(1) In 225mL Fraser basic broth, add 2.25mL sterile lithium chloride solution, 0J3mL Euprodinone sodium salt solution, 1.12mL sterile acridine yellow solution and 2.25mL sterile ferric ammonium citrate Solution (see the medium and its sterile supplements in Appendix 1 for details). After mixing to make 50% Frascr? Broth, add human samples and place in 30C to cultivate Mh for initial increase.

(2) â‘  Streak a ring of enrichment culture on Oxford agar, and take another ring to inoculate on PALCAM agar. Q plate is cultured at 35t: or 37T: 48h (Oxford agar plate cultured under aerobic conditions, PALCAM agar The plates were cultured under the aerobic conditions of microorganisms described in Section 10.1).

② Transfer 0, 1mL of enrichment broth to a test tube containing 10mL Freser base meat Yang, and place it in 351: or 37 芄 culture for 48h for secondary enrichment.

(3) Transfer the secondary enrichment solution to Oxford agar plate and PALCAM plate h according to the method in (2) â‘ .

Listeria rapid screening test

Using the appropriate ELISA kit, the primary and secondary enrichment solutions introduced on 21. 9.2.2 can be rapidly tested to rapidly screen for Listeria monocytogenes (eg, TECRA Listeria visual immunoassay) law).

Identification and verification of Listeria

(1) Listeria can hydrolyze aescin, except for Listeria monocytogenes, other Listeria can not decompose mannitol to produce acid. On Oxford agar plates, Listeria colonies are 2 to 3 mm in diameter, with dark brown or black halos (formed by the hydrolysis of escin), and the center of the colonies is often concave; The diameter is 1.5? Lnm, green with black halo (or the center of the colony is black).

(2) During verification, pick 5 typical colonies, and each colony is streaked to tryptone soy yeast extract agar (TSYEA), placed in MT: or 37T: aerobic culture for 24 to 48h, and separated on a plate To ensure the purity of the strain.

(3) Check the single colony of Sang's on the TSYEA plate, and use the colony characteristics described by Henry to isolate (see 3.4.1) o Henry believes that Listeria produces small, translucent colonies on the TYS plate, and the colonies are blue , Blue-green or blue-gray, the surface of the colony is fine granular, similar to rough frosted glass.

A single colony was picked from the TSYES plate, and inoculated into tryptone soy yeast extract broth (TSYEB) test tube and tryptone soy yeast extract agar (TSYEA) inclined surface, 25C culture 18 ~ 24h.

(4) â‘  ffl lSYEK broth culture was subjected to cell movement test and Gram stain. The Listeria spp. Exhibits somersaulting movement, which should be distinguished from the rapid linear movement of non-pathogenic Listeria after shaking culture. Listeria is a small, Gram-positive, spore-free Brevibacterium.

â‘¡The catalase and oxidase tests were carried out with TSYEB oblique and t: growths. The Listeria catalase test is positive and the oxidase test is negative o

â‘¢Inoculate the following medium with TSYEB culture to carry out verification and identification tests:

a. Inoculation of D-glucose, D-salicin, L-rhamnose, D-xylose fermentation broth (containing bromocresol purple; see Appendix 1), set 35T: or 371: redundant cultivation, daily inspection of production Acid condition (biochemical tube changed from purple to yellow).

b. Inoculate nitrate peptone water and check the production of nitrate. Incubate at 35 ° C or 37 ° C for 5 days.

c. CAMP test using Staphylococcus aureus and Rhodococcus equi (see 11,5.3). Incubate at 35 ℃ or 37 ℃ for 24h.

Optional API Listeria kit (HoMMeux). Mdauchlin found that the kit could well identify and distinguish different Listeria species.

result

Listeria monocytogenes are Gram-positive, small and slender rod-shaped bacteria. The radon peroxidase test is positive and the oxidase test is negative. Movement.

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