KR19980025375A - Enhancement culture medium and culture method for rapid identification of Listeria spp. - Google Patents

Abstract

The present invention is made by adding 0.01-2% of lithium chloride and 0.0001-1.0% of nalidixic acid to a known TSBYE medium in which 0.6% yeast extract is added to trypticase soy broth. Prepared by adding 1 to 50 mM cAMP, 1 to 200 ppm of magnesium sulfate and 0.0001 to 1.0% of acriflavin to the primary culture medium for detecting Listeria spp. And the primary culture medium for detecting Listeria spp. It is to provide a secondary enhancement culture medium and a culture method for detecting the strain of Listeria genus.

Description

Enhancement culture medium and culture method for rapid identification of Listeria spp.

1 is a view showing the growth effect of the Listeria strain according to the primary and secondary enhanced culture.

Figure 2 is a view showing the growth effect of several strains according to the primary and secondary enhancement culture.

The present invention relates to an enhanced culture medium and a culture method for the rapid identification of Listeria spp., And more particularly, to a composition and a culture method of the culture medium of Listeria spp. That can be applied to rapid growth and rapid testing.

Wastewater derived from domestic wastewater, livestock slaughterhouses and other food industries is provided as a good habitat for microorganisms due to the presence of moderate pH and large amounts of organic components. Especially, the presence of pathogenic or decaying bacteria in waterborne microorganisms It also causes diseases in the human body (Pelczar, MJ, Chan ECS and Krieg, NR, Foodborne and waterborne diseases.Microbiology; Concepts and Application, International edition, McGraw Hill Inc., pp 680-714 (1993).

Listeria monocytogenes strains, which do not form gram-positive follicles, are relatively resistant to environmental conditions such as heating, drying, etc. compared to other bacteria of the same class (Boyle, DL Sofos, J. N and Schmidt, Thermal destruction of Listeria monocytogenes in meat slurry and in ground beef.J. Food Sci, 55, 327-329 (1990), is widely found in soil, water, and plants (Ref. Welshmier, II. J., Survival of Listeria monocytogenes in soil.J. Bacteriol. 80, 316-320 (1960)) Animals are also infected through feed and cause disease (Baron, EJ, Peterson.LR and Finegold). , SM, Aerobic, non-spore-forming, gram-positive bacilli.Diagnostic Microbiology, 9th edi., Mosby Inc., pp457-462 (1994). Infected through food used as In the case of normal people with cold-like symptoms, the elderly, pregnant women, and newborns with weak immunity not only have pulmonary disease but also the central nervous system, which causes fatal onset such as meningitis and brain tumors. References: Lovett, J., Listeria monocytogenes, Foodborne Bacterial Pathogens, Doyle MP (Ed), Marcel Dekker, Inc., pp283-310 (1989); Nieman, RE and Lorber, B., Listeriosis in adults: a change pattern , report of eight cases and review of the literature, Rev. Infect, Dis, 2, 207-227 (1980).

Domestic wastewater differs according to seasons and characteristics, but in general, E. coli Proteus spp. Staphylococcus spp. Clostridium spp., Salmonella spp. Large amounts of various bacteria are detected. E. coli was 1.0 × 10 ⁴ ~ 2.0 × 10 ⁵ , Clostridium perfringen was 2.0 × 10 ² ~ 2.0 × 10 ³ , and Streptococcus spp was 1.0 × 10 ⁴ ~ 5.0 × 10 ⁴ cfu / ml and L. manocytoenes were found to be 2.0 × 10 cfu / ml (Watkins, J. and Sleath, KP, Isolation and enumeration of Listeria monocytogenes from sewage, sewage sludge and river). water, Journal of Applied Bactreriology, 50, 1-9 (1981). In the case of detection of L. monocytogenes in the sample, a relatively large number of bacteria is not only inhibited by proliferation by other bacteria in enhanced culture, but also very likely to make an error in judgment due to colonization of other bacteria in plate culture.

Therefore, it is necessary to go through the growth culture step to increase the number of cells of L. monocytogenes while inhibiting the growth of other bacteria before measurement. Conventionally, the refrigeration promotion method that stores samples at 4 ° C. for at least one week in consideration of L. monocytogenes (Rodriguez, LD, Fernandez, GS, Fernandez, JF, Garayzabal and Ferri, ER, New methodology for the isolation of Listeria microorganisms from heavily contaminated environments, Appl. Environ, Microbiol., 47, 1188- 1190 (1984); Heyes, PS, Graves, LM, Ajello, GW, Swaminathan, B., Weaver, RE, Wenger, JD, Schuchat, A., Broome, CV and the Listeria study group, Comparison of cold enrichment and US department of agriculture methods for isolating Listeria monocytogenes from naturally contaminated foods, Apl, Environ, Microbiol, 57, 2109-2113 (1991)). However, this method has a problem that it takes a long time to identify the problem caused by the microorganisms.

