JP2002253217A - Semisynthetic agar medium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a blood component-free semisynthetic agar medium capable of selecting a colony of spawn for producing pertussis vaccine and of culturing the spawn and detecting a Bordetella pertussis colony infected on the respiratory organ of a mammal. SOLUTION: This semisynthetic agar medium comprises Cohen-Wheeler liquid medium constituents, a plant-derived peptone, carbon powder and agar-agar, being characterized by containing no blood component.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a semi-synthetic agar medium suitable for selecting a seed strain for producing a pertussis vaccine and detecting B. pertussis.

[0002]

2. Description of the Related Art Vaccines are widely used to prevent infection with pathogenic microorganisms. In the production of vaccines against pathogenic bacteria such as whooping cough, these pathogenic bacteria are cultured and all the obtained cells are inactivated and used (killed cell vaccine). Is used as a vaccine material. For whooping cough, pertussis fibrous hemagglutinin (FH)
A), pertussis toxin (PT), pertussis outer membrane protein (69K-OMP),
Take out protective antigens such as pertussis cilia (Fimb)
A purified pertussis component vaccine from which endotoxin (ET) has been removed has also been put to practical use.

[0003] The BG (Bordet-Gengou) medium (Procedure for Bacteriology, University of Tokyo Institute of Infectious Diseases, Alumni Association, P.80, 1958) used for the preparation of inoculum for producing pertussis vaccine produces bovine blood. In recent years, it has become a concern to remove animal-derived raw materials due to the problem of beef scrapie (mad cow disease). In addition, since the BG medium has poor growth ability of the bacteria, it is necessary to repeat the passage for several times after selecting the colonies of the bacteria in order to obtain a large number of seeds for production. Was required.

On the other hand, semi-synthetic raw materials
Cohen-Wheeler Coal-Wheeler Semi-Synthetic Agar Medium (Proposal for Bacteriology, Academic Society of the Institute of Infectious Diseases, The University of Tokyo, Japan)
P.81, 1958), but the detection sensitivity of bacterial colonies was about 60% as compared with the BG medium, and it could not be used for colony selection of inoculum for production.

As a test method for pertussis vaccine,
The vaccine immunized mice will be infected with B. pertussis, and efficacy assessment based on the number of B. pertussis detected from the respiratory tract is being standardized by the WHO. The culture medium used for B. pertussis used here is BG
Medium has been used, but BG medium is used to
Since the preparation was performed with the addition of 20%, much labor was required for the preparation, which hindered the progress of the experiment.

Therefore, an attempt was made to use a Cohenwiller charcoal powder semi-synthetic agar medium (Proposal for Bacteriology, Gakuyukai, Institute for Infectious Diseases, University of Tokyo, p. 81, 1958) instead of the BG medium.
Compared with the G medium, the Coenwiller charcoal powder-added semi-synthetic agar medium was inferior in sensitivity of detecting colonies of bacteria to about 60%, so that it could not be used for measuring the number of pertussis bacteria in the respiratory tract infection of mice.

[0007]

SUMMARY OF THE INVENTION Beef scrapie, etc.
B to which animal blood was added for the purpose of eliminating unknown risk factors
There is a need for a semi-synthetic agar medium that can be used as a medium for selecting B. pertussis without blood components and that has improved sensitivity for detecting bacterial colonies, instead of the G medium.

Further, a semi-synthetic agar medium which can be used for producing pertussis using a semi-synthetic material instead of the BG medium, and which can reduce the number of passages and the cost of producing the inoculum by improving the growth property is provided. It has been demanded.

Further, it can be used for the measurement of the number of infectious bacteria in the respiratory tract of mice, which is about to be standardized by WHO, uses a semi-synthetic material instead of BG medium supplemented with fresh bovine blood, and uses B. pertussis. There is a need for a semi-synthetic agar medium with improved sensitivity for detecting colonies.

