双歧杆菌计数培养基的研究报告

发布时间:

2022-12-27

作者:


摘要 [目的]选择一种适用于微生态制剂中的双歧杆菌计数培养基。[方法]用改 良BBL、眦 s、改 良TJA 等选择性培养基和平板厌氧胶法对双歧杆菌进行检测,同时观察双歧杆菌和乳酸杆菌在三种培养基上生长情况。[结果]在改 良BBL 培养基上双歧杆菌生长良好,乳酸杆菌不生长。[结论 ]该培养基用 于微生态制剂中双歧杆菌计数操作简便、有效,易于推广。
关键词:双歧杆菌;计数;改 良BBL
 
近年来 双歧杆 菌 的微 生 态制 剂 产 品在 市 场上 较 为 多 见 ,主要以发酵乳和保健品的形式 出现,我国 目前尚无统一 的标准检 验方法对 双歧 杆 菌进 行 检测 ,微 生 态 活 菌制 剂 中双歧 杆菌的活菌数是该产品在保质期 内的重要质量指标。双歧菌为专性厌氧菌,检测方法繁琐。有必要选择一种能让大多数 已知的双歧杆菌在其良好生长的选择性培养基中对其进行质量控制 。
1 材 料与方 法
1.1 青春双歧杆菌、保加利亚乳酸杆菌为实验室保存菌种。
两歧 双歧杆菌 11853、嗜热 链球 菌 11855 ,北 京 生物 制 品鉴定 所提供 。
1.2 培养基及稀释液
1.2.1 培养基 改良 BBL 培养基:
多胨 8g,大豆胨 1.2g,酵母浸膏粉 1.2g;
葡萄糖 2g,可溶性淀粉 0.25g;
吐温 一80 0 .4g,L 一半胱氨酸 0 .4g,M;
7H 20 0 .2g,Nacl 2g,~ H r'0 4 lg;
硫乙醇酸钠 0 .4g,CaC 2g,牛 肉汤 2oom ];
肝 汤 2oom l,西 红柿 汁 2oral,低聚糖 3g,琼脂 8g;
调节 pH 值为 7 .0 ±0.2 ,121℃;
15r a in 灭菌后每 100m l加入 6%的氯化锂溶液 5m l,倾注平皿备用。改良TJA培养基?,MIlS 培养基【 ,厌氧胶(自配制)。
1.2.2 磷酸盐稀释液 Nai l2P44.5g.M a2HP4 6.0s,L 一半胱氨酸 0 .5g,吐温 一80 0 .5g,蒸馏水 1000m l。
2 方法与结果
2 .1 茵种的活化 将双歧杆菌接种于改 良 BBL 液体培养管中,用螺纹帽拧紧管 口,乳 酸杆菌接种于 12%脱脂 奶 中,置37℃培养,重复 2 次。
2 .2 双歧杆茵计数  以无菌操作用 hld 灭菌吸管吸取 lm l 活化后的菌液作 10 倍递增稀释 ,选择 3个合适的稀释度,每一稀释度取 0.1m l滴于预先倾注好的计数培养基平板表面,每个稀释度同时做 2 份,用灭菌 L 棒均匀涂布,并同时做稀释液的空白对照。以上操作 ~01nin 内完成。
 
将厌氧胶定量用蒸馏水溶解后倾注于涂布完菌液的改 良BBL 平 板 盖 内 ,每 盖 约 20m l,待凝 固后将 平 板 ,盖 压 紧然 后 翻转平板置 37℃培养 72h ±3h 后取 出,观察双歧杆菌菌落特征(见表 1)。选取菌落数在 30 —300 之间的平板进行计数。计数后,随机挑取 5 个菌落数进行革兰染色,显微镜检查并做触酶试验。革兰阳性,触酶阴性,无芽胞的形态不规则杆菌,呈分枝或分叉形、匙形或球菌可定为双歧杆菌。根据证实为双歧杆菌菌落计算出该平板内的双歧杆菌数 ,然后乘其稀释倍数后再换算 成每 毫升样 品中双歧 杆菌数 。
 双歧杆菌在三种培养基上的形态图表
2.3 乳酸茵在几种培养基上生长情况 将活化后的乳酸菌菌液作 10 倍递稀释后,选择合适稀释度分别接种于改 良 BBL、M RS 、改良 TJA 等 3 种培养基上,37℃72h 培养,观察菌落生长情况 (见表 2 )。
乳酸菌在三种培养基上的形态图表
2 .4 结果双歧杆菌在改 良 BBL 培养基上用平板厌 氧胶法培养后,生长 良好,菌落乳 白色,对培养菌液计数可达 1  。乳酸杆菌不生长,嗜热链球菌能够在其生长,菌落呈浅褐色。
 