Therefore, the present invention selects antibiotics that have little effect on the growth of L. monocytogenes and inhibit the growth of other bacteria, and on the other hand, by developing a medium and culture method that can shorten the culture time by using a growth promoter, a problem occurs. It is intended to contribute to the identification and treatment of the cause with a faster and more accurate identification method.

It is an object of the present invention to add 0.01-2% of lithium chloride and 0.0001-1.0% of nalidixic acid to a known TSBYE medium in which 0.6% yeast extract is added to trypticase soy broth. 1 to 50 mM cAMP, 1 to 200 ppm of magnesium sulfate and 0.001 to 1.0% of acriflavin were added to the primary culture medium for detecting Listeria spp. And the primary culture medium for Listeria spp. It is to provide a secondary enhancement culture medium and its culture method for detecting the Listeria strain prepared.

In the primary culture, the concentration of lithium chloride is preferably 0.1 to 2%, and the concentration of nalidic acid is preferably 0.001 to 1.0%. In the secondary culture, the concentration of magnesium sulfate is preferably 10 to 100 ppm, and the concentration of acriflavin is preferably 0.001 to 1.0%.

On the other hand, the culture method for the detection of Listeria strains is 6-14 hours by adding 7-11 volumes of secondary enhancement culture medium to 1 volume part of the enhanced culture medium obtained by culturing Listeria strains in the primary enhancement culture solution. It is characterized by culturing. At this time, the dose of the preferred culture medium is the case of adding 8-10ml of the secondary culture medium to the culture medium 0.8 ~ 1.2ml obtained after the culture using the first primary culture medium 5 ~ 100ml.

The listeria monocytogenes used in the present invention used serotype 1 and the other strains were Lactobacillus plantarum, Escherichia coli, Pseudomonas aeruginasa and Staphylococcus aureus. Each strain used a medium (TSBYE) added with 0.6% yeast extract to trypticase soy broth and incubated at 37 ° C. for 2 days.

The number of bacteria was used as standard plate culture method, but the selection medium for each strain was used. L. monocytogenes is polymycin acriflavin lithium chloride ceftazidime aescline, mannitol (PALCAM) Listeria selective agar, L. plantarum is Lactobacillus MRS agar, Staphylococcus aureus is Staphylococcus medium 110, Escherichia coli is MacConkey agar. Pseudomonas aeruginosa was used as Pseudomonas agar medium.

Example 1

Effect on Growth Factors

1) Effect of cAMP

5, 10, 20 mM cAMP was added to 5 mL of TSBYE medium, and then L. monocytogenes was adjusted to 10 ³ cells / mL of each cell in each medium and cultured at 37 ° C. The turbidity of the culture was measured at. In this case, when the absorbance value is 0.3, the number of cells corresponds to 1.0 × 10 ⁹ cells / ml.

cAMP is a promoter of metabolism in cells and is involved in various enzyme synthesis and regulates not only animal cells but also E. coli, Salmonella spp. It is known to affect the growth of gram-negative bacteria such as (Pastan, I, and Perlman, R., Cyclic adenosine monophosphate in bacteria, Science, 169, 339-344 (1970)). That is, cAMP promotes the synthesis of various enzymes involved in carbohydrate or protein metabolism such as intracellular galactokinase, glycerol kinase, and serinediaminease. Reported recovery. In the present invention, the listeria was confirmed by the effect of the concentration. As shown in Table 1, the number of initial cells was 10 ³ / mL, and colonies were measured after 7 hours of incubation at 37 ° C. As a result, the number of cells increased about 4 times than the control at 10 mM concentration.

TABLE 1 Effect of growth rate on cAMP in TSBYE culture

2) Effect of MgSO ₄

Adjust the concentration of MgSO ₄ to 0 (control), 1, 5, 10, 20, 30ppm with TSBYE medium containing 10mM cAMP, inoculate the initial cell concentration at 10 ³ / ml and incubate at 37 ℃ for a certain time. The absorbance was then measured at 620 nm.

Mg ions act as cofactors of enzymes and ATP activators involved in glucose metabolism and phosphorus metabolism of microorganisms, and are known to be important in cell division and protein synthesis. Purple and green bacteria also exist in chlorophyll and are known to be important factors for photosynthesis (Thimann, KV, Chapter IV Conditions of culture: media, ions, and temperature, The Life of Bacteria, 2th edition, Macmillan Company). , pp. 154-192 (1963).

In this experiment, the effect on Mg ion is different according to the strain and the required amount is known to be more positive than gram negative. In the case of Listeria, as shown in Table 2, the optimum concentration was 20ppm, and the number of cells after 7 hours of incubation increased about three times compared to the control group.

[Table 2] Growth rate effect on Mg ion in TSBYE culture containing 10mM cAMP

Example 2

Antibiotic Effects

Lithium chloride, nalidixic acid and acriflavin were prepared in TSBYE medium at different concentrations. L. plantarum, Ps. As aeruginosa and S. aureus, the initial bacterial count was adjusted to 10 ³ / ml. Each strain was shown in Table 3 by comparing the antibiotic effect by measuring the turbidity of the culture at 620nm using an absorbance 12 hours after inoculation.

