常見微生物在常見選擇性培養(yǎng)基上的菌落特征
發(fā)布時間：
2022-11-16
作者：
培養(yǎng)基制造商
常見微生物在常見選擇性培養(yǎng)基上的菌落特征
培養(yǎng)基簡介：
培養(yǎng)基是供微生物、植物和動物組織生長和維持用的人工配制的養(yǎng)料，一般都含有碳水化合物、含氮物質(zhì)、無機鹽（包括微量元素）以及維生素和水等。有的培養(yǎng)基還含有抗菌素和色素，用于各種微生物培養(yǎng)和鑒定。
一般基礎培養(yǎng)基中含有蛋白胨、牛肉浸粉、酵母浸膏、氯化鈉、瓊脂等基本營養(yǎng)萬分。
一些選擇、鑒定培養(yǎng)基根據(jù)用途不同，則需加入抑菌劑、指示劑、血液、糖等試劑，以利于需要菌的生長與鑒別。
一些需要使細菌大量生長、繁殖的培養(yǎng)基，主要成分多為氨基酸、核苷酸、無機鹽、生長因子等。
瓊脂是由石花菜或江籬(屬紅藻)中提取的一種藻膠，是由瓊脂糖和瓊膠質(zhì)組成的長鏈多糖化合物。優(yōu)點在于不能被細菌分解利用，在細菌培養(yǎng)的溫度下凝膠強度穩(wěn)定。
蛋白胨為動物蛋白經(jīng)酶消化后產(chǎn)物，能為微生物提供C源、N源、生長因子等營養(yǎng)物質(zhì)。
胰酪蛋白胨是一種優(yōu)質(zhì)蛋白胨，亦稱胰酶消化酪蛋白胨或胰蛋白胨。系采用酪蛋白經(jīng)胰酶消化后，濃縮干燥而成?？膳渲聘鞣N微生物培養(yǎng)基，用于細菌的培養(yǎng)、分離、增殖、鑒定，以及無菌試驗培養(yǎng)基、厭氧菌培養(yǎng)基等細菌生化特性試驗用培養(yǎng)基的配置。
牛肉浸膏是新鮮牛肉經(jīng)剔除脂肪，消化過濾濃縮得到的一種營養(yǎng)物質(zhì)，含有肌酸、肌酸酐、多肽類、氨基酸、核苷酸、有機酸、礦物質(zhì)及維生素的水溶性物質(zhì)。主要作用是為了補充蛋白胨以及其他氮源的營養(yǎng)不足。
酵母粉富含B族維生素、有機氮以及碳水化合物等營養(yǎng)物質(zhì)。
膽鹽是膽酸鈉鹽，是從動物的膽汁中提取的一種混合物，含膽酸、結合膽酸以及膽汁醇等，膽鹽用在培養(yǎng)基中是作為選擇性抑制劑，主要抑制革蘭氏陽性菌的生長。
培養(yǎng)基分類：
按營養(yǎng)物質(zhì)可分為：
1、天然培養(yǎng)基：指一類利用動、植物或微生物體包括其提取物制成的培養(yǎng)基。如牛肉膏蛋白胨培養(yǎng)基、麥芽汁培養(yǎng)基等。
2、組合培養(yǎng)基：又稱為合成培養(yǎng)基或綜合培養(yǎng)基，是一類按微生物的營養(yǎng)要求精確設計后用多種高純化學試劑配制成的培養(yǎng)基。如葡萄糖銨鹽培養(yǎng)基、淀粉硝酸鹽培養(yǎng)基等。
3、半組合培養(yǎng)基：一類主要以化學試劑配制，同時還加有某種或某些天然成分的培養(yǎng)基。例如，馬鈴薯蔗糖培養(yǎng)基。
按物理性狀分：
1、液體培養(yǎng)基：一類呈液態(tài)的培養(yǎng)基。
2、固體培養(yǎng)基：一類外觀呈固態(tài)的培養(yǎng)基。根據(jù)性質(zhì)又分為。固化培養(yǎng)基、非可逆性固化培養(yǎng)基、天然固態(tài)培養(yǎng)基、濾膜。
3、半固體培養(yǎng)基：指在液體培養(yǎng)基中加入少量的凝固劑而配制成的半固體狀態(tài)的培養(yǎng)基。
4、脫水培養(yǎng)基：又稱預制干燥培養(yǎng)基，指含有除水分外的一切成分的商品培養(yǎng)基。
按功能性分為：
1、選擇性培養(yǎng)基：一類根據(jù)某微生物的特殊營養(yǎng)要求或其對某化學、物理因素的抗性而設計的培養(yǎng)基，具有使混合菌樣中的劣勢菌變成優(yōu)勢菌的功能，廣泛用于菌種篩選等領域。
2、鑒別培養(yǎng)基：一類在成分中加有能與目的菌的無色代謝產(chǎn)物發(fā)生顯色反應的指示劑，從而達到只須用肉眼辨別顏色就能方便的從近似菌落中找出目的菌菌落的培養(yǎng)基。例如，伊紅美藍乳糖培養(yǎng)基（EMB）。
3、運輸培養(yǎng)基
4、保存培養(yǎng)基
5、復蘇培養(yǎng)基
6、增菌培養(yǎng)基
7、鑒定培養(yǎng)基
8、多用途培養(yǎng)基
幾種ISO、GB、SN常見選擇性培養(yǎng)基及顯色培養(yǎng)基的培養(yǎng)基配方及菌落特征分析：
一、大腸桿菌在結晶紫中性紅膽鹽葡萄糖瓊脂（VRBGA）上的菌落特征
