微生物檢查用培養(yǎng)基中膽鹽的選擇
發(fā)布時間：
2022-12-27
作者：
摘要：選用去氧膽酸鈉、牛膽鹽、3號膽鹽和5號膽鹽試驗，結(jié)果以去氧膽酸鈉抑制革蘭陽性菌效果佳，而在一定濃度內(nèi)對大腸桿菌無抑制作用，為培養(yǎng)基中膽鹽的選擇。
內(nèi)容：進行藥品微生物限度檢查及微生物常規(guī)檢查，所用膽鹽乳糖增菌液、麥康凱瓊脂、ss瓊脂等培養(yǎng)基均加入膽鹽作為抑菌劑，抑制革蘭陽性(G)苗生長，使革蘭陰性(G一)菌能生長良好膽鹽對G菌具有良好的抑菌作用，但用量過大對大腸桿菌也有抑制作用。中國藥典2000版二部附錄及衛(wèi)生部藥品衛(wèi)生檢驗方法收載了不同的膽鹽，并對各種膽鹽的用量作了規(guī)定。我們在膽鹽乳糖增菌液中按規(guī)定加入不同膽鹽，再分別加入菌液，觀察哪種膽鹽對G菌抑制作用最強，對G一菌抑制作用最弱，以期選擇最遁的膽鹽，提高控制苗的檢出率。
1材料
1．1膽鹽去氧膽酸鈉、牛膽鹽、3號膽鹽、5號膽鹽。
1．2菌種大腸桿菌~CMCC(B)441023、金黃葡萄球菌(CMCC(B)26003]、綠膿桿菌(CMCC(B)101O4]。將營養(yǎng)肉湯瓊脂斜面上菌種接種至營養(yǎng)肉湯中，36℃士1℃培養(yǎng)18h備用。
2方法與結(jié)果
2．1膽鹽對不同菌量的抑制作用用不同膽鹽配成膽鹽乳糖增苗液，其中分別加去氧膽酸鈉0．025，牛膽鹽0．13，3號膽鹽0．15，5號膽鹽O．15，每瓶裝量lOOml。大腸桿菌、金黃葡萄球菌、綠膿桿菌菌液分別用生理鹽水稀釋至1O～，10，10_。。菌液濃度：大腸桿菌依次為260個／ml，27個／ml，4個／ml；金黃葡萄球菌為192個／ml，17個／ml，3個／ml；綠膿桿菌為194個／ml，27個／ml，2個／ml。將不同稀釋級的菌液lm1分別加至上述增菌液中，以每種苗液各稀釋級接種各種膽鹽1瓶。36℃士1℃培養(yǎng)18~24h；劃線接種平板，繼續(xù)培養(yǎng)18～24h。
2．2不同濃度膽鹽的抑菌作用將不同膽鹽配成一定濃度的液體，作為原液取裝有營養(yǎng)肉湯培養(yǎng)基的滅菌試管(第1管裝量為1．8ml，其余為lm1)。第1支試管中加入不同膽鹽原液0．2ml，混勻后吸取lml加入第2管中，依次作遞倍稀釋1：2，1：4，1：8??1：512o每種膽鹽一個遞減系列濃度。去氧膽酸鈉濃度依次為8，4，2mg／ml??}牛膽鹽濃度依次為41．6，20．8．10．4mg／ml??；3號膽鹽與5號膽鹽濃度依次為48，24，12mg／ml??。每管加入金黃葡萄球菌1O稀釋苗液0．1ml。第u管加入菌液，不加膽鹽，作陽性對照管。將各管搖勻，36℃士1℃培養(yǎng)18～24h。再將試管液體轉(zhuǎn)移至培養(yǎng)皿中，傾注營養(yǎng)肉湯瓊脂15ml，連續(xù)培養(yǎng)18～24h。同法再進行一次實驗，但加入金黃葡萄球菌1O稀釋菌液，結(jié)果見表2。
3討論：4種膽鹽的抑菌作用有較大差異。去氧膽酸鈉對金黃葡萄球菌抑苗法起到抑制作用。據(jù)文獻報道，膽鹽的抑菌作用與膽酸(cA)、去氧膽酸(DCA)有關(guān)。DCA的抑菌作用比CA強得多。4種膽鹽抑菌作用的差異，可能與其所含CA與DCA的含量不同有關(guān)。
趙建英(浙江金華市藥品檢驗所金華321000)
Abstract: the test of sodium deoxycholate, bovine bile salt, No. 3 bile salt and No. 5 bile salt was used. The results showed that sodium deoxycholate was the best to inhibit Gram-positive bacteria, but it had no inhibitory effect on Escherichia coli in a certain concentration, which was the first choice for bile salt in the medium.
