Listeria test method!
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Acute infectious diseases caused by Listeria (also known as Listeria), with septicemia as the main symptom, accompanied by internal organs and central nervous system diseases. Listeria was first discovered in 1926 by a British South African scientist, Muri, in a sick rabbit. To commemorate the father of modern disinfection surgery, the British physiologist Joseph Liszt (1827-1912), was the third international microbe in 1940. The conference was named Listeria.
Listeria monocytogenes is a pathogen of zoonotic diseases. It can cause the disease of human and animal Listeria. After infection, it mainly manifests as sepsis, meningitis and mononucleosis. It is widely found in nature. The presence of Listeria monocytogenes in food is dangerous to human safety. The bacteria can still grow and reproduce in an environment of 4 ° C. It is one of the main pathogens that threaten human health in refrigerated foods. In the food hygiene microbiological examination, it must be taken seriously.
Listeria is ubiquitous in the environment and can be found in most foods. Meat, eggs, poultry, seafood, dairy products, vegetables, etc. have all been confirmed to be the source of infection with Listeria. Listeria monocytotoxicity can cause blood and brain tissue infections, and many countries have taken measures to control Listeria in food and have set standards.
Among them, Listeria monocytogenes is the only one that can cause human diseases. Listeria monocytogenes is a pathogen of zoonotic diseases. It can cause Lee's disease in humans and animals. After infection, it mainly manifests as sepsis, meningitis and mononucleosis.
Experimental procedure
1. Enrichment culture <br> Store unopened test pieces in an environment of ≤8 °C and use them within the validity period indicated on the package. In high humidity areas, it is best to return the test piece to room temperature before use.
Samples retrieved should be handled, stored, and shipped at 4 ° C. If frozen, they should be kept frozen prior to testing.
Take 25 mL of liquid or 25 g of semi-solid or solid sample into a homogenized cup containing 225 mL of non-selective reagent enrichment broth (EB) for homogenization, then transfer to a flask, incubate at 30 ° C for 4 h, add selective reagent Acridine flavin, nalidixic acid and cycloheximide were continued for 20 h and 44 h.
2. Isolation <br> After 24 h and 48 h of co-culture, EB cultures were streaked on OXA and LPM or esculin/Fe3 LPM agar plates, respectively. PALCAM agar can replace LPM agar. The OXA and PALCAM plates were incubated at 35 ° C for 24-48 h, and the LPM plates were incubated at 30 ° C for 24-48 h. Then, the LPM plate was placed on the anatomical mirror stage, and the plate was irradiated with light at an angle of 450 to illuminate the plate from below the plate, and the suspicious colonies were searched vertically through the eyepiece. Listeria has a lustrous blue or gray color on the LPM plate. A plate lined with known positive and negative bacteria was used as a control.
The opened test piece, the opening is folded back and sealed with tape.
LPM plates incorporating escin and Fe3 do not require an oblique light system, and the method of selecting suspect colonies is the same as the method of selecting suspect colonies on OXA. There is a black ring around the Listeria colonies on the OXA plate, and other bacteria can form a black ring, but the formation time is more than two days. The colony characteristics of Listeria on PALCAM and OXA plates are similar.
Five or more typical colonies were picked on PALCAM and OXA or LPM plates and streaked onto TSAYE plates, respectively, to obtain a purer, more typical single colony. Purification on TSAYE plates in food testing is necessary because colonies isolated on PALCAM and OXA or LPM plates may be contaminated with invisible microorganisms. The reason for picking 5 typical colonies is that more than one Listeria may be isolated from one sample. The 30° CTSAYE plate was cultured for 24-48 hours, and if not used for dynamic observation, it can also be cultured at 35 °C.
3. Identification Procedure <br> To prevent exposure to moisture, do not refrigerate the opened test piece. The test piece sealed with the tape is stored in a low-temperature and dry place for a period of not more than one month. Do not place the test piece in an environment where the temperature is > 25 ° C and/or the relative humidity is > 50%.
1) Observe the TSAYE plate and observe the oblique light to find the blue to blue colonies. Known bacteria were used as controls on TSAYE.
2) A typical colony was picked from a TSAYE plate cultured at 30 ° C or lower to make a wet glass slide under an oil microscope. The wet slides were made with a 0.85% saline suspension. If the amount of bacteria is too small, the cells adhere to the slide and appear non-motility. Listeria is a Brevibacterium, showing slight rotation and tumbling. Compared to the known Listeria control, the spherical, large rod-shaped and fast-moving ones are not Listeria.
