弧菌在TCBS培養(yǎng)基上的顯色原理
發(fā)布時(shí)間：
2022-11-16
作者：
TCBS瓊脂培養(yǎng)基，英文名稱(chēng)Thiosulfate citrate bile salts sucrose agar culture medium，即硫代硫酸鹽檸檬酸鹽膽鹽蔗糖瓊脂培養(yǎng)基。在水產(chǎn)養(yǎng)殖業(yè)中，經(jīng)常用于弧菌的分離與鑒定。
TCBS本身為淺綠色，用該培養(yǎng)基培養(yǎng)的弧菌顏色通常分為藍(lán)綠色、黃色和黑色三種顏色。為什么弧菌屬的細(xì)菌在TCBS培養(yǎng)基上顯示出不同的顏色呢？
TCBS培養(yǎng)基中的蛋白胨、酵母浸粉提供碳氮源、維生素和生長(zhǎng)因子；氯化鈉可刺激弧菌的生長(zhǎng)；蔗糖是可發(fā)酵的糖類(lèi)；牛膽酸鈉、牛膽粉、硫代硫酸鈉和枸櫞酸鈉及較高的pH可抑制革蘭氏陽(yáng)性菌和大腸菌群；霍亂弧菌、副溶血弧菌對(duì)酸性環(huán)境比較敏感，因此該pH值可增強(qiáng)其生長(zhǎng)；硫代硫酸鈉與檸檬酸鐵反應(yīng)作為檢測(cè)硫化氫產(chǎn)生的指示劑；溴麝香草酚蘭和麝香草酚蘭是pH指示劑；瓊脂是培養(yǎng)基的凝固劑。其中，麝香草酚藍(lán)作為pH指示劑的變色范圍為：1.2(紅)～2.8(黃) ~8.0(黃)～9.6(藍(lán))。由此可知，弧菌在TCBS培養(yǎng)上的顯色肯定與pH有關(guān)。
那么究竟弧菌是如何引起pH變化的呢？不難猜想這可能與TCBS培養(yǎng)基中的蔗糖成分有關(guān)。有的弧菌可以發(fā)酵蔗糖產(chǎn)生乳酸，pH降低，就顯示黃色，有的弧菌不能發(fā)酵蔗糖產(chǎn)酸，菌落及周?chē)捅３衷瓉?lái)的顏色。
凡是不能發(fā)酵蔗糖的弧菌就不產(chǎn)酸，只能利用檸檬酸鈉或枸櫞酸鈉作為碳源，然后生成碳酸鹽，使培養(yǎng)基pH升高，在 TCBS培養(yǎng)基上都是藍(lán)綠色（或深綠色）；能夠發(fā)酵蔗糖的弧菌可以產(chǎn)酸，使培養(yǎng)基pH降低，在TCBS培養(yǎng)基上的顏色就為黃色。如果菌株能夠產(chǎn)生H2S，則遇到培養(yǎng)基中的鐵離子就會(huì)生產(chǎn)FeS顯示黑色。
此外，筆者對(duì)以上觀點(diǎn)還做了顯色驗(yàn)證試驗(yàn)。從大連海珍品疾病防控中心的海水養(yǎng)殖動(dòng)物病原菌庫(kù)內(nèi)選取了幾株常見(jiàn)弧菌：哈維弧菌兩株、副溶血弧菌與溶藻弧菌各三株，首先在NA培養(yǎng)基進(jìn)行活化，18h后接種于TCBS培養(yǎng)基，然后于8h、20h、48h三個(gè)時(shí)間點(diǎn)進(jìn)行觀察并拍照記錄。結(jié)果如下圖所示：
結(jié)論：
1、在TCBS培養(yǎng)基中，副溶血弧菌為藍(lán)綠色，哈維弧菌和溶藻弧菌為黃色，與預(yù)期結(jié)果一致。
2、在NA培養(yǎng)基中，溶藻弧菌呈彌散型生長(zhǎng)，哈維弧菌與溶藻弧菌菌落形態(tài)較為規(guī)則。
3、在相關(guān)論壇中有的學(xué)者提到，顯黃色的菌落隨著時(shí)間的推移會(huì)將產(chǎn)生的酸也當(dāng)做碳源分解掉，顏色會(huì)由黃再變?yōu)榫G色。本人推測(cè)，這種現(xiàn)象發(fā)生在蔗糖消耗盡之后，也就是在平板厚度很薄的TCBS平板中才會(huì)發(fā)生，而本實(shí)驗(yàn)中菌株需要在培養(yǎng)箱內(nèi)培養(yǎng)48h，為了防止TCBS平板風(fēng)干，發(fā)生裂紋，因此制作時(shí)特意增加了厚度，故不會(huì)發(fā)生蔗糖耗盡的情況，因此，實(shí)驗(yàn)中也沒(méi)觀察到哈維、溶藻黃色菌落變綠的情況。
The color rendering principle of vibrio vibrio on TCBS medium
TCBS agar-agar medium, Thiosulfate citrate bile salts sucrose AGAR culture medium, namely Thiosulfate citrate gallate sucrose agar-agar culture medium. In aquaculture, it is often used for the isolation and identification of vibrio.
