微生物限度譯文usp

1、微生物限度檢驗(yàn)微生物限度檢驗(yàn)本章提供了從原材料到成品的試驗(yàn)，包括需氧微生物數(shù)量的預(yù)測(cè)和所有藥物條款中規(guī)定的微生物種類(lèi)的預(yù)防試驗(yàn)。假如自動(dòng)化的方法經(jīng)驗(yàn)證和現(xiàn)在行方法等效或有更好的結(jié)果，可以取代現(xiàn)在的試驗(yàn)。在準(zhǔn)備和實(shí)驗(yàn)中，處理樣品時(shí)遵守?zé)o菌操作。除非有特別說(shuō)明，樣品簡(jiǎn)單“培養(yǎng)”的地方，應(yīng)把容器放在 30-35的空氣中恒溫培養(yǎng) 2448 小時(shí)。術(shù)語(yǔ)“生長(zhǎng)”經(jīng)常用在此處，換言之，指明微生物的存在和預(yù)測(cè)增殖。預(yù)備試驗(yàn)預(yù)備試驗(yàn)在本章實(shí)驗(yàn)中實(shí)驗(yàn)結(jié)果的驗(yàn)證提供了大量充分的論證，用于試驗(yàn)的樣品本身在實(shí)驗(yàn)條件下不限制可能存在的微生物的生長(zhǎng)。因而，用固定成分做試驗(yàn)的預(yù)備和環(huán)境的需求，接著分別用金黃色葡萄球菌，大腸埃

2、希菌，銅綠假單孢菌和沙門(mén)菌接種檢驗(yàn)材料的稀釋樣品中?？梢酝ㄟ^(guò)加 1 毫升不少于 10濃度的肉湯培養(yǎng) 24 小時(shí)的微生物的稀釋液到實(shí)驗(yàn)材料的第一步稀釋液（PH7.2 的磷酸鹽緩沖液，大豆酪蛋白消化物培養(yǎng)基或乳糖液狀培養(yǎng)基）中和下面的實(shí)驗(yàn)過(guò)程來(lái)實(shí)現(xiàn)。在有關(guān)培養(yǎng)基中沒(méi)有微生物生長(zhǎng)，檢驗(yàn)內(nèi)容無(wú)效，需要變更程序，包括（1）對(duì)剩余的相同數(shù)量的實(shí)驗(yàn)材料增大稀釋液的體積，或（2）在稀釋液中摻入適宜的滅活劑，或（3）通過(guò)（1） （2）的共同作用，使接種物能夠生長(zhǎng)。下面是一個(gè)配料和他們的聚合物的例子，他們可以加到培養(yǎng)基中滅活樣品中的抑菌物質(zhì)：大豆蛋黃素（卵磷脂） 。0.5%；聚山梨脂 20，4.0%。作為比較，重

3、復(fù)上面描述的實(shí)驗(yàn)，用液體酪蛋白消化物-大豆蛋黃素聚山梨脂 20 培養(yǎng)基來(lái)驗(yàn)證實(shí)驗(yàn)材料中存在的防腐劑或其他抑制劑的滅活作用。如果抑菌物質(zhì)存在樣品中并且是可溶的，適宜的經(jīng)驗(yàn)證的程序可以用于薄膜過(guò)濾技術(shù)（樣品的無(wú)菌實(shí)驗(yàn)）中。如果加了適量的滅火劑和增大了稀釋液體積，仍不能恢復(fù)上面描述和不適用于薄膜過(guò)濾技術(shù)中物品的培養(yǎng)基的活力，能假定分離接種微生物的失敗原因可歸于產(chǎn)品的殺菌活力。該信息用于顯示物品不可能被給定的微生物種類(lèi)污染。為了確立物品的抑菌譜和殺菌活力，應(yīng)繼續(xù)監(jiān)測(cè)。緩沖液和培養(yǎng)基緩沖液和培養(yǎng)基培養(yǎng)基可以按下面的準(zhǔn)備，或者假若脫水培養(yǎng)基是有供貨商或分銷(xiāo)商制備，可以被應(yīng)用，他們與給定比例獲得的培養(yǎng)基想比

