化妝品衛(wèi)生規(guī)范
<span “=””>Hygienic?Standard for Cosmetics
中華人民共和國衛(wèi)生部?二○○七年一月
第四部分? ??微生物檢驗方法
Methods of Microbiological?Test
1???<span “=””>范圍?<span “=””>本規(guī)范規(guī)定了化妝品微生物學(xué)檢驗的基本要求。
本規(guī)范適用于化妝品樣品的采集、保存及供檢樣品制備。
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2???<span “=””>儀<span “=””>器和設(shè)備
2.1???<span “=””>天平。<span “=””>
2.2???<span “=””>高壓滅菌器。<span “=””>
2.3???<span “=””>振蕩器。<span “=””>
2.4???<span “=””>三角瓶，250mL<span “=””>。<span “=””>
2.5???<span “=””>玻璃珠。<span “=””>
2.6???<span “=””>玻璃棒。<span “=””>
2.7???<span “=””>刻度吸管，1mL<span “=””>、10mL<span “=””>。<span “=””>
2.8???<span “=””>研缽或均質(zhì)器。<span “=””>
2.9???<span “=””>恒溫水浴箱。<span “=””>
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3???<span “=””>培<span “=””>養(yǎng)基和試劑
3.1???<span “=””>生理鹽水<span “=””>
<span “=””>成分：氯化<span “=””>鈉???????????????????????????????????????8.5g
<span “=””>蒸餾水加<span “=””>至??????????????????????????1000mL
<span “=””>溶解后，分裝到加玻璃珠的三角瓶內(nèi)，每瓶??90mL<span “=””>，103.43kPa<span “=””>（121<span “=””>℃??15??lb<span “=””>）20min<span “=””><span “=””>
<span “=””>高壓滅菌。<span “=””>
3.2?? SCDLP?<span “=””>液體培養(yǎng)基成分：酪蛋白胨? ? ?17g?大豆蛋白胨???3g?氯化鈉? ??5g?磷酸氫二鉀??2.5g??葡萄糖? ? ?2.5g?卵磷脂? ? ??1g?吐溫??80 ? ? ?7g?蒸餾水?? 1000mL
<span “=””>制法：先將卵磷脂在少量蒸餾水中加溫溶解后，再與其它成分混合，加熱溶解，調(diào)?pH
<span “=””>為?7.2<span “=””>～7.3?<span “=””>分裝，103.43kPa<span “=””>（121<span “=””>℃??15??lb<span “=””>）20min?<span “=””>高壓滅菌。注意振蕩，使沉淀于底層的?<span “=””>吐溫?80?<span “=””>充分混合，冷卻至?25<span “=””>℃左右<span “=””>使用。
<span “=””>注：如無酪蛋白胨和大豆蛋白胨，也可用多胨代替。
3.3???<span “=””>滅菌液體石蠟。<span “=””>
3.4???<span “=””>滅菌吐溫?80<span “=””>。<span “=””>
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4???<span “=””>樣<span “=””>品的采集及注意事項
4.1???<span “=””>所<span “=””>采集<span “=””>的樣<span “=””>品<span “=””>，<span “=””>應(yīng)<span “=””>具有<span “=””>代<span “=””>表<span “=””>性<span “=””>，一<span “=””>般<span “=””>視<span “=””>每<span “=””>批化<span “=””>妝<span “=””>品<span “=””>數(shù)<span “=””>量大<span “=””>小<span “=””>，<span “=””>隨<span “=””>機抽<span “=””>取<span “=””>相<span “=””>應(yīng)<span “=””>數(shù)量<span “=””>的<span “=””>包裝單位。檢驗時，應(yīng)分別從兩個包裝單位以上的樣品中共取?10g?或?10mL。包裝量小于?20g的樣品，采樣量可適當增加樣品包裝數(shù)量。
4.2???<span “=””>供<span “=””>檢驗<span “=””>樣品<span “=””>，<span “=””>應(yīng)<span “=””>嚴<span “=””>格保<span “=””>持<span “=””>原<span “=””>有<span “=””>的包<span “=””>裝<span “=””>狀<span “=””>態(tài)<span “=””>。容<span “=””>器<span “=””>不<span “=””>應(yīng)<span “=””>有破<span “=””>裂<span “=””>，<span “=””>在<span “=””>檢驗<span “=””>前<span “=””>不<span “=””>得<span “=””>打開<span “=””>，<span “=””>防?<span “=””>止樣品被污染。
4.3???<span “=””>接到樣品后，應(yīng)立即登記，編寫檢驗序號，并按檢驗要求盡快檢驗。如不能及時檢驗，?<span “=””>樣品應(yīng)放在室溫陰涼干燥處，不要冷藏或冷凍。
4.4???<span “=””>若<span “=””>只有<span “=””>一個<span “=””>樣<span “=””>品<span “=””>而<span “=””>同時<span “=””>需<span “=””>做<span “=””>多<span “=””>種分<span “=””>析<span “=””>，<span “=””>如<span “=””>細菌<span “=””>、<span “=””>毒<span “=””>理<span “=””>、化<span “=””>學(xué)<span “=””>等<span “=””>，<span “=””>則宜<span “=””>先<span “=””>取<span “=””>出<span “=””>部分<span “=””>樣<span “=””>品?<span “=””>做細菌檢驗，再將剩余樣品做其它分析。
4.5???<span “=””>在<span “=””>檢驗<span “=””>過程<span “=””>中<span “=””>，<span “=””>從<span “=””>打開<span “=””>包<span “=””>裝<span “=””>到<span “=””>全部<span “=””>檢<span “=””>驗<span “=””>操<span “=””>作結(jié)<span “=””>束<span “=””>，<span “=””>均<span “=””>須防<span “=””>止<span “=””>微<span “=””>生<span “=””>物的<span “=””>再<span “=””>污<span “=””>染<span “=””>和擴<span “=””>散<span “=””>，?<span “=””>所用器皿及材料均應(yīng)事先滅菌，全部操作應(yīng)在無菌室內(nèi)進行，或在相應(yīng)條件下，按無菌操作?<span “=””>規(guī)定進行。
4.6???<span “=””>如檢出糞大腸菌群或其它致病菌，自報告發(fā)出之日起該菌種及被檢樣品應(yīng)保存一個月。
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5???<span “=””>供<span “=””>檢樣品的制備
5.1???<span “=””>液體樣品<span “=””>
5.1.1???<span “=””>水溶性的液體樣品，可量取?10mL?<span “=””>加到?90mL?<span “=””>滅菌生理鹽水中，如樣品少于?10mL<span “=””>，?<span “=””>仍按?10?<span “=””>倍稀釋法進行。如為?5mL?<span “=””>則<span “=””>加到?45mL?<span “=””>滅菌生理鹽水，混勻后，制成?1<span “=””>：10?<span “=””>檢液。
5.1.2???<span “=””>油性液體樣品，取樣品?10mL<span “=””>，先加?5mL?<span “=””>滅菌液體石蠟混勻，再加?10mL<span “=””><span “=””>滅菌的吐
<span “=””>溫?80<span “=””>，在?40<span “=””>℃～44<span “=””>℃水<span “=””>浴中振蕩混合?10min<span “=””>，加入滅菌的生理鹽水?75mL<span “=””>（在?40<span “=””>℃～44<span “=””>℃?<span “=””>水浴中預(yù)溫），在?40<span “=””>℃～44<span “=””>℃水浴中乳<span “=””>化，制成?1<span “=””>：10?<span “=””>的懸液。
5.2???<span “=””>膏、霜、乳劑半固體狀樣品
5.2.1???<span “=””>親水性的樣品：稱取?10g<span “=””>，<span “=””>加<span “=””>到裝有玻璃珠及?90mL<span “=””><span “=””>滅菌生理鹽水的三角瓶中，充分?<span “=””>振蕩混勻，靜置?15min<span “=””>。用其上清液作為?1:10?<span “=””>的<span “=””>檢液。
5.2.2???<span “=””>疏水性樣品：稱取?10g<span “=””>，<span “=””>放到滅菌的研缽中，加?10mL?<span “=””>滅菌液體石蠟，研磨成粘稠狀，?<span “=””>再加入?10mL?<span “=””>滅菌吐溫?80<span “=””>，研磨待溶解后，加?70mL?<span “=””>滅菌生理鹽水，在?40<span “=””>℃<span “=””>～44<span “=””>℃<span “=””>水<span “=””>浴中?<span “=””>充分混合，制成?1<span “=””>：10?<span “=””>檢液。
