资料来源：克里斯托弗·科博1，乔纳森·布莱泽1，伊丽莎白·苏特1
1大学生物科学系，瓦格纳学院，1 校园路，纽约州斯塔顿岛，10301
原核细胞能够栖息在这个星球上的几乎每一个环境。作为一个王国，它们具有巨大的代谢多样性，允许它们使用各种各样的分子来产生能量（1）。因此，在实验室中培养这些生物体时，必须在生长培养中提供制造能量所需的所有特定分子。虽然有些生物在代谢上是多种多样的，但其他生物能够在极端环境中生存，如高温或低温、碱性和酸性pH值、减少或缺氧环境，或含有高盐的环境（2，3，4）。这些生物体被称为”嗜血杆菌”，它们往往需要这些强烈的环境来增殖。当科学家寻找培育这些生物时，需要考虑到介质成分以及任何特定的环境条件，才能成功地培育出感兴趣的生物体。
科学家能够在实验室中培育出可培养的生物体，因为他们了解这些物种生长所需的具体要求。然而，可养殖生物在估计地球上的物种中所占不到1%（5）。通过基因测序检测到但无法在实验室中生长的生物体被认为是不可培养的（6）。目前，我们对这些生物体的新陈代谢和生长条件了解不够，无法在实验室中复制其环境。
挑剔的有机体位于前两者之间。这些生物是可培养的，但它们需要非常具体的生长条件，如特定的生长介质成分和/或特定的生长条件。这种属的两个例子是Neisseria sp.和血友病，这两个都需要部分分解的红血球（也称为巧克力琼脂），以及特定的生长因子和富含二氧化碳的环境（7）。如果没有所有必需的特定成分，这些生物体将根本不会生长。通常，即使满足所有要求，这些生物体的生长也很差。
与真核细胞不同，真核细胞只能在有氧或含氧环境中生长，而原核细胞则能够利用几种发酵途径进行厌氧生长，从而产生充足的能量（8）。其他原生核生物更喜欢亲微亲氧体，或减少氧气环境，甚至亲亲生物或高二氧化碳环境（9）。这些生物体更难以丰富，因为大气必须改变。经常与对含氧环境敏感的生物体工作的科学家通常会在厌氧室和孵化器中工作，在那里，大量惰性气体（如 argon）被泵送来取代氧气（10）。另一些则利用常规的密封气体包系统，利用水产生氢气和二氧化碳，以及像铂一样的催化剂来去除所有大气中的氧气。这些市售的试剂盒可产生上述任何大气条件（10）。
无论是培养病原体以确定潜在感染，还是希望确定自然环境中存在的特定细菌种类，都存在一个问题。没有一个细菌物种栖息在一个栖息地。细菌作为多细胞群落生活，从人类的皮肤到我们星球的海洋（11）。在试图分离一种细菌时，科学家必须努力排除同样居住在隔离区的其他许多生物。因此，细菌的富集生长介质往往具有两种功能。第一是使媒体有选择性。选择性剂会阻止某些物种生长，同时不抑制甚至经常促进其他物种的生长（12）。介质成分的第二个功能可能是作为差分剂工作。这种制剂可以识别一个孤立的生物体的一个特定的生化特征。通过将几种不同的选择性和差别介质与适当的生长条件配对，科学家和诊断学家能够从特定分离物中识别特定细菌物种的存在。
选择性和差别化介质有助于鉴定的一个例子是临床上重要的有机体金黄色葡萄球菌。这种有机体通常是在曼尼托盐琼脂培养的。这种介质不仅只选择生活在高盐环境中的生物体，其中包括一些克阳性物，如金黄色葡萄球菌，而且还抑制任何对盐敏感的生物体。甘醇糖是这种介质的差分成分。在所有临床上重要的金黄色葡萄球菌物种中，只有S.金黄色葡萄球菌能够发酵曼尼托。这种发酵反应产生酸作为副产品，导致介质中的红色甲基红色指示灯变黄。其他葡萄球菌物种（如金黄色葡萄球菌表皮）虽然能够生长，但会使介质呈红色。
本实验练习演示了适当的无菌技术，以及从肉汤中正确接种生长介质。它还介绍了浓缩介质上常见污染物生物的生长情况，使用气体包厌氧培养系统进行厌氧细菌，以及使用不同的选择性和差分介质对克进行推定鉴定阳性和克阴性细菌。
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.
