Protocols- Soils WG: Microbe Biodegradation Assay (June 7, 2011)
Microbe Biodegradation Capability Assay: 1st and 2nd BDM Trials
Dilution plates obtained from the mesh bag study are counted to obtain colony forming unit (CFU) numbers and
isolates streaked onto media containing minimal nutrients with biodegradable mulch pieces as the sole source of carbon
(C). BDM degraders are identified and differentiated from autotrophs by comparing growth on minimal media
containing no carbon, glucose and BDMs. Microbes capable of growing on the BDM surface are considered potential
biodegraders of the mulch. Putative mulch degraders are streaked to single isolated colonies, and then re-inoculated
onto minimal media plus BDM to confirm growth. Isolates are cultured for maximum growth so that cells/spores can be
stored in permanent cultures at both -80oC and 4oC.
Materials Required: PER SAMPLE (i.e. TN/IN)
600
40
30
20
20
1
2
1
1
1
24
24
24
16
24
16
40
40
40
30
30
1
Flat tipped toothpicks (autoclaved)
Cotton swabs (autoclaved)
Long wooden applicator sticks (autoclaved)
UV sterilized mulch squares (5 x 5 cm) of each: Biobag, Biotelo, and Spunbond PLA in sterilized containers
Autoclaved mulch pieces of cellulose control (5 x 5 cm)
L Liquid N2
Pairs of forceps
Box of P200 pipette tips
P200 pipette
Box of parafilm with scissors
25 mL fungal minimal media (FMM) agar plates with chloramphenicol (30 ug/mL)
25 mL fungal minimal media agar plates with 5 x 5 cm piece of UV sterilized mulch chloramphenicol (30 ug/mL)
25 mL fungal growth minimal media (GMM) agar plates with chloramphenicol (30 ug/mL)
25 mL M9 0 C minimal media (M9OC) agar plates with cycloheximide (50 ug/mL)
25 mL M9 0 C minimal media agar plates with 5 x 5 cm piece of UV sterilized mulch and cycloheximide (50 ug/mL)
25 mL M9 plus 0.2% glucose agar plates with cycloheximide (50 ug/mL)
PDA plus chloramphenicol (30 ug/mL) plates
1/10 X TSY plus cycloheximide (50 ug/ml) plates
1.5 mL 30% (w/v) glycerol in sterilized cryovials
1.5 mL 1/10X TSY agar slant cryovials
Sterilized Eppendorf tubes
Dissecting microscope
*see media protocol for agar, top agar and BDM agar instructions
Protocol:
Count colonies of mulch/soil dilution plates growing on TSY or PDA after incubating for 5 days at 20oC . Enter
count between 30-300 CFUs (others are labeled “TMTC” for too many to count, or “TFTC” too few to count) in Excel
spreadsheet. Back calculate to estimate CFUs per gram of mulch for fungi and bacteria. The dilution plates are kept and
used to streak individual colonies for the first BDM test. The dilution plates containing well-separated colonies are used
to streak 54 different isolates of both bacteria and fungi onto minimal media without carbon, minimal media with BDM
as sole carbon source, and minimal media plus glucose agar plates.
1
Protocols- Soils WG: Microbe Biodegradation Assay (June 7, 2011)
ONE toothpick is used to
pick up isolate and
transfer to minimal
media plates.
Sterilized toothpicks
used to pick up isolates
from dilution plates
The isolate is streaked in
the following order:
MMOC, MMOC + BDM,
and MM + C
Dilution plates
containing 30-300 CFUs
MMOC
minimal
media
agar
plates
MMOC +
BDM
minimal
media &
BDM
MM + C
growth
minimal
media
1st BDM Test:
Isolates on the dilution plates exhibiting different morphology and color [image 1] are randomly chosen from
each plate. The isolate is picked up with a sterilized flat-end toothpick [image 2]. The toothpick is then streaked (10 mm)
onto minimal media, then minimal media plus BDM, and then to growth minimal media. The same toothpick must be
used for all three minimal media plates to ensure that the same cells/spores are being transferred.