TCBS itself is light green, and the vibrio cultures grown in this medium are usually divided into three colors: blue-green, yellow and black. Why do vibrio bacteria show different colors in the TCBS medium?
Peptone and yeast extracts in TCBS medium provide carbon and nitrogen sources, vitamins and growth factors. Sodium chloride can stimulate the growth of vibrio vibrio. Sucrose is a fermentable sugar. Sodium cholate, cow-gall powder, sodium thiosulphate and sodium citrate and higher pH can inhibit gram-positive bacteria and coliform bacteria. Vibrio cholerae and vibrio parahaemolyticus are sensitive to acid environment, so the pH value can enhance their growth. Sodium thiosulphate reacts with ferric citrate as an indicator to detect hydrogen sulfide. Bromothymol and thymol are pH indicators. AGAR is the coagulant of culture medium. Among them, the color range of thymol blue as pH indicator is 1.2(red) ~ 2.8(yellow) ~8.0(yellow) ~ 9.6(blue). Therefore, the color of vibrio vibrio in TCBS culture must be related to pH.
So how does vibrio cause the pH change? It is not hard to guess that this may be related to the sucrose content in the TCBS medium. Some vibrio bacteria can ferment sucrose to produce lactic acid, and when pH drops, it shows yellow. Some vibrio bacteria cannot ferment sucrose to produce acid.
Who can't ferment sucrose vibrio don't produce acid, can only use sodium citrate and sodium citrate as carbon source, and then form carbonates, make the medium pH increases, the TCBS medium are blue-green (or dark green); Vibrio saccharum, which ferments sucrose, can produce acid, lowering the pH of the medium, and turning yellow on the TCBS medium. If the strain can produce H2S, it will produce FeS and show black when it encounters iron ions in the medium.
In addition, the author also did color verification test on the above points. From dalian Marine products the centers for disease control and prevention of mariculture animal pathogens of the rolls to select several common vibrio strains: two strains of vibrio Harvey, deputy hemolysis and dissolve various reason, v. v. in NA culture medium is activated, the first 18 h after vaccination in TCBS medium, and then in 20 h, 8 h, 48 h three time points for observation and photo record. The results are as follows:
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Conclusion:
1. In the TCBS medium, vibrio parahaemolyticus was blue-green, vibrio Harvey and vibrio alginolyticus were yellow, consistent with the expected results.
2. In the NA culture medium, vibrio alginolysis grew in a diffuse manner, and the colony morphology of vibrio Harvey and vibrio alginolyticus was relatively regular.
3. Some scholars in BBS have mentioned that the yellow colonies will decompose the generated acid as carbon source over time, and the color will change from yellow to green. I speculated that this phenomenon occurs after sugar is consumed, which is in the thickness of the plate is very thin TCBS plate occurs, the strain in this study need to be in cultivation in 48 h, in order to prevent TCBS plate dry, crack, therefore increased thickness, specially when producing reason have run out of sugar will not happen, therefore, didn't also observed in the experiment of Harvey, dissolve algae colonies yellow green.