4、有相似的成分。用給定的比例成分制備培養(yǎng)基，在水中溶解可溶性固體物，如果需要，加熱溶解完全，使用的時(shí)候加鹽酸或氫氧化鈉到溶液中是培養(yǎng)基中的 PH 適宜。在 252測(cè)定 PH 值。配方中含瓊脂時(shí)，使用含水量不超過(guò) 15%的瓊脂。配方中需用水時(shí)，使用純水。PH7.2 鹽酸緩沖液原液在 1000 毫升容量瓶中用 500 毫升水溶解 34 克磷酸二氫鉀，加氫氧化鈉（約175 毫升）調(diào) PH 到 7.20.1，加水到體積線，混勻。分裝后滅菌。冷藏儲(chǔ)存。用的時(shí)候，用水按 1：800 稀釋原液，滅菌。培養(yǎng)基除非有其他說(shuō)明，培養(yǎng)基應(yīng)該在高壓蒸汽滅菌器（見(jiàn)無(wú)菌中的蒸汽滅菌法）內(nèi)加熱滅菌，滅菌時(shí)間要參考滅菌體積。1

5、，液體酪蛋白-大豆蛋黃素-吐溫 20 培養(yǎng)基酪蛋白胰酶消化物 20 g大豆蛋黃素5 g吐溫 2040 mL水960 mL在 960 毫升水中溶解酪蛋白胰酶消化物和大豆蛋黃素，在 48-50的水浴中加熱 30分鐘使溶解完全。加 40 毫升吐溫 20，混勻，按需要的分裝。 2，大豆蛋黃素消化物瓊脂培養(yǎng)基酪蛋白胰酶消化物15.0 g大豆粉木瓜酶消化物 5.0 g氯化鈉5.0 g瓊脂15.0 g水1000 mL 滅菌后 PH：7.30.2。3，液體大豆蛋黃素培養(yǎng)基 參照無(wú)菌檢測(cè)下大豆蛋黃素消化物培養(yǎng)基的配制。4，甘露醇鹽瓊脂培養(yǎng)基酪蛋白胰酶消化物5.0 g動(dòng)物組織的胃蛋白酶的消化物 5.0 g牛肉膏1

6、.0 gD-甘露醇10.0 g氯化鈉75.0 g瓊脂15.0 g酚磺酞0.025 g水1000 mL 混勻，加熱沸騰 1 分鐘，并快速攪拌使溶解。 滅菌后 PH 值：7.40.2。5， BairdParker 瓊脂培養(yǎng)基酪蛋白胰酶消化物 10.0 g牛肉膏5.0 g酵母膏1.0 g氯化鋰5.0 g瓊脂20.0 g甘氨酸12.0 g丙酮酸鈉10.0 g水950 mL加熱并快速攪拌，沸騰 1 分鐘。滅菌，冷卻到 4050，加入 10 毫升亞碲酸鉀溶液（1：100）和 50 毫升蛋黃乳液。輕搖混勻，倒入盤(pán)子中。 （蛋黃乳液：對(duì)整個(gè)雞蛋外殼消毒，無(wú)菌打開(kāi)雞蛋，把蛋黃分離到一無(wú)菌量筒中。加無(wú)菌鹽水 TS

7、，得到一個(gè) 3：7 的鹽水蛋黃液。加到一無(wú)菌攪拌器中，高速混勻 5 秒鐘。 ）滅菌后 PH 值：6.80.2。6，VogelJohnson 瓊脂培養(yǎng)基 酪蛋白胰酶消化物 10.0 g酵母膏5.0 g甘露醇10.0 g磷酸氫二鉀5.0 g氯化鋰5.0 g甘氨酸10.0 g瓊脂16.0 g酚磺酞25.0 mg水1000 mL煮沸固體溶液 1 分鐘。 滅菌，冷卻到 4550，加入 20 毫升無(wú)菌亞碲酸鉀溶液（1：100）。滅菌后 PH 值： 7.2 0.2.7， 溴棕三甲銨瓊脂培養(yǎng)基 胰臟明膠消化物20.0 g氯化鎂1.4 g硫酸鉀10.0 g瓊脂13.6 g溴化十六烷基三甲銨 0.3 g丙三醇10

