JP2011244761A - Medium for detecting enterobacter sakazaki bacteria - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a medium for detecting Enterobactor sakazaki bacteria in an easy-to-use method.SOLUTION: Provided is a medium for detecting Enterobactor sakazaki bacteria, which contains: an α-glucosidase substrate having a detectable blue free radical; and a β-galactosidase substrate having a detectable red free radical.

Description

  The present invention relates to a medium used for detection of Enterobacter sakazaki.

  Enterobacter sakazakii (now Cronobacter sp.) Is a gram-negative yellow pigment-producing gonococci and is known as a bacterium that causes bacteremia, bacterial meningitis, and necrotizing enterocolitis in infants (See Non-Patent Document 1). Although this fungus is widely distributed in nature, especially for infant foods such as powdered milk, the eaters themselves have a high risk factor, and the current manufacturing and processing techniques are aseptic for infants. The control of this bacterium is important from the viewpoint of food hygiene and safety because it is impossible to produce milk powder.

  A general method for detecting enterobacter Sakazaki in infant formula recommended by the FDA (US Food and Drug Administration) is to incubate the specimen with EE broth (Enterobacteriaceae enrichment broth) at 36 ° C overnight. Violet red bile glucose agar (VRBG agar) is smeared or streaked, cultured at 36 ° C overnight, confirmed to be a typical colony, then transplanted to trypsoy agar medium and cultured at 25 ° C for 48-72 hours, producing yellow pigment Is identified with a simple identification kit (see Non-patent Document 2), but it is estimated that it takes at least 3 to 4 days to perform estimation judgment, and at least 5 days to complete judgment. This is a time consuming method.

  Further, as an Enterobacter Sakazaki bacteria isolation medium using a chromogenic enzyme substrate, a chromogenic plating medium or a commercially available medium for identification of Enterobacter Sakazaki bacteria described in Patent Document 1 (Cromold Enterobacter Sakazaki Agar, Merck Ltd.) ) The medium described in Patent Document 1 is a combination of a saccharide and an α-glucosidase substrate, and a combination of these with a cellobiase substrate, and the commercially available medium is an α-glucosidase substrate used alone. However, intestinal bacteria other than Enterobacter sakazaki bacteria include bacteria that use α-glucosidase substrates, so these media are supplemented with antibiotics and a culture temperature of about 45 ° C to improve selectivity. The operation was complicated.

Special table 2008-545382 gazette

Patrick Druggan and Carol Iversen, Culture media for the isolation of Cronobacter spp.International Journal of Food Microbiology, 136 (2009) 169-178. U.S. Food and Drug Administration, Isolation and Enumeration of Enterobacter sakazakii from Dehydrated Powdered Infant Homepage http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/ucm114665.htm

  It is an object of the present invention to provide a medium capable of detecting Enterobacter Sakazaki bacteria by distinguishing from other bacteria with a simple operation.

  In view of such circumstances, the present inventor has conducted intensive research. First, Enterobacter Sakazaki belongs to the coliform group and produces enzymes similar to other coliform groups, such as α-glucosidase and β-galactosidase, so it is difficult to develop a separation medium using a chromogenic enzyme substrate. It was. However, as a result of various studies, it was quite surprising that by combining an α-glucosidase substrate having a detectable blue free group and a β-galactosidase substrate having a detectable red free group, Enterobacter The present inventors have found that Sakazaki bacteria can be detected as blue colored colonies, and other coliform groups can be detected as red colored colonies.

That is, the present invention provides a medium for detecting Enterobacter Sakazaki containing an α-glucosidase substrate having a detectable blue free group and a β-galactosidase substrate having a detectable red free group. is there.
The present invention also provides a method for detecting Enterobacter sakazaki, which comprises inoculating and culturing a specimen on this medium and observing the presence or absence of blue colonies on the medium.

  By using the culture medium of the present invention, the present invention can easily detect Enterobacter Sakazaki bacteria as distinguished from other coliforms.

