Hypothesis
I hypothesise that the olfactory senses will dictate the attraction more than the species of bacteria used, as
C. elegans does not have visual senses and therefore mainly detects through chemoattraction.
Background
In recent decades, Caenorhabditis elegans has provided humans with a large variety of knowledge about the brain.
What makes C. elegans so special is that the nematode is composed of 959 somatic cells, 302 of which make up its
nervous system. This simplicity of sorts has allowed it to be the first animal to have a fully mapped out system
(Gjorgjieva et al., 2014). Using this model organism, scientists have been able to test the neural processes of
everything from responsive behaviours to communication and basic locomotive movement, with the goal of applying
this knowledge to humans in the future.
These nematodes are commonly used for behaviour analysis due to their short lifespan and limited required resources.
In order to measure these changes in behaviour, a large variety of mechanisms and measurement systems have been
utilised. Many of these pertain to sensory and locomotive behaviours, as they are the easiest to identify differences
(J. Srinivasan, professional communication, October 3rd, 2022). A significant area of research involving both of
these systems is the taxis ability of C. elegans: A way of dictating the nematode’s gait based on external gradients.
Chemotaxis, aerotaxis, and thermotaxis serve as ways with similar mechanisms in which the nematode’s seemingly
stochastic gait may be influenced and recorded for abnormalities (Thiele et al., 2009). As a result, these methods
of forced movement are typically used in laboratories to measure other factors.
For instance, locomotive behaviours were found to change under the presence of food depending on the nutritional
status of the worm, denoted as Basal Slowing Response (BSR) and Enhanced Slowing Response (ESR) (Rivard et al., 2010).
This study went beyond the normal laboratory usage of Escherichia coli strain OP50 in some cases. However, the study
conducted focuses on the presence of food as well as the feeding history of the worms. This study suggests that OP50
may not be the best food source for the worms, especially as OP50 is mainly used due to its accessibility in laboratory
environments and low lawn height (Samuel et al., 2016). Other studies’ results further this, presenting that the species
of bacteria used as food can impact variables such as lifespan and development periods (Stuhr & Curran, 2020), as well
as general attraction to the food (Shtonda & Avery, 2006).
As a result, the foods used are slowly shifting towards other bacterial groups such as CeMbio (Dirksen et al., 2020),
meaning that the research into other possible food species is increasing in importance. Most past research has
concerned itself with the nutrition of foods as well as basic attraction to them based on such. While nutrition
alone is significant in bacterial attraction, as C. elegans tend to prefer healthier foods (Shtonda & Avery, 2006),
there are also many other factors which can play a part in attraction. For instance, previous research has implicated
that the wild strain, N2, harbours an intrinsic attraction to foods from its natural habitat (Abada et al., 2009; Samuel
et al., 2016).
Procedure
The bacterial strains used in this experiment are the typical food source Escherichia coli strain OP50 as well as
Sphingobacterium multivorum strain BIGb0170. OP50 was used for the worms’ growth, however the OP50 used in assays
was started at the same time as the BIGb0170. They were both made of a single colony placed in LB broth. The
bacteria colonies were incubated at 37°C for 24 hours before being kept in 4°C until usage.
Binary Bacterial Choice Assay
In the choice assay, the bacteria and worms were grown as previously mentioned. Assays were done on 100mm petri dishes,
filled with a slight diversion from NGM medium due to its NaCl content. Two circles were placed at opposite edges of the
plate about 2 cm in diameter, each filled with 20μL of a bacterium. The plate was then dried for 30 minutes under a hood.
1µL of 1M Sodium Azide was then pipetted into each circle to paralyze the animals. The worms were washed 3 times in
Chemotaxis Buffer prior to being placed on the plate. The assay was conducted for 1 hour at 20°C in a dehumidified
room. Afterwards, the number of L4 and older worms were counted to calculate a Choice Index (CI) as in (1):
CI = (Worms on BIGb0170 - Worms on OP50)/(Total worms which moved)
Binary Bacteria and Chemotaxis Assay
In the mixture choice assay, the procedure is the same as the previous with only minor exceptions: 1M Glycerol was
pipetted onto the BIGb0170, and 1% Acetone was pipetted onto the OP50 prior to placing the worms onto the plate.
Statistical Testing and Convolutions
The prior assays were done 9 times each unless mentioned otherwise. After each assay, an unpaired t-test was
calculated using the resulting CIs. These were done as there were two “unrelated” groups made up of averages and
distinct values instead of probabilities. They would also serve to fortify the differences or unearth the true
similarities observed in the data. Normal model equations were used to give shape to the data, as in (2):
f(x) = 1/(σ√(2π)) e^(-1/2 ((x-μ)/σ)^2)
Wolfram Alpha was then utilised to assist in preforming continuous convolutions to the control data as well as the
individual chemical data obtained from Liao et al. in 2010. The resulting equation was then compressed to an area of
1 and shifted by twice the domain to account for the natural convolution manipulation.