原理：
蛋白胨和酵母粉提供碳氮源和微量元素；乳糖是可發(fā)酵的糖類；氯化鈉可維持均衡的滲透壓；膽鹽和結晶紫抑制革蘭氏陽性菌，特別抑制革蘭氏陽性桿菌和糞鏈球菌；中性紅為pH指示劑。
用法：
稱取本品 41.5g,加熱溶解于1000ml 蒸餾水中,煮沸不要超過2 分鐘。取適宜稀釋度樣品液1ml,加入到無菌平皿中心,將冷至 45 ± 0.5℃ 的 VRBA 10-15ml傾注于平皿中。小心旋轉平皿將培養(yǎng)基與樣液充分混勻。凝固后，再加 3-4ml VRBA 覆蓋 平板表層 ,翻轉平板 ,置于 36 ± 1℃培養(yǎng) 18-24 小時。無需高壓滅菌。臨用時制備,不得超過3 小時。
典型特征：
結晶紫中性紅膽鹽葡萄糖瓊脂（VRBGA）又稱作腸道菌計數(shù)瓊脂，根據(jù)SN和FDA BAM方法，腸桿菌細菌在該培養(yǎng)基上可生成有或無沉淀環(huán)的粉紅色至紅色菌落，直徑0.5mm，或更大。
二、沙門氏菌在木糖賴氨酸脫氧膽酸瓊脂（XLD）上的菌落特征
木糖賴氨酸脫氧膽酸瓊脂（XLD）：
原理：
酵母浸粉提供氮源、維生素、生長因子； 氯化鈉維持均衡的滲透壓；木塘、乳糖、蔗糖為可發(fā)酵糖類，產(chǎn)酸使苯酚紅指示劑變黃；去氧膽酸鈉抑制革蘭氏陽性菌，但不影響沙門氏菌的生長；硫代硫酸鈉可被某些細菌還原硫化氫，與檸檬酸鐵銨中的鐵鹽生成黑色硫化鐵；瓊脂是培養(yǎng)基的凝固劑；苯酚紅為pH指示劑。
用法：
稱取本品 5.7g,加熱攪拌溶解于 100ml 蒸餾水中,不要過分加熱 , 冷至 50℃左右時,傾入無菌平皿,部分開蓋干燥 2h,然后蓋上，備用。無需高壓滅菌。在 24小時內(nèi)使用。
典型特征：
木糖賴氨酸脫氧膽酸瓊脂（XLD）中的酵母粉為細菌生長提供維生素和輔助因子，木糖、乳糖和蔗糖作為可發(fā)酵的碳源。除志賀菌外，其他大多數(shù)腸桿菌均發(fā)酵木糖，加入的賴氨酸是為了鑒別沙門氏菌。沙門發(fā)酵木糖產(chǎn)酸，形成酸性環(huán)境有利于該菌產(chǎn)生脫羧酶，從而使培養(yǎng)基的pH升高向堿性轉變，但可因其他菌發(fā)酵乳糖和蔗糖產(chǎn)生大量的酸而中和，在堿性條件下，硫代硫酸鈉及檸檬酸鐵銨與沙門菌產(chǎn)生的硫代氫反應使菌落顏色呈黑色，酸性條件下下反應被抑制。酚紅作為指示劑，NaCl維持培養(yǎng)基滲透壓，去氧膽鹽鈉抑制一般革蘭陽性菌生長，也可抑制大腸桿菌生長。。根據(jù)ISO和GB、FDA BAM方法，沙門菌在木糖賴氨酸脫氧膽酸瓊脂（XLD）大多數(shù)可形成紅色帶黑心的菌落，約為2mm，光滑、濕潤、邊緣整齊，亞利桑那菌發(fā)酵乳糖，可形成帶黑心的黃色菌落。
三、金黃色葡萄球菌在甘露醇高鹽瓊脂上的菌落特征：
甘露醇高鹽瓊脂：
原理：
蛋白胨和牛肉粉提供氮源、維生素和生長因子；D—甘露醇為可發(fā)酵糖類；氯化鈉維持均衡的滲透壓；瓊脂是培養(yǎng)基的凝固劑；酚紅為pH指示劑。
用法：
稱取本品 109.0g,加入 1000ml 蒸餾水中,加熱溶解并不停攪拌煮沸 1 分鐘。121℃高壓滅菌15 分鐘，冷至 45-50℃左右時,傾入無菌平皿。
典型特征：
甘露醇高鹽瓊脂中的NaCl能夠抑制不耐鹽的非葡萄球菌生長，D-甘露醇為碳源，酚紅指示劑，凝固酶陽性菌能利用D-甘露醇產(chǎn)酸使酚紅由紅色變黃色。
四、產(chǎn)氣莢膜梭菌在哥倫比亞血瓊脂基礎的菌落特征：
哥倫比亞血瓊脂基礎：
原理：
酪蛋白胰酶消化物、肉胃酶消化物、心胰酶消化物和酵母浸出粉提供碳氮源、維生素和氨基酸；淀粉能促進奈瑟氏菌的生長和增強鏈球菌的溶血特性；氯化鈉維持均衡的滲透壓；瓊脂是培養(yǎng)基的凝固劑。
用法：
稱取本品 39克，加熱溶解于1000ml蒸餾水中，分裝，121℃高壓滅菌15分鐘，冷至45-50℃左右時，加入相當于20mg慶大霉素的無菌硫酸慶大霉素，混勻，傾入無菌平皿。
典型特征：
產(chǎn)氣莢膜梭菌在血平板上多數(shù)有雙層溶血環(huán)，內(nèi)環(huán)完全溶血，是由于θ毒素的作用，外環(huán)不完全溶血是因為α毒素所致。
五、白色念珠菌在馬鈴薯葡萄糖瓊脂培養(yǎng)基上的菌落特征：
馬鈴薯葡萄糖瓊脂培養(yǎng)基：
原理：
馬鈴薯浸出粉有助于各種霉菌的生長；葡萄糖提供能源；瓊脂是培養(yǎng)基的凝固劑。
用法：
稱取本品 46.0g,加入 1000ml 蒸餾水中,115℃高壓滅菌 20分鐘,備用。滅菌好的培養(yǎng)基僅可重復加熱熔化一次。
典型特征：
