosphate-buffered dilution water: Four 90-ml bottles per team
7. Sterile 10-ml pipettes
8. Sterile 1-ml tips (blue)
9. Sterile 0.2-ml tips (yellow)
10. Baird Parker Medium plates: 15 plates per team
11. 3M Petrifilm Rapid S. aureus Count Plates: 5 plates per team
Wednesday, October 16: Transferring 3M Petrifilm Rapid S. aureus Count Plates to
a 62 o C incubator, incubating plates 1-4 hours, placing reactive disks, incubating plates
with reactive disks at 35 or 37 o C for 1-3 hours, counting and recording colonies
1. Sterile forceps: 1 per team
2. 62 o C incubator
3. Colony counter
Thursday, October 17: Counting and recording colonies, transferring suspect S. aureus
colonies into BHI broth, inoculating agar slants of TSA, incubating BHI culture
suspension at 35 o C for 18-24 hours, incubating agar slants of TSA at room temperature
for 18-24 hours
1. Colony counters
2. Brain Heart Infusion (BHI) Broth: 0.2-0.4 ml per tubes; 3 tubes per team
3. Trypticase Soy Agar (TSA) slants: 3 slants per team
2

Tuesday, October 22: Catalase test, adding coagulase plasma to the BHI culture and
incubating at 35oC and examining periodically for clot formation, Gram stain (from TSA
slants), inoculating carbohydrate fermentation medium containing glucose (0.5%),
inoculating carbohydrate fermentation medium containing mannitol (0.5%), testing for
lysostaphin sensitivity, testing for thermostable nuclease production
1. 3% Hydrogen peroxide
2. Reconstituted coagulase plasma(rabbit) with EDTA: 1.5 ml per team
3. Gram stain materials
4. Carbohydrate fermentation medium containing glucose (0.5%): 5 tubes per team
5. Positive and negative controls (bacterial cultures that ferment and do not ferment
glucose): One set per bench
6. Carbohydrate fermentation medium containing mannitol (0.5%): 5 tubes per team
7. Positive and negative controls (bacterial cultures that ferment and do not ferment
mannitol): One set per bench
8. Sterile paraffin oil: Enough to cover 100 tubes
9. Phosphate-saline buffer: 0.2 ml per tube; 3 tubes per team
10. Phosphate-saline buffer: 0.1 ml per tube; 3 tubes per team
11. Lysostaphin (50 µg/ml) dissolved in 0.02 M phosphate-saline buffer containing
1% NaCl: 0.3 ml per team
12. DNase Test Agar with Methyl Green plates: 1 plate per team
Thursday, October 24: Read the results of carbohydrate fermentation tests. No materials
needed.
B. Preparation of sample
Using aseptic technique, weigh 25 g of sample into sterile stomacher bag. Add 225 ml
Butterfield's phosphate-buffered dilution water (1:10 dilution) and blend for 1 min at high
speed. Using the 1:10 dilution, make serial dilutions of sample for enumeration of S.
aureus.
C. Isolation and enumeration of S. aureus
1. For each dilution to be plated, aseptically transfer 1 ml sample suspension to 3 plates
of Baird-Parker agar, distributing 1 ml of inoculum equitably to 3 plates (e.g., 0.4 ml, 0.3
ml, and 0.3 ml). Spread inoculum over surface of agar plate, using sterile bent glass
streaking rod. Retain plates in upright position until inoculum is absorbed by agar (about
10 min on properly dried plates). If inoculum is not readily adsorbed, place plates upright
in incubator for about 1 h. Invert plates and incubate 45-48 h at 35°C. Select plates
containing 20-200 colonies, unless only plates at lower dilutions (>200 colonies) have
colonies with typical appearance of S. aureus. 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. Occasionally from various foods and dairy products, nonlipolytic
strains of similar appearance may be encountered, except that surrounding opaque and
clear zones are absent. Strains isolated from frozen or desiccated foods that have been
3

stored for extended periods frequently develop less black coloration than typical colonies
and may have rough appearance and dry texture.
2. For each dilution to be plated, place 1 ml of sample suspension onto center bottom film
of 3M Petrifilm Rapid S. aureus Count Plates. Refer to the “Reminders of Use for
3M Petrifilm Rapid S. aureus Count Plates” that is attached to your handout.
Incubate plates with clear side up in stacks of up to 10. Incubate at 35 o C or 37 o C for 24
hours.
3. Refer to the “Reminders of Use for 3M Petrifilm Rapid S. aureus Count
Plates” that is attached to your handout. Transfer petrifilm plates to a 62 o C incubator.
Incubate for 1-4 hours. With sterile forceps, remove the round reactive disk from the
outer square frame. Lift the top film of the Petrifilm plate and place the Petrifilm reactive
disk in the well of the plate. Lower the top film. Apply gentle pressure across the reactive
disk. Incubate plates with inserted Petrifilm reactive disks for 1-3 hours at 35 o C or 37 o C.
4. Read and record results. Refer to the “Interpretation Guide Section” that is
attached to your handout.
Dilution 10 -1
Dilution 10- 2
Dilution 10 -3
Dilution 10- 4
Dilution 10 -5
S. aureus count
5. Count and record colonies on Baird-Parker Agar plates. If several types of colonies are
observed which appear to be S. aureus on selected plates, count number of colonies of
each type and record counts separately. When plates of the lowest dilution contain 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. 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.
cfu [Plate 1 (0.4
ml)]
cfu [Plate 2 (0.3
ml)]
cfu [Plate 3 (0.3
ml)]
cfu/g food
Dilution 10 -2
4