[0010]

Means for Solving the Problems In view of the above problems, the present inventors have conducted intensive studies and, as a result, have established the technology of a semi-synthetic medium containing no blood components, and have further studied. The present inventors have found that the proliferative power of B. pertussis and the detection sensitivity of B. pertussis colonies can be improved by examining and improving additives to complete the present invention.

[0011] That is, the present invention provides: 1) a semi-synthetic agar containing the components of a Cohenwiller liquid medium, plant-derived peptone, carbon powder (charcoal) and agar (agar), and containing no blood components. 2) The medium according to 1), which is used for culturing B. pertussis; 3) The medium according to 1), wherein the plant-derived peptone is soy protein-derived peptone; 4) At least one selected from sodium glutamate, nicotinamide and cystine 5) The medium according to 1) above, which does not contain glutathione and iron sulfate; 6) the medium according to 1) above, which contains 0.3 to 2.0 w / v% of plant-derived peptone; 7) The medium according to 1), which contains 0.06 to 0.5 w / v% of carbon powder; 8) the medium according to 1), which contains 0.9 to 1.8 w / v% of agar; 9) Glutamine 10) The medium according to 4), which contains 0.01 to 0.03 w / v% of sodium; 10) the medium according to 4), which contains 0.002 to 0.004 w / v% of nicotinamide; 11) 0.002 to 0.004 w / v of cystine. 12) 0.3 to 2.0 w / v% of peptone derived from soy protein;
Cystine 0.002 to 0.004 w / v%, sodium glutamate 0.01 to 0.03 w / v%, nicotinamide 0.002 to 0.004 w / v%, carbon powder 0.06 to 0.5 w / v%, and agar
13) The medium according to 1), which is used for selecting a seed strain for producing a pertussis vaccine; and 14) The medium according to 1), which contains 0.9 to 1.8 w / v%. 15) A method for improving the growth ability of B. pertussis, which comprises culturing B. pertussis using the medium according to 1); 16) using the medium according to 1). The present invention relates to a method for improving the detection sensitivity of B. pertussis colonies, which comprises culturing B. pertussis.

"Components of the Cohenwiller liquid medium" are defined as Pathogenesis and immunity inpertussis (John Wi
ley & Sons) on page 23. Casamino acids, sodium chloride, potassium dihydrogen phosphate, magnesium chloride, soluble starch, yeast extract, calcium chloride, copper sulfate, iron sulfate and trisaminomethane. The amounts of these components are as described in the document, that is, 10 g of casamino acid, 2.5 g of sodium chloride, 0.5 g of potassium dihydrogen phosphate, 0.4 g of magnesium chloride per liter of medium.
g, soluble starch 1.5 g, yeast extract 12.5 g,
The amount may be 0.01 g of calcium chloride, 0.0005 g of copper sulfate, 0.001 g of iron sulfate and 3.0 g of trisaminomethane, but the amount may be appropriately increased or decreased, and the colony detection sensitivity and proliferation of B. pertussis may be increased. One or more components can be deleted as long as they do not adversely affect the properties and the like, and one or more additives can be added.

The "plant-derived peptone" includes, for example, soybean-derived peptone.

"Blood components" are animal defibrillated blood, serum,
It refers to plasma, whole blood, etc., and does not mean a single compound that can be separated by purifying blood, for example, amino acids, sodium chloride and the like.

"Semi-synthetic agar medium containing the components of Cohenwiller liquid medium, plant-derived peptone, carbon powder and agar, and not containing blood components" includes casamino acid, sodium chloride, phosphate Potassium dihydrogen, magnesium chloride, soluble starch, yeast extract,
Calcium chloride, copper sulfate, at least five or more components selected from iron sulfate and trisaminomethane, preferably seven or more, more preferably all ten components,
It is a semi-synthetic agar medium containing plant-derived peptone, carbon powder and agar, and no blood components. Additives may be added as needed. 0.3 to 2.0 of plant-derived peptone
w / v% is preferable, and carbon powder is 0.06 to 0.5 w / v
%, And agar is preferably contained at 0.9 to 1.8 w / v%.