白色念珠菌在馬鈴薯葡萄糖瓊脂培養(yǎng)基上的菌落特征為乳白色菌落，有突起，在加有氯霉素的馬鈴薯葡萄糖瓊脂培養(yǎng)基上，菌落表面呈奶油色。
六、綠膿桿菌在十六烷三甲基溴化銨瓊脂上的菌落特征：
十六烷三甲基溴化銨瓊脂：
原理：
蛋白胨和牛肉膏粉提供碳氮源、維生素和生長因子；氯化鈉維持均衡的滲透壓；十六烷三甲基溴化銨為選擇性抑菌劑，作為一種季銨鹽陽離子去污劑可釋放細菌細胞中的氮和磷而抑制非綠膿桿菌的細菌；瓊脂是培養(yǎng)基的凝固劑。
用法：
稱取本品 32.3g,煮沸溶解于1000ml純化水中,分裝三角瓶,121℃高壓滅菌 15 分鐘, 備用。
典型特征：
綠膿桿菌在十六烷三甲基溴化銨瓊脂上，綠膿桿菌菌落扁平無定型，向周邊擴散或略有蔓延趨勢，菌落呈灰白色，周圍有水溶性色素。
七、單增李斯特在李斯特菌選擇性瓊脂上的菌落特征
李斯特菌選擇性瓊脂：
原理：
胰蛋白胨、多價胨和牛肉粉提供氮源、維生素和生長因子；葡萄糖提供碳源；氯化鈉維持均衡的滲透壓；磷酸氫二鈉為緩沖劑；甘氨酸、氯化鋰、苯乙醇和復達欣可抑制非李斯特氏菌以外的部分革蘭氏陽性菌和革蘭氏陰性菌；瓊脂是培養(yǎng)基的凝固劑。
用法：
稱取本品 48g,加熱溶解于1000ml 蒸餾水中,分裝三角瓶,每瓶 100ml,121℃高壓滅菌 15 分鐘,冷至 50-55℃時,每瓶加入 1 支復達欣(3mg),混勻,傾入無菌平皿備用。
典型特征：
單增李斯特在李斯特菌選擇性瓊脂平板上，單核細胞增生李斯特菌培養(yǎng)后生成藍色菌落，菌落周圍有一不透明環(huán)。
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Colony characteristics of common microorganisms on common selective media
A brief introduction to the medium of culture:
The culture medium is artificial feed for the growth and maintenance of microorganism, plant and animal tissue. It usually contains carbohydrates, nitrogen containing substances, inorganic salts (including trace elements), vitamins and water. Some media also contain antibiotics and pigments, which are used for the cultivation and identification of various microorganisms.
Basic medium contains peptone, beef dip powder, yeast extract, sodium chloride, agar and other basic nutrients.
Some selections and identification media are different according to their uses. They include bacteriostat, indicator, blood, sugar and other reagents to facilitate the growth and identification of the needed bacteria.
Some media that need to grow and propagate bacteria are mostly amino acids, nucleotides, inorganic salts, growth factors, etc.
Agar is a kind of alginate extracted from gelatinous or hedgerow (red algae). It is a long chain polysaccharide compound composed of agarose and agarose. The advantage is that it can not be decomposed and utilized by bacteria, and the gel strength is stable at the temperature of bacterial culture.