8、.0 mL水1000 mL在水中溶解固體組分，加丙三醇。加熱沸騰 1 分鐘，并快速攪拌使溶解。滅菌后 PH 值： 7.2 0.2.8，假單胞菌屬瓊脂培養(yǎng)基（用于氟化熒光素的檢出） 酪蛋白胰酶消化物10.0 g動(dòng)物組織的胃蛋白酶消化物 10.0 g無(wú)水磷酸氫二鉀1.5 g硫酸鎂 (MgSO47H2O)1.5 g丙三醇10.0 mL瓊脂15.0 g水1000 mL在水中溶解固體組分，加丙三醇。加熱沸騰 1 分鐘，并快速攪拌使溶解。滅菌后 PH 值： 7.2 0.2.9，假單胞菌屬瓊脂培養(yǎng)基（用于綠膿菌素的檢出）胰臟明膠消化物 20.0 g無(wú)水氯化鎂1.4 g無(wú)水硫酸鉀10.0 g瓊脂15.0 g丙

9、三醇10.0 mL水1000 mL在水中溶解固體組分，加丙三醇。加熱沸騰 1 分鐘，并快速攪拌使溶解。滅菌后 PH 值： 7.2 0.2。.10，乳糖液狀培養(yǎng)基牛肉膏3.0 g胰臟明膠消化物 5.0 g乳糖5.0 g水1000 mL滅菌后盡可能快的冷卻。滅菌后 PH 值：6.9 0.2.11，流體亞硒酸鹽-胱氨酸培養(yǎng)基 酪蛋白胰酶消化物 5.0 g乳糖4.0 g磷酸鈉10.0 g亞硒酸鈉4.0 gL-胱氨酸10.0 mg水1000 mL最終 pH: 7.0 0.2.混勻，加熱溶解。在流動(dòng)的蒸汽中加熱 15 分鐘，不可以滅菌不可以滅菌。12，液體 Tetrathionate 培養(yǎng)基酪蛋白胰酶消化

10、物2.5 g動(dòng)物組織的胃蛋白酶消化物 2.5 g膽（汁）鹽1.0 g碳酸鈣10.0 g硫代硫酸鈉30.0 g水1000 mL加熱固體溶液至沸騰。在使用的時(shí)候，加入配制好的碘液 20 毫升（在 20 毫升水中溶解 5克碘化鉀和 6 克碘）。接著加入 10 毫升亮綠溶液（1：1000），混勻。加入亮綠溶液后不可以加熱培養(yǎng)基。13，亮綠瓊脂培養(yǎng)基 酵母膏3.0 g動(dòng)物組織的胃蛋白酶消化物 5.0 g酪蛋白胰酶消化物5.0 g乳糖10.0 g氯化鈉5.0 g蔗糖10.0 g酚磺酞80 mg瓊脂20.0 g堿性亮綠12.5 mg水1000 mL煮沸固體溶液 1 分鐘。臨用前滅菌，融化培養(yǎng)基，倒入培養(yǎng)皿中

11、，允許冷卻。滅菌后 PH 值： 6.9 0.2.14,木糖賴(lài)氨酸去氧膽酸鹽瓊脂培養(yǎng)基 木糖3.5 gL-賴(lài)氨酸5.0 g乳糖7.5 g蔗糖7.5 g氯化鈉5.0 g酵母膏3.0 g酚磺酞80 mg瓊脂13.5 g去氧膽酸鈉 2.5 g硫代硫酸鈉 6.8 g枸櫞酸鐵胺 800 mg水1000 mL最終：pH: 7.4 0.2.回旋搖動(dòng)加熱固體和水的混合物到液體沸騰。不可以過(guò)熱或滅菌 。立即轉(zhuǎn)入一水浴容器中維持水溫在 50 左右， 培養(yǎng)基一冷卻就到入培養(yǎng)皿中。15,亞硫酸鉍瓊脂培養(yǎng)基 牛肉膏5.0 g酪蛋白胰酶消化物5.0 g動(dòng)物組織的胃蛋白酶消化物 5.0 g葡萄糖5.0 g磷酸鈉4.0 g硫酸