5.3???<span “=””>固體樣品
<span “=””>稱取?10g<span “=””>，<span “=””>加到?90mL?<span “=””>滅菌生理鹽水中，充分振蕩混勻，使其分散混懸，靜置后，取上?<span “=””>清液作為?1<span “=””>：10?<span “=””>的檢液。
<span “=””>如有均質(zhì)器，上述水溶性膏、霜、粉劑等，可稱??10g??<span “=””>樣品加入??90mL<span “=””><span “=””>滅菌生理鹽水，?<span “=””>均質(zhì)?1min<span “=””>～2min<span “=””>；疏水性膏、霜及眉筆、口紅等，稱?10g?<span “=””>樣<span “=””>品，加?10mL<span “=””><span “=””>滅菌液體石蠟，
10mL?<span “=””>吐溫?80<span “=””>，70mL?<span “=””>滅菌生理鹽水，均質(zhì)?3min<span “=””>～5min<span “=””>。
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<span “=””>二、菌落總數(shù)
1???<span “=””>范圍?<span “=””>本規(guī)范規(guī)定了化妝品中菌落總數(shù)的檢驗方法。?<span “=””>本規(guī)范適用于化妝品菌落總數(shù)的測定。
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2???<span “=””>定義?<span “=””>本規(guī)范采用下列定義
<span “=””>菌落總數(shù)（Aerobic?bacterial?count<span “=””>）<span “=””>是<span “=””>指化妝品檢樣經(jīng)過處理，在一定條件下培養(yǎng)后（如?<span “=””>培養(yǎng)基成分、培養(yǎng)溫度、培養(yǎng)時間、pH?<span “=””>值、需氧性質(zhì)等），1g<span “=””>（1mL<span “=””>）<span “=””>檢樣中所含菌落的總?<span “=””>數(shù)。所得結(jié)果只包括一群本方法規(guī)定的條件下生長的嗜中溫的需氧性菌落總數(shù)。
<span “=””>測<span “=””>定菌<span “=””>落總數(shù)便<span “=””>于<span “=””>判明樣品<span “=””>被<span “=””>細菌污染<span “=””>的<span “=””>程度，是<span “=””>對<span “=””>樣品進行<span “=””>衛(wèi)<span “=””>生學(xué)總評<span “=””>價<span “=””>的綜<span “=””>合<span “=””>依<span “=””>
<span “=””>據(jù)。<span “=””>
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3???<span “=””>儀<span “=””>器和設(shè)備
3.1???<span “=””>三角瓶，250mL<span “=””>。<span “=””>
3.2???<span “=””>量筒，200mL<span “=””>。<span “=””>
3.3???pH?<span “=””>計或精密?pH?<span “=””>試紙。<span “=””>
3.4???<span “=””>高壓滅菌器。<span “=””>
3.5???<span “=””>試管：15<span “=””>×150mm<span “=””>。<span “=””>
3.6???<span “=””>滅菌平皿：直徑?9cm<span “=””>。<span “=””>
3.7???<span “=””>滅菌刻度吸管，10mL<span “=””>、1mL<span “=””>。<span “=””>
3.8???<span “=””>酒精燈。<span “=””>
3.9???<span “=””>恒溫培養(yǎng)箱：36<span “=””>℃<span “=””>±1<span “=””>℃<span “=””>。<span “=””>
3.10???<span “=””>放大鏡。<span “=””>
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4???<span “=””>培<span “=””>養(yǎng)基和試劑
4.1???<span “=””>生理鹽水：見總則中?3.1<span “=””>。<span “=””>
4.2???<span “=””>卵磷脂、吐溫?80<span “=””>—<span “=””>營養(yǎng)瓊脂培養(yǎng)基
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4.2.1 |
<span “=””>成分：蛋白胨 |
20g |
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<span “=””>牛肉膏 |
3g |
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<span “=””>氯化鈉 |
5g |
<span “=””>瓊脂????????????????????????????????????????15g
<span “=””>卵磷脂??????????????????????????????????????1g?<span “=””>吐溫??80??????????????7g?<span “=””>蒸餾<span “=””>水????????????????????????????????????????1000mL
4.2.2???<span “=””>制法：先將卵磷脂加到少量蒸餾水中，加熱溶解，加入吐溫??80<span “=””>，將其他成分（除瓊?<span “=””>脂外）加到其余的蒸餾水中，溶解。加入已溶解的卵磷脂、<span “=””><span “=””>吐溫?80<span “=””>，<span “=””>混勻，調(diào)?pH?<span “=””>值為?7.1<span “=””>～
7.4<span “=””>，加入瓊脂，103.43kPa<span “=””>（121<span “=””>℃?15?lb<span “=””>）20min?<span “=””>高壓滅菌，儲存于冷暗處備用。
4.3???0.5<span “=””>％氯化三苯四氮唑（2,3,5-triphenyl?terazolium?chloride<span “=””>，TTC<span “=””>）?<span “=””>成分：TTC??????0.5g
<span “=””>蒸餾<span “=””>水???????????????????????????????????100mL
<span “=””>溶解后過濾，103.43kPa<span “=””>（121<span “=””>℃??15 lb<span “=””>）20min?<span “=””>高壓滅菌，裝于棕色試劑瓶，置?4<span “=””>℃<span “=””>冰<span “=””>箱?<span “=””>備用。
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5???<span “=””>操<span “=””>作步驟
5.1???<span “=””>用滅菌吸管吸取?1<span “=””>：10?<span “=””>稀釋的檢液?2mL<span “=””>，分別注入到兩個滅菌平皿內(nèi)，每皿?1mL<span “=””>。另<span “=””>
<span “=””>取?1mL?<span “=””>注入到?9mL?<span “=””>滅菌生理鹽水試管中（注意勿使吸管接觸液面），更換一支吸管，并充?<span “=””>分混勻，制成?1<span “=””>：100?<span “=””>檢<span “=””>液。吸取?2mL<span “=””>，<span “=””>分別注入到兩個滅菌平皿內(nèi)，每皿?1mL<span “=””>。<span “=””>如<span “=””>樣品含?<span “=””>菌量高，還可再稀釋成?1<span “=””>：1000<span “=””>，1<span “=””>：10000<span “=””>，……等，每種稀釋度應(yīng)換?1?<span “=””>支吸管。
5.2????<span “=””>將融<span “=””>化<span “=””>并冷<span “=””>至??45<span “=””>℃<span “=””>～50<span “=””>℃<span “=””>的<span “=””>卵<span “=””>磷脂吐<span “=””>溫??80??<span “=””>營養(yǎng)瓊<span “=””>脂<span “=””>培養(yǎng)基傾注到平皿內(nèi)，每皿約15mL<span “=””>，<span “=””>隨即轉(zhuǎn)動平皿，使樣品與培養(yǎng)基充分混合均勻，待瓊脂凝固后，翻轉(zhuǎn)平皿，置?36<span “=””>℃<span “=””>±1<span “=””>℃培養(yǎng)箱<span “=””>內(nèi)培養(yǎng)??48h<span “=””>±2h<span “=””>。另取一個不加樣品的滅菌空平皿，加入約??15mL ?<span “=””>卵磷脂吐溫80?<span “=””>營養(yǎng)瓊脂培養(yǎng)基，待瓊脂凝固后，翻轉(zhuǎn)平皿，置?36<span “=””>℃<span “=””>±1<span “=””>℃<span “=””>培養(yǎng)箱內(nèi)培養(yǎng)?48h<span “=””>±2h<span “=””>，<span “=””>為空?<span “=””>白對照。
5.3???<span “=””>為便于區(qū)別化妝品中的顆粒與菌落，可在每?100mL?<span “=””>卵磷脂吐溫?80?<span “=””>營<span “=””>養(yǎng)瓊脂中加入?1mL?0.5<span “=””>％<span “=””>的?TTC?<span “=””>溶液，如有細菌存在，培養(yǎng)后菌落呈紅色，而化妝品的顆粒顏色無變化。
