02-background.qmd

# Background {#chap-background}

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## Summary {-}

The oral biome is complex. You just won't believe how vastly,
hugely,
mind-bogglingly complex it is. I mean, you may think quantum physics is complicated,
but that's just peanuts to the oral biome<!--[@adamsHitchhikersGuide2002, p.66]-->.
At any given moment, there are millions, if not billions, of microorganisms calling
your mouth 'home'. These come from a selection of at least 700 species, with
a few hundred different species living within the mouth of a single person.
Don't worry, they're harmless. Well, mostly harmless.<!--[@adamsHitchhikersGuide2002]-->
Given time, the organisms will start making themselves more and more at home.
They will find a suitable surface to attach, and start building a complex
community with communication, resource-sharing, and shelter from external
attempts to remove them, such as antibiotics and physical removal. These
sheltered communities are known as biofilms. One particular type of biofilm
is dental plaque, which starts forming on our teeth shortly after cleaning.
Proteins from our saliva will form a protective film around our teeth,
preventing damage from abrasion and acids. Specialist bacteria from the genus
*Streptococcus* have the ability to attach to the protein film, and allow
other bacteria to, in turn, attach to them. In the absence of proper oral
hygiene, the biofilm will continue to grow. Within the biofilm, there
are a range of local environments with differing levels of resources,
including oxygen, acids, and other molecules. These local environments
determine which species of bacteria are able to survive and out-compete
other bacteria for nutrients. This, in turn, determines if the biofilm
can cause damage to your teeth and gums. If acid-producing organisms
become too abundant, the surface of your teeth will start to degrade
and give rise to cavities. If alkali-producing organisms take the upper
hand, the biofilm will harden and become tartar, also known as dental calculus.
The biofilm is an intricate inter-connected system which maintains a balance,
and when changes happen to one or more parts of the system, it can result in disease.
Or calculus. To better understand this system, we try to re-create it in
our labs, because sometimes observing the real world as it is, can
be too complicated. Here, we can study the system under highly controlled conditions
to different to try and understand why it acts the way it does in disease
and health.

:::
:::

The human mouth, or oral cavity, contains many different types of surfaces
on which bacteria can attach and grow. These surfaces are both hard (teeth)
and soft (mucosa, tongue, gingiva), and are exposed to
the external environment. For this reason, the conditions within the
oral cavity can vary considerably,
resulting in a unique range of habitats for a wide variety of microbes.
In fact, the oral biome contains bacteria from over 700 different
species, some of which still haven't been named, or even cultured.
There are so many bacteria in our mouth that it's actually hard to determine
how many there are at any given time, but most estimates are in the
billions. Some like stable temperatures and lots of oxygen. Others are better
at dealing with fluctuations in temperature and oxygen availability. Some
can fend for themselves and take what they need from the environment. Others
depend on the presence of other species to break down their food into smaller
pieces. Some like acidity. Others like alkalinity. So how can they all seemingly
thrive in the same place at the same time? The answer is biofilms.

As an archaeologist, you may be wondering why you need to know all this stuff.
Dental calculus is the result of a very complex series of events that involves
the physiology of saliva, particular diets, age, genetics, and a bunch of other
things. To better understand what we see when we analyse archaeological dental
calculus to get at diet, we need to understand all of the processes that went into
forming it in the first place. Only then can we begin to fully unlock its potential
in reconstructing past diets. In any case, we all have mouths, so on some level
I'm sure this knowledge will be relevant.