12、亞鐵300 mg亞硫酸鉍指示劑8.0 g瓊脂20.0 g堿性亮綠25 mg水1000 mL最終 pH: 7.6 0.2.回旋搖動(dòng)加熱固體和水的混合物到液體沸騰。不可以過(guò)熱或滅菌 。立即轉(zhuǎn)入一水浴容器中維持水溫在 50 左右， 培養(yǎng)基一冷卻就到入培養(yǎng)皿中。16，三糖-鐵-瓊脂培養(yǎng)基 酪蛋白胰酶消化物10.0 g動(dòng)物組織的胰酶消化物 10.0 g乳糖10.0 g蔗糖10.0 g葡萄糖1.0 g硫酸亞鐵銨200 mg氯化鈉5.0 g硫代硫酸鈉200 mg瓊脂13.0 g酚磺酞25 mg水1000 mL滅菌后 PH： 7.3 0.2.17，麥康基瓊脂培養(yǎng)基 動(dòng)物膠胰酶消化物17.0 g酪蛋白胰酶消化物

13、1.5 g動(dòng)物組織胃蛋白酶消化物 1.5 g乳糖10.0 g膽鹽混合物1.5 g氯化鈉5.0 g瓊脂13.5 g中性紅30 mg結(jié)晶紫1.0 mg水1000 mL加熱固體和水的混合物沸騰 1 分鐘使溶解。滅菌后 PH：7.1 0.2.18，腸桿菌科曙紅亞甲藍(lán)瓊脂培養(yǎng)基 動(dòng)物膠胰酶消化物 10.0 g磷酸氫二鉀2.0 g瓊脂15.0 g乳糖10.0 g曙紅 Y400 mg亞甲藍(lán)65 mg水1000 mL在水中溶解動(dòng)物膠胰酶消化物，磷酸氫二鉀和瓊脂，可以冷卻。 僅僅在使用前，熔解膠狀瓊脂培養(yǎng)基，熔解時(shí)按下面的分量加入剩余的組分并混勻： 每 100 毫升膠狀瓊脂培養(yǎng)基5 mL 乳糖溶液 (1：5),

14、 2 mL 曙紅 Y 溶液 (1：50), 和 2 mL 亞甲藍(lán)溶液 (1：300)。 配制好的培養(yǎng)基允許不澄清。滅菌后 PH： 7.1 0.2.19，薩布羅右旋瓊脂培養(yǎng)基 葡萄糖40 g動(dòng)物組織胰酶消化物和酪蛋白胰酶消化物的等量混合物 10 g瓊脂15 g水1000 mL混勻，加熱沸騰使溶解。滅菌后 PH：5.6 0.2.20，馬鈴薯右旋瓊脂培養(yǎng)基 在 500 毫升蒸餾水中煮 300 克去皮切成丁的馬鈴薯,用濾布過(guò)濾，加蒸餾水至 1000 毫升，然后加入下面的成分：瓊脂15 g葡萄糖20 g加熱溶解，滅菌。滅菌后 PH： 5.6 0.2.使用時(shí)，在倒培養(yǎng)皿前，用酒石酸溶液（1：10）調(diào)整熔解

