Laura's Methods - Cuddington-Lab/documents GitHub Wiki

Detailed experimental procedures are described below that I used in my study of how stress impacts predator-prey dynamics using the nematode C. remanei and E. coli bacteria.

Nematode growth medium (1.0%, 500 ml)

a. In a 1 litre flask with a magnetic stirring rod, mix together in order, the following chemicals

488 ml DI
1.00 g NaCl
1.55 g peptone
1.50 g KH2PO4
0.33 g K2HPO4
5.00 g agar

b. Cover lid with aluminum foil and autoclave on wet cycle for 20 minutes (takes approximately 1.5 hours) and then add the following chemicals in order after agar has cooled to approximately 50°C

500 ul ampicillin
500 ul 1M CaCl Stock
500 ul MgSO4 Stock
800 ul colesterol Stock
625 ul nystatic Stock

Stock solutions

1M CaCl Stock - 14.7 g CaCl in 100 ml sterile DI
MgSO4 Stock - 24.6 g MgSO4 in 100 ml sterile DI
Colesterol Stock - 5 mg/ml in 100% ethanol, stir overnight
Nystatic Stock - 10 mg/ml in 100% ethanol, keep refrigerated
Ampicillin stock (10 ml) - dissolve 1 g of ampicillin in 10 ml of sterile DI, keep refrigerated

Sterile E. coli broth (1000 ml)

980 ml DI
10.00 g tryptone
5.00 g yeast
5.00 g NaCl

after autoclaving cool and add 1000 ml of ampicillin stock solution

Wire platinum spatula

light Bunsen burner
hold a hollow glass rod in the flame of the Bunsen burner at a 45 degree angle until glass is malleable using heat resistant gloves
place 1 cm of a 3.5 cm platinum wire into hollow of glass
use pliers to pinch glass closed around the wire
let spatula cool
use a hammer to shape the tip of the wire as desired

Creating a one week old nematode stock plate

Place 50 pregnant adult females on a 6 cm Petri plate inoculated with 50 ul of 0.8 OD600 OP50 -- GFP using a wire platinum spatula and a dissecting microscope. Flame sterilize spatula with an ethanol burner between transfers.
Plates are used one week later to remove male nematodes from for the experiment

Pouring cuvettes

Place 500 ml of 1% autoclaved agar in steamer to melt for approximately half an hour
Sterilize cuvettes and caps in UV GeneLinker (in Charles Lab, B1-371) for two STR cycles (energy, time = 180 seconds)
Remove agar from steamer and let cool. The oven should be set to 50°C to keep the agar warm if needed and place a bottle of autoclaved DI water in the oven
Add stock solutions according to procedure for making Nematode Growth Medium
Autoclave plastic pouring tube inside tin foil and open only under flow hood to prevent contamination
Whip flow hood bench with 70% ethanol
Set plate pourer (Wheaten Omnispense PLUS) to 2.5 ml on 41 revolutions per minute (RPM)
Attach a sterile, non-barrier pipette tip to the end of the plate pourer tubing
Pour one cuvette by holding the end of pipette tip a couple millimeters above the agar to minimize formation of bubbles. Discard cuvettes that have bubbles or a pronounced meniscus
Repeat step i. for the desired number of cuvettes
Run sterile DI water through plastic pouring tube to prevent agar from clogging it
Let cuvettes evaporate for 20 minutes under the flowhood before putting the caps on

Adding E. coli to cuvettes

Turn on flowhood and sterilize surface
_ _:00 Remove cultures from fridge and cuvettes from incubator
_ _:05 Bring E. coli culture to OD600 = 0.8 +/- 0.01 in a sterilized, 1 cm culture tube
_ _:10 Add 10 ul of E. coli culture to each cuvette in the flowhood. Use the 10-100 ul pipette tip, pressing as far as it goes (second stop) and discard the first aliquot into the culture tube by pressing to the first stop only. Draw a second aliquot and press to the first stop (there should only be one drop which will be attached to the pipette tip) and shake the pipette lightly to move the drop of culture to the very tip of the pipette. Apply the drop to the cuvette surface in the center, taking care not to puncture the agar.
Let the cuvettes evaporate with the lids off in the flowhood
_ _:55 Place the caps back on the cuvettes, label with the cuvette number (in order that E. coli was added)
Place cuvettes upside down in 20°C incubator, group by block
Repeat steps b-g for all time blocks