<!-- teeth? -->

## Oral biofilms {#background-biofilms}

<!-- what is it? -->
The concept of biofilms represents a recent paradigm shift in microbiology
[@costertonBacterialBiofilms1987; @costertonMicrobialBiofilms1995].
Previously, researchers believed that you could isolate the organism of
interest and learn about its growth, metabolism, etc. They assumed
bacteria would behave the same as a free-floating organism in a
lab test tube as it would in a real-world environment (such as the human mouth).
More recently researchers have discovered that the
behaviour of bacteria differs when they are part of a larger community,
compared to when they are grown in isolation.
Biofilms consist of large, intricate, multi-species
communities of bacteria enclosed in an extracellular matrix of their
own creation. The ability to produce this matrix gives the bacteria living
within it an adaptive advantage compared to free-floating (planktonic) organisms.
It equips them with resistance to both antimicrobials (such as antibiotic medication)
and immune responses from the host that would normally be detrimental to their
ability to survive [@marshDentalPlaque2005; @marshPhysiologicalApproaches1997].
Resistance to varying conditions is especially important in the oral cavity,
which is a site of frequent fluctuations in temperature, pH,
and oxygen availability<!--citation-->.
The viscoelastic nature of the biofilm provides some protection against mechanical
destruction and dislodgement caused by, for example, the tongue and
dental hygiene practices [@petersonViscoelasticityBiofilms2015].
<!--rephrase-->It also allows them to acquire nutrients from outside the biofilm,
as well as generate and distribute nutrients within the biofilm to the
various communities of bacteria residing inside [@flemmingBiofilmsEmergent2016].
Biofilms are quite persistent structures, and very few surfaces exist
that can completely prevent bacterial colonisation and biofilm formation
[@rennerPhysicochemicalRegulation2011].

### Dental plaque {#dental-plaque}

Dental calculus forms from a specific oral biofilm known as dental plaque.
After we clean our teeth, our saliva coats the surface of our teeth (enamel)
with a layer of proteins known as the dental pellicle (or acquired enamel pellicle).
The pellicle is a film that protects our teeth from both mechanical wear and
chemical decay, but in doing so, provides a viable surface for microorganisms
to attach and initiate biofilm growth<!-- in a process known as...--> [@yaoIdentificationProtein2003].
Biofilm formation goes through several, often arbitrarily defined, stages of growth.
They are arbitrary because they are defined by the researchers who study them,
but are also necessary as a foundation to explain the development of a biofilm.
Rather than thinking about the stages as occurring sequentially, you should think of
them as occurring concurrently across different areas of the tooth surface. Biofilm
formation is a very dynamic process, and is often over-simplified in visualisations
(not unlike @fig-biofilm-form).

![A simplified overview of biofilm formation stages. Created with BioRender.com.](./figures/biofilm_formation.png){#fig-biofilm-form fig-alt="Overview of biofilm formation progressing through multiple stages, including motility, adhesion, coaggregation, maturation, and mineralisation."}

<!-- pellicle and initial physicochemical attachment -->
The pellicle contains molecules (known as adhesins)
that enable specific bacteria to
attach to complementary receptors on the pellicle, in a process called adsorption,
not to be confused with absorption. The difference being that it simply attaches
to the surface of the tooth rather than being sucked into the tooth.
When the pellicle adheres to the tooth, it becomes a surface for bacterial
attachment [@yaoIdentificationProtein2003].
The first bacteria
to attach are known as early coloniser bacteria (or pioneer colonisers) and
include *Streptococcus* species (spp.), *Actinomyces* spp., and *Haemophilus* spp.
[@zijngeBiofilmArchitecture2010; @uzelMicrobialShifts2011].
The initial attachment occurs when the random movement of bacteria and the flow
of saliva brings them close enough to the pellicle to attach. Some bacteria have
a limited, often random, ability to move if they have long tail-like structures known as
flagella, but most are brought to the surface by saliva<!--cite-->.

