你真的會制備微生物培養(yǎng)基么？微生物培養(yǎng)基制備技術(shù)匯總！
發(fā)布時間：
2022-12-27
作者：
細(xì)菌檢測用總數(shù)測定微生物培養(yǎng)基
在制備培養(yǎng)基的過程中，首先要使用一些玻璃器皿，如試管、三角瓶、培養(yǎng)皿、燒杯和吸管等。這些器皿在使用前都要根據(jù)不同的情況，經(jīng)過一定的處理，洗刷干凈。有的還要進(jìn)行包裝，經(jīng)過滅菌等準(zhǔn)備就緒后，才能使用。
1、新購的玻璃器皿
除去包裝沾染的污垢后，先用熱肥皂水刷洗，流水沖凈，再浸泡于１～２％的工業(yè)鹽酸中數(shù)小時，使游離的堿性物質(zhì)除去，再以流水沖凈。對容量較大的器皿，如大燒瓶、量筒等，洗凈后注入濃鹽酸少許，轉(zhuǎn)動容器使其內(nèi)部表面均沾有鹽酸，數(shù)分鐘后傾去鹽酸，再以流水沖凈，倒置于洗滌架上將水空干，即可使用。
2、用過的玻璃器皿
凡確無病原菌或未被帶菌物污染的器皿，使用后可隨時沖洗，吸取過化學(xué)試劑的吸管，可先浸泡于清水中，待到一定數(shù)量后再集中進(jìn)行清洗。有可能被病原菌污染的器皿，必須經(jīng)過適當(dāng)消毒后，將污垢除去，用皂液洗刷，再用流水沖洗干凈。若用皂液未能洗凈的器皿，可用洗液浸泡適當(dāng)時間后再用清水洗凈。洗液的主要成份是重鉻酸鉀和濃流酸，其作用是將有機(jī)物氧化成可溶性物質(zhì)，以便沖洗。洗液有很強(qiáng)的腐蝕作用，使用時應(yīng)特別小心，避免濺到衣服、身體和其他物品上。
二、類型
在實(shí)驗室中配制的適合微生物生長繁殖或累積代謝產(chǎn)物的任何營養(yǎng)基質(zhì)，都叫做培養(yǎng)基(Media)。由于各類微生物對營養(yǎng)的要求不同，培養(yǎng)目的和檢測需要不同，因而培養(yǎng)基的種類很多。我們可根據(jù)某種標(biāo)準(zhǔn)，劃分為若干類型。
1、根據(jù)對培養(yǎng)基組成物質(zhì)的化學(xué)成分是否完全了解來區(qū)分，可分為天然、合成和半合成培養(yǎng)基。
１）天然培養(yǎng)基是指利用各種動、植物或微生物的原料，其成分難以確切知道。主要原料有：牛肉膏、麥芽汁、蛋白胨、酵母膏、玉米粉、麩皮、各種餅粉、馬鈴薯、牛奶、血清等。用這些物質(zhì)配成雖然不能確切知道它的化學(xué)成分，但一般來講，營養(yǎng)是比較豐富的，微生物生長旺盛，而且來源廣泛，配制方便，所以較為常用，尤其適合于配制實(shí)驗室常用的培養(yǎng)基。穩(wěn)定性常受生產(chǎn)廠或批號等因素的影響。
２）合成培養(yǎng)基是一類化學(xué)成分和數(shù)量完全知道的培養(yǎng)基，它是用已知化學(xué)成分的化學(xué)藥品配制而成。這類培養(yǎng)基化學(xué)成分精確、重復(fù)性強(qiáng)，但價格昂貴，而微生物又生長緩慢，所以它只適用于做一些科學(xué)研究，例如營養(yǎng)、代謝的研究。
３）在合成培養(yǎng)基中，加入某種或幾種天然成分；或者在天然培養(yǎng)基中，加入一種或幾種已知成分的化學(xué)藥品即成半合成培養(yǎng)基。例如馬鈴薯蔗糖培養(yǎng)基等。這種培養(yǎng)基在生產(chǎn)實(shí)踐和實(shí)驗室中使用最多。
２、根據(jù)物理狀態(tài)來區(qū)分，可以分為固體培養(yǎng)基、液體培養(yǎng)基和半固體培養(yǎng)基。
１）液體培養(yǎng)基 所配制的是液態(tài)的，其中的成分基本上溶于水，沒有明顯的固形物，液體培養(yǎng)基營養(yǎng)成分分布均勻，易于控制微生物的生長代謝狀態(tài)。
２）在液體培養(yǎng)基中加入適量的凝固劑即成固體培養(yǎng)基。常用作凝固劑的物質(zhì)有瓊脂、明膠、硅膠等，以瓊脂最為常用，在實(shí)際中用得十分廣泛。在實(shí)驗室中，它被用作微生物的分離、鑒定、檢驗雜菌、計數(shù)、保藏、生物測定等。
３）如果把少量的凝固劑加入到液體培養(yǎng)基中，就制成了半固體。以瓊脂為例，它的用量在0.2~1%之間。這種培養(yǎng)基有時可用來觀察微生物的動力，有時用來保藏菌種。
３、根據(jù)用途來區(qū)分，可分為選擇、增殖和鑒別培養(yǎng)基。
１）在培養(yǎng)基中加入某種物質(zhì)以殺死或抑制不需要的菌種生長的稱之為選擇培養(yǎng)基。如鏈霉素、氯霉素等抑制原核微生物的生長；而制霉菌素、灰黃霉素等能抑制真核微生物的生長；結(jié)晶紫能抑制革蘭氏陽性細(xì)菌的生長等。
２） 在自然界中，不同種的微生物常生活在一起，為了分離我們所需要的微生物，在普通培養(yǎng)基中加入一些某種微生物特別喜歡的營養(yǎng)物質(zhì)，以增加這種微生物的繁殖速度，逐漸淘汰其它微生物，稱為增殖培養(yǎng)基，常用于菌種篩選和選擇增菌中。在某種程度上講，增殖培養(yǎng)基也是一種選擇培養(yǎng)基。
