Attachment 5: MICROBIAL ANALYSIS
Microbial analyses are a voluntary part of the protocol but strongly recommended to provide data to support hydrocarbon degradation results. Several members of the RTDF group are considering Phospholipid Fatty Acid Analysis (PLFA) and 16S RNA analysis (DGGE) for a minimum of four samples from each sampling time to characterize microbial diversity over the course of the study. The proposed 4 samples will include 2 soil depths from 2 treatments within one replication of the trial. The treatments sampled will be the unvegetated plots and the standard mixture. A 50g sub-sample of each composite for the selected replication will be kept cool at 4C and sent within 24 hours to the laboratory performing the microbial analysis. Additional samples may be sent for archiving and later analysis. The microbial studies should be done on the same composites that are analyzed for hydrocarbons to allow comparison of hydrocarbon and microbial data.
The following section outlines procedures for plate counting and MPN analyses of hydrocarbon degraders.
Timing of Sampling for TPH Degraders, Identifying Number of Degraders Needed for Efficient Degradation
The Protocol calls for microbial sampling at time 0 (before fertilization) and time 3 (30 months). The time 0 sampling is intended to determine if sufficient degraders are present. The time 3 sampling is intended to see if degraders have increased in number due to rhizosphere effect (and, to a lesser degree, nutrient amendments) in phytoremediated plots. Further sampling between times 0 and 3 would (e.g., at the end of each growing season) would help determine if bacterial numbers have changed due to plant presence. Because TPH degraders tend to increase in numbers in the first few weeks after fertilization, samples should probably be taken 2 months after the last fertilization. All fertilization dates/application rates should be specified.
Suggested protocols for enumerating bacteria, actinomycetes, and fungi from RTDF field soils, and estimating degraders of specific C sources via most probable number (MPN) procedures.
Estimating bacteria, actinomycetes, and fungi.
Bacteria, actinomycetes, and fungi can be estimated by serially diluting the
soil samples, followed by spread-plating the dilutions on appropriate media,
incubation, and then counting the appropriate dilutions. Media preparation,
protocols, and comments are provided in Appendix A.
Limitations and precautions associated with plate count techniques--primarily the low percentage of soil microorganisms that are culturable and the strength of the relationship between plate counts and activity--are well recognized (Zuberer, 1994) . Never-the-less, this is a reliable and frequently used method, and the necessary equipment and materials are readily available.
Reference: Zuberer, D.A. 1994. Recovery and enumeration of viable bacteria. p. 119-144. In R.W. Weaver (ed.). Methods of soil analysis. Part 2. Microbiological and biochemical properties. SSSA Book Ser. 5. SSSA, Madison, WI.
Enumerating specific carbon degraders.
Enumerating specific carbon degraders can be done by the most probable number
(MPN) technique (Wrenn and Venosa.,1996). Media preparation, protocols, and
comments are provided in Appendix B.
MPN techniques, based on presence or absence of growth on a specific carbon source in a series of dilutions, are straight forward but labor intensive. Choice of replications, dilution ranges, and incubation vessel size can be varied to fit within resource and equipment limitations. Perhaps the most challenging issue is choice of an appropriate carbon source, and the need to make soluble carbon as uniform among carbon sources as possible. Carbon solubility can become a significant issue for more recalcitrant and insoluble carbon sources. Cyclohexanol (cycloalkane), sodium benzoate (aromatic), vegetable oil (alkane) and 30W non-detergent motor oil (complex mixture of hydrocarbons) can be evaluated to determine the number of microbes capable of degrading different fractions of petroleum hydrocarbons.
Reference: Wrenn, B.A., and A.D. Venosa. 1996. Selective enumeration of aromatic and aliphatic hydrocarbon degrading bacteria by a most-probable-number procedure. Can. J. Microbiol. 42:252-258.
