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INTRODUCTION B

The tests described in this chapter will allow determination of the absence of Burkholderia cepacia complex(Bcc),which can be detected under the conditions described.
¹ĜyԇS_BCC Ĵr™zy
The tests are designed to determine whether a substance or preparation complies with an established specification for microbiology quality and/or to evaluate whether products ----especially those for inhalation use or aqueous preparations for oral,oromucosal,cutaneous,or nasal use----contain members of the Bcc.
ԓyԇּڴ_һN|ƄǷѽ΢|˜/uaƷ؄eڷڷճĤƤwǻʹõĺˮƄǷBCC

 

GROEITH-PROMOTING AND INHIBITORY PROPERTIES OF THE MEDIA AND

SUITABILITY OF TESTS FOR ABSENCE OF Bcc
BLԼBCC m

Test each batch of ready-prepared medium and each batch of medium prepared from either dehydrated medium or ingredients.
yԇÿһAƵBԼÿһÓˮBԭƳɵB

 

Preparation of Test Strains yԇĜʂ
Use standardized stable suspensions of test strains(see Table1)NMT 5 passages removed from the original strain culture.
ʹؘʵķĜyԇþҺԭʼ겻^5 
Table1.Test Strains of Microorganisms for Growth Promotion and Suitability Testing

1 LmԜyԇľ

 

Microorganism ΢
Grow each of the test strains separately in Soy-Casein Digest Broth or on Soybean-Casein Digest Agar at 30-35 for 18-24h.
քeTSB/TSA Bÿһ30-35 18-24h.
Use Buffered sodium Chloride-peptone Solution pH7.0 or Phosphate Buffer Solution pH7.2 to make the test suspensions.Use the suspensions within 24h if stored at 2-8.If purchased,follow the suppliers introductions. If self-prepared cultures are used,follow a validated procedure (such as in Microbial Enumeration Test)for preparation. Use a challenge inoculum of NMT 100 colony-forming units(cfu) for growth promotion and suitability testing.
ʹpH7.0 Ȼc˾_ҺpH7.2 }_ҺƂҺ2o-8o 24 СrʹþҺDzُչ̵fʹBՈѭ^Cij(΢Ӌԇ)MƂʹò^100cfuMдLԇmԇ

 

NEGATIVE CONTROLS Ԍ
Include a negative control to verify the testing conditions. There must be no growth of microorganisms. A negative control is also performed when testing the products as
described in Testing of Products.
_JyԇlԌ횟o΢LڮaƷyԇڮaƷyԇ^ҲҪMԌ

 

TEST FOR GROWTH-PROMOTING PROPERTIES, SOLID MEDlA
wBĴLԇ

Perform the Surface-Spread Method (see Microbial Enumeration Test, Growth Promotion Test, Suitability of the Counting Method and Negative Controls, Suitability of the Counting Method in the Presence of Product, Recovery of Microorganisms in the Presence of Product, Plate-Count Methods), inoculating each plate with a small number(NMT 100 cfu) of the appropriate microorganism (see Table 2). Incubate at the special temperature for NMT the shortest period of time specified in the test. Growth of the microorganism comparable to that previously obtained with a previously tested and approved batch of medium occurs.
ͿҊ΢ӋLԇӋmԺԌaƷڕrӋmaƷڕr΢ƽ󷨣ӷN^100cfu ĺm΢Ҋ2ضĜضB^ضBrg̕rg΢Lcǰͨ^ǰyԇʵһB@õ΢DZ^


Table2.Microoganisms for the Growth-Promoting,Inhibitory,

and Indicative properties of the Media

2.BLƺָʾ΢



TEST FOR INHIBITORY PROPERTIES, SOLID MEDIA
wBԜyԇ
Inoculate the appropriate medium with at least 100 cfu of the appropriate microorganism. Incubate at the specified temperature for NLT the longest period of time specified in the test .Inhibition of growth of the indicated microorganisms occurs(see Table 2).
ʹ_΢ӷN100cfu mBضĜضBضBrgLrgҎ΢LҊ2


TEST FOR INDICATIVE PROPERTIESָʾԜyԇ
Perform the Surface-Spread Method (see Microbial Enumeration TestsGrowth Promotion Test, Suitability of the Counting Method and Negative Controls, Suitability of the Counting Method in the Presence of Product, Recovery of Microorganisms .In the Presence of Product, Plate-Count Methods), inoculating each plate with a small number(NMT 100 cfu) of the indicated microorganism. Incubate at the specified temperature for a period of time within the range specified in the test. Colonies are comparable in appearance and indication reactions to those previously obtained with a previously tested and approved batch of medium (see Table 2).
ͿҊ΢ӋLԇӋmԺԌaƷڕrӋmaƷڕr΢ƽ󷨣ӷN^100cfu ĺm΢Ҋ2ҎĜضԇҎķBһΕrg^ָʾcǰͨ^ǰyԇʵһB@õľஔ


