Instructor Guide
AREM Environmental Sampling Protocol
From hard surface (i.e., side walk, park bench, subway platform, door knob, etc.)
Equipment required:
Supplies required:
o Microcentrifuge
o Gloves (preferably latex-free; small, medium, &
o Vortex (with adaptor for tubes to lay flat)
large)
o Refrigerator or ice bath at 4°C
o 15mL centrifuge tubes
o 1.5 mL eppidorf tubes
o Cotton Tip swabs
o Sterile swab solution (20mL; 0.15M NaCl, 0.1%
tween)
o MoBio PowerSoil Kit (#12888)
1) Select sites for sampling
2) Record surrounding environment (i.e., sources of human influence, temperature, etc.).
3) Swab from 4 spots within a 1 m2 at the chosen sites. Note, if swabbing from smaller sites, such as shoes or doors,
you should use just one or two swabs and try to cover as much of the surface as possible.
i. Wear gloves and take precautions to avoid contaminating the site or the swabs from your skin or hair, etc.
ii. Dip a sterile swab into sterile swab solution (0.15M NaCl, 0.1% tween) and then swab an area the size of your
palm, or a little larger (~8x8 cm)
iii. Place the swab in a sterile 15 ml tube. You may need to break off the end of the swab to fit it in the tube.
iv. Repeat this with three more swabs in three nearby spots next to where you collected your first sample. *Collect
ALL of your samples within 1 m2 area but do not re-swab any area. When swabbing is complete, all swabs for
the same area are in a single 15 ml tube.
4)
Back in the lab - release sampled material from swabs by adding 2 ml of swab solution to the 15 ml tube
containing the 4 swabs and vortexing for 1 minute.
5)
While removing each swab from the tube, squeeze out all excess liquid by rolling it along the side of the tube.
6)
Check the volume remaining in the 15 ml tube, it will probably be between 0.5 and 1.5 mls remaining in the
tube and it should look like dirty water with particulate debris that came along on the swab. Transfer the liquid in
the 15 ml centrifuge tube to a sterile microcentrifuge tubes. Take note of approximately how much volume is in the
microcentrifuge tube.
7)
Spin microcentrifuge tube for 3 minutes at 14,000 x g (gravity is the same as relative centrifugal force; RCF).
This step should pellet all the debris and bacteria (and bacteria bound to debris) into the bottom of your
microcentrifuge tube.
8)
Pour off the liquid while being very careful not to dislodge the pellet. When you are done you should have
~100 ul of liquid (about a tear drop) left in the tube with the pellet of debris and bacteria.
9)
Resuspend the pellet in the remaining ~100 ul of supernatant by briefly vortexing or flicking each of the two
tubes.
10)
Follow the MoBio PowerSoil kit (#12888) protocol for isolation of genomic DNA form your sample.
From soil
Equipment required:
o Microcentrifuge
o Vortex (with adaptor for tubes to lay flat)
o Refrigerator or ice bath at 4°C
1)
2)
3)
4)
5)
6)
Supplies required:
o Gloves (preferably latex-free; small, medium, &
large)
o 50mL centrifuge tubes (one for each sample)
o 15mL centrifuge tubes (one for each sample)
o Sterile spatulas (one for each sample or ethanol and a
flame to sterilize between uses)
o MoBio PowerSoil Kit (#12888)
Select sites for sampling
Record surrounding environment (i.e., sources of human influence and plant life). Photograph the surface of the
sample site as well as the aerial view directly above the site (easily done with the new phones that have two cameras
and the camera direction can be changed without moving the phone)
Wear gloves and take precautions to avoid contaminating the site or the swabs from your skin or hair, etc.
Collect soil sample:
i.
Remove loose surface covering of the soil.
ii. Use a 50 mL tube to screw into the site and take a core sample of the soil.
iii. With a 15 mL tube scrape about 5 g of soil from the bottom of the core within the 50mL tube. This will sample
the soil about 10 cm deep. Do not over sample to collect soil from shallower sections of the core.
Back in the lab – under sterile conditions weigh 0.25 g of the sample and add to a MoBio bead-beating tube.
Follow the MoBio PowerSoil kit protocol for isolation of genomic DNA form your sample.
From water
Equipment required:
o 1L sterile bottle
o Vacuum source
o Vacuum tubing
o 2L side-arm flask
o 1L side-arm flask
o Filter funnel assembly
o Bunsen burner/ alcohol lamp
o Forceps
o 90% Ethanol
o Microcentrifuge
o Vortex (with adaptor for tubes to lay flat)
o Refrigerator or ice bath at 4°C
1)
2)
3)
4)
5)
6)
7)
8)
Supplies required:
o Gloves (preferably latex-free; small, medium, &
large)
o Sterile 1L bottles (one for each sample)
o 1% Phosphate Buffer Saline (PBS) in a squirt bottle
o Membrane filters (0.47mm with 0.45um pore size
Mixed ester nitrocellulose)
o MoBio PowerWater Kit (#14900)
Select sites for sampling
Record surrounding environment (i.e., sources of human influence, temperature, salinity, pH, etc.).
Wear gloves and take precautions to avoid contaminating the site or the swabs from your skin or hair, etc.
Collect water sample:
i. Use sterile 1L bottle to reach and collect water ~0.5m from the surface.
ii. Put sample bottle on ice to transport
Back in the lab filter water sample:
i. Set up filter funnel and vacuum source:
o Attach sterile filter funnel to 2L side-arm flask (sterilize each funnel initially by autoclaving then during
sampling place used funnel & base into a 10% bleach bath for at least 30 min then neutralize the bleach
by adding Sodium Thiosulfate crystals, rinse with 1% PBS once attached to flask)
o Use vacuum tubing to connect 2L side-arm flask to a 1L side-arm flask and vacuum tubing to connect
1L side-arm flask to vacuum source
ii.
Place filter (0.47mm with 0.45um pore size Mixed ester nitrocellulose) onto the grate on the base of the funnel
assembly with the grid side facing up.
iii.
Wet the filter with 1% PBS
iv.
Pour the sample into the filter funnel (at increments of 100mL). Record the volume that was filtered (at most it
will be is 1L)
v.
Rinse sides of funnel with 1% PBS
vi.
Remove top funnel part and with sterile forceps remove filter and place in MoBio bead-beating tube.
vii.
Empty 2L side-arm flask
Beach filter funnel assembly
Set up for next sample and process remaining samples
Follow the MoBio PowerWater kit protocol for isolation of genomic DNA form your samples in the bead-beating
tubes.