Serial passage experiments are a form of experimental evolution that is frequently used in applied sciences; for example, in vaccine development. During these experiments, molecular and phenotypic evolution can be monitored in real time, providing insights into the causes and consequences of parasite evolution. Within-host competition generally drives an increase in a parasite's virulence in a new host, whereas the parasite becomes avirulent to its former host, indicating a trade-off between parasite fitnesses on different hosts… In [serial passage experiments (SPEs)], parasites (broadly defined to include pathogens, protozoa, fungi, helminths, and small herbivores) are transferred from one host to another. During SPEs, parasites are propagated under defined conditions and their evolved characters are compared with those of the ancestral parasite. Parasite transfer is either artificial (for example, by injection) or through natural transmission in dense host cultures, relaxing the constraints on real-world infectious processes. — Ebert (1998)

Serial passage is the transfer especially of large numbers of organisms from environment to environment, such as from a broth-containing flask to a second broth-containing flask. Key toward understanding this process is that the transferred organisms are both diluted and reduced in number during their passage. Also crucial is that the transfers do not involve so few organisms that genetic bottlenecking occurs, unless the procedure is designed specifically to effect genetic bottlenecking. The result is a combination of transfer of a good representation of the genetic diversity within a population and a subsequent potential for the population to grow back to previous densities (i.e., those seen prior to transfer). The consequence of this process is selection for variants that contribute to faster population growth under experimental conditions. More complex results may be seen if organisms are allowed to reach carrying capacity (i.e., stationary phase) before transfer is undertaken. Allowing organisms to reach their carrying capacity prior to transfer, however, can have the benefit of simplifying protocols since, for example, transferring can be accomplished less often, such as once per day.

It is possible to continuously monitor evolution during serial passage, either phenotypically or genotypically by periodically placing cultures from which transfers have been removed into storage. In particular, with most microorganisms it is possible to store intermediate steps by freezing populations as well as by freezing individual isolates. This approach has been described as creating a "fossil record", i.e., revivability, of the experimental evolution experiment. Not only can individual or collective genotypes or phenotypes be determined for individual steps in protocols but so too can individuals from different time steps be co-cultured to compare competitive abilities. That is, the properties of any organisms at any point in the experiment may be compared, phenotypically or genotypically.

Impact on Environments

One can view the consequences of serial passage in terms of the impact of organisms on environments. This is as opposed to solely in terms of evolutionary impact of environments on the passaged organisms. An example is the adaptation of microorganisms to the utilization of new substrates, e.g., such as lactose by a lactic acid bacterium. An ability to utilize a new substrate has the potential to increase growth rates as well as organism end point-densities. The result, obviously, is a greater impact on the new substrate, though with greater organism numbers overall within environments – should that occur – then there also would be an increased impact on all of the other resources used by these organisms as well as increased generation of waste (e.g., lactic acid). These various consequences can viewed as comparable to an increased impact by pathogens on hosts, that is, the equivalent of the evolution of an increase in virulence: A greater potential to grow results in more pathogen individuals, such as individual bacterial cells, with more damage to host organisms even if each pathogen individual exerted the same amount of damage.

An alternative situation in which serial passage can be harnessed to modify the impact of microorganisms on environments is in terms of an applied microbial evolution towards the generation of live-attenuated vaccines. In these experiments microorganisms come to display a reduced virulence against their original host organism, such as ourselves, that is, the host organism in which the pathogen was not passaged. Vaccine development of this type represents an intentional generation of antagonistic pleiotropy via serial passage, that is, an organism that comes to be less fit in one environment (e.g., us) due to adaptation to another environment . Note nonetheless that adaptation to the new environment has occurred, which for microorganisms such as pathogens, often can result in an increased potential for exploitation of that new environment. Often this increased exploitation, in other words, is associated with increased specialization by the organism for its new environment or, equivalently, increased specialization for association with a new type or strain of host organism.

This tendency for evolution of a greater negative impact on the environment types in which passage has occurred can be understood in terms of rates of passaged-organism growth, which as noted often is what is being directly selected for in serial passage experiments (that is, selection for greater r). In other words, by growing faster, resources used by an organism or a population of organisms are potentially used up faster than those resources can be regenerated. It is also possible for microorganisms to inflict more damage to environments, or hosts, per microorganism—e.g., such that more resource is exploited per organism produced. The decreased virulence seen with attenuation similarly can be viewed as a reduced potential for individual organisms to inflict damage on their environment or a decreased potential for these same microorganisms to produce large numbers of progeny within environments that are dissimilar to those within which passaging took place.

The distinction or even apparent contradiction of serial passage leading to divergent results, increased virulence in one context versus increased attenuation in another, is a consequence of evolution occurring within an ecological context. That is, increased fitness within one environment can coincide with decreased fitness in another, a phenomenon, as noted, that is known as antagonistic pleiotropy. If these changes in fitness coincide with changes in an organism's potential to exploit or even just impact its environment, then quantitatively or qualitatively different modifications of different environments as a consequence of microorganism growth will also occur. The consequences of serial passage, along these same lines, are also considered in the chapter titled, "Virulence". Importantly, and as discussed there, pathogen fitness does not always increase at the expense of host fitness during pathogen evolution since greater pathogen virulence is not always beneficial to the pathogen. Alternatively, it can be the method of transfer itself, during serial passage, that is responsible for selecting for increased pathogen virulence (see Transmission impact, below). See Ebert (1998) for further discussion of these ideas.