３）在培養(yǎng)基中加入某種試劑或化學(xué)藥品，使難以區(qū)分的微生物經(jīng)培養(yǎng)后呈現(xiàn)出明顯差別，因而有助開快速鑒別某種微生物，稱之為鑒別培養(yǎng)基。例如用以檢查飲水和乳品中是否含有腸道致病菌的伊紅美藍(lán)就是一種常用的鑒別性培養(yǎng)基。
有些培養(yǎng)基是具有選擇和鑒別雙重作用。例如食品檢驗中常用的麥康凱培養(yǎng)基是一例。它含有膽鹽、乳糖和中性紅。膽鹽具有抑制腸道菌以外的細(xì)菌的作用（選擇性），乳糖和中性紅（指示劑）能幫助區(qū)別乳糖發(fā)酵腸道菌（如大腸桿菌）和不能發(fā)酵乳糖的腸道致病菌（如沙門氏菌和志賀氏菌）。
另外，根據(jù)營養(yǎng)成分是否“完全”，可以分為基本、完全和補(bǔ)充培養(yǎng)基，這類術(shù)語主要是用在微生物遺傳學(xué)中。根據(jù)生產(chǎn)的目的來區(qū)分，可以分為種子培養(yǎng)基和發(fā)酵培養(yǎng)基。還有專門用于培養(yǎng)病毒等寄生微生物的活組織培養(yǎng)基，如雞胚等；專門用于培養(yǎng)自養(yǎng)微生物的無機(jī)鹽培養(yǎng)基等。
三、制備的基本方法和注意事項
１、配方的選定
同一種培養(yǎng)基的配方在不同著作中常會有某些差別。因此，除所用的是標(biāo)準(zhǔn)方法，應(yīng)嚴(yán)格按其規(guī)定進(jìn)行配制外，一般均應(yīng)盡量收集有關(guān)資料，加以比較核對，再依據(jù)自己的使用目的，加以選用，記錄其來源。
２、的制備記錄
每次制備均應(yīng)有記錄，包括名稱，配方及其來源，和各種成份的牌號，最終pH值、消毒的溫度和時間制備的日期和制備者等，記錄應(yīng)復(fù)制一份，原記錄保存?zhèn)洳?，?fù)制記錄隨制好的培養(yǎng)基一同存放、以防發(fā)生混亂。
３、成分的稱取
培養(yǎng)基的各種成分必須精確稱取并要注意防止錯亂，最好一次完成，不要中斷?？蓪⑴浞街糜诎鴤?cè)，每稱完一種成分即在配方面軍做出記號，并將所需稱取的藥品一次取齊，置于左側(cè)，每種稱取完畢后，即移放于右側(cè)。完全稱取完畢后，還應(yīng)進(jìn)行一次檢查。
４、各成份的混合和溶化
培養(yǎng)基所用化學(xué)藥品均應(yīng)是化學(xué)純的。使用的蒸煮鍋不得為銅鍋或鐵鍋，以防有微量銅或鐵混入培養(yǎng)基中，使細(xì)菌不易生長。最好使用不銹鋼鍋加熱溶化，可放入大燒杯或大燒瓶中置高壓蒸汽滅菌器或流動蒸汽消毒器中蒸煮溶化。在鍋中溶化時、可先用溫水加熱并隨時擾動、以防焦化，如發(fā)現(xiàn)有焦化現(xiàn)象、該培養(yǎng)基即不能使用，應(yīng)重新制備。待大部分固體成分溶化后，再用較小火力使所有成分完全溶化，直至煮沸。如為瓊脂溶化，用另一部分水溶化其它成分，然后將兩溶液充分混合。在加熱溶化過程中，因蒸發(fā)而丟失的水分，最后必須加以補(bǔ)足。
５、pH的初步調(diào)正
因培養(yǎng)基在加熱消毒過程中、pH會有所變化，各成分完全溶解后，應(yīng)進(jìn)行pH的初步調(diào)正。例如，牛肉浸液約可降低pH0.2，而腸浸液pH卻會有顯著的升高。因此，對這個步驟，操作者應(yīng)隨時注意探索經(jīng)驗、以期能掌握最終pH，保證培養(yǎng)基的質(zhì)量。pH調(diào)整后，還應(yīng)將培養(yǎng)基煮沸數(shù)分鐘，以利培養(yǎng)基沉淀物的析出。
６、過濾澄清
液體培養(yǎng)基必須絕對澄清，瓊脂培養(yǎng)基也應(yīng)透明無顯著沉淀，因此，須要采用過濾或其它澄清方法以達(dá)到此項要求。一般液體培養(yǎng)基可用濾紙過濾法，濾紙應(yīng)折疊成折扇或漏斗形，以避免因液壓不均勻而引起濾紙破裂。
瓊脂培養(yǎng)基可用清潔的白色薄絨布趁熱過濾。亦可用中間夾有薄層吸水棉的雙層紗布過濾。新制肉、肝、血和土豆等浸液時、則須先用絨布將碎渣濾去，再用濾紙反復(fù)過濾。如過濾法不能達(dá)到澄清要求、則須用蛋清澄清法。即將冷卻至55~60°C的培養(yǎng)基放入大的三角燒瓶內(nèi)，裝入量不得超過燒瓶容量的1/2，每1000ml培養(yǎng)基加入1~2個雞蛋的蛋白，強(qiáng)力振搖3~5分鐘，置高壓蒸汽滅菌器中、121°C加熱20分鐘、取出趁熱以絨布過濾即可。
７、分裝
應(yīng)按使用的目的和要求，分裝于試管、燒瓶等適當(dāng)容器內(nèi)。分裝量不得超過容器裝盛量的2/3。容器口可用墊有防濕紙的棉塞封堵，其外還須用防水紙包扎（現(xiàn)試管一般多有用螺旋蓋者）。分裝時最好能使用半自動或電動的定量分裝器。分裝瓊脂斜面培養(yǎng)基時，分裝量應(yīng)以能形成2/3底層和1/3斜面的量為洽當(dāng)。分裝容器應(yīng)預(yù)先清洗干凈并經(jīng)干烤消毒，以利于徹底滅菌。每批應(yīng)另外分裝20ml培養(yǎng)基于一小玻璃瓶中，隨該批同時滅菌，以為測定該批培養(yǎng)基最終pH之用。
