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
- 无菌脱纤维羊血(品牌:Solarbio;产地:中国;catalog number: TX0030)
- 琼脂(品牌:Biotopped;产地:日本;catalog number: A6190)
- 氯化血红素-维生素K1(品牌:PYG;产地:日本;catalog number: 21301)
- 脑心浸液肉汤培养基(BHI)(品牌:Oxiod;产地:美国;catalog number: CM1135)
- 脑心浸液琼脂培养基(品牌:AOBOX;产地:中国;catalog number: 02-349)
- MRS肉汤培养基(品牌:Oxiod;产地:美国;catalog number: CM0359B)
- MRS琼脂培养基(品牌:Oxiod;产地:美国;catalog number: CM0359B)
- 液体沙氏培养基(品牌:海博;产地:中国;catalog number: HB0379)
- 沙氏琼脂培养基(品牌:海博;产地:中国;catalog number: HB0235)
仪器设备
- 二氧化碳孵箱(品牌:Thermo Fisher Scientific;产地:美国;catalog number: ab12345)
- 电热恒温培养箱(品牌:上海一恒;产地:中国;catalog number: DHP-9052)
- 厌氧培养罐(品牌:尤德生物;产地:中国;catalog number: UT706)
- 厌氧产气袋(品牌:梅里埃;产地:法国;catalog number: 45534)
- 恒温摇床(品牌:RADOBIO;产地:中国;catalog number: Stab S2)
- 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
- 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)。
- 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)。 - 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)。
- 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)。
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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
参考文献
- Fu, H. (2017). Clinical study of oral Candida Albicans infection screening and diagnosis. Chin J Med Guide 000(004): 616-618. (in Chinese)
- Ren, W., Chen, F., Zhang, Y., Zhang, Q., Wang, X., Liu, Y., Yuan, C., Ma, Q., Xu, T. and Zheng, S. (2014).Identification of Streptococcus mutans in carious patients’ saliva samples by MALDI-TOF mass spectrometry. J Peking Univ Health Sci 46(1): 25-29. (in Chinese)
- Zhou, X. D., Xiao, L. Y. and Xiao, X. R. (2009). Applied oral microbiology and technique. People's Medical Publishing House Co., LTD. ISBN: 9787117112857. (in Chinese)
- Zhu, L. F. and Wang, B. (2017). Platycodin D protects oral epithelial cells against infection of Candida albicans. Chin J Pathophys 33(1): 161-165. (in Chinese)
- Chen, Z. Y., Hsieh, Y. M., Huang, C. C. and Tsai, C. C. (2017). Inhibitory effects of probiotic Lactobacillus on the growth of human colonic carcinoma cell line HT-29. Molecules 22(1): 107.
- Gamarra, S., Mancilla, E., Dudiuk, C. and Garcia-Effron, G. (2015). Candida dubliniensis and Candida albicans differentiation by colony morphotype in Sabouraud-triphenyltetrazolium agar. Rev Iberoam Micol 32(2): 126–128.
- Hu, L., He, C., Zhao, C., Chen, X., Hua, H. and Yan, Z. (2019). Characterization of oral candidiasis and the Candida species profile in patients with oral mucosal diseases. Microb Pathog 134: 103575.
- Khan, M. A., Khan, S. M., Sarker, M., Hussain, R., Ahmed, M., Joarder, Y., and Hasan, M. (2013). Studies on microscopic technique and culture on Sabouraud’s dextrose agar medium for diagnosis of dermatophytes infection. KYAMC Journal, 3(1): 235-238.
- Jacouton, E., Chain, F., Sokol, H., Langella, P. and Bermúdez-Humarán, L. G. (2017). Probiotic strain Lactobacillus casei BL23 prevents colitis-associated colorectal cancer. Front Immunol 8: 1553.
- Lin, S., Deng, D. M., Roffe, S. and Gibbs, S. (2020). Differential influence of Streptococcus mitis on host response to metals in reconstructed human skin and oral mucosa. Contact Dermatitis 83(5): 347-360.
- Lu, W. X., Wu, Y. F., Xiao, L. Y., Li, M. Y., Guo, Q., Xiong, P., Jia, X. M., Xiao, X. R., Zhu, Z., Gong, Q. M. and Li, W. (2009). Preliminary study on the discrimination of putative periodontal pathogens with a metabonomics method. West China Journal of Stomatology 27(3): 310-312, 316.
- Moslemi, N., Soleiman-zadeh Azar, P., Bahador, A., Rouzmeh, N., Chiniforush, N., Paknejad, M. and Fekrazad, R. (2015). Inactivation of Aggregatibacter actinomycetemcomitans by two different modalities of photodynamic therapy using Toluidine blue O or Radachlorin as photosensitizers: an in vitro study. Lasers Med Sci 30(1): 89-94.
- Ratchapin, L. S., Boworn, K. and Natthamet, W. (2016). Antimicrobial effect of topical local anesthetic spray on oral microflora. J Dent Anesth Pain Med 16(1): 17-24.
- Sun, Y., Pan, Y. H., Sun,Y., Li, M. Y., Huang, S. B., Qiu, W., Tu, H. X. and Zhang, K. K. (2019). Effects of norspermidine on dual-species biofilms composed of Streptococcus mutans and Streptococcus sanguinis. Biomed Res Int 3: 1950790.
- Wright, C. T. and Klaenhammer, T. R. (1981). Calcium-Induced alteration of cellular morphology affecting the resistance of Lactobacillus acidophilus to freezing. Appl Environ Microbiol 41(3): 807-815.
- Yue, Y. C., Xu, X. X., Yang, B. Y., Lu, J., Zhang, S. W., Liu, L., Nassar, K., Zhang, C., Zhang, M., Pang, X. Y. and Lv, J. P. (2020). Stable colonization of orally administered Lactobacillus casei SY13 alters the gut microbiota. Biomed Res Int 3: 5281639.
- Yue, Y., Ye, K., Lu, J., Wang, X., Zhang, S., Liu, L., Yang, B., Nassar, K., Xu, X., Pang, X. and Lv, J. P. (2020). Probiotic strain Lactobacillus plantarum YYC-3 prevents colon cancer in mice by regulating the tumour microenvironment. Biomed Pharmacother 127: 110159.
- Zhang, Y., Liu, Y., Ma, Q., Song, Y., Zhang, Q., Wang, X. and Chen, F. (2014). Identification of Lactobacillus from the saliva of adult patients with caries using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. PLoS One 9: e106185.
- Zinatizadeh, N., Khalili, F., Fallah, P., Geravand, M. and Yaslianifard, S. (2018). Potential preventive effect of Lactobacillus acidophilus and Lactobacillus plantarum in patients with polyps or colorectal cancer. Arq Gastroenterol 55(4): 407-411.
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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. // 微生物组实验手册.
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. // Microbiome Protocols eBook.
Bio-101: e2003516. DOI:
10.21769/BioProtoc.2003516.