To study microorganisms and their metabolism, it is almost always desirable to bring them into the laboratory and grow them under controlled conditions. This is largely because it is necessary to have pure cultures (that is growth of only one unique type of microorganism) to be confident about assigning a characteristic or behavior to that single species. Therefore, it is necessary to create conditions where an individual microbe can grow free from other living organisms. How do scientists create suitable nutrient conditions so that microorganisms will grow? Then, once you get a group of microbes growing from a certain environment, how do you separate them from one another and create pure cultures? These are the classic methods of microbiology that were developed in the 19th century by the early microbiologists. In this section we will examine these techniques and talk about a few practical aspects of making media and isolating microbes.
We described different types of media and some of their formulations in Chapter 5 on Bacterial Nutrition. These formulations provide all the necessary nutrients that the appropriate microorganism needs to multiply. We also talked about the critical aspect of medium sterilization in Chapter 5. It is critical to remove all other life forms from a prepared medium before inoculation with the microbe to be studied if one wants to obtain interpretable results.
While it may seem at first to be a daunting task to prepare and sterilize medium, it turns out to be quite easy. In most cases media making involves dissolving the ingredients in water, adjusting the medium to the correct pH and autoclaving. If you can prepare a cake from a box mix, you have all the skills you need to successfully make growth medium. Of course, a deeper understanding about the ingredients in a medium (their nutritional role, their heat stability, which ones will react with one another at high temperature, etc.) is useful, especially in formulating a new medium or trying to troubleshoot the failure of a microbe to grow. The nuances of media-making are beyond the scope of this book, but any good introductory microbiology laboratory course will have this information.
Once a sterile medium is prepared, it is also necessary to keep unwanted microorganisms in the environment from contaminating it during handling. Such methods were developed that prevent microbes from entering a medium and these methods are collectively called aseptic technique. These procedures consist of using various barriers to prevent unwanted microbes from entering medium and making quick transfers of desirable microbes. While keeping microbes out may seem like a job for high tech equipment, it again is quite simple. In most cases flasks and tubes are capped with a cotton or foam plug or covered with a loose fitting cap or even aluminum foil. Plates of solid medium have a lid that covers the agar. For the transfer of microbes, a wire often with a loop at the end is used. Just before transfer the wire is heated to kill any microorganisms present. The loop is cooled for a few seconds and then dipped in a growth of microbes. This is then passed through the medium to be inoculated and after transfer the loop is heated again. As much as possible, the area where work is to be done is kept free of microorganisms using disinfectant and controlled airflow. Figure 30.1 contains a movie that demonstrates the most common procedures used in aseptic technique.
Figure 30.1. Flame sterilization and tube-to-tube transfer. This movie describes the techniques necessary for transferring microbes safely from one medium to another. They are simple techniques, but have to be practiced compulsively to ensure no contamination of the sample or the environment.
The extraction of a microorganism from its environment and into pure culture would seem a daunting task and it was difficult for many years after people discovered and tried to culture microbes. However, easy techniques were eventually developed that greatly simplified the isolation of pure cultures.
One important development was the formulation of solid medium and the most common solidifying agent used in microbiology is agar. Agar is a polysaccharide extracted from seaweed that has some very useful properties. When suspended in water and heated to 100 °C it melts and forms a slightly viscous solution. Agar remains molten until the temperature drops below 45°C at which point it solidifies, forming a Jello-like material. Once solidified, it will not melt until again heated to 100°C. This allows the cultivation of microorganisms at a wide range of temperatures. In addition, its lower solidification point permits the addition of heat labile compounds to media before its final distribution. By mixing agar with a liquid nutrient medium, a Jello-like material forms that can be poured into tubes or shallow dishes (called Petri plates) to create surfaces for microbes to grow on. If one places a single bacterium of most species on this solid surface, it will stay in one place and be unable to move about. Assuming the medium supports growth the microbe makes a number of identical clones of itself and these pile up on top of one another. After long enough incubation these become visible to the naked eye and form a colony. These colonies are populations of a single type of microorganism and thus achieve the stated goal, a pure culture. The question then becomes, how do you deposit a single bacterium onto the solid surface and the answer is amazingly easy. One simply dips a thin instrument, such as a metal inoculating loop or a sterile stick, into a source of microbes and then rubs it across the agar. The mechanical force of rubbing the loop or stick causes microorganisms to fall off and be deposited on the agar. By streaking in several phases, it is possible to get areas of the agar where individual bacteria have fallen off the loop and resulted in formation of isolated colonies. Now it is always possible that two different cell types happened to land on the same spot, so that the colony you see has a mixture of the progeny of both. So to be confident that you really have a pure isolate, one often restreaks colonies a second time before choosing one colony to work with. Figure 30.2 demonstrates the process of streak plates and media preparation.
Figure 30.2. Streak plates and media making. Streak plates are an easy way of mechanically separating microbes in a growth. To achieve this a sterile loop is rubbed across the surface of an agar plate.
It is also possible to obtain individual colonies by using dilution plating. A culture is repeatedly diluted in an appropriate buffer solution until a low enough concentration of cells is reached. Spreading or pouring a portion of this dilution onto a plate separates the bacteria into individual connected groups with each group of bacteria typically containing just one or hopefully no more than a few cells of a single organism. See below under viable plate counts for more on this.
Either for isolation or for subsequent analysis, it is often important to culture microbes under a gas phase that is different from that of the normal atmosphere. Though this does not refer exclusively to the removal of O2, that is such a common issue in microbiology that we will deal with that first and at greatest depth.
There are two general classes of anaerobes: obligate (or stringent) and aeroltolerant (or nonstringent). The latter can tolerate low levels of O2 for brief periods, but the former cannot. Obviously methods for handling the former group will also work for the less sensitive aerotolerant class. The standard method for handling obligate anaerobes employs an anoxic chamber, which is a flexible plastic box with gloves built into the walls so that samples can be manipulated from the outside. The internal atmosphere is maintained through the use of special gas mixes and always involves 5-10% H2. Except for H2, gasses are scrubbed of trace levels of O2 with a palladium catalyst. Samples are added and removed from the chamber through an airlock, which is a small chamber that separately opens to both the outside and the main chamber. In this way, media, tubes and bottles can be added to the airlock, the O2 removed, and then admitted to the chamber. After manipulation, the sealed bottles and tubes can be removed from the chamber through the airlock. Alternatively, samples can be incubated within the chamber itself.
Aerotolerant organisms can be handled in this way as well, but can often be handled on the bench top but with procedures that minimize the amount of O2 exposure. This might include minimizing the amount of culture surface exposed to the air, blowing inert gases over open vessels or including reducing agents in the media to remove dissolved O2.
Facultative microbes are also examined under anoxic conditions, but here the exposure of the organism to O2 is not critical. Such anaerobic environments can be created through the use of sealable jars that can hold a dozen Petri plates. These can be made anaerobic through the use of commercially available gas generators that produce H2 and CO2 (and remove O2) after water is added. Alternatively jars, bottle or tubes can be made anoxic by repeated vacuum evacuation and flushing with an inert gas. This also allows the introduction of a specific gas environment.