Media for Plate Counts
Bacteria: 0.1x Tryptic Soy Agar (TSA) Medium | |||
3.0 g Tryptic soy broth (Difco #0370-17-3) 15.0 g granulated agar |
|||
(or TSA (Difco #0369-17-6) combined TSB and agar in one product) | |||
1.0 L distilled water | |||
0.1 g cycloheximide (Sigma # C7698) in 1.0 mL methanol (added to medium after autoclaving) | |||
Autoclave @ 121°C for 15 min. After cooling to approximately 45°C, add cycloheximide.1 | |||
Reference: | Zuberer, D.A. 1994. Recovery and enumeration of viable bacteria. p. 119-144. In R.W. Weaver (ed.). Methods of soil analysis. Part 2. Microbiological and biochemical properties. SSSA Book Ser. 5. SSSA, Madison, WI. | ||
Fungi: Martin's Medium | |||
10.0 g glucose (dextrose) | |||
5.0 g peptone | |||
0.50 g KH2PO4 | |||
0.50 g K2HPO4 | |||
0.5 g MgSO4×7H2O | |||
33 mg (3.3 mL) Rose Bengal* | |||
15.0 g granulated agar | |||
1.0 L distilled water | |||
30.0 mg streptomycin sulfate (Sigma #S6501) (added after autoclaving) | |||
Autoclave @ 121°C for 15 min. After cooling to approximately 45°C, add streptomycin sulfate1. | |||
*Rose Bengal, Dissolve 1.0 g Rose Bengal in 100.0 mL deionized water. | |||
Reference: | Parkinson, D. 1994. Filamentous fungi. p. 329-350. In R.W. Weaver (ed.). Methods of soil analysis. Part 2. Microbiological and biochemical properties. SSSA Book Ser. 5. SSSA, Madison, WI. |
Actinomycetes: Starch Casein Medium | |||
10.0 g soluble starch (Fisher #S-516) | |||
0.30 g Casein Hydrolysate (Sigma #C-9386) | |||
2.0 g KNO3 | |||
2.0 g NaCl |
|||
2.0 g K2HPO4 |
|||
0.05 g MgSO4_7H2O | |||
0.02 g CaCO3 | |||
0.01 g FeSO4_7H2O | |||
15.0 g granulated agar | |||
1.0 L distilled water | |||
0.1 g cycloheximide in 1.0 mL methanol (added after
autoclaving). Boil agar, allow to cool slightly and adjust to pH 7 with HCl or NaOH. Autoclave @ 121°C for 15 min. After cooling to approximately 45°C, add cycloheximide.1 |
|||
Reference: | Wellington, E.M.H., and I.K. Toth, 1994. Actinomycetes. p. 269-290. In R.W. Weaver (ed.). Methods of soil analysis. Part 2. Microbiological and biochemical properties. SSSA Book Ser. 5. SSSA, Madison, WI. |
Dilution bottles for serial dilution: MPP Buffer | |||
0.65 g K2HPO4 | |||
0.35 g KH2PO4 | |||
0.10 g MgSO4_7H2O | |||
1.0 mL Tween 80 (Baker #7-X257)2 | |||
1.0 L distilled water | |||
2 Add 2 drops (0.1 mL) Tween 80 to -1 dilution bottles only. | |||
Reference: | Margesin, R., and F. Schinner. 1997. Laboratory bioremediation experiments with soil from a diesel-oil contaminated site-significant role of cold-adapted microorganisms and fertilizers. J. Chem. Tech. Biotechnol. 70:92-98. |
Agar Preparation
Prepare the 0.1 X TSA broth using the recipe on the media page. Add 500 mL of media to a 1000 mL Erlenmeyer flask. Autoclave @ 121_C for 15 min. After cooling to approx. 45_C, add 0.5 mL of 100 mg (0.1 g) cycloheximide in 1 mL MeOH solution to each 500 mL of media. Pour media into petri dishes.
Prepare Starch Casein medium using the recipe on the media page. Add 500 mL of media to a 1000 mL Erlenmeyer flask. After agar has boiled, allow to cool slightly and adjust to pH 7 with HCl or NaOH. Autoclave @ 121_C for 15 min. After cooling to approx. 45_C, add 0.5 mL of 100 mg (0.1 g) cycloheximide in 1 mL MeOH solution to each 500 mL of media. Pour media into petri dishes.