As bacteria approach the pellicle-coated surface of a tooth, there are both
attractive and repulsive forces at work. Repulsion because both the bacteria and
pellicle proteins have a net negative charge [@songEffectsMaterial2015],
causing electrostatic repulsive force; and attraction from van der Waals forces.
Bacteria may be more or less likely to attach depending on the distance from the
bacteria to the surface.
If the bacteria come too close to the surface,
the initial attraction (primary maximum) will most likely be overcome by
repulsion (primary maximum). Bacteria are more likely to attach when they encounter
attractive forces at a further distance (secondary minimum), ultimately
leading to a game of 'will-they-won't-they' between the bacteria and pellicle.
This initial attachment is a weak physicochemical long-distance
(10--20 nm; it's a long distance for bacteria) attraction; therefore, attachment is
initially reversible, as bacteria can become detached by salivary flow or shearing
action by the tongue [@marshDentalPlaque2016].
This model of bacterial attachment, also known as the DLVO theory, can partially
explain the aspects involved in microbial adhesion. Further explanation includes
hydrodynamic forces, where hydrophobic components of the pellicle and cell surface
interact [@bosPhysicochemistryInitial1999; @vigeantReversibleIrreversible2002].
Overcoming the repulsive forces may be in part facilitated by motility in some
organisms. The aforementioned flagellum, for example, may  give the necessary
'push' to reach a region of net attractive forces [@jinSupragingivalCalculus2002].
Additionally, the ionic strength of saliva may play a role in reducing electrostatic
repulsion with increasing ionic strength [@rennerPhysicochemicalRegulation2011].

![General structure of a bacterial cell. Common features of gram-negative bacteria on the left, and common features of gram-positive bacteria on the right. Created with BioRender.com.](figures/bacterial-structure.png){#fig-bacterial-structure fig-alt="Figure depicting the general structure of a bacterial cell. The bacterial cell is split in half with common features of gram-negative bacteria on the left side and common features of gram-positive bacteria on the right side. Common features of both, for example the nucleoid, ribosomes, flagellum, span both sides of the bacterial cell."}

<!-- direct (irreversible) attachment to surface -->
Attachment becomes stronger and colonisation becomes more solidified at a
shorter distance, as
surface molecules on the bacteria interact with complementary receptors on the
pellicle, and the interactions between bacteria and pellicle become more direct.
Some bacteria have
components on their surface that allow them to attach directly to complementary
components on the dental pellicle (adhesin-receptor interactions). These
attachments are very specific because only certain bacteria have the right
molecules on their surface [@jinSupragingivalCalculus2002].
These receptors are often carbohydrates formed by
the host, meaning us. Early colonisers are also able to attach to proteins and
enzymes present in saliva, as well as onto the surface of other bacteria already
attached to the pellicle
[@nikitkovaStarchBiofilms2013; @jinSupragingivalCalculus2002].
When bacteria come within a shorter distance of the pellicle <!-- distance in nm -->
they may also attach directly to the surface with other hair-like
structures (fimbriae) that are present on the surface of some bacteria. These
hair-like structures attach to matching receptors that
are present in the pellicle [@nobbsStreptococcusAdherence2009].

While some bacteria specialise in attaching to surfaces, not all of them possess this
ability. However, once the specialists have attached, they facilitate the adhesion of
other bacteria (secondary colonisers) by allowing them to attach to their surface (coadhesion)
rather than directly to the pellicle.
For example, *Streptococcus gordonii*
can attach to the pellicle and facilitate coadhesion with *Actinomyces naeslundii*
[@palmerCoaggregationInteractions2003].
Not all attachments involve proteins.
They can also involve carbohydrates, enzymes, and various appendages on the
surface of the bacteria, although these appendages often consist of proteins
in their structure, for example the already mentioned pili and fimbriae
[@nobbsStreptococcusAdherence2009].
This can occur on a large scale,
causing the number and types of bacteria on the tooth surface to grow, due to the
ability of different species to attach to one another (coaggregation)
[@jinSupragingivalCalculus2002; @marshDentalPlaque2006]. Coaggregation and
coadhesion are important parts of the growing oral biofilm. Most taxa don't have
the necessary morphology to attach directly to a substrate, however most oral
taxa CAN coaggregate with other species through cell-cell interactions, usually
involving polysaccharides on the bacterial-cell surfaces
[@kolenbranderOralMultispecies2010; @palmerInterbacterialAdhesion2017].
<!--Fundamental changes in the genes of bacteria can occur upon attachment to the
dental pellicle, inducing a biofilm phenotype in many bacteria that favours
coaggregation between distinct species.<!-- cite. -->
<!--Horizontal gene transfer-->