Bacteria isolates are transferred onto M90C, M90C + BDM, and M9 + 0.2% glucose. Fungal isolates are
transferred onto FMM, FMM + BDM, and GMM. Bacteria isolates are streaked 15 to a plate (3 rows of 5) on M90C and
M9 + 0.2% glucose, and 9 per plate (3 rows of 3) on M90C + BDM. Fungal isolates grow faster and are streaked 9 to a
plate (3 rows or 3) on all three plate types.
Agar dots (10 uL of either M90C or FMM top agar) are placed onto the mulch pieces to provide a water/nutrient
(but not carbon) source for initial colonization [image 3] because BDMs are hydrophobic and do not permit seepthrough from the agar below. BDM and other agar plates are prepared in advance (see media protocol). The toothpick
containing the isolate is gently rubbed on the surface of the agar dot and then onto the mulch.
1
2
3
1
1
The plates are then inverted and incubated at 20oC in the dark for 5 days. It is important to check on fungal
plates daily, because some isolates grow rapidly and can contaminate the entire plate. If rapid growth is seen, then the
plates should be parafilmed and stored at 4oC to slow growth.
Minimal media agar plates are inspected for growth after the 5 day incubation period. If the bacterium grows
on minimal media without carbon then you do not want to further test the isolate, because the bacterium is an
2
Protocols- Soils WG: Microbe Biodegradation Assay (June 7, 2011)
autotroph. Autotrophs would be difficult to study because there is not a definitive method to test if the bacterium is
making its own food or if it is utilizing C from the BDMs. Bacteria that do not grow on minimal media without carbon but
do grow on BDM plates are the isolates of interest. Fungal isolates follow the same isolation pattern, further test fungal
isolates that do not grow on FMM but do grow on the BDMs. Fungal isolates that grow on FMM may be obtaining C
from the agar used to solidify the medium.
Isolates that do grow on BDMs are streaked to single isolated colonies by picking up cells/spores from the BDM
surface or agar dot with a sterilized flat-end toothpick and streaking onto TSY (bacteria) or PDA (fungi) plates. The
toothpick is swiped across one plate quadrant multiple times (zigzag fashion) then the toothpick is discarded and a new
toothpick is used to spread cells/spores from the first quadrant to a second quadrant. This process is repeated until
cells/spores have been spread to four quadrants [figure 1]. The toothpick should gently slide across the agar surface,
avoid digging into the agar. Increase zigzag spacing with swipes so that single colonies can be isolated with ease from
later quadrants.
Single colony plates are inverted, incubated at 20oC for 5 days in the dark, and monitored starting at day 3 to
ensure isolate is growing and that the isolate is a pure culture.
Figure 1: Creation of single colonies by spreading cells/spores
Toothpick 1:
spread cells/
spores from BDM
plate in first
quadrant
Toothpick 2:
slide tip across
quadrant 1 once
then repeat
swipe pattern in
2nd quadrant
Toothpick 3:
slide across
quadrant 2 once
then zigzag with
large spaces in
3rd quadrant
Toothpick 4:
slide across
quadrant 3 once
and repeat zigzag
swiping in
quadrant 4
This plate has been incubated at 20oC for 5 days. The represents a
single colony. In quadrant 4 the single colonies are spaced out and
easily picked up by toothpicks for further transfers.
3
Protocols- Soils WG: Microbe Biodegradation Assay (June 7, 2011)
2nd BDM Test:
Single colonies are isolated from the single colony streaked plates after incubating the inverted plates for 5 days
at 20oC in the dark. A single colony is picked up with a flat-end toothpick and streaked onto a BDM plate. Again the
toothpick is rolled on the agar dot that sits on top of the BDM piece then rubbed on the mulch.