For acriflavin Ps. Aeruginasa was the most resistant and gram positive S. aureus and L. plantarum were significantly affected. The concentration of acriflavin used in the experimental results was 0.1 mg / ml, which inhibited the growth of L. monocytogenes, and incubated for 7 hours with the initial bacterial count of 10 ³ / ml. it was not added) showed a 10 ³ / ml is less than about 50% 5.0 × 10 ³ / ml reduced the number of bacteria in.

In the case of nalidic acid, S. sureus is the most resistant and Ps. aeruginosa and L. plantarum were found to be relatively small and as a result Ps. Concentrations of more than 0.5 mg / ml were required to inhibit aeruginosa, S. aureus and L. plantarum.

LiCl is Ps. S. aureus and L. plantarum strains were more resistant than aeruginosa and were found to be resistant to three strains at concentrations above 1.0 mg / ml.

TABLE 3 Growth Inhibitory Effects on Acriflavin, Nalidic Acid and LiCl in Several Strains

Example 3

Selection of the Promotion Culture Act

The promotion culture method was divided into 1st and 2nd. In the primary enhancement culture, 10 ml of a culture solution containing LiCl (1.0 mg / ml) nalidic acid (0.5 mg / ml) in TSBYE was incubated for 8 hours. The initial bacterial count was 2 / ml for Listeria monocytogenes, 2.0 × 10 ⁵ / ml for Lactobacillus plantarum, 2.6 × 10 ² / ml for Escherichia coli, and Staphylococcus. Aureus was 1.1 × 10 ² / ml and Pseudomonas aeruginosa was 8 × 10 ¹ / ml. The control group was cultured only in L. monocytogenes in TSBYE.

In secondary culture, 9 ml of TSBYE mixed with cAMP (10 mM), MgSO ₄ (20 ppm), LiCl (1.0 mg / ml), nalidic acid (0.5 mg / ml), and acriflavin (0.05 mg / ml) Cells were counted 2, 4 and 6 hours after 1.0 ml of primary culture was transplanted.

In the present invention, the use of selective antibiotics is used to shorten the enhancement culture time. In the first propagation culture, the applicability to the culture medium is increased, and then the acriflavin is added to the secondary culture medium to increase the selectivity. L. monocytogenes is shown in Fig. 1, in L. palntarum, S. aureus, E. coli and Ps. aeruginosa is shown in Figure 2, respectively.

As shown in FIG. 1, up to 8 hours of the primary enhancement culture time did not show a significant difference from the pure culture control group, but the growth rate of the acriflavin was decreased during the secondary enhancement culture (the control group was TSBYE). Only L. monocytogenes was cultured in the culture medium, Sample 1 was cultured only in Listeria in the invented culture, and Sample 2 means mixed culture with Listeria and 4 other strains.

However, as shown in Figure 2, it can be seen that it is much higher than the growth rate of other strains. At this time, L. plantarum and E. coli decreased in secondary culture and S. aureus had bacteriostatic effect in Ps. aeruginose showed some proliferation, respectively. Immunization with secondary culture media is considered to be a more efficient way of promoting culture with increased selectivity and a more abundant supply of nutrients. In this experiment, it took about 14 hours for Listeria to reach 10 ⁶ / ml in mixed culture with other bacteria.

Claims (8)

Listeria strain consisting of 0.01-2% lithium chloride and 0.0001-1.0% Nalidixic acid added to a known TSBYE medium containing 0.6% yeast extract in trypticase soy broth Primary enhancement culture for detection. The method according to claim 1, wherein the concentration of lithium chloride is the primary culture medium for the detection of Listeria strains, characterized in that 0.1 to 2%. According to claim 1, wherein the concentration of nalidic acid is a primary enhancement culture medium for detecting Listeria strains, characterized in that 0.001 ~ 1.0%. 2 for the detection of Listeria strain prepared by adding 1-50 mM cAMP, 1-200 ppm magnesium sulfate and 0.0001-1.0% of acriflavin to the primary enhancement culture medium for detecting the Listeria strain of claim 1 Primary enhancement culture. According to claim 4, wherein the concentration of magnesium sulfate secondary culture for the detection of Listeria strains, characterized in that 10 ~ 100ppm. The secondary enhancement culture medium for detecting Listeria strains according to claim 4, wherein the concentration of acriflavin is 0.001 to 1.0%. 6 to 14 hours of culture medium for 6 days to the culture of the Listeria strain to detect the list of strains of Listeria strains 7 to 11 parts of the secondary culture medium is added for 6 hours to the culture medium for 6 to 14 hours A culture method for detecting Listeria spp. Characterized by Sikkim. 8. The method of claim 7, wherein the amount of the enhancement culture medium is detected in Listeria genus, characterized in that 8-10 ml of the secondary culture medium is added to 0.8-1.2 ml of the culture medium obtained after the culture using the primary culture medium 5-100 ml. Culture method for.