Examples of the α-glucosidase substrate having a detectable blue free group for use in the present invention include α-glucopyranoside or a salt thereof capable of releasing a blue chromogenic compound. Bromo-4-chloro-3-indoxyl-α-D-glucopyranoside, 5-bromo-3-indoxyl-α-D-glucopyranoside, 4-chloro-3-indoxyl-α-D-glucopyranoside, 3-India Examples include xyl-α-D-glucopyranoside, 6-bromo-2-naphthyl-α-D-glucopyranoside.
Of these, 5-bromo-4-chloro-3-indoxyl-α-D-glucopyranoside is preferred.
The concentration of the α-glucosidase substrate having a detectable blue free group is preferably 0.001 g / L to 5 g / L at detection, particularly 0.01 g / L to 0.25 g / L, and more preferably 0.05 g / L to 0.15 g. / L is preferred.

The β-galactosidase substrate having a detectable red free group includes β-galactopyranoside or a salt thereof capable of releasing a red chromogenic compound, specifically, for example, 5-bromo- 6-chloro-3-indoxyl-β-D-galactopyranoside, 6-chloro-3-indoxyl-β-D-galactopyranoside, 5-iodo-3-indoxyl-β-D-galacto And pyranoside.
Of these, 5-bromo-6-chloro-3-indoxyl-β-D-galactopyranoside is preferred.
The concentration of the β-galactosidase substrate having a detectable red free group is preferably 0.001 g / L to 5 g / L at the time of detection, particularly 0.01 g / L to 0.25 g / L, more preferably 0.05 g / L to 0.15 g. / L is preferred.

  The medium of the present invention preferably further contains inorganic salts from the viewpoint of improving the growth of Enterobacter sakazaki and coliforms. Examples of such inorganic salts include sodium chloride, disodium hydrogen phosphate, potassium nitrate and the like, and among these, potassium nitrate is preferable. The concentration of the inorganic salts is preferably 0.01 g / L to 10 g / L at detection, particularly preferably 0.1 g / L to 5 g / L, and more preferably 0.5 g / L to 2 g / L.

Furthermore, it is preferable that the medium of the present invention contains a bile salt and / or an ionic surfactant from the viewpoint of suppressing Gram-positive bacteria. Examples of such bile salts include sodium cholate, sodium deoxycholate, and sodium taurocholate, and examples of the ionic surfactant include sodium dodecyl sulfate (SDS), Tergitol 4, and Tergitol 7. The concentration of the bile salt is preferably 0.01 g / L to 10 g / L at detection, particularly preferably 0.1 g / L to 3 g / L, and more preferably 0.5 g / L to 1.5 g / L. The concentration of the ionic surfactant is preferably 0.001 g / L to 10 g / L at detection, particularly preferably 0.01 g / L to 5 g / L, and more preferably 0.1 g / L to 0.5 g / L.
It is preferable that the medium of the present invention further contains components usually used in the medium, for example, amino acids such as tryptophan, organic acid salts such as sodium pyruvate, nutrients such as yeast extract, vitamins and the like.

  The form of the medium of the present invention is not particularly limited, and an agar medium, a liquid medium, a simple medium described in JP-A-8-140664, and the like can be used.

In the method of the present invention, the medium of the present invention is inoculated with a specimen and cultured, and the presence or absence of blue colonies on the medium is observed to detect Enterobacter sakazaki. By using the medium of the present invention, The bacteria can be clearly detected by two different colors such that Bacter sakazaki bacteria can be detected by blue colonies and the total number of coliforms can be detected by the sum of blue colonies and red colonies.
In addition, since β-galactosidase is used preferentially in coliforms other than Enterobacter Sakazaki, β-galactosidase is preferentially used. Enterobacter Sakazaki uses both substrates, but the blue color derived from the α-galactosidase substrate is stronger, so even if both are used at the same time, only a deep blue color is obtained. Therefore, according to the method of the present invention, Enterobacter Sakazaki bacteria can be clearly detected even in E. coli groups other than Enterobacter Sakazaki bacteria.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these.
Example 1
Preparation of Medium Each medium having the composition shown in Table 1 was prepared as follows.