<!--  extracellular matrix formation and biofilm maturation -->
As the biofilm formed by early colonisers grows through continued multiplication and
coadhesion/coaggregation, the diversity of the biofilm increases. The proportion of
early-colonising streptococci gradually decreases while there is an increase of
*Tannerella forsythia*, *Actinomyces* spp., and *Fusobacterium nucleatum*
[@zijngeBiofilmArchitecture2010].
*F. nucleatum* is a bacterium also known as the 'bridging species', as it's
believed to play an important part in linking together early and late coloniser
species---including *Prevotella* spp., *S. gordonii*, and *Porphyromonas gingivalis*---
which might not otherwise be able to coaggregate
[@kolenbranderAdhereToday1993; @kolenbranderOralMultispecies2010].
The increasing diversity of bacteria adhering to a surface results in communities
of bacteria with the ability to communicate with each other, distribute
nutrients, and alter the local environment for more favourable conditions. <!-- elaborate with examples, e.g. anaerobes, fastidious organisms? -->
This is made possible by the presence of an extracellular matrix, formed by the
production of polymers by certain bacterial species [@marshMicrobiologyDental2010].
Microenvironmental changes can allow species to survive in otherwise unfavourable
environments; for example, the survival of many obligate anaerobes in an environment
which is largely aerobic (oxygen continuously enters the oral cavity as we
breathe). Bacteria with the ability to
consume oxygen and produce carbon dioxide allow bacteria with a lower oxygen
tolerance to thrive [@marshDentalPlaque2005]. In fact, dental plaque
predominantly consists of obligate and facultative anaerobes and is especially
true for periodontitis-associated
biofilms, which tend to be dominated by more species with a lower oxygen tolerance
than their non-periodontitis counterparts [@curtisRoleMicrobiota2020].
A pH balance may be maintained by species that are able to consume acidic metabolic
products produced by other species, and convert them to weaker acids.
*Veillonella* spp. especially [@marshDentalPlaque2005].
Metabolic products of some bacteria are used by others
as nutrients. By-products of urea metabolism can be used by some organisms,
who further break down the by-products, which can be used by yet other organisms
[@flemmingBiofilmsEmergent2016].
Working as a community can increase survivability in the harsh and dynamic
environment of the oral cavity, with rapid changes in pH, oxygen, nutrient
availability, etc; though, extended fluctuations
in environmental conditions can alter the composition of biofilms
[@huangEffectArginine2017; @huangFactorsAssociated2012].

<!-- erosion, detachment, dispersion -->
Perhaps ironically, an important part of the maturation of a biofilm is the
removal of bacteria from the biofilm itself. Removal can occur through both
internal and external mechanisms. It's likely that there is a continuous
loss of microbes near/on the surface of the biofilm caused by shear forces
from saliva and mechanical removal by the tongue. There can be multiple
motivating factors involved in the active detachment by bacteria, including
increasingly adverse conditions within the biofilm, such as nutrient depletion
or an unfavourable local environment. If sufficiently adverse conditions persist,
certain bacteria may make the active decision to 'peace out'. Dispersion of
bacteria from a biofilm requires production of matrix-degrading enzymes, and,
as such, not all bacteria can actively disperse from a biofilm
[@petrovaEscapingBiofilm2016]. The detached bacteria then colonise other parts
of the biofilm, making the biofilm a highly dynamic structure undergoing
continuous remodelling [@flemmingBiofilmsEmergent2016].