Examples of the additives include sodium glutamate, nicotinamide, and cystine. It is preferable to add one or more selected from these, preferably two, more preferably all three. good. Sodium glutamate is 0.01 to 0.03 w / v%,
Nicotinamide is 0.002-0.004 w / v%, cystine is
The content is preferably 0.002 to 0.004 w / v%.

Preferred compounds not contained in the medium include glutathione, iron sulfate and the like. By not containing these compounds, the medium components can be sterilized by heating, and the productivity of the semi-synthetic agar medium can be improved.

The components of the semi-synthetic agar medium are casamino acid 0.5 to 2.0 w / v%, sodium chloride 0.2 to 0.6
w / v%, potassium dihydrogen phosphate 0.03 to 0.08 w / v%, magnesium chloride 0.02 to 0.08 w / v%, soluble starch 0.05
To 0.3 w / v%, yeast extract 0.1 to 0.4 w / v%, calcium chloride 0.0005 to 0.002 w / v%, copper sulfate 0.00005 to
0.00015w / v%, soy protein-derived peptone 0.3 to 2.0w
/ v%, carbon powder 0.06-0.5w / v%, agar 0.9-1.8w / v
%, Trisaminomethane 0.3 to 0.9 w / v%, sodium glutamate 0.01 to 0.03 w / v%, nicotinamide 0.
002 to 0.004 w / v% and cystine 0.002 to 0.004 w / v
v%, especially casamino acid 1.2 w / v
%, Sodium chloride 0.25w / v%, potassium dihydrogen phosphate 0.0
5w / v%, magnesium chloride 0.04w / v%, soluble starch 0.1
5w / v%, yeast extract 0.2w / v%, calcium chloride 0.001w / v
%, Copper sulfate 0.000075w / v%, soy protein-derived peptone 0.5
w / v%, carbon powder 0.125 w / v%, agar 1.35 w / v%, trisaminomethane 0.6 w / v%, sodium glutamate 0.02 w / v%, nicotinamide 0.003 w / v% and cystine 0.003 w / More preferably, it contains v%.

If necessary, an antibiotic may be added to the medium of the present invention. Examples of such an antibiotic include cephem antibiotics (for example, cephalexin, cefaclor,
Cefloxazine, etc.), penicillin antibiotics (eg,
Amoxicillin, ampicillin, cyclacillin, etc.),
Tetracycline antibiotics (eg, doxycycline, minocycline, etc.) and the like. The compounding amount may be any range that does not affect the growth of B. pertussis and that can suppress the growth of various bacteria and improve the detection of B. pertussis colonies. When is used, 10 to 30 μg / mL is preferable, and 10 to 15 μg / mL is particularly preferably used.

The medium of the present invention is excellent in terms of improvement in detection sensitivity and proliferative properties, and is used as a medium for producing pertussis vaccine, selecting inoculum, and detecting B. pertussis in mammalian respiratory organs. It is advantageous to use. Further, by culturing B. pertussis using the medium of the present invention, it is possible to improve the proliferation power of B. pertussis and the detection sensitivity of B. pertussis colonies.

Here, mammals include humans, monkeys, dogs,
Rabbits, rats, guinea pigs, mice and the like can be mentioned, and among them, mice which are to be standardized in the WHO evaluation system are preferably used.

