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 Flat tipped toothpicks (autoclaved)

40 Cotton swabs (autoclaved)

30 Long wooden applicator sticks (autoclaved)

20 UV sterilized mulch squares (5 x 5 cm) of each: Biobag, Biotelo, and Spunbond PLA in sterilized containers

20 Autoclaved mulch pieces of cellulose control (5 x 5 cm)

1 L Liquid N2

2 Pairs of forceps

1 Box of P200 pipette tips

1 P200 pipette

1 Box of parafilm with scissors

24 25 mL fungal minimal media (FMM) agar plates with chloramphenicol (30 ug/mL)

24 25 mL fungal minimal media agar plates with 5 x 5 cm piece of UV sterilized mulch chloramphenicol (30 ug/mL)

24 25 mL fungal growth minimal media (GMM) agar plates with chloramphenicol (30 ug/mL)

16 25 mL M9 0 C minimal media (M9OC) agar plates with cycloheximide (50 ug/mL)

24 25 mL M9 0 C minimal media agar plates with 5 x 5 cm piece of UV sterilized mulch and cycloheximide (50 ug/mL)

16 25 mL M9 plus 0.2% glucose agar plates with cycloheximide (50 ug/mL)

40 PDA plus chloramphenicol (30 ug/mL) plates

40 1/10 X TSY plus cycloheximide (50 ug/ml) plates

40 1.5 mL 30% (w/v) glycerol in sterilized cryovials

30 1.5 mL 1/10X TSY agar slant cryovials

30 Sterilized Eppendorf tubes

1 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.

ONE toothpick is used to pick up isolate and transfer to minimal media plates.

The isolate is streaked in the following order: MMOC, MMOC + BDM, and MM + C

Dilution plates containing 30-300 CFUs

Sterilized toothpicks used to pick up isolates from dilution plates

MMOC + BDM minimal media & BDM

MM + C

growth minimal media

MMOC

minimal media agar plates

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 seep-through 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.

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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 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 2: slide tip across quadrant 1 once then repeat swipe pattern in 2nd quadrant

Toothpick 4: slide across quadrant 3 once and repeat zigzag swiping in quadrant 4

Toothpick 3: slide across quadrant 2 once then zigzag with large spaces in 3rd quadrant

Toothpick 1: spread cells/ spores from BDM plate in first quadrant

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.

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

Place autoclaved filter paper disc onto agar with sterilized forceps

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 fungal isolate.

The lawn plate only requires one toothpick. Pick up a single colony, streak it across plate from left to right.

Rotate the plate 90o and streak again from left to right.

Rotate the plate again and streak from left to right. Two lawn plates are required per bacterium isolate.

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

FUNGI

BACTERIA

2 Cryovials at -80oC

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Cryovial agar slant at 4oC

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Agar plug at 4oC (Eppendorf tube)

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Filter paper disc at 4oC (Eppendorf tube)

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