ソース: クリストファー・P・コルボ1, ジョナサン・F・ブレイズ1, エリザベス・スーター1
1ワーグナーカレッジ生物科学科、1キャンパスロード、スタテンアイランドニューヨーク、10301
原核細胞は、この惑星上のほぼすべての環境に生息することができる。王国として、彼らは大きな代謝多様性を持ち、エネルギー生成(1)のために多種多様な分子を使用することができます。したがって、研究室でこれらの生物を培養する際には、エネルギーを作るために必要なすべての分子と特定の分子を成長培養培養剤に提供する必要があります。代謝的に多様な生物もあれば、高温または低温、アルカリ性および酸性pH、還元または酸素欠乏環境、または高塩を含む環境(2,3,4)などの極端な環境で生き残ることができる生物もあります。「極端な好酸球」と呼ばれていますが、これらの生物はしばしば増殖するためにこれらの強烈な環境を必要とします。科学者がそのような生物を成長させようとするとき、メディア成分だけでなく、特定の環境条件は、関心のある生物を正常に栽培するために考慮する必要があります。
科学者たちは、これらの種が成長する必要がある特定の要件を理解しているので、研究室で培養可能な生物を成長させることができます。しかし、培養可能な生物は、地球上に生息すると推定される種の1%未満を占めている(5)。遺伝子シーケンシングで検出したが、研究室では増殖できない生物は、培養不能と考えられている(6)。現時点では、これらの生物の代謝と成長条件について十分に知られていない。
断固たる生物は前者の2人の間のどこかに横たわっている。これらの生物は培養可能であるが、特定の成長培養培養成分や特定の成長条件など、非常に特定の成長条件を必要とする。このような属の2つの例は、部分的に分解された赤血球(チョコレート寒天とも呼ばれる)、ならびに特定の成長因子および二酸化炭素が豊富な環境(7)を必要とするネイセリアsp.およびヘモフィラスsp.である。必要な特定の成分のすべてがなければ、これらの生物は全く成長しません。多くの場合、すべての要件を満たしていても、これらの生物は不十分に成長します。
好気性、または酸素を含む酸素でしか増殖できない真核細胞とは異なり、原核細胞は、いくつかの発酵経路を用いて嫌気的に増殖し、十分なエネルギーを生成することができる(8)。他の原核生物は、微好気性、または酸素環境の低下、あるいはカプノフィリック、または高い二酸化炭素環境を好む(9)。これらの生物は、大気を変えなければならないので、豊かにするのがより難しい。酸素化された環境に敏感な生物と頻繁に働く科学者は、通常、嫌気性チャンバーとインキュベーターで動作し、アルゴンなどの重い不活性ガスが酸素を置き換えるためにポンプで送り込まれる(10)。また、水を利用して水素や二酸化炭素を発生させる密閉ガスパケットシステムや、パラジウムのような触媒を使用して大気中の酸素をすべて除去するシステムもあります。これらの市販キットは、上記の大気条件(10)のいずれかを作成することができる。
潜在的な感染を決定するために病原体を培養するか、自然環境に存在する細菌の特定の種を同定しようとしているかどうか、1つの問題が存在する。1つの生息地に生息する細菌種は一つもない。バクテリアは、人間の皮膚から地球の海まで、あらゆる場所で多細胞共同体として生きています(11)。1種の細菌を単離しようとする際、科学者は、孤立した地域に生息する他の多数の生物を排除するために働かなければならない。このため、細菌に対する濃縮成長培分は、多くの場合、2つの機能を行う。1 つ目は、メディアを選択的にする方法です。選択的剤は、いくつかの種が成長するのを防ぎ、他の種を阻害せず、しばしば他の種の成長を促進する(12)。媒体成分の第2の機能は、差分剤として働くことであってもよい。このような薬剤は、単離された生物の特定の生化学的特徴の同定を可能にする。適切な成長条件と一緒にいくつかの異なる選択的および差分媒体を組み合わせることで、科学者および診断士は、特定の単離物から特定の細菌種の存在を識別することができる。
同定を支援する選択的および差分媒体の一例は、臨床的に有意な生物黄色ブドウ球菌の場合である。この生物は、典型的にはマンニトール塩寒天上で培養される。この媒体は、ブドウ球菌のようないくつかのグラム陽性を含む高塩環境に住むことができる生物だけを選択するだけでなく、塩に敏感な任意の生物を阻害します。マンニトール糖は、この媒体の差分成分である。臨床的に有意なブドウ球菌種の中で、マンニトールを発酵できるのはS.aureusのみである。この発酵反応は、媒体中の赤色の赤色指標が黄色に変わる副産物として酸を生成します。他のブドウ球菌種(表皮ブドウ球菌など)は成長することができるが、メディアを赤色のままにする。
このラボ演習では、適切な無菌技術と、スープからの成長培った培宿の適切な接種を示します。また、濃縮培養媒体上の一般的な汚染物質生物の成長、嫌気性細菌に対するガスパッケージ嫌気培養システムの使用、およびグラムの推定同定のための異なる選択的および差動媒体の使用を紹介します。陽性およびグラム陰性の細菌。
Mannitol Salt Agar (MSA): This medium is selective for gram positive organisms that are able to survive in 6.5% sodium chloride. The gram-negative organisms Escherichia coli and Proteus vulgaris should not be able to grow on this medium because of the high salt concentration. S. epidermidis and S. aureus should be able to grow. The media is differential between the two because the S. aureus is able to ferment the mannitol – turning the methyl red indicator bright yellow due to the production of acid as a fermentation by-product. S. epidermidis should maintained the pink color on the plate.