The pertussis bacterium (Bordetella pertussis) used in the present invention includes pertussis fibrous hemagglutinin, pertussis outer membrane protein, pertussis cilia or pertussis toxin, which are protective components of pertussis-derived infection.
There is no particular limitation as long as one or more types can be produced. For example, pertussis I phase Higashihama strain [Infection and Immunity (Infection and Immunity)
6, Volume 89, (1972)] (The strain was stored at the National Institute of Health. (NIHJ 1052), and deposited on August 13, 1980 at the Fermentation Research Institute, under the accession number IFO 14073. ), Pertussis I phase Yamaguchi strain, pertussis I phase
Known strains such as 18-323 strain and 165 pertussis I phase bacterium can be mentioned. Above all, H. pertussis I phase Higashihama strain (IFO 14073) is conveniently used in terms of productivity.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in more detail with reference to the following examples, which, however, are not intended to limit the scope of the present invention. Good. In the following examples, "medium before improvement-1", "medium before improvement-2" and "medium of the present invention"
Was adjusted with the following composition per liter of medium. Medium component before improvement -1 Casamino acid 10.0 g (1.0 w / v%) Sodium chloride 2.5 g (0.25 w / v%) Potassium dihydrogen phosphate 0.5 g (0.05 w / v%) Magnesium chloride 0. 4g (0.04w / v%) Soluble starch 1.5g (0.15w / v%) Yeast extract 12.5g (1.25w / v%) Calcium chloride 0.01g (0.001w / v%) Copper sulfate 0.00075g ( 0.000075 w / v%) Iron sulfate 0.001 g (0.0001 w / v%) Trisaminomethane 3.0 g (0.3 w / v%) Carbon powder 5.0 g (0.5 w / v%) Agar 20.0 g (2.0 w / v%) Medium component before improvement -2 Casamino acid 10.0 g (1.0 w / v%) Sodium chloride 2.5 g (0.25 w / v%) Potassium dihydrogen phosphate 0.5 g (0.05 w / v%) Magnesium chloride 0.4 g (0.04 w / v%) Soluble starch 1.5 g (0.15 w / v%) Yeast extract 2.0 g (0.2 w / v%) Calcium chloride 1g (0.001w / v%) Tryptophan 0.001g (0.0001w / v%) Cystine 0.025g (0.0025w / v%) Copper sulfate 0.0005g (0.00005w / v%) Iron sulfate 0.01g (0.001w / v%) Peptone derived from milk casein 5.0 g (0.5 w / v%) Carbon powder 5.0 g (0.5 w / v%) Agar 20.0 g (2.0 w / v%) Semi-synthetic agar medium of the present invention Casamino acid 12.0 g (1.2 w / v%) Sodium chloride 2.5 g (0.25 w / v%) Potassium dihydrogen phosphate 0.5 g (0.05 w / v%) Magnesium chloride 0.4 g (0.04 w / v%) Soluble Starch 1.5g (0.15w / v%) Yeast extract 2.0g (0.2w / v%) Calcium chloride 0.01g (0.001w / v%) Copper sulfate 0.00075g (0.000075w / v%) Trisaminomethane 6.0 g (0.6 w / v%) Peptone derived from soy protein 5.0 g (0.5 w / v%) Carbon powder 1.25 g (0.125 w / v%) Agar 13 5g (1.35w / v%) sodium glutamate 0.2g (0.02w / v%) Nicotinamide 0.03g (0.003w / v%) cystine 0.03g (0.003w / v%)

[0024]

[Example] Example 1 A 0.02 mL (about 200 CFU / dish) of the same dilution was inoculated on a plate medium obtained by dispensing 15 mL of each of the above-mentioned culture media into a 9 cm-diameter dish, and cultured at 35 ° C for 4 days. Was counted, and the ratio to the number of colonies detected in the BG medium was evaluated. The detection rate of bacterial colonies in the unmodified Cohenwiller semi-synthetic agar medium (before modification-1) prepared by adding carbon powder and agar to the Cohenwiller liquid medium is 26.3% of that of the BG medium. The detection rate of the bacterial colonies in the medium (before modification-2) by the preparation method described in the summary was 62.9% of that of the BG medium. On the other hand, the detection rate in the semi-synthetic agar medium of the present invention was 94.2%, which was not significantly different from the BG medium, and showed the same bacterial colony detection rate.
In addition, 10 bil of B. pertussis was inoculated on a plate medium prepared by dispensing 200 mL of each of the above-mentioned media in a Le-type culture bottle, and cultured at 35 ° C. for 48 hours. The ratio to the amount of bacteria grown on the medium was evaluated. Unmodified Cohenwiller semi-synthetic agar medium prepared by adding carbon powder and agar to Cohenwiller liquid medium (before modification-1)
Was 280% with respect to the BG medium, and the growth rate in the medium (before improvement-2) according to the preparation method described in the Proposal for Bacteriology was 347% of the BG medium. In contrast, the growth rate in the semi-synthetic agar medium of the present invention is 478%,
It showed the highest proliferation.