15、冷卻到 45 的培養(yǎng)基至 PH 3.5 0.1.。不可以再加熱 pH 3.5 的培養(yǎng)基。取樣取樣按論文中要求的預(yù)備 10-mL or 10-gs 樣品用于實(shí)驗(yàn)。操作過(guò)程操作過(guò)程 通過(guò)與樣品物理特性相適應(yīng)的方法準(zhǔn)備待測(cè)樣品，不能改變樣品中本來(lái)存在的微生物的種類(lèi)和數(shù)量，獲得一個(gè)溶液，或全部的懸浮液，或一個(gè)適于實(shí)驗(yàn)過(guò)程的形狀的部分。對(duì)于一個(gè)在可察覺(jué)的范圍內(nèi)溶解但不完全溶解的固體，把這個(gè)固體變成適度的細(xì)粉狀，在特定的媒介物中做成懸浮液，按下面所示的程序進(jìn)行：總的需氧微生物計(jì)數(shù)，金黃色葡萄球菌實(shí)驗(yàn)，銅綠假單胞菌實(shí)驗(yàn)，沙門(mén)菌和大腸桿菌實(shí)驗(yàn)。對(duì)于液體樣品，包括真溶液，水中的懸浮液或含水酒精（酒精含量不少于

16、30%）；和在90 毫升 PH7.2 的磷酸鹽緩沖液中能快速并完全溶解的固體樣品，按下面所示的程序進(jìn)行：總的需氧微生物計(jì)數(shù)，金黃色葡萄球菌實(shí)驗(yàn)，銅綠假單胞菌實(shí)驗(yàn)，沙門(mén)菌和大腸桿菌實(shí)驗(yàn)。For water-immiscible fluids, ointments, creams, and waxes, prepare a suspension with the aid of a minimal quantity of a suitable, sterile emulsifying agent (such as one of the polysorbates), using a mechanica

17、l blender and warming to a temperature not exceeding 45 , if necessary, and proceed with the suspension as directed under Total Aerobic Microbial Count, and under Test for Staphylococcus aureus and Pseudomonas aeruginosa and Test for Salmonella species and Escherichia coli.For a fluid specimen in ae

18、rosol form, chill the container in an alcohol-dry ice mixture for approximately 1 hour, cut open the container, allow it to reach room temperature, permit the propellant to escape, or warm to drive off the propellant if feasible, and transfer the quantity of test material required for the procedures

19、 specified in one of the two preceding paragraphs, as appropriate. Where 10.0 g or 10.0 mL of the specimen, whichever is applicable, cannot be obtained from 10 containers in aerosol form, transfer the entire contents from 10 chilled containers to the culture medium, permit the propellant to escape,

20、and proceed with the test on the residues. If the results of the test are inconclusive or doubtful, repeat the test with a specimen from 20 more containers.Total Aerobic Microbial Count For specimens that are sufficiently soluble or translucent to permit use of the Plate Method, use that method; oth

21、erwise, use the Multiple-Tube Method. With either method, first dissolve or suspend 10.0 g of the specimen if it is a solid, or 10 mL, accurately measured, if the specimen is a liquid, in pH 7.2 Phosphate Buffer, Fluid SoybeanCasein Digest Medium, or Fluid Casein DigestSoy Lecithin-Polysorbate 20 Me

22、dium to make 100 mL. For viscous specimens that cannot be pipeted at this initial 1:10 dilution, dilute the specimen until a suspension is obtained, i.e., 1:50 or 1:100, etc., that can be pipeted. Perform the test for absence of inhibitory (antimicrobial) properties as described under Preparatory Te

23、sting before the determination of Total Aerobic Microbial Count. Add the specimen to the medium not more than 1 hour after preparing the appropriate dilutions for inoculation.PLATE METHOD Dilute further, if necessary, the fluid so that 1 mL will be expected to yield between 30 and 300 colonies. Pipe

24、t 1 mL of the final dilution onto each of two sterile petri dishes. Promptly add to each dish 15 to 20 mL of SoybeanCasein Digest Agar Medium that previously has been melted and cooled to approximately 45 . Cover the petri dishes, mix the sample with the agar by tilting or rotating the dishes, and a

25、llow the contents to solidify at room temperature. Invert the petri dishes, and incubate for 48 to 72 hours. Following incubation, examine the plates for growth, count the number of colonies, and express the average for the two plates in terms of the number of microorganisms per g or per mL of speci

