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INTRODUCTION ½éÉÜ

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.
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The tests are designed to determine whether a substance or preparation complies with anestablished specification for microbiology quality and/or to evaluate whetherproducts ----especially those for inhalation use or aqueous preparations for oral,oromucosal,cutaneous,or nasal use----contain members of the Bcc.
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GROEITH-PROMOTING AND INHIBITORY PROPERTIES OF THE MEDIA AND

SUITABILITY OF TESTS FOR ABSENCE OF Bcc
ÅàÑø»ù´ÙÉú³¤ºÍÒÖÖÆ×÷ÓÃÒÔ¼°BCC ·½·¨ÊÊÓÃÐÔ

Test each batch of ready-prepared medium and each batch of medium prepared from either dehydrated medium or ingredients.
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Preparation of Test Strains ²âÊÔ¾úÖêµÄ×¼±¸
Use standardized stable suspensions of test strains(see Table1)NMT 5 passages removed from the original strain culture.
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Table1.Test Strains of Microorganisms for Growth Promotion and Suitability Testing

±í1 ´ÙÉú³¤¼°ÊÊÓÃÐÔ²âÊԵľúÖê

Microorganism ΢ÉúÎï
Grow each of the test strains separately in Soy-Casein Digest Broth or on Soybean-Casein Digest Agar at30¡æ-35¡æfor 18-24h.
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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¡æ-8¡æ.Ifpurchased,follow the supplier¡¯s introductions. If self-prepared cultures are used,follow a validated procedure (such as in Microbial Enumeration Test)for preparation. Use achallenge inoculum of NMT 100 colony-forming units(cfu) for growth promotion and suitability testing.
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NEGATIVE CONTROLS ÒõÐÔ¶ÔÕÕ
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.
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TEST FOR GROWTH-PROMOTING PROPERTIES, SOLID MEDlA
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Perform the Surface-Spread Method (see Microbial Enumeration Test, GrowthPromotion Test, Suitability of the Counting Method and Negative Controls, Suitability of the Counting Method in the Presence of Product, Recovery of Microorganisms in thePresence 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 specialtemperature for NMT the shortest period of time specified in the test. Growth of the microorganism comparable to that previously obtained with a previously tested andapproved batch of medium occurs.
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Table2.Microoganisms for the Growth-Promoting,Inhibitory,

and Indicative properties of the Media

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TEST FOR INHIBITORY PROPERTIES, SOLID MEDIA
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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).
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TEST FOR INDICATIVE PROPERTIESָʾÐÔ²âÊÔ
Perform the Surface-Spread Method (see Microbial Enumeration TestsGrowthPromotion Test, Suitability of the Counting Method and Negative Controls, Suitability ofthe Counting Method in the Presence of Product, Recovery of Microorganisms .In thePresence of Product, Plate-Count Methods), inoculating each plate with a small number(NMT 100 cfu) of the indicated microorganism. Incubate at the specified temperature fora period of time within the range specified in the test. Colonies are comparable inappearance and indication reactions to those previously obtained with a previouslytested and approved batch of medium (see Table 2).
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Suitability of the Test Method ²âÊÔ·½·¨ÊÊÓÃÐÔ
The ability of the test to detect Bcc in the presence of the product to be tested mustbe established. The incubation time for the method suitability should not exceed theshortest incubation period specified. Suitability must be confirmed if there is a change intesting performance or a change in the product that may affect the outcome of the test.
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For each new product to be tested, perform the sample preparation as described inTesting of Products. At the time of mixing, add each test strain in the prescribed growthmedium. Inoculate the test strains individually. Use a number of microorganismsequivalent to NMT 100 cfu in the inoculated test preparation.
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Perform the test as described in Testing of Products, using the shortest incubationperiod prescribed. Bcc microorganisms must be detected with the indication reactionsdescribed in Interpretation.
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Any antimicrobial activity of the product necessitates a modification of the testprocedure (see Microbial Enumeration Test, Growth Promotion Test, Suitability ofthe Counting Method and Negative Controls, Suitability of the Counting Method in thePresence of Product,

