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口腔常见微生物的培养方法   

张翼飞张翼飞*卢洪叶卢洪叶*张倩张倩陈智滨陈智滨 陈峰陈峰  (*contributed equally to this work)
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摘要:口腔微生物实验常见菌包括:链球菌属、乳酸杆菌属、白色念珠菌、牙龈卟啉单胞菌、具核梭杆菌、中间普雷沃菌以及伴放线聚集杆菌等。其基本培养条件分别是:口腔链球菌需在37℃含5%CO2或厌氧条件下,静置培养在BHI培养基中;乳酸杆菌需在37℃含5%CO2或厌氧条件下,静置培养于MRS培养基中;口腔白色念珠菌需37℃,150 r/min摇瓶培养于沙氏培养基或静置培养于沙堡弱培养基平皿;牙龈卟啉单胞菌、具核梭杆菌、中间普雷沃菌等牙周可疑致病菌需在37℃下,用BHI血肉汤培养基在80%N2、10%H2和10%CO2混合气体下厌氧培养;伴放线聚集杆菌需在37℃含5~10% CO2下,用BHI血肉汤培养基培养。

关键词: 口腔链球菌属, 口腔乳酸杆菌属, 白色念珠菌, 牙龈卟啉单胞菌, 具核梭杆菌, 中间普雷沃菌, 伴放线聚集杆菌, 培养条件

材料与试剂

  1. 无菌脱纤维羊血(品牌:Solarbio;产地:中国;catalog number: TX0030)
  2. 琼脂(品牌:Biotopped;产地:日本;catalog number: A6190)
  3. 氯化血红素-维生素K1(品牌:PYG;产地:日本;catalog number: 21301)
  4. 脑心浸液肉汤培养基(BHI)(品牌:Oxiod;产地:美国;catalog number: CM1135)
  5. 脑心浸液琼脂培养基(品牌:AOBOX;产地:中国;catalog number: 02-349)
  6. MRS肉汤培养基(品牌:Oxiod;产地:美国;catalog number: CM0359B)
  7. MRS琼脂培养基(品牌:Oxiod;产地:美国;catalog number: CM0359B)
  8. 液体沙氏培养基(品牌:海博;产地:中国;catalog number: HB0379)
  9. 沙氏琼脂培养基(品牌:海博;产地:中国;catalog number: HB0235)

仪器设备

  1. 二氧化碳孵箱(品牌:Thermo Fisher Scientific;产地:美国;catalog number: ab12345)
  2. 电热恒温培养箱(品牌:上海一恒;产地:中国;catalog number: DHP-9052)
  3. 厌氧培养罐(品牌:尤德生物;产地:中国;catalog number: UT706)
  4. 厌氧产气袋(品牌:梅里埃;产地:法国;catalog number: 45534)
  5. 恒温摇床(品牌:RADOBIO;产地:中国;catalog number: Stab S2)
  6. 立式压力蒸汽灭菌器(品牌:上海申安;产地:中国;catalog number: LDZX-30KBS)

实验步骤

一、培养方法

  1. 口腔链球菌(Streptococcus),包括血链球菌(Streptococcus sanguinis)、变异链球菌(Streptococcus mutans)、唾液链球菌(Streptococcus salivarius)、酿脓链球菌(Streptococcus pyogenes)、轻型链球菌(Streptococcus mitis)等的培养。
    该方法已被广泛应用于多个研究并得到了有效验证(Fu, 2013; Zhu and Wang, 2017; Sun et al., 2019;Ren et al., 2014)。
    1.1
    培养基的选择:选用脑心浸液肉汤(brain heart infusion, BHI)培养基和BHI琼脂培养基。
    1.2
    菌株的活化与复壮:取20 μl冻存于甘油管中的保种液,接种于1 ml的BHI肉汤培养基中,于5%二氧化碳孵箱(或厌氧培养罐)中,37 ℃下静置培养24 h,活化菌株。将过夜培养的口腔链球菌采用三区划线的方式转接于BHI琼脂培养基上,37 ℃培养48 h,以此连续活化3次。
    1.3
    菌株扩大培养:从培养皿上挑取单菌落,接种到1 ml的BHI肉汤培养基中,放置到5%二氧化碳孵箱中(或厌氧培养罐中),37 ℃下静置培养12 h。
    1.4
    菌种的保存:常采用冷冻保存法或液氮保存法(Zhou et al., 2009)。

  2. 口腔乳酸杆菌属(Lactobacillus),包括嗜酸乳酸杆菌(Lactobacillus acidophilus)、干酪乳酸杆菌(Lactobacillus casei)等的培养。
    该方法已被广泛应用于多个研究并得到了有效验证(Wright and Klaenhammer, 1981;Zhang et al., 2014)。
    2.1
    培养基的选择:通常选用MRS肉汤培养基(De Man, Rogosa and Sharpe Broth)和MRS琼脂培养基(Ratchapin et al., 2016; Zinatizadeh et al., 2018; Lin et al., 2020)。
    2.2
    菌株的活化与复壮:取20 μl冻存于甘油管中的保种液,接种于1 ml的MRS肉汤培养基中,于37 ℃条件下静置培养24 h,活化菌株(Chen et al., 2017; Yue et al., 2020)。将过夜培养的乳酸杆菌采用三区划线的方式转接于MRS琼脂培养基上,37 ℃培养48 h,以此连续活化3次。
    2.3
    菌株扩大培养:从培养皿上挑取单菌落,接种到1 ml的MRS肉汤培养基中,放置到5%二氧化碳孵箱中(或厌氧培养罐中),37 ℃下培养18 h。
    2.4
    菌种的保存:常用的有冷冻保存法或冷冻干燥法。