Dilution 10 -3
Dilution 10 -4
Dilution 10 -5
D. Coagulase test and Gram staining
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°C. Retain slant cultures at room temperature for ancillary or repeat tests in case
coagulase test results are questionable. Take 100 µl of BHI culture suspensions and
keep it in a sterile tube for thermostable nuclease production test. Add 0.5 ml
reconstituted coagulase plasma with EDTA to the BHI culture and mix thoroughly.
Incubate at 35°C and examine periodically over 6 h period for clot formation. Only firm
and complete clot that stays in place when tube is tilted or inverted is considered positive
for S. aureus. Partial clotting, formerly 2+ and 3+ coagulase reactions, must be tested
further (4). 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 TEST TM , Trisum Corp.,
Taipei, Taiwan) may be substituted for the coagulase test if a more rapid procedure is
desired.
Colony 1
Colony 2
Colony 3
E. Ancillary tests
Coagulase activity (clot formation)
1. Catalase test. Use growth from TSA slant for catalase test on glass slide or spot plate,
and illuminate properly to observe production of gas bubbles.
Colony 1
Colony 2
Colony 3
Catalase activity (gas bubbles)
2. Anaerobic utilization of glucose. 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 37°C. Acid is produced
anaerobically if indicator changes to yellow throughout tube, indicating presence of S.
5

aureus. Run controls simultaneously (positive and negative cultures and medium
controls).
Colony 1
Colony 2
Colony 3
Positive control
Negative control
Anaerobic utilization of glucose (yellow
color)
3. Anaerobic utilization of mannitol. Repeat 2, above, using mannitol as carbohydrate
in medium. S. aureus is usually positive but some strains are negative. Run controls
simultaneously.
Colony 1
Colony 2
Colony 3
Positive control
Negative control
Anaerobic utilization of mannitol (yellow
color)
4. Lysostaphin sensitivity. 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 x 100 mm) containing 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°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
(clearing of turbidity)
Tube 1 (Phosphate-saline
buffer + lysostaphin)
Control (turbidity)
Tube 2 (Phosphate-saline
buffer)
Colony 1
Colony 2
6

Colony 3
5. Thermostable nuclease production. Please note that we will perform II. Section I is
an alternative and is included for your information.
I. This test is claimed to be as specific as the coagulase test but less subjective, because it
involves a color change from blue to bright pink. It is not a substitute for the coagulase
test but rather is a supportive test, particularly for 2+ coagulase reactions. Prepare
microslides by spreading 3 ml toluidine blue-deoxyribonucleic acid agar on the surface of
each microscope slide. When agar has solidified, cut 2 mm diameter wells (10-12 per
slide) in agar and remove agar plug by aspiration. Add about 0.01 ml of heated sample
(15 min in boiling water bath) of broth cultures used for coagulase test to well on
prepared slide. Incubate slides in moist chamber 4 h at 35°C. Development of bright pink
halo extending at least 1 mm from periphery of well indicates a positive reaction.
II. Heat (15 min in boiling water bath) BHI culture suspensions (100 µl each). Mark
bottom of a DNase Test Agar with Methyl Green plate into 3 equal sections with a
marker and label each section. Spot 50 µl of heated sample on the plate (do not spread).
Incubate plate at 4 o C for overnight. Development of a clearing (loss of blue-green color)
around growth indicates a positive reaction.
Colony 1
Colony 2
Colony 3
Thermostable nuclease production (loss of
blue-green color around growth)
F. Some typical characteristics of 2 species of staphylococci and the micrococci,
which may be helpful in their identification, are shown in Table 1.
7

