Fonte: Christopher P. Corbo1, Jonathan F. Blaize1, Elizabeth Suter1
1 Dipartimento di Scienze Biologiche, Wagner College, 1 Campus Road, Staten Island NY, 10301
Le cellule procariotiche sono in grado di abitare quasi ogni ambiente su questo pianeta. Come regno, possiedono una grande diversità metabolica, che consente loro di utilizzare un’ampia varietà di molecole per la generazione di energia (1). Pertanto, quando si coltivano questi organismi in laboratorio, tutte le molecole necessarie e specifiche necessarie per produrre energia devono essere fornite nei mezzi di crescita. Mentre alcuni organismi sono metabolicamente diversi, altri sono in grado di sopravvivere in ambienti estremi come alte o basse temperature, pH alcalino e acido, ambienti ridotti o privi di ossigeno o ambienti contenenti sale elevato (2,3,4). Definiti “estremofili”, questi organismi spesso richiedono questi ambienti intensi per proliferare. Quando gli scienziati cercano di coltivare tali organismi, i componenti dei media e le eventuali condizioni ambientali specifiche devono essere presi in considerazione per coltivare con successo gli organismi di interesse.
Gli scienziati sono in grado di coltivare organismi coltivabili in laboratorio perché comprendono i requisiti specifici di cui quelle specie hanno bisogno per crescere. Tuttavia, gli organismi coltivabili rappresentano meno dell’1% delle specie stimate sul pianeta (5). Gli organismi che abbiamo rilevato mediante sequenziamento genico ma che non sono in grado di crescere in laboratorio sono considerati incoltibili (6). In questo momento, non sappiamo abbastanza sul metabolismo e sulle condizioni di crescita di questi organismi per replicare il loro ambiente in laboratorio.
Gli organismi meticolosi si trovano da qualche parte tra i primi due. Questi organismi sono coltivabili, ma richiedono condizioni di crescita molto specifiche, come componenti specifici dei mezzi di crescita e / o condizioni di crescita specifiche. Due esempi di tali generi sono Neisseria sp. e Haemophilus sp., entrambi i quali richiedono globuli rossi parzialmente scomposti (noto anche come agar al cioccolato), nonché fattori di crescita specifici e un ambiente ricco di anidride carbonica (7). Senza tutti i componenti specifici richiesti, questi organismi non cresceranno affatto. Spesso, anche con tutte le loro esigenze, questi organismi crescono male.
A differenza delle cellule eucariotiche, che sono in grado di crescere solo in un ambiente aerobico o contenente ossigeno, le cellule procariotiche sono in grado di crescere anaerobamente utilizzando diverse vie di fermentazione per generare ampia energia (8). Altri procarioti preferiscono un ambiente microaerofilo, o a ridotto ossigeno, o anche un ambiente capnofilo o ad alta anidride carbonica (9). Questi organismi sono più difficili da arricchire, poiché l’atmosfera deve essere alterata. Gli scienziati che lavorano frequentemente con organismi sensibili a un ambiente ossigenato normalmente lavorano in una camera anaerobica e in un incubatore, dove un gas pesante e inerte come l’argon viene pompato per spostare l’ossigeno (10). Altri fanno uso di sistemi di pacchetti di gas sigillati convenzionalmente disponibili che utilizzano acqua per generare idrogeno e anidride carbonica, insieme a un catalizzatore come il palladio per rimuovere tutto l’ossigeno atmosferico. Questi kit disponibili in commercio possono creare una qualsiasi delle condizioni atmosferiche sopra menzionate (10).
Sia che si coltivi un agente patogeno per determinare una potenziale infezione o che si cerchi di identificare una specifica specie di batteri presenti in un ambiente naturale, esiste un problema. Nessuna specie batterica abita un habitat. I batteri vivono come comunità multicellulari ovunque, dalla pelle degli esseri umani agli oceani del nostro pianeta (11). Quando si tenta di isolare una specie di batteri, gli scienziati devono lavorare per escludere i numerosi altri organismi che abitano anche l’area isolata. Per questo motivo, i mezzi di crescita arricchiti per i batteri spesso svolgono due funzioni. Il primo è quello di rendere i media selettivi. Un agente selettivo impedirà ad alcune specie di crescere, pur non inibendo e spesso anche promuovendo altre a crescere (12). La seconda funzione degli ingredienti dei media può essere quella di lavorare come agenti differenziali. Tali agenti consentono l’identificazione di una particolare caratteristica biochimica di un organismo isolato. Accoppiando diversi mezzi selettivi e differenziali insieme a condizioni di crescita appropriate, scienziati e diagnostici sono in grado di identificare la presenza di specifiche specie batteriche da un particolare isolato.
Un esempio di mezzo selettivo e differenziale che aiuta nell’identificazione è nel caso dell’organismo clinicamente significativo Staphylococcus aureus. Questo organismo è tipicamente coltivato su mannitolo sale agar. Questo mezzo non solo seleziona solo gli organismi che possono vivere in un ambiente ad alto contenuto di sale, che includono alcuni gram positivi come lo Staphylococcus, ma inibisce anche tutti gli organismi sensibili al sale. Lo zucchero di mannitolo è il componente differenziale di questo mezzo. Di tutte le specie di Staphylococcus clinicamente significative, solo S. aureus è in grado di fermentare il mannitolo. Questa reazione di fermentazione produce acido come sottoprodotto che fa ingiallire l’indicatore rosso metile rosso nel mezzo. Altre specie di Staphylococcus (come lo Staphylococcus epidermidis)sebbene in grado di crescere, lasceranno i media di colore rosso.
Questo esercizio di laboratorio dimostra una corretta tecnica asettica, nonché una corretta inoculazione dei mezzi di crescita dal brodo. Introduce inoltre la crescita di organismi contaminanti comuni sui mezzi di arricchimento, l’uso di un sistema di coltura anaerobica a pacchetto di gas per batteri anaerobici e l’uso di diversi mezzi selettivi e differenziali per l’identificazione presuntiva di batteri gram positivi e gram-negativi.
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.