  The medium of the present invention is added to 1 liter of purified water in a volume of 1 liter, sterilized by autoclaving at 121 ° C for 15 minutes, cooled to about 50 ° C, and then dispensed into a plastic petri dish (90 mm) at a rate of 20 ml. It left still until it hardened, and the culture medium for enterobacter Sakazaki bacteria detection of this invention was produced. As a control, 5-bromo-6-chloro-3-indoxyl-β-D-galactopyranoside was removed from the culture medium of the present invention, and an α-glucosidase substrate was used in the same manner.

X-GAL agar medium is dissolved in 1 liter of purified water in 1 liter of purified water, sterilized by autoclaving at 121 ° C for 15 minutes, cooled to about 50 ° C, and then dispensed in 20 ml portions into a plastic petri dish (90 mm). The culture medium was allowed to stand until it hardened, dispensed in 20 ml portions into a plastic petri dish, and allowed to stand until the culture medium solidified.
VRBG agar medium (Difco & BBL Manual, Manual of Microbiological Culture Media 2003, Becton Dickinson and Company pages 613-615) add 1 liter to 1 liter of purified water, dissolve at 100 ° C for 20 minutes, and stir well. Thereafter, 20 ml each was dispensed into a plastic petri dish (90 mm) and allowed to stand until the medium was solidified.
Tryptosoy agar medium (TSA) is added to 1 liter of purified water in a volume of 1 liter, autoclaved at 121 ° C for 15 minutes under high-pressure steam sterilization, and then dispensed in 20 ml portions into a plastic petri dish (90 mm), until the medium solidifies I put it.

Test of the strain The test strain was pre-cultured with tryptic soy bouillon for 24 hours, and this was diluted 10-fold with sterile 0.05% agar-saline and microplanter method (Sakuma Seisakusho website: http: // www .sakumajp.com / category / 1220255.html) and the Miles-Misra method (New Bacterial Culture Studies Course-up- <Second Sales> pages 182-192, Modern Publishing Co., Ltd., 1986) were inoculated.

  Each strain was tested using a microplanter and cultured at 35 ° C. and 42 ° C. for 24 hours. The results are shown in Table 2.

  In the culture medium of the present invention, good growth of Enterobacter Sakazaki bacteria and blue to black color development were observed. In addition, coliforms other than Enterobacter Sakazaki bacteria were found to form a red-purple colony. Furthermore, the detection ability of Enterobacter Sakazaki was the same in the 35 ° C culture and the 42 ° C culture, but in the 35 ° C culture, the coliform group formed a red colony and was found to show good growth. In addition, in the control product to which the α-glucosidase substrate was added alone, it was confirmed that K. pneumoniae, which is an Escherichia coli group other than Enterobacter Sakazaki, exhibited a blue color.

Example 2
Each strain was tested and cultured at 35 ° C. and 42 ° C. for 24 hours by the Miles-Misra method. The results are shown in Table 3.

  As shown in Table 3, good growth of Enterobacter Sakazaki bacteria and blue to black color development were observed in the culture medium of the present invention. In addition, coliforms other than Enterobacter Sakazaki bacteria were found to form a red-purple colony.

Claims (7)

  A culture medium for detecting Enterobacter sakazaki comprising an α-glucosidase substrate having a detectable blue free group and a β-galactosidase substrate having a detectable red free group.   2. The medium according to claim 1, wherein the α-glucosidase substrate having a detectable blue free group is α-glucopyranoside or a salt thereof capable of releasing a blue chromogenic compound.   The medium according to claim 1 or 2, wherein the β-galactosidase substrate having a detectable red free group is β-galactopyranoside or a salt thereof capable of releasing a red chromogenic compound.   Furthermore, the culture medium of any one of Claims 1-3 which contains inorganic salts.   The medium according to any one of claims 1 to 4, further comprising a bile salt and / or an ionic surfactant.   A method for detecting Enterobacter sakazaki, comprising inoculating a sample according to any one of claims 1 to 5 and culturing the sample, and observing the presence or absence of a blue colony on the medium.   Inoculating and cultivating the sample in the medium according to any one of claims 1 to 5, observing the presence or absence of blue colonies on the medium and observing the total number of coliforms and the sum of blue and red colonies A method for detecting Enterobacter Sakazaki and total coliforms.