８、滅菌
一般培養(yǎng)基可采用121°C高壓蒸汽滅菌15分鐘的方法。在各種培養(yǎng)基制備方法中，如無特殊規(guī)定，即可用此法滅菌。
某些畏熱成分，如糖類，應(yīng)另行配成20%或更高的濃液，以過濾或間歇滅菌法消毒，以后再用無菌操作技術(shù)、定量加于培養(yǎng)基。明膠培養(yǎng)基亦應(yīng)用較低溫度滅菌。血液、體液和抗生素等則應(yīng)以無菌操作技術(shù)抽取和加入于經(jīng)冷卻約50°C左右的培養(yǎng)基中。
瓊脂斜面應(yīng)在滅菌后立即取出，冷至55℃-60℃時，擺置成適當(dāng)斜面，待其自然凝固。
９、質(zhì)量測試
制備好以后，應(yīng)仔細(xì)檢查一遍，如發(fā)現(xiàn)破裂、水分浸入、色澤異常、棉塞被培養(yǎng)基沾染等、均應(yīng)挑出棄去。并測定其最終pH。
將全部培養(yǎng)基放入36±1°C恒溫箱培養(yǎng)過夜，如發(fā)現(xiàn)有菌生長，即棄去。
用有關(guān)的標(biāo)準(zhǔn)菌株接種1~2管或瓶培養(yǎng)基，培養(yǎng)24~48小時，如無菌生長或生長不好。應(yīng)追查原因并重復(fù)接種一次，如結(jié)果仍同前，則該批培養(yǎng)基即應(yīng)棄去，不能使用。
10、保存
應(yīng)存放于冷暗處，最好能放于普通冰箱內(nèi)。放置時間不宜超過一周，傾注的平板不宜超過3天。每批產(chǎn)品均必須附有該批培養(yǎng)基制備記錄副頁或明顯標(biāo)簽。
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Do you actually make microbial culture? Microbial culture medium preparation technology summary!
Cleaning of glassware
In the process of preparing the culture medium, some glassware, such as test tube, triangular bottle, culture dish, beaker and straw, should be used first. These utensils should be washed and cleaned according to different conditions before use. Some must be packed, sterilized and ready to use.
1. Newly purchased glassware
After packaging contamination to remove the dirt, with hot, soapy water scrub, rinse water, then soak in 1 to 2% of the industrial hydrochloric acid for hours, remove free alkaline substances, then rinse with running water. The capacity of the larger vessels, such as large flask, measuring cylinder, wash after injection of a few of concentrated hydrochloric acid, turn the container make its internal surface participation of hydrochloric acid, pour to hydrochloric acid, a few minutes later again to rinse water, pour in washing dry water, can be used.
2. Used glassware