So far, the picture of biofilm formation is one of peaceful coexsistence,
collaboration, and even neighbourly interspecies actions. A basis for
this cooperation is increased overall benefits to the communities
[@renduelesMechanismsCompetition2015].
However, competition between bacteria still exists within the biofilm.
The metabolic
by-products produced by some bacteria may be toxic for others, allowing the
producers to gain a competitive advantage. The aforementioned acid-production
by some bacteria can cause
unfavourable conditions for species that prefer more neutral pH environments,
particularly in the absence of the secondary feeders that would normally neutralise
these compounds. A more direct example of bacterial competition is the ability
of bacteria to produce substances that are toxic to other bacteria. These are
often proteins or peptides termed bacteriocins, and can either inhibit or even
kill other bacteria [@grahamEnterococcusFaecalis2017; @dawBacteriocinsNature1996].
*S. sanguinis* and *S. gordonii* can produce H~2~O~2~ that is toxic to *S. mutans*,
a member of their own genus. *S. mutans* can, in turn, produce mutacin, which
inhibits the growth of *S. sorbrinus*. There is no love lost among these close
relatives [@chenSpecificGenes1999].
In addition to H~2~O~2~, oral streptococci can produce lactate
by consuming carbohydrates, giving them a competitive advantage
over acid-sensitive species by altering the local environment. Some species are
resistant to specific metabolic by-products that others consider toxic, and
may even consider them a delicacy (so to speak). *Veillonella* spp. are
an example of organisms that thrive under these conditions, allowing both
streptococci and *Veillonella* spp. to accumulate in the biofilm and create
a favourable environment to select species [@edlundUncoveringComplex2018].
These are simplistic examples, and often competition involves more interactions
between multiple species taking on various roles of 'sensing', 'mediating',
and 'killing' [@renduelesMechanismsCompetition2015]. Competition between and within
species will ultimately shape the wider biofilm communities. 

<!-- time-span of initial formation and maturation. Climax communities by 24 hours. -->
<!-- segway to biofilm mineralisation, as opposed to demineralisation of enamel. -->

### Dental calculus {#dental-calculus}

<!-- biofilm mineralisation -->
The exact mechanism of dental calculus formation is not fully
understood, but involves processes of biomineralisation and crystal formation within dental plaque.
The main mineral components of calculus are crystals containing various combinations
of calcium and phosphate ions. Other salts are also present, but the bulk of the
crystals are made up of calcium phosphates.
Initial mineralisation of dental plaque is a chemical process in which equilibrium
of minerals in saliva and gingival crevicular fluid tips towards saturation with
regard to calcium and phosphate, causing an increase of precipitation relative to
dissolution. This means that when the concentration
of ions increases and tips the balance between dissolution
and precipitation, salts will accumulate within and on the surface of the biofilm.
An increase in concentration of minerals within the biofilm reaches a critical
threshold (supersaturation) and
nucleation is triggered within the plaque matrix, initiating crystal growth.
This may or may not involve spontaneous
(or homogenous) nucleation, as it's unclear whether mineral concentrations are
sufficient to cause spontaneous nucleation, or whether other biochemical
processes act as a catalyst [@omelonReviewPhosphate2013]. That it's a chemical
process can be shown by the ability to produce calculus deposits in germ-free
rats [@theiladeGermfreeCalculus1964; @glasBiophysicalStudies1962]. However, it's
unclear how the germ-free calculus compares to conventional calculus, and, to my
knowledge there have only been studies on rats.
Just because calculus growth can be induced in sterile conditions doesn't
mean bacteria are not an essential part of the process.
Bacteria are inevitably part of the scaffolding of dental calculus in humans,
since, as I mentioned in the beginning of this chapter, our mouths are full
of bacteria, and dental plaque is essentially built by bacteria.
Mineralisation does seem to start in the biofilm matrix between
microorganisms, but they are eventually also mineralised along with the biofilm matrix
[@friskoppUltrastructureNondecalcified1983].
There are pockets of living bacteria within dental calculus. These pockets and
the layer of plaque that covers the surface of dental calculus are likely what
cause the correlation between calculus presence and periodontal disease
[@tanBacterialViability2004].
While the process can be explained by chemistry, the conditions leading up to
and surrounding the process are both chemical and biological in nature,
and certainly involve bacteria.
<!-- @bertranMineralizationDNA2013-->