26、men. If no microbial colonies are recovered from the dishes representing the initial 1:10 dilution of the specimen, express the results as “l(fā)ess than 10 microorganisms per g or per mL of specimen.”MULTIPLE-TUBE METHOD Into each of fourteen test tubes of similar size place 9.0 mL of sterile Fluid Soy

27、beanCasein Digest Medium. Arrange twelve of the tubes in four sets of three tubes each. Put aside one set of three tubes to serve as the controls. Into each of three tubes of one set (“100”) and into a fourth tube (A) pipet 1 mL of the solution or suspension of the specimen, and mix. From tube A, pi

28、pet 1 mL of its contents into the one remaining tube (B) not included in a set, and mix. These two tubes contain 100 mg (or 100 L) and 10 mg (or 10 L) of the specimen, respectively. Into each of the second set (“10”) of three tubes pipet 1 mL from tube A, and into each tube of the third set (“1”) pi

29、pet 1 mL from tube B. Discard the unused contents of tubes A and B. Close well, and incubate all of the tubes. Following the incubation period, examine the tubes for growth: the three control tubes remain clear and the observations in the tubes containing the specimen, when interpreted by reference

30、to Table 1, indicate the most probable number of microorganisms per g or per mL of specimen.Table 1. Most Probable Total Count by Multiple-Tube Method Observed Combinations of Numbers of Tubes Showing Growth in Each SetNo. of mg (or mL) of Specimen per Tube100(100 L)10 (10 L)1(1 L)Most ProbableNumbe

31、r ofMicroorgan-isms per g or per mL3331100 33211003315003302003232903222103211503209031316031212031170310403039530260Observed Combinations of Numbers of Tubes Showing Growth in Each SetMost ProbableNumber ofMicroorgan-isms per g or per mLNo. of mg (or mL) of Specimen per Tube100(100 L)10 (10 L)1(1 L

32、)3014030023Test for Staphylococcus aureus and Pseudomonas aeruginosa To the specimen add Fluid SoybeanCasein Digest Medium to make 100 mL, mix, and incubate. Examine the medium for growth, and if growth is present, use an inoculating loop to streak a portion of the medium on the surface of VogelJohn

33、son Agar Medium (or BairdParker Agar Medium, or MannitolSalt Agar Medium) and of Cetrimide Agar Medium, each plated on petri dishes. Cover and invert the dishes, and incubate. If, upon examination, none of the plates contains colonies having the characteristics listed in Tables 2 and 3 for the media

34、 used, the test specimen meets the requirements for freedom from Staphylococcus aureus and Pseudomonas aeruginosa. Table 2. Morphologic Characteristics of Staphylococcus aureus on Selective Agar Media Selective MediumCharacteristic Colonial MorphologyGram StainVogel-JohnsonAgar MediumBlack Surrounde

35、d by yellow zonePositive cocci(in clusters)Mannital-SaltAgar MediumYellow colonies with yellow zonesPositive cocci (in clusters)Baird-ParkerAgar MediumBlack, shiny, surrounded by clear zones 2 to 5 mmPositive cocci (in clusters)Table 3. Morphologic Characteristics of Pseudomonas aeruginosa on Select

36、ive and Diagnostic Agar Media Selective MediumCharacteristic Colonial MorphologyFluorescence in UV LightOxidase TestGram StainCentrimide Agar MediumGenerally greenishGreenishPositiveNegative rodsPseudomonas Agar Medium for Detection of FluorescinGenerally colorless to yellowishYellowishPositiveNegat

37、ive rodsPseudomonas Agar Medium forDetection of PyocyaninGenerally greenishBluePositiveNegative rodsCoagulase Test (for Staphylococcus aureus) With the aid of an inoculating loop, transfer representative suspect colonies from the agar surfaces of the VogelJohnson Agar Medium (or BairdParker Agar Med

38、ium, or MannitolSalt Agar Medium) to individual tubes, each containing 0.5 mL of mammalian, preferably rabbit or horse, plasma with or without suitable additives. Incubate in a water bath at 37 , examining the tubes at 3 hours and subsequently at suitable intervals up to 24 hours. Test positive and

