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Resuscitation fluids of VBNC salmonella and preparation method thereof and resuscitation method

A technology of Salmonella and resuscitation solution, applied in the biological field, can solve problems such as many shortcomings, inconvenient material collection, and failure to resuscitate VBNC bacteria, and achieve the effect of less raw materials and easy operation

Inactive Publication Date: 2009-05-06
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Generally speaking, the recovery effect of this method is better, but there are many shortcomings.
(1) There are many types of materials involved in the resuscitation fluid, at least 8 different ingredients are required; (2) Some materials are inconvenient to obtain, such as sheep blood, which needs to be freshly collected, and is used now; (3) The concentration ratio of various ingredients needs to be adjusted. Accurate enough, otherwise the recovery efficiency will be seriously reduced, and sometimes even VBNC bacteria will not be recovered

Method used

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  • Resuscitation fluids of VBNC salmonella and preparation method thereof and resuscitation method
  • Resuscitation fluids of VBNC salmonella and preparation method thereof and resuscitation method
  • Resuscitation fluids of VBNC salmonella and preparation method thereof and resuscitation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1: the preparation of VBNC Salmonella pullorum CVCC578 standard strain (S578)

[0025] Take the number of cultivable bacteria as 10 6 ~10 7 Put 1 mL of CFU / mL Salmonella pullorum CVCC578 standard strain (S578) bacteria solution into a sterile centrifuge tube, centrifuge at 8000r / min for 3min, discard the supernatant, and then fully wash the cell pellet twice with sterilized normal saline, each time Average 8000r / min, centrifuge for 2min, discard the supernatant, then connect the cell pellet to VBNC induction solution, cultivate under aerobic conditions at 4°C, regularly measure the number of cultivable bacteria, the number of viable bacteria and the total number of bacteria, and wait for cultivability When the bacterial count is zero, it proves that Salmonella enters the VBNC.

Embodiment 2

[0026] Embodiment 2: preparation and inspection of resuscitation fluid of VBNC Salmonella

[0027] Bovine serum and double-distilled water (sterilized by high-pressure steam) are gently stirred and mixed according to the ratio of 1:1 to 3, and then filtered and sterilized with a microporous filter to obtain a resuscitation solution;

[0028] Prepare the SS agar plate according to the conventional method, evenly spread the filter-sterilized serum on the SS agar plate, place it upside down in the incubator, and culture it anaerobically or aerobically at 37°C for 1 to 2 days. No colonies grow, indicating that the resuscitation fluid qualified;

[0029] The resuscitation solution was aliquoted and stored at -20°C for later use.

Embodiment 3

[0030] Example 3: VBNC Salmonella resuscitation

[0031] Take 2 mL of the bacteria that entered the VBNC in Example 1, centrifuge at 8000r / min for 5min, discard the supernatant, wash the thallus with sterilized physiological saline, centrifuge at 8000r / min for 5min, discard the supernatant, precipitate twice, and finally dissolve the bacterium precipitate In 300 μl of resuscitation solution prepared by bovine serum and sterilized pure water at 1:3, use a micro-sampler to blow and mix evenly, place it in the hole of a thermally programmed cycler, and set the temperature and time according to program 1 in Table 1. , to heat the bacteria; after heat treatment, pour the bacterial solution into the SS liquid medium without anaerobic culture, put it into the air bath shaking shaker, 220r / min, and cultivate for 12h;

[0032] Take 10 μl of recovered bacteria and spread evenly on the SS agar plate, place it upside down in an incubator, and incubate anaerobically at 37°C for 24 hours; r...

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PUM

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Abstract

The invention discloses a recovery liquid for VBNC salmonella, a method for preparing the same and a reclaiming method. The recovery liquid consists of 1 portion of serum and 1 to 3 portions of sterile water which are mixed and stirred evenly and are filtered for sterilization through a millipore filter. The reclaiming method comprises the following steps: placing the salmonella in a VBNC into the recovery liquid and adopting an incremental temperature raising mode in pores of a thermal program control circulator, wherein the recovery temperature rise and an incubation time program of the thermal program control circulator are at a temperature of between 5 and 37 DEG C, and the incubation time is between 2 minutes and 1.5 hours; and then inoculating the salmonella in an SS liquid culture medium, and culturing the salmonella in a gas bath shaking table at a speed of between 200 and 220 revolutions per minute. The invention overcomes the defects that the prior art uses a plurality of materials, is inconvenient to get materials and the like; the invention only uses one material to recover the salmonella which is in the VBNC for nearly one year through procedural temperature rise; and few materials are used and the operation is simple and convenient, the concentration of the recovered salmonella can reach 10<8> CFU per milliliter in 12 hours, and each recovery index (time, bacterial population and the like) is greatly improved compared with that of the prior art.

Description

technical field [0001] The invention belongs to the field of biological technology, and specifically relates to a resuscitation solution of Salmonella in a "living non-culturable state" and a preparation method and a resuscitation method thereof. Background technique [0002] The recovery of the viable non-culturable state (Viable but Non-culturable, VBNC) of bacteria refers to the process of restoring the unculturable bacteria to a culturable state, and is often regarded as the most objective evaluation of VBNC for the identification and detection of VBNC bacteria. Basic technical means. At present, the methods used for VBNC Salmonella recovery at home and abroad are mainly derived from an article entitled "Resuscitation of Salmonella enterica Serovar Typhimurium and Enterohemorrhagic Escherichia coli from the Viablebut Nonculturable State by Heat" published in Applied and Environmental Microbiology by Reissbrodt et al. -Stable Enterobacterial Autoinducer". The main mater...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12R1/42
Inventor 李影钱爱东段锐王伟利
Owner JILIN AGRICULTURAL UNIV
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