Beef extract is a nutritive substance obtained from fresh beef, which is extracted by digestion and filtration. It contains water soluble substances of creatine, creatinine, polypeptide, amino acids, nucleotides, organic acids, minerals and vitamins. The main role is to supplement peptone and other nitrogen sources of undernourishment.
Yeast powder is rich in B vitamins, organic nitrogen and carbohydrates.
Bile salt is a sodium cholic acid, a mixture extracted from the bile of animals, containing cholic acid, conjugated cholic acid and bile alcohol. Bile salts are used as selective inhibitors in the culture medium, which mainly inhibit the growth of Gram-positive bacteria.
Culture medium classification:
According to the nutrient substance, it can be divided into:
1. Natural medium: a kind of culture medium made from animal, plant or microorganism, including its extract. Such as beef paste peptone medium, wort medium, etc.
2. The combination medium, also known as the synthetic medium or the comprehensive medium, is a kind of medium prepared with a variety of high purity chemical reagents after the precise design of the nutritional requirements of microbes. Such as glucose ammonium salt medium, starch nitrate medium and so on.
3, half combination medium: a kind of medium which is mainly prepared with chemical reagents, plus some or some natural ingredients. For example, the potato sucrose medium.
According to the physical character:
1. Liquid medium: a liquid medium.
2. Solid medium: a medium with solid appearance. According to the nature, it is divided into. Solidified medium, non reversible curing medium, natural solid medium and filter membrane.
3. Semisolid medium: a semisolid medium made up of a small amount of coagulant in a liquid medium.
4, dehydrating medium: also known as prefabricated dry medium, refers to contain all ingredients except water for commercial media.
