GB 4789.41-2016National food safety standard - Microbiological Examination of Food Hygiene - Examination of Enterobacteriaceae (English PDF)

2.1 Enterobacteriaceae

The oxidase-negative aerobic or facultative anaerobic Gram-negative non-spore- bearing bacillus, which ferments glucose producing acid under given conditions.

2.3 most probable number; MPN

An indirect enumeration method based on Poisson's distribution.

6.1 Specimen dilution

Test specimen

6.1.1 Solid and semi-solid specimens. take 25 g of specimen to place into a sterile homogeneous cup containing 225 ml of BPW, homogenize for 1 min ~ 2 min at 8 000 r/min ~ 10 000 r/min, or place into a homogeneous bag containing 225 ml of BPW,

6.1.2 Liquid specimens. use a sterile pipette to absorb 25 ml of specimen to pour into a sterile conical flask (in which an appropriate quantity of sterile glass beads are provided in the flask in advance) containing 225 ml of BPW, mix up, and make homogeneous specimen solutions of 1.10.

6.2 Pout plate and culture

6.2.1 In accordance with the estimation of the contamination of specimens and

relevant limit requirements, select 2 ~ 3 homogeneous specimen solutions (liquid specimens may be raw solutions) of appropriate continuous dilution and inoculate the solutions of each dilution degree to 2 sterile plates. And Meanwhile, absorb 1 ml of

BPW each to add to two sterile plates as blank control.

6.2.4 Turn over the plate after the upper-layer agar solidifies on the VRBGA plate and culture for 18 h ~ 24 h at 36°C ± 1°C.

6.3 Enumeration and confirmation of typical colonies

6.3.1 The typical colonies of Enterobacteriaceae are pink to red or violet colonies with or without precipitation rings. Select the plates with typical colony count between

15 CFU ~ 150 CFU and without spreading growth of colonies and only enumerate the typical colonies. The colony counting is expressed by the colony-forming unit, CFU.

6.3.5 Confirmation of typical colonies.

6.4 Calculation of results

6.4.1 General principles

If the plate counts of typical colonies of two continuous dilution degrees are within the appropriate counting range, calculate in accordance with Formula (1). See A.1, A.2,

A.3 and A.4 for the examples.

6.4.2 Small colony counts

If all the typical colony counts of the plate of minimum dilution degree (including the raw solution of the liquid specimen) are less than 15 CFU and there are confirmed

Enterobacteriaceae colonies, then multiply the confirmed colony count by the minimum dilution factor.

6.4.3 Special circumstances

If all the typical colony counts on the plate of the first dilution degree are greater than

150 CFU and there are confirmed Enterobacteriaceae colonies; and if there is no confirmed Enterobacteriaceae colony on the plate of the second dilution degree or the typical colony counts are not between 15 CFU ~ 150 CFU, then multiply the confirmed colony count by the first dilution factor. See A.5 for the example.

9.1 Specimen dilution

As in 6.1.

9.2 Inoculation and culture

9.2.1 Nonselective pre-enrichment

In accordance with the estimation of the contamination of specimens and relevant limit requirements, select 3 homogeneous specimen solution of appropriate continuous dilution degrees, 3 test tubes for each dilution degree and altogether 9 test tubes of

BPW, and culture for 18 h ± 2 h at 36°C ± 1°C.

9.3 Confirmation of typical colonies

See 6.3 for the typical colonies.

B.1 Buffered peptone water (BPW)

B.2 Buffered glucose brilliant green bile broth (EE broth)

B.3 Crystal violet neutral red bile salt glucose agar (VRBGA)

B.4 Nutrient agar (NA)

B.5 Glucose agar

B.6 Gram stain solutions

B.6.1 Crystal violet stain solution

Annex C

Retrieval Table of Enterobacteriaceae Most Probable

Numbers (MPNs)

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