Most Probable Number Method for Staphylococcus spp.
The most probable number (MPN) method (2) is recommended for routine surveillance
of products in which small numbers of S. aureus are expected and in foods expected to
contain a large population of competing species. Please note that we will not perform
MPN. The following is included for your information.
A. Materials--Same as for Direct Plate Count Method, above. In addition: Trypticase
(tryptic) soy broth (TSB) containing 10% NaCl and 1% sodium pyruvate.
B. Preparation of sample--Same as for Direct Plate Count Method, above.
C. Determination of MPN
Inoculate 3 tubes of TSB containing 10% NaCl and 1% sodium pyruvate (A, above) with
1 ml portions of decimal dilutions of each sample. Highest dilution must give negative
endpoint. Incubate tubes 48 ± 2 h at 35°C. Using 3 mm loop, transfer 1 loopful from each
tube showing growth (turbidity) to plate of Baird-Parker medium with properly dried
surface. Vortex-mix tubes before streaking if growth is visible only on bottom or sides of
tubes. Streak inoculum to obtain isolated colonies. Incubate plates 48 h at 35°C. From
each plate showing growth, transfer at least 1 colony suspected to be S. aureus to BHI
broth (see C and D of Direct Plate Count Method, above). Continue procedure for
identification and confirmation of S. aureus (D and E, Direct Plate Count, above).
Report S. aureus/g as MPN/g, according to tables in Appendix 2, MPN Determination.
References:
1. AOAC INTERNATIONAL. 1995. Official Methods of Analysis, 16th ed., sec. 975.55.
AOAC INTERNATIONAL, Arlington, VA.
8

2. AOAC INTERNATIONAL. 1995. Official Methods of Analysis, 15th ed., sec. 987.09.
AOAC INTERNATIONAL, Arlington, VA.
3. Bennett, R.W., M. Yeterian, W. Smith, C.M. Coles, M. Sassaman, and F.D. McClure.
1986. Staphylococcus aureus identification characteristics and enterotoxigenicity. J. Food
Sci. 51:1337-1339.
4. Sperber, W.H., and S.R. Tatini. 1975. Interpretation of the tube coagulase test for
identification of Staphylococcus aureus. Appl. Microbiol. 29:502-505.
9

Media and Reagents:
From The Compendium of Analytical Methods for the Analysis of Food and Agricultural Products
(a) Baird-Parker medium (egg tellurite glycine pyruvate agar, ETGPA).—(1) Basal
medium.—Suspend 10.0 g tryptone, 5.0 g beef extract, 1.0 g yeast extract, 10.0 g sodium
pyruvate, 12.0 g glycine, 5.0 g LiCl· 2O, and 20.0 g agar in 950 mL H 2 O. Heat to bp
with frequent agitation to dissolve ingredients completely. Dispense 95 mL portions into
screw-cap bottles. Autoclave 15 min at 121°C. Final pH should be 7.0 ± 0.2 at 25°C.
Store 1 month at 4 ± 1°C.
(b) Enrichment.—Bacto EY tellurite enrichment (Difco Laboratories or equivalent) or
prepare as follows: Soak fresh eggs ca 1 min in dilution of saturated HgCl 2 solution (1 +
1000). Aseptically crack eggs and separate yolks from whites. Blend yolk and
physiological saline solution, (b), (3 + 7, v/v) in high-speed blender ca 5 s. To 50 mL egg
yolk emulsion, add 10 mL filter-sterilized 1% potassium tellurite solution (w/v). Mix and
store at 4 ±
(c) Tryptic (trypticase) soy agar (TSA).—Suspend 15.0 g trypticase peptone (pancreatic
digest of casein), 5.0 g phytone peptone (papaic digest of soya meal), 5.0 g NaCl, and
15.0 g agar in l L H 2 O. Heat with agitation to dissolve agar. Dispense 200 mL portions
into flasks. Autoclave 15 min at 121°C. Final pH should be 7.3 ± 0.2. Pour 15–18 mL
medium into sterile 15 100 mm Petri dishes.1°C.
(d) Brain-heart infusion (BHI) broth.—Dissolve infusion from 200 g calf brain and from
250 g beef heart, 10.0 g proteose peptone or Gelysate, 5.0 g NaCl, 2.5 g
Na 2 HPO 4·12H 2 O, and 2.0 g glucose in 1 L H 2 O, heating gently if necessary. Dispense
5 mL portions into 16 150 mm test tubes and autoclave 15 min at 121°C. Final pH
should be 7.4 ± 0.2.
(e) Desiccated coagulase plasma (rabbit) with EDTA.—Reconstitute according to
manufacturer's directions. If not available, reconstitute desiccated coagulase plasma
(rabbit) and add Na 2 H 2 EDTA to final concentration of 0.1% in reconstituted plasma.
(f) Butterfield's buffered phosphate diluent.—(1) Stock solution.—Dissolve 34.0 g
KH 2 PO 4 in 500 mL H 2 O, adjust to pH 7.2 with ca 175 mL 1M NaOH, and dilute to 1 L.
Store in refrigerator. (2) Diluent.—Dilute 1.25 mL stock solution to 1 L with H 2 O.
Prepare dilution blanks with this solution, dispensing enough to allow for losses during
autoclaving. Autoclave 15 min at 121°C.
(g) DNase test agar with Methyl Green. See attached.
10