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Microbiology

Enrichment Cultures: Culturing Aerobic and Anaerobic Microbes on Selective and Differential Medias

Overview

Source: Christopher P. Corbo1, Jonathan F. Blaize1, Elizabeth Suter1
1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island NY, 10301

Prokaryotic cells are able to inhabit nearly every environment on this planet. As a kingdom, they possess a great metabolic diversity, allowing them to use a wide variety of molecules for energy generation (1). Therefore, when cultivating these organisms in the lab, all necessary and specific molecules required to make energy must be provided in the growth media. While some organisms are metabolically diverse, others are able to survive in extreme environments such as high or low temperatures, alkaline and acidic pH, reduced or oxygen absent environments, or environments containing high salt (2,3,4). Termed "extremophiles", these organisms often require these intense environments to proliferate. When scientists look to grow such organisms, the media components as well as any specific environmental conditions all need to be taken into account in order to successfully cultivate the organisms of interest.

Scientists are able to grow culturable organisms in the lab because they understand the specific requirements that those species need to grow. However, culturable organisms account for less than 1% of species estimated to be on the planet (5). Organisms that we have detected by gene sequencing but are not able to grow in the lab are considered unculturable (6). At this time, we do not know enough about the metabolism and growth conditions of these organisms to replicate their environment in the lab.

Fastidious organisms lie somewhere in between the former two. These organisms are culturable, but they require very specific growth conditions, such as specific growth media components and/or specific growth conditions. Two examples of such genera are Neisseria sp. and Haemophilus sp., both of which require partially broken-down red blood cells (also known as chocolate agar), as well as specific growth factors and an environment rich in carbon dioxide (7). Without all of the required specific components, these organisms will not grow at all. Often, even with all of their requirements, these organisms grow poorly.

Unlike eukaryotic cells, which are only able to grow in an aerobic, or oxygen containing, environment, prokaryotic cells are able to grow anaerobically using several fermentation pathways to generate ample energy (8). Other prokaryotes prefer a microaerophilic, or reduced oxygen environment, or even a capnophilic, or high carbon dioxide environment (9). These organisms are more challenging to enrich for, since the atmosphere must be altered. Scientists that frequently work with organisms sensitive to an oxygenated environment would normally work in an anaerobic chamber and incubator, where a heavy, inert gas such as argon is pumped in to displace the oxygen (10). Others make use of conventionally available sealed gas packet systems that use water to generate hydrogen and carbon dioxide, along with a catalyst like palladium to remove all atmospheric oxygen. These commercially available kits can create any of the above-mentioned atmospheric conditions (10).

Whether cultivating a pathogen to determine potential infection or looking to identify a specific species of bacteria present in a natural environment, one problem exists. No one bacterial species inhabits one habitat. Bacteria live as multicellular communities everywhere from the skin of humans to the oceans of our planet (11). When attempting to isolate one species of bacteria, scientists must work to exclude the numerous other organisms that are also inhabiting the isolated area. For this reason, enriched growth media for bacteria often carry out two functions. The first is to make the media selective. A selective agent will prevent some species from growing, while not inhibiting and often even promoting others to grow (12). The second function of media ingredients may be to work as differential agents. Such agents allow for the identification of a particular biochemical feature of an isolated organism. By pairing several different selective and differential medias along with appropriate growth conditions, scientists and diagnosticians are able to identify the presence of specific bacterial species from a particular isolate.

One example of a selective and differential media aiding in to identification is in the case of the clinically significant organism Staphylococcus aureus. This organism is typically cultured on mannitol salt agar. This media not only selects for only organisms which can live in a high salt environment, which include some gram positives like Staphylococcus, but it also inhibits any organisms sensitive to salt. The mannitol sugar is the differential component of this medium. Of all the clinically significant Staphylococcus species, only S. aureus is able to ferment mannitol. This fermentation reaction produces acid as a by-product which causes the red methyl red indicator in the media to turn yellow. Other Staphylococcus species (such as Staphylococcus epidermidis) although able to grow, will leave the media red in color.

This lab exercise demonstrates proper aseptic technique, as well as proper inoculation of growth media from broth. It also introduces the growth of common contaminant organisms on enrichment media, the use of a gas package anaerobic culture system for anaerobic bacteria, and the use of different selective and differential medias for the presumptive identification of gram positive and gram-negative bacteria.

Procedure

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1. Preparation

  1. Before beginning, wash hands thoroughly and put on appropriately sized gloves.
  2. Sterilize work surface with 5% sodium hypochlorite (bleach) and dry thoroughly.
  3. Place an inoculating loop in an empty 120 mL Erlenmeyer flask so that it does not touch the bench top while working.

2. Growth Media and Cultures

  1. Gather four plates of Mannitol Salt Agar (MSA), Eosin Methylene blue agar (EMB), and eight tryptic soy agar (TSA) from the refrigerator (these can be purchased commercially).
  2. Place plates onto the cleaned work area.
  3. Gather the following broth cultures: Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Proteus vulgaris. Use caution: as these are aerobic organisms, the caps of the tubes should be slightly loose.
  4. Place all cultures in a test tube rack on the cleaned work surface.