3) Pick a typical colony for catalase experiment, and Listeria is positive for catalase.
4) The culture of 16-24 h was subjected to Gram staining, and the Listeria was Gram-positive bacilli. However, Gram stains in old cultures change, and the cells can be spherical. On the stained glass slides, the bacteria have a tendency to be arranged in a grid shape, which is easy to be mistaken for diphtheria.
4. Collect environmental samples with applicator sticks, sponges, or other sampling equipment. The liquid of the wet sampling equipment should be ≤ 10 mL. The humectant can be sterile water, Buffered Peptone Water (BPW), or a neutralization buffer such as Letheen Broth or Dey/Engley (DE) Neutralizing Broth.
5) Pick up typical colonies inoculated in TSBYE broth tube and incubate at 35 °C for 24 hours for sugar fermentation and other biochemical project experiments. The TSBYE broth can be stored for several days at 4 ° C or it can be inoculated repeatedly.
6) Pick a typical colony on the TSAYE plate to 5% sheep blood or horse blood agar plate, avoid touching the bottom of the plate and rupture the agar when pricking, and set a positive control (monocyte proliferation Lister Bacteria and Listeria monocytogenes and a negative control (Listinella linburg) were cultured for 48 h at 35 °C. Listeria monocytogenes is a narrow beta-hemolytic ring.
7) Observing the punctured blood agar plate under bright light, Listeria monocytogenes and Listeria monocytogenes produce a clear β-hemolytic ring around the puncture site, Listeria innocua No hemolysis occurs, and Listeria monocytogenes produces a large hemolytic ring with obvious boundaries. Do not attempt to distinguish between species, but to record the characteristics of the hemolysis reaction, the CAMP test can distinguish the hemolytic reaction between them.
8) Nitrate reduction test (optional). The TSBYE broth culture was inoculated into the nitrate broth, and after incubation at 35 ° C for 5 days, 0.2 mL of reagent A was added, and then 0.2 mL of reagent B was added and mixed, and a purple-red reaction was observed, indicating that the nitrate had been reduced, such as No color appeared, add a small amount of zinc powder in the test tube, and place it for 1 hour. If purple color appears, it indicates that the nitrate is still present and is not reduced by bacteria. Only Listeria monocytogenes reduces nitrates, so the distinction must be made between the isolation of Listeria from Listeria. There is also an equivalent test in this test, that is, after adding 0.2 mL of reagent A, 0.2 mL of reagent C is added, and an orange color indicates that the nitrate has been reduced. If no color reaction occurs, a small amount of zinc powder is also added, and if orange appears, the nitrate is not reduced.
5. Add 5 mL of the buffered protein buffer (BPW) solution to the collected sample in a sterile environment at 20-30 °C. Do not share Petrifilm EL test strips with Vermont medium (UVM), Fraser broth, Listeria enrichment medium (LEB) or Listeria monocytogenes (BLEB).
9) The TSBYE culture was punctured into SIM and MTM tubes and cultured at room temperature for 7 days. The daily observation of Listeria showed typical umbrella growth. Umbrella growth is more typical in MTM. At the same time, the TSBYE culture at 30 °C showed bacteria flipping under the oil microscope.
10) Inoculate TSAYE broth culture in 0.5% (w/v) glucose, maltose, esculin, mannitol, rhamnose, xylose fermentation tubes (optional inverted fermentation tube), and culture at 35 ° C for 7 days. The positive reaction of Listeria has no acid production, and the case of Listeria fermented rhamnose and xylose is shown in the table below. All Listeria can ferment glucose, esculin and maltose, and no mannitol can be fermented except Listeria. If the colony pigment is evident on the OXA, PALCAM or safariin/Fe3 LPM separation plates, the escin assay may be avoided.
6. Mix, wriggle or rotate the mixture of sample and BPW for approximately one minute. The sample was placed at room temperature (20-30 ° C) for 1 h and no longer than 1.5 h for repairing the damaged Listeria.
The TSBYE broth culture was inoculated into 3 mL TSBYE broth, cultured at 35 ° C for 24 h, inoculated with 2 TSAYE agar slants, and cultured at 35 ° C for 24 h. Wash the stalk fungus with 3mL 0.01mol/l phosphate buffer solution, heat the bacterial suspension in a water bath at 80 °C for 1 hour, centrifuge at 30 at 500 r/min, discard 2 mL of supernatant, and mix the remaining liquid with the precipitate. The bacterial suspension was subjected to a serological slide agglutination test.
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