Content: drug microbial limit test and microbial routine examination, bile salt lactose increasing liquid, Mai Kang Kai agar, SS Agar and other culture medium added bile salt as bacteriostat, inhibiting the growth of gram-positive (G) seedlings, so that gram-negative (G I) bacteria can grow well and have good bacteriostasis on G bacteria, but the dosage is too much. Large Escherichia coli also has a inhibitory effect. The appendix of the second part of the Chinese Pharmacopoeia 2000 and the hygienic inspection methods of the Ministry of Health contain different kinds of bile salts and stipulate the dosage of various kinds of bile salts. We add different bile salts in the bile salt lactose adding liquid according to the regulations, and then add bacteria liquid to observe which bile salts have the strongest inhibitory effect on G bacteria, and the weakest to G one bacteria, in order to choose the most isolated bile salts and improve the detection rate of the control seedlings.
1 material
1.1 bile salt sodium deoxycholate, ox bile salt, 3 bile salt, 5 bile salt.
1.2 strains Escherichia coli ~CMCC (B) 441023, Staphylococcus aureus (CMCC (B) 26003], Pseudomonas aeruginosa (CMCC (B) 101O4]). The nutrient broth on the agar slant was inoculated into the nutrient broth, and 18h was cultured at 36 C for 1 degrees.
2 methods and results
2.1 the inhibitory effect of bile salt on different bacteria amounts was mixed with bile salt lactose solution with different bile salts, including sodium deoxycholate 0.025, bovine bile salt 0.13, No. 3 bile salt 0.15, 5 bile salt O.15, and each bottle was lOOml. Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa were diluted with normal saline to 1O ~ 10, 10_ respectively. Bacterial concentration: Escherichia coli was 260 / ml, 27 / ml, 4 / ml; Staphylococcus aureus was 192 / ml, 17 / ml, 3 / ml; Pseudomonas aeruginosa was 194 / ml, 27 / ml, 2 / ml. The bacteria solution LM1 of different dilution levels was added to the above enrichment solution, and each bile solution was inoculated with 1 bottles of bile salts at each dilution stage. 18~24h was cultured at 36 C for 1 degrees, and 18 to 24h by streak inoculation. The results were shown in Table 1.
Table 1 inhibitory effects of bile salts on different bacteria
2.2 the Bacteriostasis of different concentrations of bile salts combined different bile salts to a certain concentration of liquid. As the original liquid, the sterilization test tube containing the medium of nourishing broth was taken (first tubes were 1.8ml, and the rest were LM1). First different bile salts were added to the first tubes. After mixing, LML was added to the second tubes, and the doubling dilution 1:2, 1:4, 1:8... 1:512o each of the bile salts had a decreasing series of concentration. The concentration of sodium deoxycholate was 8, 4, 2mg / ml...} the concentration of bovine bile salt was 41.6, 20.8.10.4mg / ml... 3 bile salt and 5 bile salt concentration were 48, 24, 12mg / ml respectively. Each tube was added with Staphylococcus aureus 1O diluted 0.1ml solution. Tube u added bacteria solution, without bile salt, positive for care. Each tube was shaken well, and 36 C was cultivated for 18 ~ 24h at 1 centigrade. Then transfer the test tube liquid into the culture dish, pour the nutrient Broth Agar 15ml, and continuously cultivate 18 to 24h. Another experiment was carried out with the same method, but Staphylococcus aureus 1O diluted bacteria solution was added. The result is shown in Table 2.
Table 2 inhibitory effect of different concentrations of bile salts on inhibition of golden grape grape castor
3 discussion: the bacteriostatic effects of 4 kinds of bile salts are quite different. Sodium deoxycholate inhibits the inhibition of Staphylococcus aureus. It is reported that the bacteriostatic action of bile salts is related to cholic acid (cA) and deoxycholic acid (DCA). The Bacteriostasis of DCA is much stronger than that of CA. The difference in bacteriostatic action between 4 kinds of bile salts may be related to the difference in the content of CA and DCA.