3. Transferring Cultures and Incubation

  1. Stand or sit at the bench with all of the materials in reach.
  2. To use aseptic technique to transfer the bacteria to the plate, first flame the loop until it is glowing orange - and then allow it to cool in the air.
  3. While the loop cools, take the broth culture of E. coli, and then open the cap with using your pinky finger.
  4. Flame the opening of the tube quickly. This prevents contamination.
  5. Dip the loop into the tube and streak the organism onto the first quadrant of an EMB plate.
  6. After the first streak is performed, flame the loop, and then perform a second streak crossing the first streak only once or twice. This will allow for single colony isolation and to determine if there is any contamination.
  7. Repeat this until all four quadrants of the plate have been streaked.
  8. Now, transfer each of the rest of the four organisms in the same streak plating manner so that the final product is one MSA plate, one EMB plate, and two separate TSA plates per organism.
  9. Once all organisms have been transferred, flame the loop one final time and put it away.
  10. Place 1 TSA plate with each organism into the gas packet, and then shake a gas sachet before placing it into the chamber alongside the plates. Seal tightly.
  11. Incubate all plates at 37°C overnight.
  12. Sterilize the work surface one final time with 5% bleach.

4. Reading and Recording Results

  1. Remove the plates from the incubator and place them onto a clean lab bench
  2. Take note of the growth as of the organisms on each plate in your lab notebook. In particular, take detailed notes on:
    1. Numbers and size of colonies (if present) on each plate, for each plated strain
    2. Appearance of the agar surrounding any colonies growing on the MSA plates
    3. Appearance of any colonies growing on the EMB plates
    4. Differences in growth between the TSA plates grown outside versus inside the gas package

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.

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Results

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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.

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Applications and Summary

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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.

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References

  1. Fernandez, L. A. Exploring prokaryotic diversity: there are other molecular worlds. Molecular Microbiology, 55 (1), 5-15 (2005).
  2. Grattieri, M., Suvira, M., Hasan, K., & Minteer, S. D. Halotolerant extremophile bacteria from the Great Salt Lake for recycling pollutants in microbial fuel cells. Journal of Power Sources, 356, 310-318 (2017).
  3. Wendt-Potthoff K. & Koschorreck, M. Functional Groups and Activities of Bacteria in a Highly Acidic Volcanic Mountain Stream and Lake in Patagonia, Argentina. Microbial Ecology, 1, 92 (2002).
  4. Lee, L. S., Goh, K. M., Chan, C. S., Annie Tan, G. Y., Yin, W.-F., Chong, C. S., & Chan, K.-G. Microbial diversity of thermophiles with biomass deconstruction potential in a foliage-rich hot spring. Microbiology Open, 7 (6), e00615 (2018)
  5. Ito, T., Sekizuka, T., Kishi, N., Yamashita, A., & Kuroda, M. Conventional culture methods with commercially available media unveil the presence of novel culturable bacteria. Gut Microbes, 10 (1), 77-91. (2019)
  6. Vartoukian, S. R., Palmer, R. M., & Wade, W. G. Strategies for culture of "unculturable" bacteria. FEMS Microbiology Letters, 309 (1), 1-7. (2010)
  7. Harris, T. M., Rumaseb, A., Beissbarth, J., Barzi, F., Leach, A. J., & Smith-Vaughan, H. C. Culture of non-typeable Haemophilus influenzae from the nasopharynx: Not all media are equal. Journal of Microbiological Methods, 137, 3-5. (2017)
  8. Wang, Y.-Y., Ai, P., Hu, C.-X., & Zhang, Y.-L. Effects of various pretreatment methods of anaerobic mixed microflora on biohydrogen production and the fermentation pathway of glucose. International Journal of Hydrogen Energy, 36 (1), 390-396. (2011)
  9. Pascual, A., Basco, L. K., Baret, E., Amalvict, R., Travers, D., Rogier, C., & Pradines, B. Use of the atmospheric generators for capnophilic bacteria Genbag-CO2 for the evaluation of in vitro Plasmodium falciparum susceptibility to standard anti-malarial drugs. Malaria Journal, 10, 8 (2011).
  10. Summanen, P., McTeague, M., Väisänen, M.-L., Strong, C., & Finegold, S. Comparison of Recovery of Anaerobic Bacteria Using the Anoxomat®, Anaerobic Chamber, and GasPak®Jar Systems. Anaerobe, 5, 5-9. (1999)
  11. de la Fuente-Núñez, C., Reffuveille, F., Fernández, L., & Hancock, R. E. Bacterial biofilm development as a multicellular adaptation: antibiotic resistance and new therapeutic strategies. Current Opinion in Microbiology, 16, 580-589. (2013)
  12. Possé, B., De Zutter, L., Heyndrickx, M., & Herman, L. Novel differential and confirmation plating media for Shiga toxin-producing Escherichia coli serotypes O26, O103, O111, O145 and sorbitol-positive and -negative O157. FEMS Microbiology Letters, 282 (1), 124-131. (2008)
Enrichment Cultures: Culturing Aerobic and Anaerobic Microbes on Selective and Differential Medias
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