The main source of minerals in the
oral cavity is saliva, which enters the mouth through salivary glands. The three
main paired glands are the parotid, sublingual, and submandibular glands, located
by the cheeks, under the tongue, and under the lower jaw bone, respectively.
Saliva contains sodium (Na), potassium (K), calcium (Ca), chlorine (Cl),
bicarbonate (buffer), and inorganic phosphate (Pi)
[@dawesEffectsDiet1970; @doddsHealthBenefits2005], and the
locations of the glands contribute to the pattern of dental calculus deposits
within the mouth, which commonly grow on the buccal portion of maxillary (upper) molars
and the lingual portion of mandibular (lower) incisors
[@jinSupragingivalCalculus2002; @whiteDentalCalculus1997].
Salivary pH also affects saturation
of salts, which in turn is influenced by salivary flow rates. Increased flow rate
of saliva will increase salivary pH, which reduces dissolution and increases
precipitation of calcium and phosphate. This is an important mechanism that protects
our teeth against demineralisation of the enamel caused by caries. Protection is
provided by the exchange of calcium and phosphate from saliva to enamel
[@dahlenMicrobiologicalStudy2010].
Saliva further acts as a buffer for the oral cavity, reducing
the impact of short-term drops in pH caused by metabolic byproducts of acid-producing
bacteria [@doddsHealthBenefits2005; @jinSupragingivalCalculus2002]. Higher rates of salivary
flow are also likely to contribute to an increase in calcium and phosphate secretion
in addition to pH, all contributing to an environment favouring plaque mineralisation.
Metabolic byproducts produced by bacteria can also affect local pH, both pushing towards
alkaline conditions as well as acidic. A major cause of acidic pH is metabolism
of overabundant dietary sugars and starch, especially the metabolic activity of
*Streptococcus mutans*, known to be one of the main culprits behind dental caries
[@bowenOralBiofilms2018; @extercateAAA2010; @duarteInfluencesStarch2008].

Conversely, alkaline conditions can
be generated by metabolism of various products that can either be directly or
indirectly linked to diet. One such product is urea. Urea is present in saliva,
and its concentration depends on multiple factors. One of these factors is
a high-protein diet, which increases levels of urea in serum and saliva
[@lieverseDietAetiology1999].
Hydrolysis of urea produces ammonia and causes a rise in pH.
Bacteria possess the ability to produce ammonia from urea, <!--either directly or using enzymes ureases?-->
which is further used by ammonia-oxidising organisms and converted to nitrite
[@flemmingBiofilmsEmergent2016; @wongCalciumPhosphate2002; @sissonsPHResponse1994].
In a similar way, arginine can be broken down to ammonia and increase in pH.
Another pathway to alkalinity is through enzymatic activity. Saliva contains
proteases which specialise in breaking down proteins into smaller
components such as ammonia, and increased protease activity in saliva
may therefore cause an increase in calculus production [@jinSupragingivalCalculus2002].

There are also a number of inhibitors and promoters of mineralisation present in
the oral cavity, originating both from saliva and bacteria. Substances known
to promote plaque mineralisation through hydroxyapatite formation and deposition,
calcium-phospholipid-phosphate complexes (CPLX), are present in bacteria<!--rephrase-->.
*Corynebacterium matruchotii* (formerly *Bacterionema matruchotii*) accumulates
calcium within its cell structure, and has therefore received a lot of attention
in biomineralisation studies
[@takazoeCalciumHydroxyapatite1970; @enneverIntracellularCalcification1960, in @enneverMicrobiologicCalcification1967; @boyan-salyersRelationshipProteolipids1980].
Biomineralisation is not a feature unique to *Corynebacterium matruchotii*.
Even species associated with caries may induce calcification under the right conditions
and after cell death [@moorerCalcificationCariogenic1993; @sidawayMicrobiologicalStudy1978a].
Inhibitors of biomineralisation include salivary proline-rich polypeptides, small amino
acids important for the immune system; and statherin, a protein that controls the
precipitation of calcium phosphate in saliva [@jinSupragingivalCalculus2002].