Where no pathogens or have not been with fungus pollution of the vessel, may at any time after flushing, draw a chemical reagent of straws, can soak in the water, until a certain number on again after cleaning. Utensils that may be contaminated by pathogenic bacteria must be properly disinfected before removing dirt, washing them with soap and rinsing them with running water. If you can't clean the container with soap solution, you can soak it for an appropriate time before rinsing it with clean water. The main components of the lotion are potassium dichromate and concentrated acid, whose function is to oxidize organic matter into soluble substances for washing. The lotion has a strong corrosive effect and should be used with special care to avoid splashing on clothes, body and other articles.
Type of culture medium
Any nutrient substrate prepared in the laboratory that is suitable for microbial growth and reproduction or for the accumulation of metabolic products is called Media. There are many kinds of media because of the different nutritional requirements of various microorganisms and the different purposes of culture and testing. We can classify a wide variety of media into several types according to certain criteria.
1. According to the complete understanding of the chemical composition of the medium, the medium can be divided into natural medium, synthetic medium and semi-synthetic medium.
1) natural medium natural medium refers to the use of various raw materials of animals, plants or microorganisms, whose ingredients are difficult to know accurately. The main ingredients used for this culture medium are beef paste, malt juice, peptone, yeast paste, corn meal, bran, various kinds of cake powder, potato, milk, serum, etc. With these substances as the medium doesn't know exactly what the chemical composition of it, but in general, nutrition is rich, microbes thrive, and source widely, convenient preparation, so the more commonly used, especially suitable for the preparation laboratory commonly used medium. The stability of the medium is often affected by factors such as the production plant or batch number.
2) synthetic media the synthetic media is a kind of medium with known chemical composition and quantity. This type of culture medium has precise and repeatable chemical composition, but is expensive, and microbes grow slowly, so it is only suitable for scientific research, such as nutrition and metabolism.