NOTE: If colonies are small, growth on this medium may require additional incubation for a total of up to 48 hours at optimal temperature – here, 37°C.
Eosin Methylene Blue agar (EMB): This medium is selective for gram negative organisms, so Escherichia coli and Proteus vulgaris plates should exhibit growth. The eosin and methylene blue dyes are toxic to gram positive cells so neither Streptococcus species should grow. The outer membrane of gram-negative cells prevents the dyes from entering the cells. This media is differential because it allows for one to test for the ability of the organism to ferment lactose. E. coli turns a bright purple color (often with a green metallic sheen if cultivated long enough) due to the fermentation of lactose in the media. The P. vulgaris, although able to grow, does not ferment lactose (however it is able to ferment other sugars).
Tryptic Soy Agar (TSA): This medium is non-selective, so all of the study species should grow. However, comparing the aerobic versus anaerobic conditions, the plates from the gas package should display less growth (and smaller colonies). This is because none of the bacteria grown in the demonstration are obligate aerobes, but their optimal growth condition does include oxygen.
Different bacterial species are able to grow in different environments and are able to use different carbon sources as a way of generating energy. When working with these as cultures in the lab, it is important to know the components of the growth media being worked with and to match the growth media to the bacterial species. Scientists and diagnosticians can also exploit the varying biochemical reactions as a way to isolate different species from others and as a way to distinguish and identify bacteria in a mixed environment.
Bacteria are able to inhabit almost every environment on Earth, from desert tundra to tropical rainforests. This ability to colonize vastly different niches is due to their adaptability and vast metabolic diversity, which allows them to utilize a wide variety of molecules for energy generation. It is this massive array of diversity which leads to the phenomenon that less than 1% of the bacterial species on the planet are considered culturable and these are only possible due to an understanding of their specific metabolic and environmental needs.
Performing manipulations of media and environment in the laboratory not only allows researchers to experiment to find the optimal conditions for culturing a species of interest, but it also enables enrichment, the process of changing conditions to select for specific species from a mixed culture. Some microbial species are generalists and able to tolerate a wide variety of states or environments. Such organisms may grow readily under laboratory conditions, but they may also be prevented from growing if given an extreme habitat – which can help if the goal is to enrich for organisms from a mixed culture which are tolerant to this condition.
Fastidious organisms can be culturable but only when specific conditions are met. Neisseria or Haemophilus species, for example, require media containing partially broken down red blood cells and a high carbon dioxide concentration, which may also discourage the growth of other species. Extremophiles are named for their preference for extreme conditions, such as very low or high temperatures, reduced or oxygen absent conditions, or in the presence of high salt. These conditions are likely intolerable to most other microbes.
To further enrich for an organism of interest, some media types contain indicators which give insight into the metabolism of the organism. Mannitol Salt Agar inhibits the growth of organisms sensitive to high salt. Gram negative bacteria typically cannot survive, but the gram positive Staphylococcus genus are able to thrive. In addition, the MSA agar indicates any colonies able to ferment mannitol because the acid byproducts of fermentation will turn the methyl red indicator in the media to a bright yellow. This can allow for more specific selection of a species.