It's likely that multiple biomineralisation events occur under various conditions,
resulting in a heterogeneous calculus composition with crystals of various stages
of growth [@friskoppComparativeScanning1980; @friskoppUltrastructureNondecalcified1983].
The differing susceptibility of bacteria to calcification is also a contributor
to the heterogeneous composition. Overall, plaque mineralisation is a complex
interaction between conditions in the local environment, availability
of minerals, the equilibrium between precipitation and dissolution, balance
between nucleation promoters and inhibitors. 

## Oral biofilm models {#background-biofilm-models}

<!-- what is it -->
Biofilm models are a way of studying the growth and development of
biofilms. By creating models that replicate the conditions and complexity
(to some extent) of biofilms in a lab, models allow researchers to conduct
various experiments to test the efficacy of treatments on the growth and
pathogenicity of biofilms. There are many choices to be made when growing a biofilm,
such as the composition of the initial oral microbial community, nutrient content
and availability, and the makeup of the atmosphere in which the model is situated.
As such, biofilm models can differ widely in their complexity and ability to
mimic conditions in a human mouth. A choice of model can be made based on the
end-goals of the research, or in some cases the choice is made for you based on
(a lack of) available equipment and financial constraints.
All models must have a defined biome containing a substratum and
nutrients. The substratum is a surface on which the biofilm is intended
to form and grow. For oral biofilm models the environment is the oral cavity
and the substrata are the teeth, tongue, mucosa, or whatever the model is the
biofilm supposed to be mimicking. The simplest models
generally involve multiwell plates (e.g., 6-, 24-, and 98-well plates) with
a substratum, usually glass cover-slips or hydroxyapatite discs, placed at
the bottom of the well. Similar models suspend the substrata from a lid
to promote active attachment of bacteria to the substrata [@extercateAAA2010].
When the substrata are attached to a lid instead of the multiwell plates, it
allows samples to be periodically transferred between solutions/media
if necessary, adding more flexibility to the experimental setup.

Next, an inoculate is chosen. This can be anything from
a single species of bacterium (pure culture), to multiple select species (defined
consortium), to all organisms occurring naturally within a system (microcosm)
[@mcbainBiofilmModels2009].
The purpose of the inoculate is to initiate biofilm formation by allowing the
bacteria to adsorb to the substrata, ideally in the presence of a conditioning
film, such as saliva. For pure cultures and defined consortia, the inoculate may
come from saliva or another oral site, such as dental plaque. The bacteria of
interest are then isolated using selective media, essentially providing ideal
growing conditions to certain types of bacteria, promoting their growth and
eliminating others [e.g. @bassonEstablishmentCommunity1996]. Alternatively,
the bacteria can be acquired directly from companies like the American Type
Culture Collection (ATCC). For microcosms, the inoculate is often the
saliva itself, or dental plaque, in its (mostly) raw form.
The inoculate is added to the wells to initiate biofilm formation on the
substrata as described [above](#dental-plaque).
As such, the content of the inoculate influences the complexity of the
biofilm microbiome as well as the interactions between
the communities within the biofilm [@roderStudyingBacterial2016].
It's not always possible to use donated saliva as a growth medium for the duration
of the experiment, especially if the experiment lasts more than a few days.
Media with salivary components can be created as a substitute for long lasting
experiments.
There are many different recipes for media floating around out there,
but most of them are generally a mixture containing mucin, proteins, minerals
commonly found in saliva, and a buffer to maintain pH
[@tianUsingDGGE2010; @extercateAAA2010; @sissonsMultistationPlaque1991; @prattenVitroStudies1998; @shellisSyntheticSaliva1978].