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6???<span “=””>菌<span “=””>落計數(shù)方法
<span “=””>先用肉眼觀察，點數(shù)菌落數(shù)，然后再用放大?5?<span “=””>倍～10?<span “=””>倍的放大鏡檢查，以防遺漏。記?<span “=””>下各平皿的菌落數(shù)后，求出同一稀釋度各平皿生長的平均菌落數(shù)。若平皿中有連成片狀的菌?<span “=””>落或花點樣菌落蔓延生長時，該平皿不宜計數(shù)。若片狀菌落不到平皿中的一半，而其余一半?<span “=””>中菌落數(shù)分布又很均勻，則可將此半個平皿菌落計數(shù)后乘以?2<span “=””>，以代表全皿菌落數(shù)。
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7???<span “=””>菌<span “=””>落計數(shù)及報告方法
7.1???<span “=””>首先選取平均菌落數(shù)在?30?<span “=””>個～300?<span “=””>個之間的平皿，作為菌落總數(shù)測定的范圍。當只有?<span “=””>一個稀釋度的平均菌落數(shù)符合此范圍時，即以該平皿菌落數(shù)乘其稀釋倍數(shù)（見表?1?<span “=””>中例?1<span “=””>）<span “=””>。
7.2???<span “=””>若有兩個稀釋度，其平均菌落數(shù)均在?30?<span “=””>個～300?<span “=””>個之間，<span “=””><span “=””>則應(yīng)求出兩菌落總數(shù)之比值?<span “=””>來決定，若其比值小于或等于?2<span “=””>，<span “=””>應(yīng)<span “=””>報告其平均數(shù)，若大于?2??<span “=””>則報告其中稀釋度較低的平皿
<span “=””>的菌落數(shù)（見表?1?<span “=””>中例?2?<span “=””>及例?3<span “=””>）<span “=””>。
7.3???<span “=””>若所有稀釋度的平均菌落數(shù)均大于??300??<span “=””>個<span “=””>，則應(yīng)按稀釋度最高的平均菌落數(shù)乘以稀釋?<span “=””>倍數(shù)報告之（見表?1?<span “=””>中<span “=””>例?4<span “=””>）<span “=””>。
7.4???<span “=””>若所有稀釋度的平均菌落數(shù)均小于?30?<span “=””>個，則應(yīng)按稀釋度最低的平均菌落數(shù)乘以稀釋倍?<span “=””>數(shù)報告之（見表?1?<span “=””>例?5<span “=””>）<span “=””>。
7.5???<span “=””>若所有稀釋度的平均菌落數(shù)均不在?30?<span “=””>個～300?<span “=””>個之間，其中一個稀釋度大于?300?<span “=””>個，?<span “=””>而相鄰的另一稀釋度小于?30?<span “=””>個時，則以接近?30?<span “=””>或?300?<span “=””>的平均菌落數(shù)乘以稀釋倍數(shù)報告之（見
<span “=””>表?1?<span “=””>中例?6<span “=””>）<span “=””>。
7.6???<span “=””>若所有的稀釋度均無菌生長，報告數(shù)為每?g?<span “=””>或每?mL?<span “=””>小<span “=””>于?10CFU<span “=””>。<span “=””>
7.7???<span “=””>菌落計數(shù)的報告，菌落數(shù)在?10?<span “=””>以內(nèi)時，按實有數(shù)值報告之，大于?100?<span “=””>時<span “=””>，<span “=””>采用二位有?<span “=””>效數(shù)字，在二位有效數(shù)字后面的數(shù)值，應(yīng)以四舍五入法計算。為了縮短數(shù)字后面零的個數(shù)，?<span “=””>可用?10?<span “=””>的指數(shù)來表示（見表?1?<span “=””>報告方式欄）。在報告菌落數(shù)為“不可計”時，應(yīng)注明樣品的?<span “=””>稀釋度。
<span “=””>
|
1 |
1365 |
164 |
20 |
<span “=””>─ |
16400 |
16000?<span “=””>或?1.6<span “=””>×104 |
||||
|
2 |
2760 |
295 |
46 |
1.6 |
38000 |
38000?<span “=””>或?3.8<span “=””>×104 |
||||
|
3 |
2890 |
271 |
60 |
2.2 |
27100 |
27000?<span “=””>或?2.7<span “=””>×104 |
||||
|
4 |
<span “=””>不可計 |
4650 |
513 |
<span “=””>─ |
513000 |
510000?<span “=””>或?5.1<span “=””>×105 |
||||
|
5 |
27 |
11 |
5 |
<span “=””>─ |
270 |
270?<span “=””>或?2.7<span “=””>×102 |
||||
|
6 |
<span “=””>不可計 |
305 |
12 |
<span “=””>─ |
30500 |
31000?<span “=””>或?3.1<span “=””>×104 |
||||
|
7 |
0 |
0 |
0 |
<span “=””>─ |
<span “=””>＜１×10 |
|
*CFU<span “=””>：菌落形成單位。
<span “=””>
<span “=””>
<span “=””>三、糞大腸菌群<span “=””>
<span “=””>
1???<span “=””>范圍?<span “=””>本規(guī)范規(guī)定了化妝品中糞大腸菌群的檢驗方法。?<span “=””>本規(guī)范適用于化妝品中糞大腸菌群的檢驗。
<span “=””>
2????<span “=””>定義?<span “=””>本規(guī)范采用下列定義
<span “=””>糞大腸菌群（Fecal?coliforms<span “=””>）<span “=””><span “=””>系<span “=””>一群需氧及兼性厭氧革蘭氏陰性無芽胞桿菌，在?44.5
<span “=””>℃±0.5<span “=””>℃培<span “=””>養(yǎng)?24h<span “=””>～48h?<span “=””>能發(fā)酵乳糖產(chǎn)酸并產(chǎn)氣。?<span “=””>該菌直接來自糞便，是重要的衛(wèi)生指示菌。
<span “=””>
3???<span “=””>儀器<span “=””>
3.1???<span “=””>恒溫水浴箱或隔水式恒溫箱：44<span “=””>℃<span “=””>±0.5<span “=””>℃<span “=””>。<span “=””>
3.2???<span “=””>溫度計。<span “=””>
3.3???<span “=””>顯微鏡。<span “=””>
3.4???<span “=””>載玻片。<span “=””>
3.5???<span “=””>接種環(huán)。<span “=””>
3.6???<span “=””>電磁爐。<span “=””>
3.7???<span “=””>三角瓶，250mL<span “=””>。<span “=””>
3.8???<span “=””>試管：15<span “=””>×150mm<span “=””>。<span “=””>
3.9???<span “=””>小倒管。<span “=””>
3.10???pH?<span “=””>計<span “=””>或?pH?<span “=””>試紙。<span “=””>
3.11???<span “=””>高壓滅菌器。<span “=””>
3.12???<span “=””>滅菌吸管，10mL<span “=””>、1mL<span “=””>。<span “=””>
3.13???<span “=””>滅菌平皿：直徑?90mm<span “=””>。<span “=””>
<span “=””>
4???<span “=””>培<span “=””>養(yǎng)基和試劑
4.1???<span “=””>雙倍乳糖膽鹽（含中和劑）培養(yǎng)基?<span “=””>成分：蛋白胨?????40g
<span “=””>豬膽鹽????????????????????????????????????????10g
<span “=””>乳糖????????????????????????????????????????????10g
0.4<span “=””>％溴甲酚<span “=””>紫<span “=””>水溶<span “=””>液???????????????? 5mL?<span “=””>卵磷脂????????????????? ?2g?<span “=””>吐溫??80??????????????????????????????????????????????14g?<span “=””>蒸餾<span “=””>水??? 1000mL
<span “=””>制法：將卵磷脂、吐溫?80?<span “=””>溶解到少量蒸餾水中。將蛋白胨、膽鹽及乳糖溶解到其余的?<span “=””>蒸餾水中，加到一起混勻，調(diào)?pH?<span “=””>到?7.4<span “=””>，<span “=””>加入?0.4<span “=””>％溴甲酚紫水溶液，混勻，分裝試管（每?<span “=””>支試管中加一個小倒管）。68.95kPa<span “=””>（115<span “=””>℃??10?lb<span “=””>）20min?<span “=””>滅菌。
4.2???<span “=””>伊紅美蘭（EMB<span “=””>）<span “=””>瓊脂<span “=””>
<span “=””>成分：蛋白胨????????????????????????????????????????10g
<span “=””>? ? ? ??乳糖??????????????????????????????????????????? 10g
<span “=””>?磷酸氫二鉀??? ? 2g?<span “=””>瓊脂? ? ? ? ? ? ? ? ? ? ? ???20g