Suitability of the Test Method yԇm
The ability of the test to detect Bcc in the presence of the product to be tested must be established. The incubation time for the method suitability should not exceed the shortest incubation period specified. Suitability must be confirmed if there is a change in testing performance or a change in the product that may affect the outcome of the test.
횽ڴyaƷڵr™zyBCC ԇԵBrg^ҎBrgyԇܰl׃߮aƷl˿ӰyԇY׃t횴_Jm
For each new product to be tested, perform the sample preparation as described in Testing of Products. At the time of mixing, add each test strain in the prescribed growth medium. Inoculate the test strains individually. Use a number of microorganisms equivalent to NMT 100 cfu in the inoculated test preparation.
ÿһҪyԇ®aƷծaƷyԇķMИƷʂڻϵrҎLBмÿyԇքeӷNԇڽӷNԇƄʹһЩஔڲ^100 cfu ΢
Perform the test as described in Testing of Products, using the shortest incubation period prescribed. Bcc microorganisms must be detected with the indication reactions described in Interpretation.
ծaƷyԇķʹҎBrgMМyԇBCC ܙzyڽָʾԷ
Any antimicrobial activity of the product necessitates a modification of the test procedure (see Microbial Enumeration Test, Growth Promotion Test, Suitability of the Counting Method and Negative Controls, Suitability of the Counting Method in thePresence of Product,

Neutralization/Removal of Antimicrobial Activity).
ԓaƷκοԶҪ޸ĜyԇҊ΢ӋLԇӋmԺԌaƷڕrӋm־Ե/ȥ 
 

TESTING OF PRODUCTS aƷyԇ


Sample Preparation and Pre-Incubation ƷƂAB
Prepare a sample using a 1-in-10 dilution of NLT 1 g of the product to be examined.Use 10ml or the quantity corresponding to 1 g or 1ml to inoculate a suitable amount(determined as described in Suitability of the Test method) of Soybean-Casein Digest Broth or an appropriate dilution of Soybean-Casein Digest Broth as determined during method suitability (for example ,a 1:10 dilution may be required when conducting optional testing of pharmaceutical waters). Then mix and incubate at 30o-35ofor 48-72h.
110 ϡጵIJ1g ĴyƷƂ乩ԇҺ10ml ஔ1g 1mlĺmģyԇmԴ_ԇҺӷNTSB ڷmԴ_mweTSB УMˎˮxzyrҪ110 ϡጣȻϼ30-35 B48-72h.


Selection and subculture x^mB
Subculture by streaking on a plate of Burkholderia cepacia selective agar(BCSA),and
incubate 30o-35o for 48-72h.
BCSA τ^mB30o-35o B48-72h


Interpretation
The possible presence of Bcc is indicated by the growth of greenish-brown colonies with yellow halos, or white colonies surrounded by a pink-red zone on BCSA .Any growth on BCSA is confirmed by identification tests. See Microbial Characterization,Identification,and Strain Typingfor additional information.
BCC ܴڵָʾLSɫhľGɫBCSA @ʾۼtɫ^İɫκBCSA ϵľҪͨ^byԇ_JϢҊ΢b;.
The product complies with the test if clones of the types described are not present or if the confirmatory identification tests are negative.
͵ľ䲻ߴ_JbyԇtaƷϜyԇ


RECOMMEND CULTURE MEDIA ]B
[Note-This section is given for information.][ע:݃H]The following solutions and culture media have been found satisfactory for the purposes for which they are prescribed in the tests in this Pharmacopeia . Other media may be used provided that their suitability can be demonstrated.
ҺBѱlFϱˎyԇҎĿ܉CBmtʹB


Stock Buffer Solution 侏_Һ
Transfer 34 g of potassium dihydrogen phosphate to a 1000-ml volumetric flask,dissolve in 500 ml of Purified Water adjust with sodium hydroxide to a pH of 7.20.2,add Purified Water to volume,and mix. Dispense in containers,and sterilize. Store at a temperature of 2o-8o.
34 DƵ1000 ƿܽ500 ˮÚc{pH ֵ7.20.2뼃ˮbضȞ2-8


Phosphate Butter Solution pH 7.2 pH7.2 }_Һ
Prepare a mixture of purified Water and Stock Buffer Solution (800:1 v/v),and sterilize.
ʂ伃ˮ̓侏_Һ(800:1 v/v)


Buffered Sodium Chloride-Peptone Solution pH7.0 pH7.0 Ȼc-˾_Һ
Prepare Buffered Sodium Chloride-Peptone Solution pH 7.0 as directed in table 3.Sterilize in an autoclave using a validated cycle.
3 ʾƂpH 7.0 Ȼc-˾_Һڜʹý^Cij

Table 3 3

 

Soybean-Casein Digest Broth ˴✫

Prepare Soybean-Casein Digest Broth as directed in table 4 .Adjust the pH so that after sterilization it is 7.30.2 at 25o.Sterilize in an autoclave using a validated cycle.
4 ƂTSB{pH ʹÜ25o 7.30.2.ڜʹý^Cij

Table 4 4

 

Burkholderia cepacia Selective Agar [˻¾xԭ֬
Prepare BCSA as directed in Table 5. When preparing media in-house,first prepare the base ingredients without the antibiotics. Adjust the pH so that after sterilization it is 6.80.3 at 25o.Sterilize in an autoclave using a validated cycle.Cool the base medium to 45o-50o and add a 1% solution of the sterile filtered antibiotics ,mix,and pour into the plates.
5 ƂBCSAڌҜʂBrȜʂ]пصĻɷ{pH ʹÜ25o 6.80.2.ڜʹý^CijABs45-501%ğo^VҺϺƽ

Table 5 5

 

 

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