GB 4789.10-2016National food safety standard - Food microbiological examination - Staphylococcus aureus (English PDF)

2.1 Constant temperature incubator. 36 °C ± 1 °C.

2.2 Refrigerator. 2 °C ~ 5 °C.

2.3 Constant temperature water bath. 36 °C ~ 56 °C.

2.4 Balance. with division of 0.1 g.

2.5 Homogenizer.

2.6 Oscillator.

2.7 Sterile pipette. 1 mL (with 0.01 mL scale), 10 mL (with 0.1 mL scale) or micro pipette

and tip.

2.8 Sterile Erlenmeyer flask. with capacity of 100 mL and 500 mL.

2.9 Sterile Petri dish. with diameter of 90 mm.

2.10 Spreader.

2.11 pH meter or pH colorimetric tube or precision pH test paper.

3.2 Blood agar plate. see A.2.

3.3 Baird-Parker agar plate. see A.3.

3.4 Brain and heart infusion broth (BHI). see A.4.

3.5 Rabbit plasma. see A.5.

3.6 Diluent. phosphate buffer. see A.6.

3.7 Nutrient agar slant. see A.7.

3.8 Gram's stain. see A.8.

3.9 Sterile saline. see A.9.

Method I Qualitative examination of Staphylococcus aureus

5.1 Sample treatment

WEIGH 25 g of sample to a sterile homogeneous cup containing 225 mL of 7.5 %

sodium chloride broth, HOMOGENIZE for 1 min to 2 min at 8000 r/min to 10000 r/min;

or PLACE in a sterile homogeneous bag containing 225 mL of 7.5 % sodium chlorine

broth, BEAT with a bag mixer for 1 min ~ 2 min. If the sample is liquid, pipette 25 mL

of sample to a sterile Erlenmeyer flask (it may preset an appropriate number of sterile

glass beads in the flask) containing 225 mL of 7.5 % sodium chloride broth.

5.2 Enrichment

PLACE the homogenized sample above at 36 °C ± 1 °C to culture for 18 h ~ 24 h.

Staphylococcus aureus is growing turbidly in 7.5 % sodium chloride broth.

5.3 Separation

The enriched culture is inoculated into Baird-Parker plate and blood plate. The blood

plate shall be cultured at 36 °C ± 1 °C for 18 h ~ 24 h. The Baird-Parker plate shall be

cultured at 36 °C ± 1 °C for 24 h ~ 48 h.

5.4 Preliminary identification

Staphylococcus aureus on the Baird-Parker plate is round, smooth-surface, raised,

moist, with colony diameter of 2 mm ~ 3 mm, color of gray black to black color, shiny,

and usually light (non-white) edge, around the opaque circle (precipitation) and a clear

band outside. When the inoculating needle touches the colony, there is a butter-like

sticky feeling.

5.5 Confirmation identification

5.5.1 Stain microscopic examination. Staphylococcus aureus is Gram's positive cocci,

arranged in grape-like, no spores, no capsule, with the diameter of about 0.5 µm ~ 1 µm.

5.5.2 Plasma coagulase test. PICK at least 5 suspicious colonies (select all when there

are less than 5) on Baird-Parker plate or blood plate; INOCULATE them to 5 mL of BHI

and nutrient agar slants respectively; CULTURE at 36 °C ± 1 °C for 18 h ~ 24 h.

5.6 Staphylococcal enterotoxin examination (optional)

For identification of suspicious food poisoning samples or Staphylococcus aureus

strains producing staphylococcal enterotoxin, Staphylococcal enterotoxin shall be

tested according to Annex B.

6.1 Result determination. when complying with 5.4, 5.5, it can be determined as

Staphylococcus aureus.

6.2 Result report. Staphylococcus aureus is detected or not detected in 25 g (mL) of

sample.

8.1 Sample dilution

8.1.1 Solid and semi-solid sample. WEIGH 25 g of sample in a sterile homogeneous

cup containing 225 mL of phosphate buffer or saline, HOMOGENIZE at 8000 r/min ~

8.1.2 Liquid sample. PIPETTE 25 mL of sample with a sterile pipette in a sterile

Erlenmeyer flask containing 225 mL of phosphate buffer or saline (an appropriate

number of sterile glass beads shall be preset in the flask), MIX well, to make into 1.10

sample homogenate.

8.1.3 PIPETTE 1 mL of 1.10 sample homogenate with a 1 mL sterile pipette or micro

pipette and slowly INJECT it along the tube wall to a sterile test tube containing 9 mL

of phosphate buffer or saline (note that the pipette or tip shall not touch the surface of

the diluent), SHAKE the test tube or USE a 1mL sterile straw to repeatedly blow to

make it evenly mixed, to make into 1.100 sample homogenate.

8.2 Sample inoculation

According to the estimation of the contamination status of the sample, select sample

homogenates of 2 ~ 3 appropriate dilutions (for liquid sample, it can include the original

solution).

8.3 Culture

Under normal circumstances, after spreading, the plate stands for 10 min; if the sample

homogenate is not easy to absorb, the plate can be placed in an incubator at 36 °C ±

8.4 Counting and confirmation of typical colony

8.4.1 Staphylococcus aureus on the Baird-Parker plate is round, smooth-surface,

raised, moist, with colony diameter of 2 mm ~ 3 mm, color of gray black to black color,

shiny, and usually light (non-white) edge, around the opaque circle (precipitation) and

a clear band outside. When the inoculating needle touches the colony, there is a butter-

like sticky feeling.

8.4.2 SELECT the plate with typical Staphylococcus aureus colonies, and the total

number of colonies on all three plates of the same dilution is between 20 CFU ~ 200

CFU, to count the number of typical colonies.

8.4.3 SELECT at least 5 suspected colonies (select all when there are less than 5)

from typical colonies for identification test.

9.1 If the number of typical colonies on only one dilution plate is between 20 CFF and

9.2 If the number of typical colonies on the lowest dilution plate is less than 20 CFU,

the typical colonies on the dilution plate shall be counted and calculated as equation (1).

9.3 If the number of typical colonies on a certain dilution plate is greater than 200 CFU,

and there is not typical colony on the next dilution plate, the typical colonies on the

dilution plate shall be counted and calculated as equation (1).

9.4 If the number of typical colonies on a certain dilution plate is greater than 200 CFU,

and the number of typical colonies on the next dilution plate is within the range of 20

CFU ~ 200 CFU, the typical colonies on the dilution plate shall be counted and

calculated as equation (1).

9.5 If the number of typical colonies of 2 consecutive dilution plates is between 20 CFU

~ 200 CFU, calculated as equation (2).

9.6 Calculation equation

12.1 Sample dilution

CARRY out according to 8.1.

12.2 Inoculation and culture

12.2.1 According to the estimation of the contamination status of the sample, select

sample homogenates of 3 appropriate dilutions (for liquid sample,

12.2.2 USE inoculating loop to take a loop of inoculum from the cultured 7.5 % sodium

chloride broth, and transplant it on Baird-Parker plate and culture at 36 °C ± 1 °C for

12.3 Confirmation of typical colony

CARRY out according to 8.4.1, 8.4.3.