Procedure for plating soil
Materials: | |||
dilution bottles (95 mL, 90 mL, and 45 mL volumes as needed)
top loading balance weighing boats shaker table 10 mL disposable glass pipets pipet bulb alcohol lamp (or gas burner) 100 µl Eppendorf pipet pipet tips glass spreading bars (or disposable hockey sticks (Midwest Scientific #LLS-50)) glass bowl (2) inoculation turntable plates w/media matches |
|||
Preparation: | |||
Dilution Bottles: | |||
To allow for volume loss during autoclaving, initial dilution volumes should be measured to 97, 92, and 47 mL to achieve final volumes of 95, 90, and 45 mL of buffer, respectively. For -1 (95 mL) dilution bottles, add 3 to 5 glass beads and two drops (0.1 mL) Tween 80. For -2 (90 mL) and -3 and higher (45mL) dilution bottles use MPP Buffer without Tween 80. Cap all dilution bottles loosely and autoclave @ 121_C for 15 min. | |||
Glass Spreading Bars: | |||
In autoclave bags, autoclave clean glass spreading bars. Flame with alcohol and store in sterile glass bowl. | |||
Procedure: | |||
Weigh out 10 g of field moist soil to be plated. Place soil in -1 dilution bottle. | |||
Shake dilution bottle on horizontal shaker table for 5 minutes. Remove from shaker. | |||
Open -2 bottle and sterilize bottle mouth and cap in flame from alcohol lamp or gas burner. | |||
Shake -1 dilution bottle 50 times by hand (full 90-degree arc). | |||
Pipet 10 mL from -1 bottle into -2 bottle. Cap -2 bottle. Dispose of 10 mL pipet in waste container. | |||
Shake -2 dilution bottle on horizontal shaker table for 5 minutes. Remove from shaker. | |||
Shake -2 dilution bottle 50 times by hand. If desired, plate -2 dilution on appropriate media plates. | |||
Place pipet tip on 100 µl Eppendorf pipet. | |||
Rinse pipet tip with solution from -2 bottle 3 times. Dispense 100 µl solution onto media plates. Dispose of pipet tip in waste container. | |||
Place plate, without lid, on inoculation turntable. | |||
Holding sterile spreading bar lightly on surface of media, spin inoculation turntable, making sure to spread suspension evenly. Place used glass spreader bar in unused glass bowl (or other suitable container). | |||
Place lids on plates and incubate plates upside down at 25_C. | |||
Open -3 dilution bottle and sterilize bottle mouth and lid in flame from alcohol lamp or gas burner. | |||
Shake -2 dilution bottle 50 times by hand. | |||
Pipet 5 mL from -2 bottle into -3 bottle (45 mL dilution bottle). Cap -2 bottle. Dispose of 5 mL pipet in waste container. | |||
Shake -3 dilution bottle 50 times by hand. | |||
Plate -3 dilution, or dilute to -4. | |||
Continue in this manner, plating where appropriate to media. | |||
Notes: Only -1 and -2 dilution bottles get shaken on shaker table. Only use sterile pipets or pipet tips. Do not forget to flame cap and bottle each time it is opened. |
|||
Soil Moisture Determination | |||
1. Determine moisture content of soil by drying a known amount of soil @ 105°C to a constant weight. |
Clean-up Procedures:
Reading Plates
Fungi:
Martin's medium plates are to be read 3 and 10 days after inoculation.
Bacteria: 0.1 X TSA plates are to be read 2 and 7 days after
inoculation.
Actinomycetes: Starch Casein plates are to be read 14 days after
inoculation.
Values are calculated and reported as log10 CFU/g dry soil.