2<span “=””>％伊紅水<span “=””>溶液?????????????????????????20mL
0.5<span “=””>％<span “=””>美<span “=””>藍水<span “=””>溶<span “=””>液??????????????????????13mL
<span “=””>蒸餾<span “=””>水?????????????????????????????????1000mL
<span “=””>制法：先將瓊脂加到?900mL?<span “=””>蒸<span “=””>餾水中，加熱溶解，然后加入磷酸氫二鉀蛋白胨，混勻，?<span “=””>使之溶解。再以蒸餾水補足至?1000mL<span “=””>。<span “=””>校正?pH?<span “=””>值<span “=””>為?7.2<span “=””>～7.4<span “=””>，<span “=””>分裝于三角瓶內(nèi)，103.43kPa
<span “=””>（121<span “=””>℃??15??lb<span “=””>）15min?<span “=””>高壓滅菌備用。臨用時加入乳糖并加熱融化瓊脂。冷至?60<span “=””>℃<span “=””>左右無?<span “=””>菌操作加入滅菌的伊紅美藍溶液，搖勻。傾注平皿備用。
4.3???<span “=””>蛋白胨水（作靛基質(zhì)試驗用）?<span “=””>成分：蛋白胨（或胰蛋白胨）??20g
<span “=””>氯化鈉??????????????????????????????????????????5g
<span “=””>蒸餾<span “=””>水?????????????????????????????????1000mL
<span “=””>制法：將上述成分加熱融化，調(diào)?pH?<span “=””>值為?7.0<span “=””>～7.2<span “=””>，<span “=””>分裝小試管，103.43kPa<span “=””>（121<span “=””>℃??15
lb<span “=””>）15min?<span “=””>高壓滅菌。<span “=””>
4.4???<span “=””>靛基質(zhì)試劑<span “=””>
<span “=””>柯凡克試劑：將?5g?<span “=””>對二甲氨基苯甲醛溶解于?75mL?<span “=””>戊醇中，然后緩慢加入濃鹽酸?25mL<span “=””>。?<span “=””>試驗方法：接種細菌于蛋白胨水中，于??44<span “=””>℃±0.5<span “=””>℃培<span “=””>養(yǎng)??24h<span “=””>±2h<span “=””>。沿管壁加柯凡克試
<span “=””>劑?0.3mL<span “=””>～0.5mL<span “=””>，輕搖試管。陽性者于試劑層顯深玫瑰紅色。?<span “=””>注：蛋白胨應(yīng)含有豐富的色氨酸，每批蛋白胨買來后，應(yīng)先用已知菌種鑒定后方可使用。
4.5???<span “=””>革蘭氏染色液：
4.5.1???<span “=””>染液制備<span “=””>
4.5.1.1???<span “=””>結(jié)晶紫染色液：<span “=””>
<span “=””>結(jié)<span “=””>晶<span “=””>紫????????????????????????????????????????????????????1g
95<span “=””>％<span “=””>乙醇????????????????????????????????????????????20mL
1<span “=””>％<span “=””>草酸銨水溶液????????????????????????????????80mL
<span “=””>將結(jié)晶紫溶于乙醇中，然后與草酸銨溶液混合。<span “=””>
4.5.1.2???<span “=””>革蘭氏碘液：<span “=””>
<span “=””>碘???????????????????????????????????????????????????????????1g?<span “=””>碘<span “=””>化<span “=””>鉀???????????2g?<span “=””>蒸餾水加至?????????????????????????????????????????????????????300mL
<span “=””>將碘與碘化鉀先進行混合，加入蒸餾水少許，充分振搖，待完全溶解后，再加蒸餾水至
300mL<span “=””>。<span “=””>
4.5.1.3???<span “=””>脫色液：95<span “=””>％乙醇。<span “=””>
4.5.1.4???<span “=””>復(fù)染液：<span “=””>
<span “=””>（1<span “=””>）沙黃復(fù)染液：<span “=””>
<span “=””>沙<span “=””>黃??????????????????????????????????????????????0.25g
95<span “=””>％乙醇??????????????????????????????????????????10mL?<span “=””>蒸<span “=””>餾水????90mL?<span “=””>將沙黃溶解于乙醇中，然后用蒸餾水稀釋。
<span “=””>（2<span “=””>）<span “=””>稀石碳酸復(fù)紅液：稱取堿性復(fù)紅?10g<span “=””>，<span “=””>研細，加?95<span “=””>％乙醇?100mL<span “=””>，<span “=””>放置過夜，濾?<span “=””>紙過濾。取該液?10mL<span “=””>，<span “=””>加?5<span “=””>％石碳酸水溶液?90mL?<span “=””>混合，即為石碳酸復(fù)紅液。再取此液?10mL?<span “=””>加水?90mL<span “=””>，<span “=””>即為稀石碳酸復(fù)紅液。
4.5.2???<span “=””>染色法<span “=””>
4.5.2.1???<span “=””>將涂片在火焰上固定，滴加結(jié)晶紫染色液，染?1min<span “=””>，水洗。<span “=””>
4.5.2.2???<span “=””>滴加革蘭氏碘液，作用?1min<span “=””>，水洗。<span “=””>
4.5.2.3???<span “=””>滴<span “=””>加?95<span “=””>％乙醇脫色，約?30s<span “=””>，<span “=””>或?qū)⒁掖嫉螡M整個涂片，立即傾去，再用乙醇滴滿整?<span “=””>個涂片，脫色?10s<span “=””>，水洗。
4.5.2.4???<span “=””>滴加復(fù)染液，復(fù)染?1min<span “=””>，水洗，待干，鏡檢。
4.5.3?????<span “=””>染色結(jié)果?<span “=””>革蘭氏陽性菌呈紫色，革蘭氏陰性菌呈紅色。
<span “=””>注：如用?1:10?<span “=””>稀釋石碳酸復(fù)紅染色液作復(fù)染，復(fù)染時間僅需?10s<span “=””>。
<span “=””>
5???<span “=””>操<span “=””>作步驟
5.1???<span “=””>取?10mL??1<span “=””>：10?<span “=””>稀<span “=””>釋的檢液，加到?10mL?<span “=””>雙倍乳糖膽鹽（含中和劑）培養(yǎng)基中，置?44
<span “=””>℃±0.5<span “=””>℃培<span “=””>養(yǎng)箱中培養(yǎng)?24h<span “=””>～48h<span “=””>，如不產(chǎn)酸也不產(chǎn)氣，則報告為糞大腸菌群陰性。<span “=””>
5.2???<span “=””>如產(chǎn)酸產(chǎn)氣，劃線接種到伊紅美藍瓊脂平板上，置??36<span “=””>℃<span “=””>±1<span “=””>℃<span “=””>培<span “=””>養(yǎng)??18h<span “=””>～24h<span “=””>。同時取?<span “=””>該培養(yǎng)液?1<span “=””>～2?<span “=””>滴接種到蛋白胨水中，置?44<span “=””>℃<span “=””>±0.5<span “=””>℃<span “=””>培養(yǎng)?24h<span “=””>±2h<span “=””>。
<span “=””>經(jīng)培養(yǎng)后，在上述平板上觀察有無典型菌落生長。糞大腸菌群在伊紅美藍瓊脂培養(yǎng)基上?<span “=””>的典型菌落呈深紫黑色，圓形，邊緣整齊，表面光滑濕潤，常具有金屬光澤。也有的呈紫黑?<span “=””>色，不帶或略帶金屬光澤，或粉紫色，中心較深的菌落，亦常為糞大腸菌群，應(yīng)注意挑選。
5.3???<span “=””>挑取上述可疑菌落，涂片作革蘭氏染色鏡檢。
5.4???<span “=””>在蛋白胨水培養(yǎng)液中，加入靛基質(zhì)試劑約??0.5mL<span “=””>，觀察靛基質(zhì)反應(yīng)。陽性者液面呈玫?<span “=””>瑰紅色；陰性反應(yīng)液面呈試劑本色。
<span “=””>
<span “=””>
6???<span “=””>檢<span “=””>驗結(jié)果報告?<span “=””>根據(jù)發(fā)酵乳糖產(chǎn)酸產(chǎn)氣，平板上有典型菌落，并經(jīng)證實為革蘭氏陰性短桿菌，靛基質(zhì)試
<span “=””>驗陽性，則可報告被檢樣品中檢出糞大腸菌群。
<span “=””>
<span “=””>
<span “=””>四、銅綠假單胞菌<span “=””>
1???<span “=””>范圍?<span “=””>本規(guī)范規(guī)定了化妝品中銅綠假單胞菌的檢驗方法。?<span “=””>本規(guī)范適用于化妝品中銅綠假單胞菌的檢驗。
2???<span “=””>定義?<span “=””>本規(guī)范采用下列定義。
