Current print version = text and graphics
Click here for a print version containing text only

( 1of 21) Basic Methods Used in Soil Biology

• FACTORS IN THE FORMULATION OF CULTURE MEDIA
• MEDIA FOR MICROBIAL GROWTH
• SUBCULTURING ORGANISMS
• CULTURAL CONDITIONS
• COUNTING ORGANISMS FROM SOIL OR WATER
• CHARACTERIZATION OF MICROORGANISMS
• SEROLOGY

Reference Sources

• Gerhardt et al. (1994) Methods for general and molecular microbiology, ASM Washington.
• Weaver et al. (1994) Methods of soil analysis: Microbiological and Biochemical properties. SSSA Book Series 5. Madison, Wi
• Somasegaran and Hoben (1994) Handbook for rhizobia. Springer Laboratory New York

( 2 of 21) Factors Involved in Selecting and Preparing Culture Media

• Nutrients needed and the mechanism by which they are obtainedbacteria vs. protozoa; photosynthetic vs. heterotrophic nutrition

• Purpose of culture

• Routine vs. experimental; selective medium

• Solid or liquid medium

• Need for pH stability using buffers

• Heat stability affecting sterilization: Heat or filter sterilization, irradiation

(3 of 21) Chemical and Macromolecular Composition of a Gram Negative Bacteria

• Chemical (% dry weight basis):

  • Carbon, 50; Oxygen, 20; Nitrogen, 14; Hydrogen, 8; Phosphorus, 3; Potassium, 2; Sulfur, 1; Calcium, Magnesium, Chlorine, each 0.5; Iron, 0.2; Trace elements 0.3

• Macromolecular (% dry weight basis):

  • Protein, 55; RNA, 20.5; Lipid, 9; LPS, 3.4; Murein, 2.5; Glycogen, 2.5; Metabolites and vitamins, 2.9

• One cell weighs 9.5 x 10^-13 g, but includes 1,050 proteins and 400 messenger RNAs

(4 of 21) Nutrient Composition of “Average” Bacterial and Plant Cells

From Neidhardt et al. (1990); Marschner (1995)

(5 of 21) Microorganisms Can Obtain the Energy They Need in Different Ways

• Microbes derive their energy in a number of different ways:

  • by photosynthesis

  • chemoautotrophically using S, Fe, ammonia, etc

  • heterotrophically using a range of compounds that include:

    • complex carbohydrates (starch, pectin)
    • monosaccharides( glucose, lactose, ribose)
    • sugar alcohols (glycerol)
    • dicarboxylic acids (succinic acid)
    • mino acids (glutamic acid)
    • aromatic compounds (protocatechuic acid)

• These differences are important both in defining culture medium and in strain identification

(6 of 21) Components of Normal Culture Media

• Energy source: glucose, starch, sulfur or light

• Nitrogen source: inorganic compounds such as ammonium sulfate or potassium nitrate, or organic compounds such as urea, peptone or casein

• Vitamins or growth factors: blood, yeast powder, individual vitamins (often heat sensitive) or soil extract

• Essential minerals: P, K, Mg, Ca, S, etc.

• Buffers for pH control: MES, HEPES, TRIS Addatives: Agar, Gelrite, selective agents, etc.

(7 of 21) Complex Culture Medium, e.g. BYMA

• Used for the routine culture of non-fastidious organisms, e.g. Rhizobium

• Medium composition is not well defined i.e. Nitrogen supply is from amino acids, amides, protein, etc., in yeast extract

• Will grow many organisms, often with copious gum production which can interfere with centrifugation, extraction of DNA, etc.