Transmission Impact

There are at least two phenomena involved in serial passage experiments: Population growth of the microorganisms and the movement that occurs during passage (i.e., the dilution/transmission step). There can be differences in both the quality and intensity of selection imposed at each of these steps. At an extreme, movement can impose no selection (e.g., transfer of a thoroughly homogenized, log-phase liquid culture to fresh media). In that case selection is entirely on growth rate (and particularly so if transfer takes place prior to a culture's stationary or death phases). Alternatively, it is possible for transfer to take place by more complicated means, as is often seen with natural forms of transmission of microorganisms between hosts. There also can be requirements for organisms to do something in addition to growing, such as to form spores in order to survive transfers. In these latter cases there can be conflicts between growth-rate rapidity and transmissibility. These conflicts can be viewed as resulting from pleiotropies, i.e., single genes that are responsible, in some manner, for determining both growth rate and transmission ability. Note that a straightforward means of detecting such conflicts is to perform serial transfer experiments in which selection is not imposed or at least is only minimally imposed during the transmission step. In these cases, if there are tradeoffs between population growth rates and transmission ability, then a consistent result should be evolution of increased replication ability at the expense of transmission ability under the non-contrived, i.e., "normal" transmission route.

These ideas can be generalized to consideration of growth and transmission as different life stages, and many organisms have multiple life stages. These multiple life stages could, for example, include a lag phase, a log phase, a stationary phase, and then a transmission stage. In such cases, the fitness component of different stages could trade off with one another, with improvements in one stage resulting in costs in another (Ebert, 1998). These observations can be summarized with consideration that organisms are complicated, that the simultaneous "perfection" of two or more adaptations can be impossible to achieve, and, finally, that at least some of these conflicts can be brought to light through serially staged schemes of experimental evolution.

In systems in which selection on transmission is completely relaxed there still will be selection operating on transmission. That is, in a serial passage experiment it is necessary for an individual organism to be transferred in order for that organism to survive and reproduce. The primary distinction is that without selection operating explicitly on the transmission step then the probability of transmission is simply a numbers game: Those individuals that produce the most transmittable progeny, all else held constant, will have the highest likelihood of being transmitted. In this case we again could describe a pleiotropy in which both growth rate and transmission likelihood are controlled by the same genes, except in this instance the same genes can be simultaneously optimized for both traits, with alleles giving rise to faster population growth also resulting in higher rates of transfer. In other words, it is possible for there to be no conflict, that is, no antagonism between the evolution of faster growth rates and the evolution of more effective transmission. This in a sense can only happen to the extent that the transmission step does not impinge on the growth step, which can be the case given transfer from mid log-phage cultures to fresh media or mid-infection-stage transmission of pathogens (or parasites) between hosts such as intravenously.

Yet another complication on these musings is that even with low stringency on which organisms are transferred during a given transmission step, there still can be selection operating. For example, if transmission is made solely as a broth sampling then bacteria that have happened to form biofilms may be excluded (that is, unless the latter are suspended into the broth phase prior to sampling). Even transmission that seemingly is selective only on organism growth rate, or organism frequency within an environment, thus can be more complicated in its effect than expected.

Avoiding Evolution During Transfer

Organism adaptation during laboratory serial passage has been taking place for as long as microorganisms have been propagated by microbiologists, and intentional adaptation to specific conditions has been going on for almost as long (e.g., generation of live-attenuated vaccines). By contrast, once it was understood that unintended evolution, such as leading to organism domestication, was both possible and potentially problematic, effort was extended to avoid such evolution during laboratory propagation. Unfortunately, avoiding evolution technically is not possible. This is because any time organism reproduction is involved there will be selection for faster reproduction. Similarly, any time survival is challenged, then there will be selection too for those organisms that better resist that challenge. Evolution can be reduced, however, and these strategies of evolution reduction generally form one basis of microbiology's pure culture technique.

We can break down processes required to avoid microorganism evolution into a series of rules or strategies. The first rule is that serial transfer must be avoided. Instead, stock cultures should be initiated with single organisms, or at least the product of single organisms (e.g., colonies). This minimizes the potential for selection to operate since a genotype displaying higher fitness would need to increase in frequency very rapidly in order to have a reasonable probability of being the single organism that is transferred (e.g., of millions or billions). Note though that this strategy is less effective at protecting populations from evolving if mutation rates are substantial, such as one sees with RNA viruses or general mutator strains.

A second consideration is duration of growth in the course of a single passage. That is, by supplying sufficient rounds of replication, or just ample time, selection may act sufficiently robustly that cultures will come to be dominated by more-fit variants. To avoid this, it has become standard practice in microbiology to minimize the passage of the stock cultures and also to store such strains under conditions in which, at least ideally, neither selection nor metabolism operate. Such conditions are met or at least approximated by freezing to low temperatures, such as are attained in -80°C freezers or under liquid nitrogen (the "approximation" comes into play should freezing, thawing, or extended storage itself impart selection on cultures).

The third strategy, essentially as just noted, is to avoid subjecting cultures to either intended or unintended selection or, for that matter, mutagenesis. In short, everything that one would employ in the course of an experimental evolution study to increase the potential for organisms to evolve, particularly in terms of natural selection, is what one should not do in order to avoid unintended evolution by stock organisms.