Moving male nematodes to cuvettes

_ _:00 Remove block of nine cuvettes from incubator and photograph using fluorescence microscope
_ _:05 Inspect cuvettes for irregularities such as bubbles or punctures made when adding E. coli and discard such cuvettes (so that eight cuvettes remain per block). There is one extra cuvette marked in the random order than can be used to replace a discarded
_ _:10 Open and cover cuvettes with their caps.
For modified agar treatments, create a 1 mm puncture in the center of the agar. For nematode present treatments, move seven male nematodes onto the agar surface. Split number of nematodes moved so that the agar is touched three times. For nematode absent treatments mimic the disturbance caused by moving nematodes by touching the wire spatula to the surface of the agar three times. Do not unintentionally puncture the agar.
_ _:50 Seal the caps tightly back onto the cuvettes. Record how many minutes the cuvettes lids were removed for ("Time" on data sheet)
Return cuvettes upside down to incubator
Repeat for steps a. to f. for eight blocks

Applying stress

_ _:00 Turn on flowhood
Sterilize flowhood surface with ethanol
_ _:00 Remove cuvettes for the appropriate block from the incubator
Count number of nematodes on the sides of the cuvette (above agar surface) and record number
_ _:10 Take fluorescent photos of two clear sides of cuvette. Side "a" has a triangle at the top, side "b" does not. Take photos with the cuvette lid on the left (will be the opposite on the computer screen).
_ _:20 open lids of all 8 cuvettes under the flowhood and place in order
Sterilize the forceps with ethanol and lighter flame
Firmly apply a square filter paper to the appropriate high stress treatment cuvettes in order (place paper in sideways, then tilt up to cover surface of agar, use a gentle s motion to press down in two directions)
_ _:25 Put lids of the cuvettes back on
Turn off flowhood and light
_ _:50 Turn on flowhood. Open all cuvettes
Remove filter papers using sterilized forceps, place in ethanol for later disposal. Take care not to damage the agar.
_ _:55 Recap cuvettes, weigh (tare to empty cuvette with lid), count number of worms remaining in the cuvette, and place back in incubator upside down for 24 hours
Turn off flowhood and light
Count number of nematodes remaining in all nematode present treatments
Weigh cuvettes standardized to an empty cuvette with a cap and record
Repeat steps a -- p. until all blocks have been completed

Homogenizing the cuvettes

_ _:00 Remove cuvettes of the first time block from incubator and take fluorescent photos of each (two sides per cuvette)
Fill 24 sterile culture tubes with 4.95 ml of sterile saline and 8 with 4.50 ml of saline
Label culture tubes according to cuvette number and dilution. Dilutions for high stress treatments are labelled as 10-2, 10-4, 10-5, 10-6 and dilutions for low stress treatments are labelled as 10-2, 10-4, 10-6, 10-7 (these are not the calculated final dilutions used when estimating colony forming units per ml but are labelled in this manner for convenience during the homogenization procedure). Dilutions 10-2, 10-4, 10-6 are made in culture tubes with 4.95 ml of saline and 10-7 and 10-5 dilutions are made with 4.50 ml of saline
Flame sterilize metal stirring rod with ethanol and lighter flame
Aseptically add 7 sterile glass beads (3mm) to first cuvette followed by 1.0 ml of sterile saline solution using the 100-1000 ul pipette
Use paddle end of stirring rod to detach agar from the walls of the first cuvette. Run the stirring rod along each of the four edges of the cuvette and one diagonally through the middle of the agar. Make sure no agar is stuck to the rod when removing the it
Flame sterilize metal stirring rod
Vortex cuvette for 15 seconds upright than 15 seconds up-side-down on speed 8
Tap cuvette 10 times on bench to remove bubbles
Take two 50 ul aliquots of the homogenized cuvette, drawing from the bottom of the cuvette to prevent agar from blocking the pipette tip, and place in 10-2 dilution
Vortex 10-2 culture tube for 10 seconds on speed 8 then transfer 50 ul aliquot to 10-4 tube
Vortex 10-4 culture tube for 10 seconds on speed 8 then transfer 50 ul aliquot to 10-6 tube
For high stress treatments vortex culture tube 10-4 once and transfer 500 ul aliquot into 10-5 tube. For low stress treatments vortex culture tube 10-6 once and transfer 500 ul into 10-7 tube
Flame sterilize metal stirring rod with ethanol and lighter flame
Repeat steps e-n for each cuvette in the block
Label 16 Petri plates with the appropriate cuvette number and dilution (10-6 and 10-7 for high stress, 10-7 and 10-8 for low stress)
Plate bacteria using the appropriate culture tube by vortexing then pipetting 100 ul onto the surface of an agar petri. For example, to plate 10-6 take 100 ul from the 10-5 culture tube. Spread the bacteria using a sterilized glass hockey stick twice around the plate but do not spread to the edges of the plate. Start with the highest dilution (least amount of bacteria) and use the same pipette tip and the same sterilized hockey stick. Sterilize the hockey stick after plating both dilutions.
Repeat step q for all cuvettes in the block (total of 16 plates with 2 plates per cuvette)
Let plates evaporate upright then place in empty sleeve and into 30°C incubator
Repeat steps a-s until all temporal blocks are completed
Incubate plates for 23 hours at 30°C

