Procedure

1. Prepping E. coli
a. Using a serological pipet, take 0.2 ml of E. coli broth and transfer to two sterile vials of nutrient broth
b. Incubate for 24 hours before placing onto agar plate
2. Prepping Nematode Growth Medium agar (NGM)
a. Lay out sterile plates (do not leave plates open)
b. Loosen cap of NGM bottle (do not remove it)
c. Place bottle into microwave and heat for 3 minutes
d. Pour Agar onto plate
3. Prepping Nematode Growth Medium with Ciprofloxacin
a. Repeat steps 2a to 2c
b. Weigh out 0.00675g on scale
c. Repeat steps 2d
4. Seeding NGM plates with E. coli
a. Apply 0.5 ml of E. coli OP50 liquid culture onto NGM petri plate using a sterile serological pipette
b. Allow E. coli OP50 lawn to grow overnight at room temperature
5. Subculturing C. elegans on NGM Plate
a. With sterile scalpel cut out small block of agar and place onto a new agar with block faced down (such that worms contact the new plate)
b. Incubate at 37℃ for 24 hours and repeat every two weeks
6. Adding L. Rhamnosus GG
a. Break open capsule using exacto knife
b. Weigh out .025g on scale using a scooper with a scale and dilute with 500ml of deionized water
c. Spread out over petri dish after 2 days of growth using a glass stir rod in trials with Lactobacillus rhamnosus
7. Tracking lifespan of C. elegans
8. Measuring Size of C elegans
a. Using a ring stand and selfie stick, prop up phone and align camera with left eyepiece of microscope
b. Align microscope viewing to the middle of each quadrant
c. Rotate knob three full rotations (once in each direction)
i. Take picture of C. elegans using phone
d. Overlay transparent mm grid over picture in Google Photos
e. Count and measure each worm within the picture
9. Disposal

Materials
❏ 70% ethanol ❏ Microwave
❏ Non-Latex gloves ❏ Weigh boat
❏ Mask ❏ Analytical Balance
❏ Lighter ❏ Scoopula
❏ Bunsen Burner ❏ Spreader
❏ Serological pipette 1ml (8) ❏ Scalpel (8)
❏ Serological pipette pump 1ml (1) ❏ Lactobacillus Rhamnosus GG pills (0.025g)
❏ Escherichia coli K12 (I Vial) ❏ 1000 ml beaker
❏ Nutrient Broth (4 vials) ❏ 500 ml graduated cylinder
❏ Incubator ❏ Glass stir rod
❏ Petri Dishes (8) ❏ Autoclave
❏ Nematode Growth Medium (405ml or 3 bottles) ❏ Protractor
❏ Caenorhabditis Elegans (1 original plate) ❏ Inverted microscope
❏ Ciprofloxacin (0.00675g) ❏ Selfie stick
❏ Sharpie ❏ Ring stand
❏ Autoclave bags ❏ Phone
❏ Bleach ❏ Platinum Transfer pick

Discussion & Conclusion

1st Hypothesis: The slope of C. elegan amounts, limited size range, and higher
percentage of shorter lengths was a factor for C. elegans with Ciprofloxacin. This
reflects findings found in prior research studies conducted with humans wherein
the biodiversity of their GI tract was negatively altered by the presence of
Ciprofloxacin.
2nd Hypothesis: The results of my study affirm the first principle of my second
hypothesis. The amount of C. elegans was overall less than the amount quantified
for the control group yet comparable percentages of the size categories of the
control group and L. rhamnosus GG suggest an overall progressive growth curve
and similar pattern. However, the C. elegans with L. rhamnosus GG had not
experienced a prolonged lifespan which was the precedent set in the research
study conducted by Oh, Park, Jun Son, and Kim.
Third: Hypothesis: My data results refute my third hypothesis stating C. elegans
Conclusion
would experience standard development when given both Ciprofloxacin and L.
rhamnosus GG. The amount and progression of it’s amount values followed the
trend set by Ciprofloxacin. However, the hypothesis was addressing growth
development rather than rate. The percentage of the amount of C. elegans were
higher for longer length values (0.55-1.0mm). The percentages exceed the
standards set by the control group therefore suggesting higher development rates
but lower amounts. This contradicts prior research studies where patients had
altered biodiversity levels when given ciprofloxacin and probiotic treatment.

Impact
As shown above, antibiotics and probiotics may critically impact the state of our gut
microbiomes. Research pertaining to gut microbiomes is critical in understanding our own unique
needs that are directly connected to our health. With a wide accessibility and various types of
probiotics, information regarding probiotics and antibiotics can be applicable to anyone who will
or has contracted a disease, especially gastrointestinal diseases. Continuing research on the
internal state of the gut microbiome in response to external variables is imperative in preventing
further misconceptions or damage to our well-being.

Abstract/Endorsement/References