According to the function, it is divided into:
1, selective medium: a kind of medium based on the special nutritional requirements of a microorganism or its resistance to some chemical and physical factors. It has the function of making the inferior bacteria in the mixed bacteria into the dominant bacteria, and is widely used in the field of strain screening.
2, differential medium: a class of indicators that add color reaction to the colorless metabolites of the target bacteria in the composition, so as to find the medium of the colony of the target bacteria easily from the approximate colony only with the naked eye color. For example, eosinylene lactose medium (EMB).
3. Transport medium
4. Preservation medium
5, resuscitation medium
6, increase the culture medium
7, identification medium
8, multipurpose medium
Several ISO, GB, SN common medium and color medium culture medium formula and colony characteristics analysis:
1. Colony characteristics of Escherichia coli on crystal violet Neutral Red Bile Glucose Agar (VRBGA).
Principle:
Peptone and yeast powder provide carbon and nitrogen sources and trace elements; lactose is fermentable sugar; sodium chloride can maintain a balanced osmotic pressure; bile salts and crystal violet inhibit Gram-positive bacteria, especially gram positive bacilli and Streptococcus faecalis; neutral red is a pH indicator.
Usage:
Take this product 41.5g and dissolve it in 1000ml distilled water and boil it for no more than 2 minutes. The suitable dilution sample 1ml was added to the sterile dish center, and the VRBA 10-15ml, which was cold to 45 + 0.5 C, was poured into the pan. Carefully rotate the dish and mix the medium with the sample. After solidification, 3-4ml VRBA was used to cover the surface of the flat plate, and the plate was placed at 36 + 1 degrees for 18-24 hours. No autoclave is needed. The preparation shall not exceed 3 hours at the time of use.
Typical features:
Crystal violet neutral red gall salt glucose agar (VRBGA) is also called intestinal bacteria count agar. According to the SN and FDA BAM methods, Enterobacteriaceae bacteria can produce pink to red colonies with or without precipitation ring on this medium, with a diameter of 0.5mm or larger.
Two. Colony characteristics of Salmonella on xylose lysine deoxycholate agar (XLD)
Xylose lysine deoxycholic acid agar (XLD):
Principle:
Yeast extract provides nitrogen sources, vitamins and growth factors; sodium chloride maintains a balanced osmotic pressure; wood ponds, lactose and sucrose are fermentable sugars; acids make the phenol red indicator yellow; sodium deoxycholate inhibits Gram-positive bacteria, but does not affect the growth of Salmonella; sodium thiosulfate can be reduced to hydrogen sulfide by certain bacteria. The ferric salt of ferric ammonium citrate is used to form black iron sulfide; agar is the coagulant of the medium; phenol red is pH indicator.
Usage:
Call this product 5.7g, heated and dissolve in the 100ml distilled water, do not overheat, cold to about 50 degrees, dip into the sterile flat, partially open the dry 2h, and then cover, spare. No autoclave is needed. It is used in 24 hours.
Typical features:
The yeast powder in the xylose lysine deoxycholic acid agar (XLD) provides vitamins and cofactors for the growth of bacteria, and xylose, lactose and sucrose as a fermentable carbon source. In addition to Shigella, most other Enterobacteriaceae ferment xylose, and lysine is added to identify Salmonella. The acid environment of xylose fermentation is beneficial to the production of decarboxylase by the formation of acid environment, which makes the increase of pH in the medium to alkaline, but can be neutralized by a large amount of acid produced by the fermentation of lactose and sucrose by other bacteria. Under alkaline conditions, the reaction of sodium thiosulfate and ammonium citrate with Salmonella thiosulphic reaction causes colony color. It is black, and the reaction under acidic condition is inhibited. Phenol red as an indicator, NaCl maintains osmotic pressure of the medium, sodium deoxycholate inhibits the growth of common gram-positive bacteria, and inhibits the growth of E. coli. According to the ISO and GB and FDA BAM methods, most of Salmonella in xylose lysine deoxycholic acid agar (XLD) can form a colony of red with black heart, about 2mm, smooth, moist and tidy, Arizona fermented lactose, and can form a yellow colony with black heart.