Media Preparation
MPP Buffer | ||
(same as described in Appendix A.) | ||
Bushnell-Haas Negative Control (5 tubes/soil sample) | ||
3.26 g Bushnell-Haas (BH) medium (Difco#0578-17-3) 1.0 L deionized H2O (each tube contains 4.5 mL of BH medium) |
||
Dextrose Positive Control (5 tubes/soil sample) | ||
3.26 g BH medium 10.0 g Dextrose (Fisher #D-16) 1.0 L deionized H2O (each tube contains 4.5 mL of Dextrose medium) |
||
Motor Oil (40 tubes/soil sample) | ||
3.26 g BH medium 1.0 L deionized H2O (one drop Coastal 30W non-detergent motor oil added to each of 40 test tube containing 4.5 mL BH medium/tube) Reference: Walker, J.D., and R.R. Colwell.1976. Enumeration of petroleum-degrading microorganisms. Appl. Environ. Microbiol. 31:198-207. |
||
Vegetable Oil (40 tubes/soil sample) |
||
3.26 g BH medium 1.0 L deionized H2O (one drop Wesson vegetable oil added to each of 40 test tube containing 4.5 mL BH medium/tube) |
||
Sodium Benzoate (40 tubes/soil sample) | ||
3.26 g BH medium 6.90 g Sodium Benzoate (Fisher#S-299) 1.0 L deionized H20 (each tube contains 4.5 mL of Sodium Benzoate medium) Reference Modified from: Mesarch, M.B., and L. Nies. 1997. Modification of heterotrophic plate counts for assessing the bioremediation potential of petroleum-contaminated soils. Environ. Tech. 18:639-646. |
||
Cyclohexanol (40 tubes/soil sample) | ||
3.26 g BH medium 1.0 L deionized H2O (one drop cyclohexanol (Aldrich#10,589-9) added to each of 40 test tube containing 4.5 mL BH medium/tube) |
Materials (for one soil sample)
170 | test tubes (6-10mL) | |
5 | test tube racks | |
170 | test tube caps | |
Aluminum foil | ||
1 | multi-channel pipetter | |
40 | sterile tips for multi-channel pipetter | |
8 | sterile multi-channel pipetter basins | |
1 | aluminum weigh dish | |
25 | mL BH negative control medium | |
25 mL | Dextrose positive control medium | |
200 mL | Motor Oil medium | |
200 mL | Vegetable Oil medium | |
200 mL | Sodium Benzoate medium | |
200 mL | Cyclohexanol medium | |
1 | 95 mL MPP dilution bottle (10-1 dilution) | |
1 | 90 mL MPP dilution bottle (10-2 dilution) | |
7 | 45 mL MPP dilution bottles (10-3 through
10-9 dilutions) (total of 9 MPP dilution bottles per sample) |
|
9 | stoppers for dilution bottles | |
1 | wide tip 10-mL sterile pipette | |
7 | 5-mL sterile pipettes | |
1 | pipette bulb |
MPN Media Preparation per Sample
Procedure:
Preparation of MPN Dilutions | |||
See Appendix A. | |||
Inoculation of Media | |||
|
|||
Reading Tubes | |||
|
|||
Medium BH - Negative Dextrose Cyclohexanol Benzoate Motor Oil Vegetable Oil |
Weeks of incubation 5 5 5 6 8 8 |
||
|
|||
Reference: Woomer, P.L. 1994. Most probable number counts. p. 59-79. In R.W. Weaver (ed.). Methods of soil analysis. Part 2. Microbiological and biochemical properties. SSSA Book Ser. 5. SSSA, Madison, WI. | |||
Clean-up Procedures:
See Appendix A. | ||
Note: The MPN determination method appears suitable for adaptation to microtiter plate methodology. References: | ||
Haines, J.R., B.A. Wrenn, E.L. Holder, K.L. Strohmeier, R.T. Herrington, and A.D. Venosa. 1996. Measurement of hydrocarbon-degrading microbial populations by a 96-well plate most probable number procedure. J. Indust. Microbiol. 16:36-41. | ||
Wrenn, B.A., and A.D. Venosa. 1996. Selective enumeration of aromatic and aliphatic hydrocarbon degrading bacteria by a most-probable-number procedure. Can. J. Microbiol. 42:252-258. |
1 To determine temperature of media after autoclaving, use stick on thermometers. DO NOT autoclave thermometer.