Another common enrichment medium, Eosin Methylene Blue, contains eosin and methylene blue dyes, which are toxic to gram positive organisms. It also contains lactose and bacteria on these plates which can ferment this will produce acids that lower the pH encouraging dye absorption. These colonies take up large amounts of pigment and appear dark and metallic. In this lab, you will grow four different test organisms across three different media conditions and then under aerobic versus anaerobic conditions before observing their development.
Before beginning the experiment, thoroughly wash your hands and dry them, before putting on appropriately sized laboratory gloves. Then, sterilize the work surface with 5% bleach, wiping it down thoroughly. Next, take a sterile inoculating loop and place it handle down into an empty 125 milliliter flask so that it does not touch the bench surface. Then, from the refrigerator, gather four plates of Mannitol Salt Agar, or MSA, four plates of Eosin Methylene Blue agar, EMB, and eight Tryptic Soy Agar, or TSA, plates. TSA medium is a non-selective growth medium which will be used for the two different environmental conditions. Finally, gather your cultures of interest in a tube rack. Here, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermis, and Proteus vulgaris will be grown.
To begin, light a Bunsen burner, which will be used to sterilize the tools. Then, place one MSA plate, one EMB plate, and two TSA plates close at hand. Then, select one of the bacterial cultures. You will inoculate all four of these plates with the first culture. With your free hand, pick up the inoculating loop and then sterilize it in the flame of the burner until it glows orange for a couple of seconds. Allow the loop to cool in the air. Then, open the broth culture tube and quickly flame the opening. Dip the loop into the culture and then streak the organism onto the first quadrant of the first plate. Then flame sterilize the loop again and streak the second quadrant. Repeat this action of flame sterilization and then streaking to complete the third and fourth quadrants. Streaking in this manner should give isolated colonies and also allow for confirmation that the culture is not contaminated.
Now, replace the lid and label the bottom of the plate with the name of the bacteria, media type, date, and your initials. Then, repeat the streak plating using the same bacterial culture for each of the remaining three plates taking care to label them each time. Now that the first culture has been streaked, repeat these steps for the other bacteria to obtain one inoculated MSA plate, one EMB plate, and two TSA plates for each species. Once all of the organisms have been transferred, flame the loop one final time.
To determine which organisms can grow in a reduced oxygen environment, open up a sealed gas chamber system and place one set of four TSA bacteria plates inside. Then, place an anaerobic condition sachet into the chamber and seal it tightly. Finally, place all of the plates, including those inside the sealed gas chamber system, into a 37 degree Celsius incubator overnight. Going forward, check the plates every 24 to 48 hours to give the colonies time to grow and metabolize any indicator reactants.
To assess how well the different bacterial species responded to each growth condition, first examine the plates for growth and record which species were able to produce colonies on each media type and in the anaerobic versus aerobic condition. Note the color of the organisms growing as well as the sizes and shape of the colonies.
The mannitol salt agar medium is selective for gram positive organisms which are able to survive in 6. 5% sodium chloride. In this experiment, this meant that the gram negative E. coli and P. vulgaris did not grow due to the high salt concentrations. S. epidermis and S. aureus were able to grow, however, confirming that they are gram positive. Additionally, there is a clear difference between the two species because the S. aureus is able to ferment mannitol turning the methyl red indicator in the media to a bright yellow due to the acid byproducts of fermentation. This was not seen in the case of S. epidermis.
The EMB medium on the other hand is selective for gram negative organisms because the eosin and methylene blue dyes are toxic to gram positive cells. The outer membrane of gram negative bacteria prevents these toxic dyes from entering the cells, meaning they are able to grow. Moreover, this medium indicates whether the bacterial species present is able to ferment lactose. Here, E. coli colonies turn a dark purple color, sometimes with a green metallic sheen indicating fermentation. P. vulgaris grows on this medium but does not ferment lactose and so appears a light pinkish to purple from being in the presence of the dye. In the anaerobic condition, the bacterial species on TSA media should still grow but may do so very poorly compared to those with ample oxygen. This is because none of the test species are obligate anaerobes.
Experiments like this to enrich the growth environment can help to favor and isolate a specific species from a mixed sample. They can also help determine the optimal growth conditions for different bacterial species in a laboratory setting, thus aiding further research.