  3. 常见牙周致病菌的培养
    主要牙周致病菌包括:牙龈卟啉单胞菌(Porphyromonas gingivalis)、具核梭杆菌(Fusobacteria nucleatum)、中间普雷沃菌(Prevotella intermedia)以及伴放线聚集杆菌(Aggregatibacter actinomycetemcomitans)等。该方法已被广泛应用于多个研究并得到了有效验证 (Moslemi et al., 2015; Lu et al., 2009;Li et al., 2019)。
    3.1
    培养基的选择:通常选用BHI血肉汤培养基和BHI血琼脂平板培养基(用于厌氧菌培养,最好新鲜配置,避免放置时间过长而渗入大量氧气)。
    3.2
    菌种复苏:从吸取20 μl保种液,接种到BHI血琼脂平板培养基上,放入厌氧培养袋中,并将袋置入5%二氧化碳孵箱(厌氧袋法);也可直接置于含有80%N2、10%H2和10%CO2混合气体的厌氧培养罐中(厌氧培养罐法),37 ℃下培养5-7 d。
    3.3
    菌株扩大培养:从培养皿上挑取1-3个单菌落,接种到2 ml的BHI血肉汤培养基中,放到厌氧产氧袋中,将袋置入37 ℃的空气孵箱中,培养48 h。
    3.4
    菌种的保存:常用的有冷冻保存法或冷冻干燥法。
    注:另外一个重要的牙周致病菌伴放线聚集杆菌(Aggregatibacter actinomycetemcomitans)是微需氧菌,生长需要5~10%的二氧化碳条件下,其他操作步骤同上。

  4. 口腔白色念珠菌(Candida albicans)的培养
    该方法已被广泛应用于多个研究并得到了有效验证(Khan et al., 2013; Gamarra et al., 2015;Hu et al., 2019)。
    4.1
    培养基的选择:通常选用沙氏(Sabouraud)琼脂培养基和沙氏液体培养基。
    4.2
    菌种复苏:吸取20 μl甘油保种液,接种到沙氏琼脂培养板上,放置到5%二氧化碳孵箱中,37 ℃下培养48 h(Jacouton et al., 2017)。
    4.3
    菌株扩大培养:从培养皿上挑取单菌落,接种到1 ml的沙氏液体培养基中,37 ℃下150 r/min摇瓶培养48 h(Yue et al., 2020)。
    4.4
    菌种的保存:常用的有冷冻保存法或冷冻干燥法。


二、 培养基的配制
注:培养基的配制及灭菌需要参考产品包装上的说明,不同品牌的同一产品配制比例及灭菌温度和时间可能不一样。

  1. BHI肉汤培养基:称取3.7 g BHI肉汤干粉,溶于100 ml蒸馏水中,121 ℃高压灭菌15 min,待冷却至常温,备用。
  2. BHI血肉汤培养基:将3.7 g BHI 肉汤干粉,溶于100 ml蒸馏水中。121 ℃高压灭菌15 min,降至50 ℃。以1:10和1:100的浓度加入无菌脱纤维羊血(冻融的羊血效果更佳)和氯化血红素-维生素K,震荡混合均匀。冷却至常温,放入4 ℃冰箱,避光保存,备用。
  3. BHI琼脂培养基:称取5.2 g BHI琼脂干粉,溶于100 ml蒸馏水中,121 ℃高压灭菌15 min,待温度降至50℃左右倒入无菌培养皿中,待凝固,备用。
  4. BHI血琼脂平板培养基:将BHI粉、琼脂粉与蒸馏水以3.7 g:2 g:100 ml的比例混合。121℃高压灭菌15 min,降至50 ℃。以1:10和1:100的浓度加入无菌脱纤维羊血(冻融的羊血效果更佳)和氯化血红素-维生素K,震荡混合均匀。倾注于无菌平板上,待其冷却凝固,放入4 ℃冰箱,避光保存,备用。
  5. MRS肉汤培养基:称取5.2 g MRS肉汤干粉,溶于100 ml蒸馏水中,115 ℃高压灭菌20 min,待冷却至常温,备用。
  6. MRS琼脂培养基:称取6.62 g MRS琼脂粉,溶于100 ml蒸馏水中,115 ℃高压灭菌20 min,待温度降至50 ℃左右倒入无菌培养皿中,待凝固,备用。
  7. 沙氏液体培养基:称取5 g液体沙氏培养基干粉,溶于100 ml蒸馏水中,115  ℃高压灭菌20 min,待冷却至常温,备用。
  8. 沙氏固体培养基:称取6.5 g液体沙氏琼脂培养基干粉,溶于100 ml蒸馏水中,115 ℃高压灭菌20 min,待温度降至50  ℃左右倒入无菌培养皿中,待凝固,备用。


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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:张翼飞, 卢洪叶, 张倩, 陈智滨, 陈峰. (2021). 口腔常见微生物的培养方法. Bio-101: e2003516. DOI: 10.21769/BioProtoc.2003516.
How to cite: Zhang, Y. F., Lu, H. Y., Zhang, Q., Chen, Z. B. and Chen, F. (2021). Culture Method of Oral Common Microorganisms. Bio-101: e2003516. DOI: 10.21769/BioProtoc.2003516.
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