 
南京市疾病预防控制中心,江苏:陈晓蔚 ,丁洁 ,王炜 ,贾力敏

Study on the culture medium of Bifidobacterium counting
[Objective] to select a counting medium for Bifidobacterium in microecological preparations. [Methods] the bifidobacteria were detected by modified BBL, canthus s, improved TJA and other selective medium, and the growth of bifidobacteria and Lactobacillus on three kinds of medium. [results] bifidobacteria grew well on modified BBL medium and Lactobacillus did not grow. [Conclusion] the culture medium used for counting Bifidobacterium in microecological preparation is simple, effective and easy to popularize.
Key words: bifidobacteria; counting; improved BBL
In recent years, the microecological products of bifidobacteria are more common in the market, mainly in the form of fermented milk and health care products. There is no unified standard in China at present.
The detection of bifidobacteria was carried out by the method of quasi test. The number of Bacillus Bifidobacterium in the microecological living bacteria preparation is the important quality index of the product in the shelf life. Bifidobacterium
Specific anaerobes, the testing method is tedious. It is necessary to select a selective medium in which most known bifidobacteria can be controlled.
1 materials and methods
1.1 Bifidobacterium youth and Lactobacillus Bulgaria are the bacteria for preservation in the laboratory.
Bifidobacterium Bifidobacterium 11853 and Streptococcus thermophilus 11855, provided by Beijing biologics identification.
1.2 medium and diluent
1.2.1 medium improved BBL medium: peptone 8g, soybean peptone 1.2g, yeast extract powder 1.2g, glucose 2G, soluble starch 0.25g, Twain 80 0.4g, L
Cysteine 0.4g, M. 7H 20 0.2g, Nacl 2G, ~ H r'0 4 LG, sodium glycolate 0.4g, CaC 2G, beef soup 2oom, liver soup 2oom, tomato juice, oligosaccharide, agar, 7 + 0.2121. Modified TJA medium, MIlS medium, anaerobic gel (self formulated).
1.2.2 Phosphate diluent Nai l2P44.5g.M a2HP4 6.0s, L-cysteine 0.5g, Tween 1800.5g, distilled water 1000ml.
2 methods and results
The activation of 2.1 strains of bacilli inoculated Bifidobacterium in the modified BBL liquid culture tube, tightened the pipe mouth with threaded cap, inoculated lactobacillus in 12% skimmed milk, and placed 37 C for 2 times.
2.2 the count of the bifidus strain was 10 times increasing dilution with the bacteria liquid activated by LM L in the sterile HLD sterilizing suction tube, selecting 3 appropriate dilution degrees, and dropping 0.1M L on the surface of a pre poured count medium plate, each dilution at the same time, 2 copies at the same time, and evenly coated with the sterilized L rod. At the same time, a blank control of the diluent was made. The above operation is completed within ~01nin.
The anaerobic adhesive was dissolved in the distilled water, and then poured into the modified BBL flat cover, covering about 20m L. After solidification, the flat plate was pressed and the plate was pressed and then turned over to 37 C for 72h + 3H to observe the colony characteristics of bifidobacteria (see Table 1). The number of plates with a colony number between 30 and 300 was counted. After counting, 5 colonies were randomly selected for Gram staining, microscopic examination and contact enzyme test. Gram-positive, enzyme-negative, sporeless, irregular-shaped bacilli, branching or bifidobacteria, spoon-shaped or coccus can be classified as bifidobacteria. The number of bifidobacteria in the plate was calculated and then converted into the number of bifidobacteria in every millilitre of the sample by the dilution multiple of the Bacillus Bifidobacterium.
 
After 2.3 Lactobacillus on several medium growth conditions, the lactic acid bacteria liquid was diluted at 10 times, and the suitable dilution degree was selected on 3 medium of improved BBL, M RS and improved TJA, respectively. The growth of colony was observed at 37, and the growth of colony was observed (see Table 2).
 
2.4 the growth of bifidobacteria on the improved BBL medium was good, the colony was white, and the count of the culture liquid could reach 1. Lactobacillus does not grow, Streptococcus thermophilus can grow in its growth, and its colony is light brown.