[Table 1]

Example 2 Seed cells (2 inoculated at 10 bil, cultured at 35 ° C. for 2 days) with the semi-synthetic agar medium of the present invention dispensed into a culturing bottle.
, 7th, and 10th passage), the growth of the bacteria pre-cultured and main-cultured is the same as that of the BG medium, and the serotype of the passage has mutations within the range of investigation up to the 10th generation. Did not. In addition, the production of B. pertussis infection protective antigen was equivalent to that of the BG medium.

[Table 2]

Example 3 In the semi-synthetic agar medium of the present invention, a large amount of inoculum for production can be obtained by one colony selective culture in two subcultures, and long-term production using the same lot of inoculum can be performed. Made it possible. Therefore, the obtained bacterial solution was dispensed into vials containing 27 mL of the Japanese Pharmacopoeia, and the stability up to 60 months after storage at −70 ° C. was investigated and confirmed. As a result, the inoculum produced on the semi-synthetic agar medium of the present invention had both a viable cell rate and an increased amount of the inoculum by preliminary culture (inoculation of 1 bil / mL inoculum in a Cohen Willer liquid medium at 35 ° C. for 24 hours with stirring). It was confirmed that there was no tendency to decrease due to storage, and it was stable.

[Table 3]

[0027]

According to the present invention, unknown risk factors such as bovine scrapie can be avoided by using the semi-synthetic agar medium of the present invention. In addition, the growth ability of B. pertussis and the detection sensitivity of B. pertussis colonies can be improved, so that the experiment of spraying B. pertussis in mice can be efficiently performed. According to the present invention, the time, equipment, and man-hours required for inoculum production can be significantly reduced.

Claims (16)

[Claims] (1) A semi-synthetic agar medium comprising a Cohenwiller liquid medium component, plant-derived peptone, carbon powder and agar, and free of blood components. 2. The medium according to claim 1, which is used for culturing B. pertussis. 3. The medium according to claim 1, wherein the plant-derived peptone is a soy protein-derived peptone. 4. The medium according to claim 1, which contains at least one selected from sodium glutamate, nicotinamide and cystine. 5. The medium according to claim 1, which does not contain glutathione and iron sulfate. 6. The medium according to claim 1, comprising 0.3 to 2.0 w / v% of plant-derived peptone. 7. The medium according to claim 1, containing 0.06 to 0.5 w / v% of carbon powder. 8. The medium according to claim 1, comprising 0.9 to 1.8 w / v% of agar. (9) sodium glutamate in an amount of 0.01 to 0.03;
The medium according to claim 4, which contains w / v%. 10. A nicotinamide containing 0.002 to 0.004 w /
The medium according to claim 4, which contains v%. 11. The medium according to claim 4, containing 0.002 to 0.004 w / v% of cystine. 12. A soy protein-derived peptone 0.3 to 2.0.
w / v%, cystine 0.002 to 0.004 w / v%, sodium glutamate 0.01 to 0.03 w / v%, nicotinamide 0.0
2. The medium according to claim 1, comprising 02 to 0.004 w / v%, carbon powder 0.06 to 0.5 w / v%, and agar 0.9 to 1.8 w / v%. 13. The medium according to claim 1, which is used for selecting a seed strain for producing a pertussis vaccine. 14. The medium according to claim 1, which is used for detecting B. pertussis in a mammal's respiratory tract. 15. A method for improving the proliferation of B. pertussis, comprising culturing B. pertussis using the medium according to claim 1. 16. A method for improving the detection sensitivity of B. pertussis colonies, comprising culturing B. pertussis using the medium according to claim 1.