<span “=””>銅<span “=””>綠<span “=””>假<span “=””>單胞<span “=””>菌<span “=””>（Pseudomonas??aeruginosa<span “=””>）<span “=””>屬<span “=””>于假<span “=””>單<span “=””>胞<span “=””>菌<span “=””>屬，<span “=””>為<span “=””>革<span “=””>蘭<span “=””>氏陰<span “=””>性<span “=””>桿<span “=””>菌<span “=””>，氧化?<span “=””>酶陽性，能產(chǎn)生綠膿菌素。此外還能液化明膠，還原硝酸鹽為亞硝酸鹽，在?42<span “=””>℃<span “=””>±1<span “=””>℃條件?<span “=””>下能生長。
<span “=””>該菌對人有致病力，可使傷處化膿，引起敗血癥等。
<span “=””>
3???<span “=””>儀器<span “=””>
3.1???<span “=””>培養(yǎng)箱：42<span “=””>℃<span “=””>±1<span “=””>℃<span “=””>、36<span “=””>℃<span “=””>±1<span “=””>℃<span “=””>。<span “=””>
3.2???<span “=””>三角瓶，250mL<span “=””>。<span “=””>
3.3???<span “=””>試管：15<span “=””>×150mm<span “=””>。<span “=””>
3.4???<span “=””>滅菌平皿：直徑?90mm<span “=””>。<span “=””>
3.5???<span “=””>滅菌刻度吸管，10mL<span “=””>、1mL<span “=””>。<span “=””>
3.6???<span “=””>顯微鏡。<span “=””>
3.7???<span “=””>載玻片。<span “=””>
3.8???<span “=””>接種針、接種環(huán)。<span “=””>
3.9???<span “=””>電磁爐。<span “=””>
3.10???<span “=””>高壓滅菌器。<span “=””>
<span “=””>
4???<span “=””>培<span “=””>養(yǎng)基和試劑
4.1?? SCDLP?<span “=””>液體培養(yǎng)基?<span “=””>見總則中?3.2<span “=””>。
4.2???<span “=””>十六烷基三甲基溴化銨培養(yǎng)基
<span “=””>成分：牛肉膏??????????????????????????????????????? ??3g?<span “=””>蛋白胨?????????????????10g?<span “=””>氯化鈉?????????????????????????????????????????????? ??5g?<span “=””>十六烷基<span “=””>三<span “=””>甲基溴化<span “=””>銨????? ????????????????????????????????????????????0.3g?<span “=””>瓊脂????????????????????20g?<span “=””>蒸餾<span “=””>水????????????????????????????????????????????1000mL
<span “=””>制法：除瓊脂外，將上述成分混合加熱溶解，調(diào)?pH?<span “=””>為?7.4<span “=””>～7.6<span “=””>，加入瓊脂，68.95kPa
<span “=””>（115<span “=””>℃??10?lb<span “=””>）20min?<span “=””>滅<span “=””>菌后，制成平板備用。<span “=””>
4.3???<span “=””>乙酰胺培養(yǎng)基<span “=””>
<span “=””>成分：乙酰<span “=””>胺?????????????????????????????????????10.0g?<span “=””>氯化<span “=””>鈉??? ??5.0g?<span “=””>無水磷酸<span “=””>氫<span “=””>二<span “=””>鉀?????????????????????????????????????????1.39g
<span “=””>無水磷酸<span “=””>二<span “=””>氫<span “=””>鉀???????????????????????0.73g?<span “=””>硫酸<span “=””>鎂<span “=””>（MgSO4.7H2O<span “=””>）???? ??????????????????????????????????????????? ??0.5g?<span “=””>酚<span “=””>紅?? ???0.012g?<span “=””>瓊<span “=””>脂???????????? ????????????????????????????????????????????20g?<span “=””>蒸餾<span “=””>水???1000mL
<span “=””>制法：除瓊脂和酚紅外，將其它成分加到蒸餾水中，加熱溶解，調(diào)?pH?<span “=””>為?7.2<span “=””>，加入瓊?<span “=””>脂、酚紅，103.43kPa<span “=””>（121<span “=””>℃??15?lb<span “=””>）20min?<span “=””>高壓滅菌后，制成平板備用。
4.4???<span “=””>綠膿菌素測定用培養(yǎng)基
<span “=””>成分<span “=””>：<span “=””>蛋白<span “=””>胨????????????????????????????????????????20g?<span “=””>氯化<span “=””>鎂???????????1.4g?<span “=””>硫酸<span “=””>鉀?? ??????????????????????????????????????????????????10g?<span “=””>瓊<span “=””>脂???????????????18g?<span “=””>甘油（化<span “=””>學(xué)<span “=””>純<span “=””>）????????????????????????????????????????????10g?<span “=””>蒸餾<span “=””>水????1000mL
<span “=””>制法：將蛋白胨、氯化鎂和硫酸鉀加到蒸餾水中，加溫使其溶解，調(diào)?pH?<span “=””>至?7.4<span “=””>，加入?<span “=””>瓊脂和甘油，加熱溶解，分裝于試管內(nèi)，68.95kPa<span “=””>（115<span “=””>℃??10?lb<span “=””>）20min<span “=””><span “=””>高壓滅菌后，制成
<span “=””>斜面?zhèn)溆谩?/p>
4.5???<span “=””>明膠培養(yǎng)基<span “=””>
<span “=””>成分<span “=””>：<span “=””>牛肉<span “=””>膏??????????????????????????????????????????3g?<span “=””>蛋白<span “=””>胨??????????????????5g?<span “=””>明<span “=””>膠? ????????????????????????????????????????????120g?<span “=””>蒸餾<span “=””>水??1000mL
<span “=””>制法：取各成分加到蒸餾水中浸泡?20min<span “=””>，<span “=””>隨時攪拌加溫使之溶解，調(diào)?pH?<span “=””>至?7.4<span “=””>，<span “=””>分<span “=””>裝?<span “=””>于試管內(nèi)，經(jīng)?68.95kPa<span “=””>（115<span “=””>℃??10?lb<span “=””>）20min?<span “=””>滅<span “=””>菌后，直立制成高層備用。
4.6???<span “=””>硝酸鹽蛋白胨水培養(yǎng)基
<span “=””>成分<span “=””>：<span “=””>蛋白<span “=””>胨????????????????????????????????????????10g?<span “=””>酵母<span “=””>浸<span “=””>膏???????????????????? ??3g?<span “=””>硝酸<span “=””>鉀??????????????????????????????????????? ??2g?<span “=””>亞硝酸<span “=””>鈉?????????????0.5g?<span “=””>蒸餾<span “=””>水????????????????????????????????????????1000mL
<span “=””>制法：將蛋白胨和酵母浸膏加到蒸餾水中，加熱使之溶解，調(diào)?pH?<span “=””>為?7.2<span “=””>，煮沸過濾后?<span “=””>補足液量，加入硝酸鉀和亞硝酸鈉，溶解混勻，分裝到加有小倒管的試管中，68.95kPa<span “=””>（115
<span “=””>℃??10?lb<span “=””>）20min?<span “=””>滅菌后備用。
4.7???<span “=””>普通瓊脂斜面培養(yǎng)基<span “=””>
<span “=””>成分<span “=””>：<span “=””>蛋白<span “=””>胨????????????????????????????????????????10g?<span “=””>牛肉<span “=””>膏????????????????3g?<span “=””>氯化<span “=””>鈉?????????????????????????????????????????????????5g?<span “=””>瓊<span “=””>脂?????????????15g?<span “=””>蒸餾<span “=””>水???????????????????????????????????????????1000mL
<span “=””>制法：除瓊脂外，將其余成分溶解于蒸餾水中，調(diào)?pH?<span “=””>為?7.2<span “=””>～7.4<span “=””>，<span “=””>加入瓊脂，加熱溶?<span “=””>解，分裝試管，103.43kPa<span “=””>（121<span “=””>℃??10?lb<span “=””>）20min?<span “=””>高壓滅菌后，制成斜面?zhèn)溆谩?/p>
<span “=””>