Three. Staphylococcus aureus colonies on mannitol high salt agar:
Mannitol high salt agar:
Principle:
Peptone and beef powder provide nitrogen sources, vitamins and growth factors; D - mannitol is fermentable sugar; sodium chloride maintains a balanced osmotic pressure; agar is the coagulant of culture medium; phenol red is an indicator of pH.
Usage:
This product is called 109.0g, added to 1000ml distilled water, heated and dissolved, stirring and boiling for 1 minutes. 121 degree high pressure sterilization for 15 minutes, cold to 45-50 degrees Celsius, pour into sterile dish.
Typical features:
NaCl in mannitol high salt agar can inhibit non salty non staphylococcal growth, D- mannitol as a carbon source, phenol red indicator, coagulase positive bacteria can make use of D- mannitol to produce acid to make red from red to yellow.
Four. Colony characteristics of Clostridium perfringens on Blood Agar Base in Columbia:
Columbia Blood Agar Base:
Principle:
The casein trypsin digests, meat and stomach enzyme digests, cardiac pancreatin digests and yeast extract powder provide carbon and nitrogen sources, vitamins and amino acids; starch can promote the growth of Neisseria bacteria and enhance the hemolytic characteristics of streptococcus; sodium chloride maintains a balanced osmotic pressure; agar is the coagulant of culture medium.
Usage:
39 grams of this product, heat dissolved in 1000ml distilled water, separate, 121 degrees centigrade sterilization 15 minutes, cold to 45-50 degrees, add the equivalent of 20mg gentamicin, sterile gentamicin sulfate, mixed, into a sterile flat dish.
Typical features:
Most of the Clostridium perfringens have double hemolytic ring on the blood plate, and the inner ring is completely hemolytic. It is due to the effect of theta toxin. The incomplete hemolysis of the outer ring is caused by alpha toxin.
Five. Colony characteristics of Candida albicans on potato dextrose agar medium:
Potato glucose agar medium:
Principle:
Potato leach powder helps the growth of various molds; glucose provides energy; agar is the coagulant of the medium.
Usage:
The product is called 46.0g, added to 1000ml distilled water, and sterilized at 115 C for 20 minutes. The medium with good sterilization can be reheated and melted once.
Typical features:
The colony characteristics of Candida albicans on the potato glucose agar medium were milky white colonies, and were protruding. On the potato glucose agar medium with chloramphenicol, the colony surface was coloured with milk oil.
Six. The colony characteristics of Pseudomonas aeruginosa on sixteen alkane three methyl ammonium bromide agar:
Sixteen alkanes and three methyl ammonium bromide agar:
Principle:
Peptone and beef extract provide carbon and nitrogen sources, vitamins and growth factors; sodium chloride maintains a balanced osmotic pressure; sixteen alkanes and three methyl ammonium bromide are selective bacteriostat, which can release nitrogen and phosphorus in bacterial cells and inhibit bacteria from Pseudomonas aeruginosa as a quaternary ammonium salt decontamination agent; agar is a coagulant of culture medium.
Usage:
This product is called 32.3g, boiled in 1000ml purified water, separated into triangular bottles, and sterilized at 121 C for 15 minutes.
Typical features:
Pseudomonas aeruginosa was on sixteen alkane three methyl ammonium bromide agar, the colony of Pseudomonas aeruginosa flattened and amorphous, spread to the periphery or slightly spread, the colony was gray white, and there were water soluble pigments around it.
Seven. Single colony Lester's colony characteristics on Lester selective agar.
Selective agar of Lester bacteria:
Principle:
Tryptone, polypeptone and beef powder provide nitrogen sources, vitamins and growth factors; glucose provides carbon source; sodium chloride maintains a balanced osmotic pressure; sodium hydrogen phosphate two is a buffer; glycine, lithium chloride, benzyl alcohol and dahdahin can inhibit some gram-positive and Gram-negative bacteria other than Listeria. Agar is the coagulant of the medium.
Usage:
The product was called 48g. The heating was dissolved in 1000ml distilled water and divided into a triangulation. Each bottle was sterilized at 121 degrees centigrade for 15 minutes. When cold to 50-55, each bottle was added to 1 Fu Daxin (3mg).
Typical features:
On the selective agar plate of Lester's bacteria, mononuclear cells grew on mononuclear cells, and Lester grew into blue colonies. There was an opaque ring around the colony of List Rand.