Neutralization/Removal of Antimicrobial Activity).
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TESTING OF PRODUCTS ²úÆ·²âÊÔ

Sample Preparation and Pre-Incubation ÑùÆ·ÖƱ¸ºÍÔ¤ÅàÑø
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 DigestBroth or an appropriate dilution of Soybean-Casein Digest Broth as determined duringmethod suitability (for example ,a 1:10 dilution may be required when conductingoptional testing of pharmaceutical waters). Then mix and incubate at 30o-35ofor 48-72h.
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Selection and subculture Ñ¡ÔñºÍ¼ÌÐøÅàÑø
Subculture by streaking on a plate of Burkholderia cepacia selective agar(BCSA),and
incubate 30o-35o for 48-72h.
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Interpretation ½âÊÍ
The possible presence of Bcc is indicated by the growth of greenish-brown colonieswith yellow halos, or white colonies surrounded by a pink-red zone on BCSA .Any growthon BCSA is confirmed by identification tests. See MicrobialCharacterization,Identification,and Strain Typingfor additional information.
BCC ¿ÉÄÜ´æÔÚµÄָʾÐÔÌØÕ÷ΪÉú³¤´øÓлÆÉ«¹â»·µÄÂÌ×ØÉ«¾úÂ䣬»òÔÚBCSA ÏÔʾ±»·ÛºìÉ«ÇøÓò°üΧµÄ°×É«¾úÂä¡£ÈκÎÔÚBCSA ÉϵľúÂäÐèҪͨ¹ý¼ø¶¨²âÊÔÈ·ÈÏ¡£¸ü¶àÐÅÏ¢¼û΢ÉúÎïÌØÐÔ¡¢¼ø¶¨ºÍ¾úÖê·ÖÐÍ.
The product complies with the test if clones of the types described are not presentor if the confirmatory identification tests are negative.
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RECOMMEND CULTURE MEDIA ÍƼöµÄÅàÑø»ù
[Note-This section is given for information.][×¢:±¾½ÚÄÚÈݽö¹©²Î¿¼¡£]The following solutions and culture media have been found satisfactory for thepurposes for which they are prescribed in the tests in this Pharmacopeia . Other mediamay be used provided that their suitability can be demonstrated.
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Stock Buffer Solution ´¢±¸»º³åÒº
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 7.2¡À0.2,addPurified Water to volume,and mix. Dispense in containers,and sterilize. Store at atemperature of 2o-8o.
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Phosphate Butter Solution pH 7.2pH7.2 Á×ËáÑλº³åÒº
Prepare a mixture of purified Water and Stock Buffer Solution (800:1 v/v),andsterilize.
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Buffered Sodium Chloride-Peptone Solution pH7.0pH7.0 ÂÈ»¯ÄÆ-µ°°×ëË»º³åÒº
Prepare Buffered Sodium Chloride-Peptone Solution pH 7.0 as directed in table 3.Sterilize in an autoclave using a validated cycle.
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Table 3 ±í3

Soybean-Casein Digest Broth ÒÈÀÒëË´ó¶¹ÈâÌÀ

Prepare Soybean-Casein Digest Broth as directed in table 4 .Adjust the pH so thatafter sterilization it is 7.3¡À0.2 at 25o.Sterilize in an autoclave using a validated cycle.
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Table 4 ±í4

Burkholderia cepacia Selective Agar Ñó´Ð²®¿Ë»ô¶ûµÂ¾úÑ¡ÔñÐÔÇíÖ¬
Prepare BCSA as directed in Table 5. When preparing media in-house,first preparethe base ingredients without the antibiotics. Adjust the pH so that after sterilization it is6.8¡À0.3 at 25o.Sterilize in an autoclave using a validated cycle.Cool the base medium to45o-50o and add a 1% solution of the sterile filtered antibiotics ,mix,and pour into theplates.
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Table 5 ±í5

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