Saline solution 0.9% (15 ml per cuvette)

dissolve 9 g of NaCl in 1000 ml of sterile DI

Dilution series

Bacteria, sterile saline
100 ul from cuvette into 4.95 ml = label as 10-2
50 ul from 10-2 into 4.95 ml = label as 10-4
500 ul from 10-4 into 4.50 ml = label as 10-5 *high stress - plate 0.1 ml*
50 ul from 10-4 into 4.95 ml = label as 10-6 *high stress, low stress - plate 0.1 ml*
500 ul from 10-6 into 4.50 ml = label as 10-7 *low stress - plate 0.1 ml*

Agar for colony plate count (1 L, 15 ml per plate)

10.00 g tryptone
5.00 g yeast
5.00 g NaCl
15.00 g agar
1000 ul of ampicillin stock solution after autoclaving and cooled to 50°C

Cleaning Nematode Stocks

Select a nematode culture that is overpopulated but has E. coli lawn remaining and does not have dauer larvae (i.e. population is not starved). The most appropriate plates for bleaching will have a high population of pregnant females
Pipette 0.5 ml of 5% bleach onto the plate
Gently slide the plate in a figure eight motion to distribute the bleach across the plate
Use a sterile spatula to detach nematodes from the agar surface if necessary
Tilt the plate to one side so that the bleach pools on one side
Pipette 0.05 ml of bleach onto a fresh Petri plate, inoculated overnight with 0.05 ml of OD600 E. coli culture
Wait for the bleach to absorb into the agar
Label petri plate with date bleached and store upside down at room temperature
Plate will be ready to harvest pregnant females from in 1-2 weeks, depending on the number of pregnant females in your old culture

E. coli culture

Remove old E. coli culture from the fridge, blank E. coli broth, and sterilized 16 mm by 100 mm culture tube (with metal cap)
Label the empty culture tube with the date and type of culture
Turn on Bunsen burner and flame sterilize loop by holding it at a 45 degree angle in the flame. Let the loop cool
Flame top of empty culture tube
Use pipette to add 10 ml of blank E. coli broth into empty culture tube
Vortex old E. coli culture
Transfer one loop of E. coli culture from the old broth to the new broth
Turn off Bunsen burner and put away with loop in the cupboard
Put the old E. coli culture and unused blank E. coli broth back in the fridge to be later autoclaved and then disposed of
Put the new E. coli culture on the shaker at 175 RPM overnight for 18 hours
Check the OD600 of the E. coli culture using a 10 mm culture tube filled with 2 ml of the broth. If the OD600 is less than 0.800, then shake the culture for an additional hour or until the OD reaches 0.800
Put the E. coli culture in the fridge for later use

Microscope camera settings

Take photo of side with arrow first (a) then side without arrow (b)
Take two photos of clear side of cuvette only
Lid of cuvette on the photographer's left
Brightness -0.50
Contrast 1.00
(Symbol that looks like a line plot) 1.00
Exposure 1.20 s, 122%
Colour offset -2.00
Saturation 0.2
Framestart 0/168
Framesize 1384/720
4.8 zoom
48.0 mm Field of View (FOV)
0.3x lense

Summary of experiment

Synchronize age of nematode stock plates
Fill cuvettes with 1% agar and store upside down in 20°C incubator
Add 10 µl of OP50-GFP and incubate for 24 hours at 20°C
Place 7 male nematodes on each cuvette
Incubate for 4 days at 20°C
Add filter paper and remove for high stress treatments
Incubate for 24 hours at 20°C
Homogenize cuvettes, perform dilution series, and plate bacteria
Incubate at 30°C for 23 hours
Photograph plates and count CFUs using ImageJ