3) in the synthetic medium, some or more natural ingredients are added to the semi-synthetic medium; Or in a natural medium, a semi-synthetic medium is formed by the addition of one or more chemicals with known ingredients. For example, potato sucrose culture medium and so on. This medium is most used in production practice and laboratory.
2. According to the physical state of the medium, it can be divided into solid medium, liquid medium and semi-solid medium.
1) prepared by liquid medium is a liquid medium, including the composition of soluble in water, basically have no obvious solids, liquid medium nutrients distribution uniform, easy to control the growth of microbial metabolic state.
2) solid medium is formed by adding appropriate coagulant into liquid medium. The materials commonly used as coagulants are agar-agar, gelatin, silica gel, etc. Solid medium is widely used in practice. In the laboratory, it is used for the separation, identification, detection of hybrid bacteria, counting, storage, and bioassay of microorganisms.
3) semi-solid medium if a small amount of coagulant is added to the liquid medium, the semi-solid medium is made. Take agar-agar for example. Its dosage is between 0.2 and 1%. The medium is sometimes used to observe the dynamics of microbes, and sometimes to preserve species.
3. According to the use of the medium, it can be divided into selective medium, proliferative medium and differential medium.
1) selective medium a medium in which a substance is added to kill or inhibit the growth of unwanted strains, called selective medium. For example, streptomycin and chloramphenicol inhibit the growth of prokaryotes. The growth of eukaryotes can be inhibited by mycostatin and griseofulvin. Crystal violet can inhibit the growth of gram-positive bacteria.
2) proliferation medium in nature, different species of microbes often live together, in order to separate micro-organisms, we need add some microorganisms in ordinary medium particularly fond of nutrients, in order to increase the microbe propagation velocity, phase out other microorganisms, this medium is called proliferation medium, the medium is often used to increase the fungus strains screening and choices. To some extent, the proliferation medium is also an alternative medium.
3) the differentiation medium adds a certain reagent or chemical drug to the medium, which makes the indistinguishable microbes show obvious difference after culture, thus facilitating the rapid identification of certain microorganisms. Such a medium is called differential medium. For example, a commonly used differential medium is the irus blue medium used to check for intestinal pathogens in drinking water and dairy products.
Some mediums have a dual function of selection and identification. For example, the commonly used McConkey medium in food testing is an example. It contains bile salt, lactose and neutral red. Bile salts can inhibit the role of intestinal bacteria of bacteria (optional), lactose and neutral red (indicator) can help distinguish lactose fermentation intestinal bacteria such as e. coli and not ferment lactose intestinal pathogenic bacteria such as salmonella and hayes bacteria).
In addition, depending on whether the nutrient content of the culture medium is "complete", it can be divided into basic culture medium, complete culture medium and supplementary culture medium. Such terms are mainly used in microbial genetics. According to the purpose of production, the culture medium can be divided into seed medium and fermentation medium. There are also living tissue culture media, such as chicken embryos, which are specially used for cultivating parasitic microorganisms such as viruses. Inorganic salt culture medium specially used for cultivating self-cultured microorganisms.
Basic methods and precautions for preparing media
1. Selection of medium formula
The formulations of the same medium often differ from one work to another. Are used in addition to the standard method, therefore, should be strictly in accordance with the provisions of preparation, generally all should try to collect the relevant information, comparing to check, then according to their purpose, to choose, record its source.
2. Preparation record of culture medium
Every time the preparation of culture medium are should be recorded, including the name of culture medium, formula and its source, and a variety of ingredients of brand, the final pH value, the date of the preparation of sterilization temperature and time and preparation, etc., a copy of records shall be, the original records for future reference, duplicate records with good medium together, to prevent confusion.
3. Weighing of medium ingredients
The ingredients of the culture medium must be accurately weighed and attention should be paid to prevent derangement, preferably once, without interruption. Formula can be placed alongside side, after each said a component that is on the army to mark, and the required according to take drugs make even at a time, on the left side, each said after the completion, or move on the right side. After the complete weighing is finished, an inspection should be carried out.
