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.


Appendix A. Plate Counts

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

  1. Prepare the Martin's Medium using the recipe above. (You will need 2 Erlenmeyer flasks for autoclaving each liter of media.) Add 500 mL media to a 1000-mL Erlenmeyer flask. Autoclave @ 121°C for 15 min. After cooling to approximately 45_C, add 15 mg streptomycin sulfate to each 500 mL of media. Pour media (approx. 20 mL) into petri dishes. (500 mL of medium/sample.)

    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:

  1. All glassware and dilution bottles should be autoclaved at 121°C for 15 min prior to cleaning or disposal. After autoclaving, glass pipets can be disposed of in waste glass container. Used pipet tips and other materials may be disposed of in appropriate waste containers after autoclaving. Rinse dilution bottles in sink, making sure to catch any soil waste and glass beads in fine sieve. Rinse glass spreading bars, wash bars and dilution bottles in dishwasher. Autoclave to reuse spreading bars. Refill dilution bottles with MPP buffer and autoclave for later use.

Reading Plates

  1. Incubate plates in the dark at room temperature (_25_C).

    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.

Appendix B: MPN Tubes

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

  1. Prepare the MPP buffer using direction from Appendix A. You will need 9 dilution bottles and 9 stoppers per soil sample.

  2. BH negative control: Prepare medium using recipe on media page, add 4.7 mL of medium to each of 5 test tubes. To allow for volume loss during autoclaving, initial tube volumes should be measured to 4.7 mL to achieve final volumes of 4.5 mL of medium. The 10-2 dilution will be the only dilution inoculated for BH negative control medium. Cover test tubes with foil and autoclave @ 121_C for 15 min.

  3. Dextrose positive control: Prepare medium using recipe on media page, add 4.7 mL of medium to each of 5 test tubes. The 10-3 dilution will be the only dilution inoculated for Dextrose positive control medium. Cover test tubes with foil and autoclave @ 121_C for 15 min.

  4. Motor oil medium: Prepare medium using recipe on media page, add 4.7 mL of medium to each of 40 test tubes. After the medium has been added to the tubes, add one drop of 30W non-detergent motor oil to each test tube. Cover test tubes with foil and autoclave @ 121_C for 15 min.

  5. Vegetable oil medium: Prepare medium using recipe on media page, add 4.7 mL of medium to each of 40 test tubes. After the medium has been added to the tubes, add one drop of Wesson vegetable oil to each test tube. Cover test tubes with foil and autoclave @ 121_C for 15 min.

  6. Sodium Benzoate medium: Prepare medium using recipe on media page, add 4.7 mL of medium to each of 40 test tubes. Cover test tubes with foil and autoclave @ 121_C for 15 min.

  7. Cyclohexanol medium: Prepare media using recipe on media page, add 4.7 mL of medium to each of 40 test tubes. After medium has been added to test tubes, add one drop of cyclohexanol to each test tube. Cover test tubes with foil and autoclave @ 121_C for 15 min.

  8. Autoclave 170 test tube caps @ 121_C for 15 min.

Procedure:

  Preparation of MPN Dilutions
    See Appendix A.
     
  Inoculation of Media
 
  1. Motor oil, Vegetable oil, Sodium Benzoate, and Cyclohexanol media: 0.5 mL of each dilution (10-2 - 10-9) will be added respectively to each of 5 test tubes. Begin by shaking the dilution bottle to ensure even dispersion. Then pour an appropriate amount of the dilution into a sterile pipetter basin. Add 0.5 mL of the dilution to the media. (It is best to add, for example, the 10-2 dilution to all of the media and then add the 10-3 dilution and so on. It is quicker and uses fewer pipette tips). Cover tubes with sterile caps after adding inoculant.
  2. BH: 0.5 mL of the 10-2 dilution will be added to each of five test tubes. To save space, you can put the neg. control tubes in the same test tube rack with the dextrose samples. Cover tubes with sterile caps after adding inoculant.
  3. Dextrose: 0.5 mL of the 10-3 dilution will be added to each of five test tubes. Cover tubes with sterile caps after adding inoculant.
     
Reading Tubes
 
  1. Incubate in the dark at room temperature (_25_C).

  2. Read the MPN tubes on the following schedule:
    Medium
BH - Negative
Dextrose
Cyclohexanol
Benzoate
Motor Oil
Vegetable Oil
Weeks of incubation
            5
            5
            5
            6
            8
            8
 
  1. Microbial growth or a positive reading is indicated by turbidity in the tube(s). Vortexing the tubes is helpful to discern microbial growth. When positive tubes are determined, the MPN value can be determined using appropriate MPN tables (Woomer, 1994).
  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.

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