More complicated models make use of increasingly sophisticated equipment to
mimic the oral environment. Another level of model complexity can be added
by adjusting the rate at which nutrients are dispersed through the system,
and the overall nutrient supply. Nutrient distribution can be continuous,
semi-continuous, or batch cultures, with the latter providing a finite amount of
nutrients in a closed system. An example of a batch culture model is a biofilm
grown on an agar plate, which has a finite amount of resources
[@kearnsMasterRegulator2005]. Once the nutrients in the agar have been depleted,
that's it. At the other end of the spectrum is a system with a pump attached to
a reservoir that can continuously supply the biofilm with growth medium, similar
to salivary flow. In between the former options is the semi-continuous supply of
nutrients. This can, for example, be the multiwell plate model with a lid, where
the samples can be periodically transferred to new plates containing fresh growth
medium [@extercateAAA2010]. Other parameters that can be controlled to more closely
simulate conditions in the oral cavity are pH and gas phase, as can be done
with the multistation artificial mouth. This system gives researchers control
over a large number of parameters using multiple chambers with complete control
over the flow of treatment and/or nutrient conditions---environmental conditions
such as pH, temperature, and gas phase---and access to real-time measurements
[@sissonsArtificialPlaque1997].

The duration of an experiment depends on the scope of the study. If the purpose
is to learn more about initial biofilm formation and prevention, it may
only be necessary to grow the biofilms for a few hours to 48 hours
[@extercateAAA2010; @dibdinDiffusionSugars1981]. If, instead, the goal is to
learn more about biofilm maturation and calcification, the experiments can run
for days or even weeks
[@sissonsMultistationPlaque1991; @wongCalciumPhosphate2002; @filocheFluorescenceAssay2007].

Models developed for studying oral biofilms include, in increasing complexity,
the ACTA active attachment model [@extercateAAA2010], Calgary biofilm device
[@ceriCalgaryBiofilm1999], modified Robbins device [@honraetModifiedRobbins2006],
constant depth film-fermenter [@petersConstantDepth1988],
and the multistation artificial mouth [@sissonsMultistationPlaque1991]
representing the upper echelon of complexity.
Summaries of biofilm models, including benefits and limitations of the various types,
can be found in reviews by McBain -@mcbainBiofilmModels2009, Tan and colleagues
-@tanAllTogether2017, and Røder and colleagues -@roderStudyingBacterial2016.

It might be tempting to think that the goal should always be to mimic the oral
environment as closely as possible. However, there are benefits to more
simplistic models, as well as limitations to the more sophisticated models.
Benefits of pure cultures and defined consortia are reproducibility between
experiments and more control over physiological and factors and making it easier
to take various measurements. Microcosms have the benefit of more closely
mimicking the complexity of the organisms' natural environment [@mcbainBiofilmModels2009].
However, even microcosms can be limited in their ability to recreate the complexity and
diversity of the oral microbiome [@tianUsingDGGE2010].
Alternatives to *in vitro* models are *in situ* models which usually involve
growing plaque on a removable surface inside the mouth of a willing participant.
These models add a level of realism, as they are grown inside an actual oral
cavity, and can reflect biogeographical differences in biofilm composition caused
by differing conditions across the oral cavity. They also come with additional
difficulties and reduced control over experimental parameters
[@marshRoleMicrobiology1995; @zeroSituCaries1995].

Reiterating a point made in the [Introduction](#chap-intro), and
[Discussion](#chap-discussion), and probably somewhere in the articles as well,
the benefit
of using an oral biofilm model over naturally occurring dental calculus in the
mouth of a research participant, is the control that it provides to tweak every
aspect of the system, from the quantity and quality of nutrients available, to the
amount of enzymes and bacterial species present. Plus, the added ethical benefit
of not needing to ask someone to give up their oral hygiene regime for a few weeks.
The following chapters, [Chapter 3](#byoc-valid) and [Chapter 4](#byoc-starch),
provide a small glimpse of what a model looks like, and how it might be used to
inform archaeological research.

## References cited {.unnumbered}