39、negative controls simultaneously with the unknown specimens. If no coagulation in any degree is observed, the specimen meets the requirements of the test for absence of Staphylococcus aureus. Oxidase and Pigment Tests (for Pseudomonas aeruginosa) With the aid of an inoculating loop, streak represent

40、ative suspect colonies from the agar surface of Cetrimide Agar Medium on the agar surfaces of Pseudomonas Agar Medium for Detection of Fluorescin and Pseudomonas Agar Medium for Detection of Pyocyanin contained in petri dishes. If numerous colonies are to be transferred, divide the surface of each p

41、late into quadrants, each of which may be inoculated from a separate colony. Cover and invert the inoculated media, and incubate at 35 2 for not less than three days. Examine the streaked surfaces under UV light. Examine the plates to determine whether colonies having the characteristics listed in T

42、able 3 are present. Confirm any suspect colonial growth on one or more of the media as Pseudomonas aeruginosa by means of the oxidase test. Upon the colonial growth place or transfer colonies to strips or disks of filter paper that previously has been impregnated with N,N-dimethyl-p-phenylenediamine

43、 dihydrochloride: if there is no development of a pink color, changing to purple, the specimen meets the requirements of the test for the absence of Pseudomonas aeruginosa. The presence of Pseudomonas aeruginosa may be confirmed by other suitable cultural and biochemical tests, if necessary.Test for

44、 Salmonella species and Escherichia coli To the specimen, contained in a suitable vessel, add a volume of Fluid Lactose Medium to make 100 mL, and incubate. Examine the medium for growth, and if growth is present, mix by gently shaking. Pipet 1-mL portions into vessels containing, respectively, 10 m

45、L of Fluid SeleniteCystine Medium and Fluid Tetrathionate Medium, mix, and incubate for 12 to 24 hours. (Retain the remainder of the Fluid Lactose Medium.)Test for Salmonella Species By means of an inoculating loop, streak portions from both the selenite-cystine and tetrathionate media on the surfac

46、e of Brilliant Green Agar Medium, XyloseLysineDesoxycholate Agar Medium, and Bismuth Sulfite Agar Medium contained in petri dishes. Cover and invert the dishes, and incubate. Upon examination, if none of the colonies conforms to the description given in Table 4, the specimen meets the requirements o

47、f the test for absence of the genus Salmonella. Table 4. Morphologic Characteristics of Salmonella Species on Selective Agar Media Selective MediumCharacteristic Colonial MorphologyBrilliant GreenAgar MediumSmall, transparent, colorless or pink to white opaque (frequently surrounded by pink to red z

48、one)Xylose-Lysine-DesoxycholateAgar MediumRed, with or without black centersSelective MediumCharacteristic Colonial MorphologyBismuth SulfiteAgar MediumBlack or greenIf colonies of Gram-negative rods matching the description in Table 4 are found, proceed with further identification by transferring r

49、epresentative suspect colonies individually, by means of an inoculating wire, to a butt-slant tube of Triple SugarIronAgar Medium by first streaking the surface of the slant and then stabbing the wire well beneath the surface. Incubate. If examination discloses no evidence of tubes having alkaline (

50、red) slants and acid (yellow) butts (with or without concomitant blackening of the butt from hydrogen sulfide production), the specimen meets the requirements of the test for the absence of the genus Salmonella.* Test for Escherichia coli By means of an inoculating loop, streak a portion from the re

51、maining Fluid Lactose Medium on the surface of MacConkey Agar Medium. Cover and invert the dishes, and incubate. Upon examination, if none of the colonies conforms to the description given in Table 5 for this medium, the specimen meets the requirements of the test for absence of Escherichia coli. Table 5. Morphologic Characteristics of Escherichia coli on MacConkey Agar Medium Gram StainCharacteristic Colonial MorphologyNegative rods(cocco-bacilli)Brick-red; may have surrounding zone of precipitated bileIf colonies matching the descri