This is the 2nd BDM test and this step is required to confirm that the isolate is truly a potential biodegrader of the
BDM mulch. Two lawn plates are also made with single colonies, for subsequent storage. The lawn plates are for
growing the isolate uniformly over nutrient media to maximize growth. Lawn plates are incubated along with the
inoculated 2nd BDM plates at 20oC for 5 days.
Bacterial lawn plates are made by streaking single colonies onto 1/10 X TSY with cycloheximide (50 ug/mL)
[figure 2]. Agar slants in cryovials are also inoculated with bacterial isolates and allowed to grow for short-term storage
at 4oC. Fungal lawn plates are made by streaking a single colony onto PDA with chloramphenicol (30 ug/mL)[figure 3].
In addition, an autoclaved filter paper disc is placed onto the agar of the fungal lawn plates, near the site of initial
inoculum. When the paper is covered with mycelium/spores, it is removed with sterilized forceps, placed into a
sterilized Eppendorf tube and air dried overnight (cap off), and then sealed and stored at 4oC.
After 5 days of incubation at 20oC, the 2nd BDM plates are then rated using a dissecting microscope to visualize
bacterial/fungal growth and characterize morphology. If the isolate grows on the BDM then the lawn plates are used to
make permanent glycerol stocks of the isolate to be stored at -80oC for future testing.
Figure 2: Bacterial Lawn Plate
Figure 3: Fungal Lawn Plate
The lawn plate only
requires one toothpick.
Pick up a single colony,
streak it across plate
from left to right.
Place autoclaved filter
paper disc onto agar
with sterilized forceps
Rotate the plate 90o
and streak again from
left to right.
The lawn plate only
requires one toothpick.
Pick up single colony
and streak toothpick
across plate from left
to right.
Rotate the plate again
and streak from left to
right. Two lawn plates
are required per
bacterium isolate.
Rotate the plate again
and streak from left to
right. Two lawn plates
are required per fungal
isolate.
4
Protocols- Soils WG: Microbe Biodegradation Assay (June 7, 2011)
Preparation of Isolates for Long-Term Storage:
FOR BACTERIA:
Glycerol stocks: Sterilized cotton swabs are wiped across the lawn plate, picking up cells/spores. After the whole
plate has been swabbed, the swab is swirled into the glycerol to deposit cells/spores. One cryovial per lawn plate (i.e.
two cryovials per isolate) is made, using a new swab for each plate. After the glycerol stock is made the cryovial is
immediately mixed by shaking or vortexing, and transferred to a container with liquid N2. The liquid N2 flash freezes the
sample because slow cooling creates more cellular damage. After all glycerol stocks are made the pre-labeled cryovials
are transferred from the liquid N2 to storage boxes and placed in a -80oC freezer. A pair of long forceps and mesh
strainer can be used to pick up cryovials floating in the liquid N2.
Agar slants: Agar slants are incubated at 20oC for 5 days and then stored at 4oC.
FOR FUNGI:
Glycerol stocks: Glycerol stocks are made from the lawn plates just like bacteria (instructions above).
Spores on filter discs: The filter paper discs are removed from the agar surface with sterilized forceps and placed
into a pre-labeled Eppendorf tube. The lid is left open and the filter paper is air dried in a closed biohazard safety
cabinet with no airflow (to minimize cross-contamination) overnight. After 8 hours the Eppendorf tube is closed and
Parafilm is used to seal the top. The tube is then placed in a storage box and stored at 4oC.
Agar plugs: An agar plug is removed from the lawn plate with a sterilized razor (prior to cotton swabbing). The
agar piece is approximately 0.5 x 0.5 mm. The agar containing the isolate is then transferred to a pre-labeled Eppendorf
tube, closed, sealed, then placed in a storage box and stored at 4oC.
STORAGE
2 Cryovials at -80oC
Cryovial agar slant at 4oC
Agar plug at 4oC (Eppendorf tube)
Filter paper disc at 4oC (Eppendorf tube)
FUNGI
X
BACTERIA
X
X
X
X
5