5???<span “=””>操<span “=””>作步驟
5.1???<span “=””>增菌培養(yǎng)：取?1:10?<span “=””>樣品稀釋液?10mL?<span “=””>加到?90mL??SCDLP?<span “=””>液體培養(yǎng)基中，置?36<span “=””>℃<span “=””>±1<span “=””>℃<span “=””>
<span “=””>?<span “=””>培養(yǎng)??18h<span “=””>～24h<span “=””>。如有銅綠假單胞菌生長，培養(yǎng)液表面多有一層薄菌膜，培養(yǎng)液常呈黃綠色?<span “=””>或藍綠色。
5.2???<span “=””>分<span “=””>離培<span “=””>養(yǎng)：<span “=””>從<span “=””>培<span “=””>養(yǎng)<span “=””>液的<span “=””>薄<span “=””>膜<span “=””>處<span “=””>挑取<span “=””>培<span “=””>養(yǎng)<span “=””>物<span “=””>，劃<span “=””>線<span “=””>接<span “=””>種<span “=””>在十<span “=””>六<span “=””>烷<span “=””>三<span “=””>甲基<span “=””>溴<span “=””>化<span “=””>銨<span “=””>瓊脂<span “=””>平<span “=””>板?<span “=””>上，置?36<span “=””>℃<span “=””>±1<span “=””>℃<span “=””>培養(yǎng)?18h<span “=””>～24h<span “=””>。<span “=””>凡<span “=””>銅綠假單胞菌在此培養(yǎng)基上，其菌落扁平無定型，向周?<span “=””>邊擴散或略有蔓延，表面濕潤，菌落呈灰白色，菌落周圍培養(yǎng)基常擴散有水溶性色素，此培?<span “=””>養(yǎng)基選擇性強，大腸艾希氏菌不能生長，革蘭氏陽性菌生長較差。
<span “=””>在缺乏十六烷三甲基溴化銨瓊脂時也可用乙酰胺培養(yǎng)基進行分離，將菌液劃線接種于平?<span “=””>板上，放?36<span “=””>℃±1<span “=””>℃培養(yǎng)?24h<span “=””>±2h<span “=””>，銅綠假單胞菌在此培養(yǎng)基上生長良好，菌落扁平，邊緣?<span “=””>不整，菌落周圍培養(yǎng)基略帶粉紅色，其它菌不生長。
5.3???<span “=””>染<span “=””>色鏡<span “=””>檢：<span “=””>挑<span “=””>取<span “=””>可<span “=””>疑的<span “=””>菌<span “=””>落<span “=””>，<span “=””>涂片<span “=””>，<span “=””>革<span “=””>蘭<span “=””>氏染<span “=””>色<span “=””>，<span “=””>鏡<span “=””>檢為<span “=””>革<span “=””>蘭<span “=””>氏<span “=””>陰性<span “=””>者<span “=””>應(yīng)<span “=””>進<span “=””>行氧<span “=””>化<span “=””>酶?<span “=””>試驗。
5.4???<span “=””>氧<span “=””>化酶<span “=””>試驗<span “=””>：<span “=””>取<span “=””>一<span “=””>小塊<span “=””>潔<span “=””>凈<span “=””>的<span “=””>白色<span “=””>濾<span “=””>紙<span “=””>片<span “=””>放在<span “=””>滅<span “=””>菌<span “=””>平<span “=””>皿內(nèi)<span “=””>，<span “=””>用<span “=””>無<span “=””>菌玻<span “=””>璃<span “=””>棒<span “=””>挑<span “=””>取銅<span “=””>綠<span “=””>假?<span “=””>單胞菌可疑菌落涂在濾紙片上，然后在其上滴加一滴新配制的?1<span “=””>％二甲基對苯二胺試液，在
15s<span “=””>～30s?<span “=””>之<span “=””>內(nèi)，出現(xiàn)粉紅色或紫紅色時，為氧化酶試驗陽性；若培養(yǎng)物不變色，為氧化酶試?<span “=””>驗陰性。
5.5???<span “=””>綠膿菌素試驗：取可疑菌落?2?<span “=””>個～3?<span “=””>個，分別接種在綠膿菌素測定培養(yǎng)基上，置?36<span “=””>℃<span “=””>
<span “=””>±1<span “=””>℃培養(yǎng)??24h<span “=””>±2h<span “=””>，加入氯仿??3mL<span “=””>～5mL<span “=””>，充分振蕩使培養(yǎng)物中的綠膿菌素溶解于氯仿液?<span “=””>內(nèi)，待氯仿提取液呈藍色時，用吸管將氯仿移到另一試管中并加入?1mol/L?<span “=””>的鹽酸?1mL?<span “=””>左右，?<span “=””>振蕩后，靜置片刻。<span “=””><span “=””>如<span “=””>上層鹽酸液內(nèi)出現(xiàn)粉紅色到紫紅色時為陽性，表示被檢物中有綠膿?<span “=””>菌素存在。
5.6???<span “=””>硝<span “=””>酸鹽<span “=””>還原<span “=””>產(chǎn)<span “=””>氣<span “=””>試<span “=””>驗：<span “=””>挑<span “=””>取<span “=””>可<span “=””>疑的<span “=””>銅<span “=””>綠<span “=””>假<span “=””>單胞<span “=””>菌<span “=””>純<span “=””>培<span “=””>養(yǎng)物<span “=””>，<span “=””>接<span “=””>種<span “=””>在硝<span “=””>酸<span “=””>鹽<span “=””>胨<span “=””>水培<span “=””>養(yǎng)<span “=””>基?<span “=””>中，置?36<span “=””>℃<span “=””>±1<span “=””>℃<span “=””>培養(yǎng)?24h<span “=””>±2h<span “=””>，<span “=””>觀察結(jié)果。凡在硝酸鹽胨水培養(yǎng)基內(nèi)的小倒管中有氣體者，?<span “=””>即為陽性，表明該菌能還原硝酸鹽，并將亞硝酸鹽分解產(chǎn)生氮氣。
5.7???<span “=””>明<span “=””>膠液<span “=””>化試<span “=””>驗<span “=””>，<span “=””>取<span “=””>銅綠<span “=””>假<span “=””>單<span “=””>胞<span “=””>菌可<span “=””>疑<span “=””>菌<span “=””>落<span “=””>的純<span “=””>培<span “=””>養(yǎng)<span “=””>物<span “=””>，穿<span “=””>刺<span “=””>接<span “=””>種<span “=””>在明<span “=””>膠<span “=””>培<span “=””>養(yǎng)<span “=””>基內(nèi)<span “=””>，<span “=””>置<span “=””>
36<span “=””>℃<span “=””>±1<span “=””>℃<span “=””>培<span “=””>養(yǎng)?24h<span “=””>±2h<span “=””>，<span “=””>取出放冰箱?10min<span “=””>～30min<span “=””>，<span “=””>如仍呈溶解狀或表面溶解時即為明膠?<span “=””>液化試驗陽性；如凝固不溶者為陰性。
5.8??42<span “=””>℃<span “=””>生<span “=””>長試<span “=””>驗<span “=””>：<span “=””>挑<span “=””>取可<span “=””>疑<span “=””>的<span “=””>銅<span “=””>綠假<span “=””>單<span “=””>胞<span “=””>菌<span “=””>純培<span “=””>養(yǎng)<span “=””>物<span “=””>，<span “=””>接種<span “=””>在<span “=””>普<span “=””>通<span “=””>瓊脂<span “=””>斜<span “=””>面<span “=””>培<span “=””>養(yǎng)基<span “=””>上<span “=””>，?<span “=””>放在?42<span “=””>℃<span “=””>±1<span “=””>℃<span “=””>培養(yǎng)箱中<span “=””>，<span “=””>培養(yǎng)?24h<span “=””>～48h<span “=””>，<span “=””>銅綠假單胞菌能生長，為陽性，而近似的熒光假?<span “=””>單胞菌則不能生長。
<span “=””>
6???<span “=””>檢<span “=””>驗結(jié)果報告?<span “=””>被檢樣品經(jīng)增菌分離培養(yǎng)后，經(jīng)證實為革蘭氏陰性桿菌，氧化酶及綠膿菌素試驗皆為陽
<span “=””>性者，即可報告被檢樣品中檢出銅綠假單胞菌；如綠膿菌素試驗陰性而液化明膠、硝酸鹽還?<span “=””>原產(chǎn)氣和?42<span “=””>℃生長試驗三者皆為陽性時，仍可報告被檢樣品中檢出銅綠假單胞菌。<span “=””>
<span “=””>五、金黃色葡萄球菌<span “=””>
1???<span “=””>范圍?<span “=””>本規(guī)范規(guī)定了化妝品中金黃金色葡萄球菌的檢驗方法。?<span “=””>本規(guī)范適用于化妝品中金黃色葡萄球菌的檢驗。
<span “=””>
2???<span “=””>定義?<span “=””>本規(guī)范采用下列定義