4. Mixing and dissolution of various components of the culture medium
All the chemicals used in the media should be chemically pure. The cooking pot used shall not be a copper or iron pot, in case any trace of copper or iron is mixed into the culture medium, making bacteria difficult to grow. It is best to use stainless steel pot to heat melting, can put in the large beaker or large beaker in the high pressure steam sterilizer or flow steam sterilizer in the boiling dissolution. When dissolved in a pot, warm water can be used to heat it up and disturb it at any time to prevent coking. If coking is found, the medium cannot be used and should be prepared again. When most of the solid ingredients are dissolved, use a small amount of heat to dissolve them completely until they boil. In the case of AGAR dissolving, dissolve the other ingredients in another part of water and mix the two solutions thoroughly. In the process of heating and melting, the water lost by evaporation must be finally replenished.
5. Initial pH adjustment of the medium
Since the pH of the medium will change in the process of heating and disinfection, the initial pH adjustment should be carried out after all components of the medium are completely dissolved. For example, beef leach decreased pH by about 0.2, while intestinal leach pH increased significantly. Therefore, for this step, operators should always pay attention to exploring experience in order to master the final pH of the medium and ensure the quality of the medium. After pH adjustment, the culture medium should be boiled for several minutes to facilitate the precipitation of the culture medium.
6. Medium filtration and clarification
Liquid media must be absolutely clarified and AGAR media should be transparent without significant precipitation. Therefore, filtration or other clarification methods are required to meet this requirement. In general, liquid media can be filtered by filter paper. Filter paper should be folded into folding fan or funnel shape to avoid cracking of filter paper due to hydraulic inhomogeneity.
AGAR medium can be filtered by hot while using clean white flannelette. It can also be filtered by double-layer gauze with a thin layer of absorbent cotton in the middle. When new meat, liver, blood and potatoes are soaked, the residue must be filtered out with a flannelette before repeated filtration with filter paper. If the filtration method cannot meet the clarification requirements, the egg white clarification method should be used. Is cooled to 55 ~ 60 ° C medium into the big triangle flask, loading amount shall not exceed the flask capacity 1/2, add 1 ~ 2 eggs per 1000 ml medium protein, strong vibration wave 3 ~ 5 minutes, buy high pressure steam sterilizer, 121 ° C in the heat for 20 minutes, remove the strike to lint filter.
7. Separation of media
The separation of medium shall be carried in suitable containers such as test tubes and flasks according to the purpose and requirements of use. The loading capacity shall not exceed 2/3 of the container loading capacity. The mouth of the container can be sealed with a cotton plug with dampproof paper, and it must be wrapped with waterproof paper. It is best to use semi-automatic or electric quantizer when assembling. When the AGAR slanting medium is separately loaded, the amount of separation should be consistent with the amount that can form 2/3 of the bottom layer and 1/3 of the slanting medium. The container should be cleaned in advance and sterilized by dry roasting to facilitate complete sterilization of the culture medium. Each batch of culture medium should be separately packed with 20ml culture based on a small glass bottle. The culture medium should be sterilized simultaneously with the batch of culture medium for the purpose of determining the final pH of the batch of culture medium.
8. Sterilization of culture medium
General medium can be used 121 ° C high pressure steam sterilization method for 15 minutes. In all kinds of culture medium preparation methods, if there is no special provisions, this method can be sterilized.
Some heat - repellent ingredients, such as sugar, should be mixed with 20% or higher concentration to be sterilized by filtration or intermittent sterilization. Gelatin culture medium is also used to sterilize at low temperature. Blood, body fluids and antibiotics should be to aseptic operating technology such as extraction and join in about 50 ° C or so by the cooling medium.
AGAR slant should be removed immediately after sterilization, cold to 55 ℃ to 60 ℃, cant pieces into the appropriate, with its natural coagulation.
9. Quality test of the medium
After each batch of culture medium is prepared, it should be carefully examined, such as rupture, water immersion, color abnormality, cotton plug contamination by culture medium, etc., should be picked out and discarded. And its final pH was measured.
Put all medium in 36 + 1 ° C for incubator culture overnight, if found to have bacteria growth, namely abandon.
Inoculate 1 to 2 tubes or bottles of culture medium with the relevant standard strains for 24 to 48 hours, such as sterile growth or poor growth. The cause should be traced and repeated. If the results are the same, the culture medium should be discarded and unusable.
10. Preservation of culture medium
The culture medium should be stored in cold and dark places, preferably in a general refrigerator. It should not be placed for more than a week, and the plate medium should not be poured for more than 3 days. Each batch of culture medium must be accompanied by a copy of the preparation record or a clear label.
From the food lab service