Fuente: Christopher P. Corbo1, Jonathan F. Blaize1, Elizabeth Suter1
1 Departamento de Ciencias Biológicas, Wagner College, 1 Campus Road, Staten Island NY, 10301
Las células procaquerías son capaces de habitar casi todos los ambientes de este planeta. Como reino, poseen una gran diversidad metabólica, lo que les permite utilizar una amplia variedad de moléculas para la generación de energía (1). Por lo tanto, al cultivar estos organismos en el laboratorio, todas las moléculas necesarias y específicas necesarias para hacer energía deben ser proporcionadas en los medios de crecimiento. Mientras que algunos organismos son metabólicamente diversos, otros son capaces de sobrevivir en ambientes extremos como altas o bajas temperaturas, pH alcalino y ácido, ambientes reducidos o ausentes de oxígeno, o ambientes que contienen alta sal (2,3,4). Llamados “extremofílicos”, estos organismos a menudo requieren que estos ambientes intensos proliferen. Cuando los científicos buscan cultivar estos organismos, los componentes de los medios de comunicación, así como las condiciones ambientales específicas, deben tenerse en cuenta para cultivar con éxito los organismos de interés.
Los científicos son capaces de cultivar organismos cultivables en el laboratorio porque entienden los requisitos específicos que esas especies necesitan para crecer. Sin embargo, los organismos culturables representan menos del 1% de las especies que se estima que están en el planeta (5). Los organismos que hemos detectado por secuenciación genética pero que no son capaces de crecer en el laboratorio se consideran inculturables (6). En este momento, no sabemos lo suficiente sobre el metabolismo y las condiciones de crecimiento de estos organismos para replicar su entorno en el laboratorio.
Los organismos fastidiosos se encuentran en algún lugar entre los dos primeros. Estos organismos son culturables, pero requieren condiciones de crecimiento muy específicas, como componentes específicos de los medios de crecimiento y/o condiciones de crecimiento específicas. Dos ejemplos de estos géneros son Neisseria sp. y Haemophilus sp., que requieren glóbulos rojos parcialmente descompuestos (también conocidos como agar de chocolate), así como factores de crecimiento específicos y un entorno rico en dióxido de carbono (7). Sin todos los componentes específicos necesarios, estos organismos no crecerán en absoluto. A menudo, incluso con todos sus requisitos, estos organismos crecen mal.
A diferencia de las células eucariotas, que sólo son capaces de crecer en un ambiente aeróbico, u oxígeno que contiene, las células procasinoticas son capaces de crecer anaeróbicamente utilizando varias vías de fermentación para generar una amplia energía (8). Otros prokaryotes prefieren un ambiente microaerofílico, o de oxígeno reducido, o incluso un ambiente conofílico, o de dióxido de carbono alto (9). Estos organismos son más difíciles de enriquecer, ya que la atmósfera debe ser alterada. Los científicos que trabajan con frecuencia con organismos sensibles a un entorno oxigenado normalmente trabajarían en una cámara anaeróbica e incubadora, donde un gas pesado e inerte como el argón se bombea para desplazar el oxígeno (10). Otros hacen uso de sistemas de paquetes de gas sellados disponibles convencionalmente que utilizan agua para generar hidrógeno y dióxido de carbono, junto con un catalizador como el paladio para eliminar todo el oxígeno atmosférico. Estos kits disponibles comercialmente pueden crear cualquiera de las condiciones atmosféricas antes mencionadas (10).
Ya sea cultivando un patógeno para determinar una posible infección o buscando identificar una especie específica de bacterias presentes en un entorno natural, existe un problema. Ninguna especie bacteriana habita en un hábitat. Las bacterias viven como comunidades multicelulares en todas partes, desde la piel de los seres humanos hasta los océanos de nuestro planeta (11). Al intentar aislar una especie de bacteria, los científicos deben trabajar para excluir los numerosos otros organismos que también habitan el área aislada. Por esta razón, los medios de crecimiento enriquecidos para las bacterias a menudo llevan a cabo dos funciones. La primera es hacer que los medios sean selectivos. Un agente selectivo evitará que algunas especies crezcan, sin inhibir y a menudo incluso promover áotras para crecer (12). La segunda función de los ingredientes de los medios de comunicación puede ser trabajar como agentes diferenciales. Estos agentes permiten la identificación de una característica bioquímica particular de un organismo aislado. Al emparejar varios medios selectivos y diferenciales diferentes junto con condiciones de crecimiento adecuadas, los científicos y diagnósticos son capaces de identificar la presencia de especies bacterianas específicas de un aislado particular.
Un ejemplo de un medio selectivo y diferencial que ayuda a la identificación es en el caso del organismo clínicamente significativo Staphylococcus aureus. Este organismo se cultiva típicamente en agar sal de manitol. Este medio no sólo selecciona para sólo los organismos que pueden vivir en un ambiente de alta sal, que incluyen algunos gram positivos como Staphylococcus,sino que también inhibe cualquier organismo sensible a la sal. El azúcar de manitol es el componente diferencial de este medio. De todas las especies clínicamente significativas de Staphylococcus, sólo S. aureus es capaz de fermentar manitol. Esta reacción de fermentación produce ácido como subproducto que hace que el indicador rojo metilo rojo en los medios se vuelva amarillo. Otras especies de Staphylococcus (como Staphylococcus epidermidis)aunque capaces de crecer, dejarán los medios de color rojo.
Este ejercicio de laboratorio demuestra una técnica aséptica adecuada, así como la inoculación adecuada de los medios de crecimiento del caldo. También introduce el crecimiento de organismos contaminantes comunes en medios de enriquecimiento, el uso de un sistema de cultivo anaeróbico de paquete de gas para bacterias anaeróbicas, y el uso de diferentes medios selectivos y diferenciales para la identificación presunta de gramos bacterias positivas y gramnegativas.
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.