<span “=””>金黃色葡萄球菌（Staphylococcus aureus<span “=””>）<span “=””>為革蘭氏陽性球菌，呈葡萄狀排列，無芽胞，?<span “=””>無莢膜，能分解甘露醇，血漿凝固酶陽性。
<span “=””>該菌是葡萄球菌中對人類致病力最強的一種，能引起人體局部化膿性病灶，嚴重時可導(dǎo)?<span “=””>致敗血癥。
<span “=””>
3???<span “=””>儀<span “=””>器和設(shè)備
3.1???<span “=””>顯微鏡。<span “=””>
3.2???<span “=””>恒溫培養(yǎng)箱：36<span “=””>℃<span “=””>±1<span “=””>℃<span “=””>。<span “=””>
3.3???<span “=””>離心機。<span “=””>
3.4???<span “=””>滅菌吸管，1mL<span “=””>、10mL<span “=””>。<span “=””>
3.5???<span “=””>滅菌試管：15<span “=””>×150mm<span “=””>。<span “=””>
3.6???<span “=””>載玻片。<span “=””>
3.7???<span “=””>酒精燈。<span “=””>
<span “=””>
4???<span “=””>培<span “=””>養(yǎng)基和試劑
4.1?? SCDLP??<span “=””>液體培養(yǎng)基?<span “=””>見總則中?3.2<span “=””>。
4.2???7.5<span “=””>％的氯化鈉肉湯
<span “=””>成分<span “=””>：<span “=””>蛋白<span “=””>胨??????????????????????????????????10g?<span “=””>牛肉<span “=””>膏???????????3g?<span “=””>氯化<span “=””>鈉???? ??????????????????????????????????????????75g
<span “=””>蒸餾水加<span “=””>至?????????????????????????1000mL
<span “=””>制法：將上述成分加熱溶解，調(diào)?pH?<span “=””>為?7.4<span “=””>，分裝，103.43kPa<span “=””>（121<span “=””>℃??15??lb<span “=””>）15min?<span “=””>高?<span “=””>壓滅菌。
4.3?? Baird Parker?<span “=””>平板
<span “=””>成分：胰蛋白<span “=””>胨???????????????????????????????????10g?<span “=””>牛肉<span “=””>膏??? ??5g?<span “=””>酵母<span “=””>浸<span “=””>膏????? ??1g?<span “=””>丙酮<span “=””>酸<span “=””>鈉??????????????????????????????10g?<span “=””>甘氨<span “=””>酸????12g?<span “=””>氯化鋰（LiCl?6H2O<span “=””>）????????????????????????? ??5g
<span “=””>瓊<span “=””>脂??????????????????????????????????????????20g
<span “=””>蒸餾<span “=””>水?????????????????????????????????950mL
pH7.0<span “=””>±0.2
<span “=””>增菌劑的配制：30<span “=””>％卵黃鹽水?50mL?<span “=””>與除菌過濾的?1<span “=””>％亞碲酸鉀溶液?10mL?<span “=””>混合，保存?<span “=””>于冰箱內(nèi)。
<span “=””>制法：將各成分加到蒸餾水中，加熱煮沸完全溶解，冷至??25<span “=””>℃±1<span “=””>℃校正??pH<span “=””>。分裝每
<span “=””>瓶?95mL<span “=””>，103.43kPa<span “=””>（121<span “=””>℃??15?lb<span “=””>）高<span “=””>壓滅菌?15min<span “=””>。<span “=””>臨用時加熱溶化瓊脂，每?95mL?<span “=””>加入?<span “=””>預(yù)熱至?50<span “=””>℃<span “=””>左右的卵黃亞碲酸鉀增菌劑?5mL<span “=””>，<span “=””>搖勻后傾注平板。培養(yǎng)基應(yīng)是致密不透明的。?<span “=””>使用前在冰箱貯存不得超過?48h<span “=””>±2h<span “=””>。
4.4???<span “=””>血瓊脂培養(yǎng)基
<span “=””>成分：營養(yǎng)瓊<span “=””>脂????????????????????????????100mL
<span “=””>脫纖維羊血（或兔血）??????????????10mL
<span “=””>制法：將營養(yǎng)瓊脂加熱融化，待冷至??50<span “=””>℃左右無<span “=””>菌操作加入脫纖維羊血，搖勻，制成?<span “=””>平板，置冰箱內(nèi)備用。
4.5???<span “=””>甘露醇發(fā)酵培養(yǎng)基
<span “=””>成分：蛋白胨????????????????????????????????????????10g?<span “=””>氯化鈉??? ??5g?<span “=””>甘露醇???10g?<span “=””>牛肉膏??????????????????????????????????????? ??5g
0.2<span “=””>％麝香草<span “=””>酚<span “=””>藍溶<span “=””>液???????????????12mL
<span “=””>蒸餾<span “=””>水?????????????????????????????????1000mL
<span “=””>制法：將蛋白胨、氯化鈉、牛肉膏加到蒸餾水中，加熱溶解，調(diào)?pH7.4<span “=””>，<span “=””>加入甘露醇和?<span “=””>指示劑，混勻后分裝試管中，68.95kPa<span “=””>（115<span “=””>℃??10?lb<span “=””>）20min?<span “=””>滅菌備用。
4.6???<span “=””>兔（人）血漿制備
<span “=””>取?3.8<span “=””>％<span “=””>檸檬酸鈉溶液，103.43kPa<span “=””>（121<span “=””>℃??15??lb<span “=””>）30min?<span “=””>高壓滅菌，1?<span “=””>份加兔（人）全<span “=””>
<span “=””>血?4?<span “=””>份，混勻靜置；2000rpm<span “=””>～3000rpm?<span “=””>離心?3min<span “=””>～5min<span “=””>。血球下沉，取上面血漿。<span “=””>
<span “=””>
5???<span “=””>操<span “=””>作步驟
5.1???<span “=””>增菌：取?1<span “=””>：10?<span “=””>稀<span “=””>釋的樣品接種到?90mL?SCDLP?<span “=””>液體培養(yǎng)基中，置?36<span “=””>℃<span “=””>±1<span “=””>℃<span “=””>培<span “=””>養(yǎng)箱，?<span “=””>培養(yǎng)?24h<span “=””>±2h<span “=””>。
<span “=””>注：如無此培養(yǎng)基也可用?7.5<span “=””>％<span “=””>氯化鈉肉湯。
5.2???<span “=””>分離：自上述增菌培養(yǎng)液中，取?1<span “=””>～2??<span “=””>接種環(huán)，劃線接種在<span “=””>Baird?Parker?<span “=””>氏培養(yǎng)基，如?<span “=””>無此培養(yǎng)基也可劃線接種到血瓊脂平板，置?36<span “=””>℃<span “=””>±1<span “=””>℃<span “=””>培養(yǎng)?24h<span “=””>～48h<span “=””>。<span “=””>在<span “=””>血瓊脂平板上菌落?<span “=””>呈金黃色，大而突起，圓形，不透明，表面光滑，周圍有溶血圈。在?Baird?Parker?<span “=””>氏培養(yǎng)基?<span “=””>上為圓形，光滑，凸起，濕潤，直徑為??2mm<span “=””>～3mm<span “=””>，顏色呈灰色到黑色，邊緣為淡色，周?<span “=””>圍為一混濁帶，在其外層有一透明帶。用接種針接觸菌落似有奶油樹膠的軟度。偶然會遇到?<span “=””>非脂肪溶解的類似菌落，但無混濁帶及透明帶。挑取單個菌落分純在血瓊脂平板上，置??36
<span “=””>℃±1<span “=””>℃培養(yǎng)?24h<span “=””>±2h<span “=””>。<span “=””>
5.3???<span “=””>染<span “=””>色鏡<span “=””>檢：<span “=””>挑<span “=””>取<span “=””>分<span “=””>純菌<span “=””>落<span “=””>，<span “=””>涂<span “=””>片，<span “=””>進<span “=””>行<span “=””>革<span “=””>蘭氏<span “=””>染<span “=””>色<span “=””>，<span “=””>鏡檢<span “=””>。<span “=””>金<span “=””>黃<span “=””>色葡<span “=””>萄<span “=””>球<span “=””>菌<span “=””>為革<span “=””>蘭<span “=””>氏?<span “=””>陽性菌，排列成葡萄狀，無芽胞，無夾膜，致病性葡萄球菌，菌體較小，直徑約為?0.5ìm<span “=””>～
1ìm<span “=””>。
