雙歧桿菌計數(shù)培養(yǎng)基的研究報告
發(fā)布時間：
2022-12-27
作者：
摘要 [目的]選擇一種適用于微生態(tài)制劑中的雙歧桿菌計數(shù)培養(yǎng)基。[方法]用改 良BBL、眥 s、改 良TJA 等選擇性培養(yǎng)基和平板厭氧膠法對雙歧桿菌進行檢測，同時觀察雙歧桿菌和乳酸桿菌在三種培養(yǎng)基上生長情況。[結(jié)果]在改 良BBL 培養(yǎng)基上雙歧桿菌生長良好，乳酸桿菌不生長。[結(jié)論 ]該培養(yǎng)基用 于微生態(tài)制劑中雙歧桿菌計數(shù)操作簡便、有效，易于推廣。
關鍵詞：雙歧桿菌；計數(shù)；改 良BBL
近年來 雙歧桿 菌 的微 生 態(tài)制 劑 產(chǎn) 品在 市 場上 較 為 多 見 ，主要以發(fā)酵乳和保健品的形式 出現(xiàn)，我國 目前尚無統(tǒng)一 的標準檢 驗方法對 雙歧 桿 菌進 行 檢測 ，微 生 態(tài) 活 菌制 劑 中雙歧 桿菌的活菌數(shù)是該產(chǎn)品在保質(zhì)期 內(nèi)的重要質(zhì)量指標。雙歧菌為專性厭氧菌，檢測方法繁瑣。有必要選擇一種能讓大多數(shù) 已知的雙歧桿菌在其良好生長的選擇性培養(yǎng)基中對其進行質(zhì)量控制 。
1 材 料與方 法
1．1 青春雙歧桿菌、保加利亞乳酸桿菌為實驗室保存菌種。
兩歧 雙歧桿菌 11853、嗜熱 鏈球 菌 11855 ，北 京 生物 制 品鑒定 所提供 。
1．2 培養(yǎng)基及稀釋液
1．2．1 培養(yǎng)基 改良 BBL 培養(yǎng)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；
吐溫 一80 0 ．4g，L 一半胱氨酸 0 ．4g，M；
7H 20 0 ．2g，Nacl 2g，~ H r'0 4 lg；
硫乙醇酸鈉 0 ．4g，CaC 2g，牛 肉湯 2oom ]；
肝 湯 2oom l，西 紅柿 汁 2oral，低聚糖 3g，瓊脂 8g；
調(diào)節(jié) pH 值為 7 ．0 ±0．2 ，121℃；
15r a in 滅菌后每 100m l加入 6％的氯化鋰溶液 5m l，傾注平皿備用。改良TJA培養(yǎng)基?，MIlS 培養(yǎng)基【 ，厭氧膠(自配制)。
1．2．2 磷酸鹽稀釋液 Nai l2P44．5g．M a2HP4 6．0s，L 一半胱氨酸 0 ．5g，吐溫 一80 0 ．5g，蒸餾水 1000m l。
2 方法與結(jié)果
2 ．1 茵種的活化 將雙歧桿菌接種于改 良 BBL 液體培養(yǎng)管中，用螺紋帽擰緊管 口，乳 酸桿菌接種于 12％脫脂 奶 中，置37℃培養(yǎng)，重復 2 次。
2 ．2 雙歧桿茵計數(shù) 以無菌操作用 hld 滅菌吸管吸取 lm l 活化后的菌液作 10 倍遞增稀釋 ，選擇 3個合適的稀釋度，每一稀釋度取 0．1m l滴于預先傾注好的計數(shù)培養(yǎng)基平板表面，每個稀釋度同時做 2 份，用滅菌 L 棒均勻涂布，并同時做稀釋液的空白對照。以上操作 ~01nin 內(nèi)完成。
將厭氧膠定量用蒸餾水溶解后傾注于涂布完菌液的改 良BBL 平 板 蓋 內(nèi) ，每 蓋 約 20m l，待凝 固后將 平 板 ，蓋 壓 緊然 后 翻轉(zhuǎn)平板置 37℃培養(yǎng) 72h ±3h 后取 出，觀察雙歧桿菌菌落特征(見表 1)。選取菌落數(shù)在 30 —300 之間的平板進行計數(shù)。計數(shù)后，隨機挑取 5 個菌落數(shù)進行革蘭染色，顯微鏡檢查并做觸酶試驗。革蘭陽性，觸酶陰性，無芽胞的形態(tài)不規(guī)則桿菌，呈分枝或分叉形、匙形或球菌可定為雙歧桿菌。根據(jù)證實為雙歧桿菌菌落計算出該平板內(nèi)的雙歧桿菌數(shù) ，然后乘其稀釋倍數(shù)后再換算 成每 毫升樣 品中雙歧 桿菌數(shù) 。
2．3 乳酸茵在幾種培養(yǎng)基上生長情況 將活化后的乳酸菌菌液作 10 倍遞稀釋后，選擇合適稀釋度分別接種于改 良 BBL、M RS 、改良 TJA 等 3 種培養(yǎng)基上，37℃72h 培養(yǎng)，觀察菌落生長情況 (見表 2 )。
2 ．4 結(jié)果雙歧桿菌在改 良 BBL 培養(yǎng)基上用平板厭 氧膠法培養(yǎng)后，生長 良好，菌落乳 白色，對培養(yǎng)菌液計數(shù)可達 1 。乳酸桿菌不生長，嗜熱鏈球菌能夠在其生長，菌落呈淺褐色。
南京市疾病預防控制中心，江蘇：陳曉蔚 ，丁潔 ，王煒 ，賈力敏
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.