5.4???<span “=””>甘<span “=””>露醇<span “=””>發(fā)酵<span “=””>試<span “=””>驗<span “=””>：<span “=””>取上<span “=””>述<span “=””>分<span “=””>純<span “=””>菌落<span “=””>接<span “=””>種<span “=””>到<span “=””>甘露<span “=””>醇<span “=””>發(fā)<span “=””>酵<span “=””>培養(yǎng)<span “=””>基<span “=””>中<span “=””>，<span “=””>在培<span “=””>養(yǎng)<span “=””>基<span “=””>液<span “=””>面上<span “=””>加<span “=””>入<span “=””>
2mm<span “=””>～3mm?<span “=””>的滅菌液體石蠟，置?36<span “=””>℃<span “=””>±1<span “=””>℃<span “=””>培養(yǎng)?24h<span “=””>±2h<span “=””>，<span “=””>金黃色葡萄球菌應(yīng)能發(fā)酵甘露醇?<span “=””>產(chǎn)酸。
5.5???<span “=””>血漿凝固酶試驗：吸取?1:4?<span “=””>新鮮血漿?0.5mL<span “=””>，<span “=””>放入滅菌小試管中，加入待檢菌?24h<span “=””>±2h?<span “=””>肉湯培養(yǎng)物?0.5mL<span “=””>。<span “=””>混勻，放?36<span “=””>℃<span “=””>±1<span “=””>℃<span “=””>恒溫箱或恒溫水浴中，<span “=””>每半小時觀察一次，6h?<span “=””>之內(nèi)?<span “=””>如呈現(xiàn)<span “=””>凝塊<span “=””>即為陽<span “=””>性。<span “=””>同時以<span “=””>已知<span “=””>血漿凝<span “=””>固酶<span “=””>陽性和<span “=””>陰性<span “=””>菌株肉<span “=””>湯培<span “=””>養(yǎng)物及<span “=””>肉湯<span “=””>培<span “=””>養(yǎng)基<span “=””>各
0.5mL<span “=””>，分別加入滅菌?1:4?<span “=””>血漿?0.5mL<span “=””>，混勻，作為對照。
<span “=””>
6???<span “=””>檢<span “=””>驗結(jié)果報告?<span “=””>凡在上述選擇平板上有可疑菌落生長，經(jīng)染色鏡檢，證明為革蘭氏陽性葡萄球菌，并能發(fā)酵甘露醇產(chǎn)酸，血漿凝固酶試驗陽性者，可報告被檢樣品檢出金黃色葡萄球菌。
<span “=””>六、霉菌和酵母菌<span “=””>
1???<span “=””>范圍?<span “=””>本規(guī)范規(guī)定了化妝品中霉菌和酵母菌數(shù)的檢測方法。?<span “=””>本規(guī)范適用于各種化妝品中霉菌和酵母菌的計數(shù)。
<span “=””>
2???<span “=””>定義?<span “=””>本規(guī)范采用下列定義。
<span “=””>霉菌和酵母菌數(shù)測定（Determination?of?molds?and?yeast count<span “=””>）<span “=””>是指化妝品檢樣在一定條?<span “=””>件下培養(yǎng)后，1g?<span “=””>或?1mL?<span “=””>化妝品中所污染的活的霉菌和酵母菌數(shù)量，藉以判明化妝品被霉菌?<span “=””>和酵母菌污染程度及其一般衛(wèi)生狀況。
<span “=””>本方法根據(jù)霉菌和酵母菌特有的形態(tài)和培養(yǎng)特性，在虎紅培養(yǎng)基上，置?28<span “=””>℃<span “=””>±2<span “=””>℃培養(yǎng)72h<span “=””>，計算所生長的霉菌和酵母菌數(shù)。
<span “=””>
3???<span “=””>儀<span “=””>器和設(shè)備
3.1???<span “=””>培養(yǎng)箱：28<span “=””>℃<span “=””>±2<span “=””>℃<span “=””>。<span “=””>
3.2???<span “=””>振蕩器。<span “=””>
3.3???<span “=””>天平。<span “=””>
3.4???<span “=””>三角瓶，250mL<span “=””>。<span “=””>
3.5???<span “=””>試管：15<span “=””>×150mm<span “=””>。<span “=””>
3.6???<span “=””>平皿：直徑?9cm<span “=””>。<span “=””>
3.7???<span “=””>吸管，1mL<span “=””>、10mL<span “=””>。<span “=””>
3.8???<span “=””>量筒，200mL<span “=””>。<span “=””>
3.9???<span “=””>酒精燈。<span “=””>
3.10???<span “=””>高壓滅菌器。<span “=””>
<span “=””>
4???<span “=””>培<span “=””>養(yǎng)基和試劑
4.1???<span “=””>生理鹽水?<span “=””>見總則中?3.1<span “=””>。
4.2???<span “=””>虎紅（孟加拉紅）培養(yǎng)基
<span “=””>成分：蛋白胨??????????????????????????????????????? ??5g?<span “=””>葡萄糖??????????10g?<span “=””>磷酸二氫鉀?????????????????????????????????????????????? ??1g?<span “=””>硫酸鎂（含?7H2O<span “=””>）?????0.5g?<span “=””>瓊脂????????????????????????????????????????????20g
1/3000?<span “=””>虎紅溶液????????????????????100mL
<span “=””>（<span “=””>四氯四碘熒光素）<span “=””>
<span “=””>蒸餾<span “=””>水?????????????????????????????????1000mL
<span “=””>氯<span “=””>霉<span “=””>素???????????????????????????????????100mg
<span “=””>制法：將<span “=””>上<span “=””>述各成分<span “=””>（<span “=””>除虎紅外<span “=””>）<span “=””>加入蒸餾<span “=””>水<span “=””>中溶解后<span “=””>，<span “=””>再加入虎<span “=””>紅<span “=””>溶液。分<span “=””>裝<span “=””>后，103.43kPa<span “=””>（121<span “=””>℃??15 lb<span “=””>）20min??<span “=””>高壓滅菌，另用少量乙醇溶解氯霉素，過濾溶解后加入培<span “=””>養(yǎng)基中，若無氯霉素，使用時每?1000mL?<span “=””>加鏈霉素?30mg<span “=””>。
<span “=””>
5???<span “=””>操<span “=””>作步驟
5.1???<span “=””>樣品稀釋?<span “=””>見菌落總數(shù)測定中?6.1<span “=””>。
5.2???<span “=””>取??1<span “=””>：10<span “=””>、1<span “=””>：100<span “=””>、1<span “=””>：1000??<span “=””>的<span “=””>檢液各??1mL??<span “=””>分別注入滅菌平皿內(nèi)，每個稀釋度各用??2
<span “=””>個平皿，注入融化并冷至?45<span “=””>℃±1<span “=””>℃<span “=””>左右的虎紅培養(yǎng)基，充分搖勻。凝固后，翻轉(zhuǎn)平板，置<span “=””>
28<span “=””>℃<span “=””>±1<span “=””>℃<span “=””>培<span “=””>養(yǎng)??72h<span “=””>±2h<span “=””>，計數(shù)平板內(nèi)生長的霉菌和酵母菌數(shù)。若有霉菌蔓延生長，為避免?<span “=””>影響其它霉菌和酵母菌的計數(shù)時，于?48h<span “=””>±2h?<span “=””>應(yīng)及時將此平板取出計數(shù)。
5.3???<span “=””>計<span “=””>算方<span “=””>法：<span “=””>先<span “=””>點<span “=””>數(shù)<span “=””>每個<span “=””>平<span “=””>板<span “=””>上<span “=””>生長<span “=””>的<span “=””>霉<span “=””>菌<span “=””>和酵<span “=””>母<span “=””>菌<span “=””>菌<span “=””>落數(shù)<span “=””>，<span “=””>求<span “=””>出<span “=””>每個<span “=””>稀<span “=””>釋<span “=””>度<span “=””>的平<span “=””>均<span “=””>菌?<span “=””>落數(shù)。判定結(jié)果時，應(yīng)選取菌落數(shù)在?5?<span “=””>個～50?<span “=””>個范圍之內(nèi)的平皿計數(shù)，乘以稀釋倍數(shù)后，?<span “=””>即為每?g<span “=””>（或每?mL<span “=””>）檢樣中所含的霉菌和酵母菌數(shù)。其它范圍內(nèi)的菌落數(shù)報告應(yīng)參照菌落?<span “=””>總數(shù)的報告方法報告之。
5.4???<span “=””>每?g<span “=””>（<span “=””>或每?mL<span “=””>）化妝品含霉菌和酵母菌數(shù)以?CFU/g<span “=””>（mL<span “=””>）表示。
<span “=””>
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