• Composition: Mannitol…..10 g; Yeast extract……0.5 g¹ CaCl₂.2H₂O….0.2 g; K₂HPO₄…..0.5 g MgSO₄.7H₂O….0.2 g; FeCl₃.6H₂O……0.01 g NaCl, 0.1 g; Agar 20 g Distilled water 1000 mL; pH 7.0

¹ Yeast extract levels greater than 1 g L^-1 can be toxic

(8 of 21) Synthetic Culture Media, e.g. KMA

• Medium in which all components are defined

• Most used in experiments where interaction between unknown media components might affect experimental results : for example in studies of pH tolerance

• Also used in growing out mating mixes that include donor strains which have specific vitamin and amino-acid requirements

• Composition: Glycerol, 5.0 g; K₂SO₄, 0.13 g; K₂HPO₄, 0.10; Na glutamate, 0.22 g; MgSO₄.7H₂O, 0.08 g; CaCl₂.2H₂0, 0.143 g; FeEDTA, 0.04 g; Vitamin solution, 1. 0 mL; Micronutrient soln, 1.0 mL

(9 of 21) Additives to Normal Culture Medium Packaging Culture Media

• Additives:

  • Agar, gelrite or other gelling agent

  • substances making the medium selective for particular organisms

    • antibiotics that are selective for particular mutants or marked strains
    • fungicides that can limit growth of contaminant organisms from soil, root, or water samples
    • higher salt or heavy metal concentrations that will make the medium selective

• Packaging:

  • Media may be dispensed either before or after autoclaving into:

    • Petri dishes
    • Tubes (deeps/slants)
    • Media flasks

(10 of 21) Methods of Sterilization

• Autoclaving: heating at 121^oC for 20 mins

  • Used to sterilize culture media, sterilize water or stock solutions or other heat stable liquids

• Pasteurizing: heating to 160-170^oF

  • Used to remove pathogens, but not necessarily to sterilize

  • When repeated on successive days can be used to sterilize somewhat heat sensitive material

• Dry-heat sterilization: heating in an oven at 172^oC for 90 mins

  • Used to sterilize glassware, pipets, containers, syringes; not used for anything containing media

• Filtration: passing solutions through membranes of less than 0.2 µ pore size

  • Used for heat labile solutions (vitamins, antibiotics, stock solutions

(11 of 21) Subculturing Organisms on Agar Plates

• Aim is to progressively dilute out the culture until you have isolated colonies

• Colonies in this region should be uniform in size, shape and color with no evidence of comtaminants

NOTE: The air around us, our hands, etc., all contain organisms. To subculture we need all materials to be sterile, and must work in a clean room or laminar flow hood

(12 0f 21) Oxygen Requirements of Microorganisms

• Obligate aerobe: O₂ essential for aerobic respiration. Micrococcus

• Facultative aerobe: Grows better with O₂, but can undertake both aerobic and anaerobic respiration, and fermentation. Rhizobium

• Microaerophilic: O₂ is essential for respiration but at levels below those in the air. Azospirillum

• Aerotolerant anaerobe: O₂ not required and doesn’t improve growth. Fermentative. Streptococcus

• Obligate anaerobe: O₂ harmful, grows via anaerobic respiration or fermentation. Clostridium

(13 of 21) Temperature in the Growth of Organisms

(14 of 21) Methods for Maintaining Organisms

• Petri dishes dry out rapidly (3-4 weeks) so can only be stored for any time if wrapped in parafilm and kept in refrigerator

• Slant cultures may survive up to one year in refrigerator, longer if culture covered with sterile mineral oil

• Non-active and/or very important cultures may be suspended in sucrose/peptone as a cryoprotectant, then dried under vacuum. In this form may last 25-30 years without subculture. Ideal for shipping between laboratories.

• Non-active or very important cultures may be suspended in 15% glycerol and nutrient salt solution and frozen at -70^oC

• Porcelain beads

• Spore producing fungi

(15 of 21) Counting the Number of Organisms in Soil

• Essentially impossible to do

• Sampling methods for larger organisms such as nematodes different from those needed for bacteria and fungi

• Total counts based on microscopy, optical density, serology include both living and dead organisms and so tend to overestimate “active” population

• Viable counts based on growing the organisms seldom recover more than 20% of the population, and those recovered vary with medium and growth conditions used

• Most probable number (MPN) counts

• Indirect methods: Soil fumigation; Functional diversity with FAME or CHO

• Some organisms, e.g. AM mycorrhizal fungi not yet grown in culture

(16 of 21) Counting Soil Organisms

• Total count:

  • Soil shaken in water or buffer, colloids floculated then aliquots filtered onto membranes and stained

  • For cultures where numbers important, Petroff Hausser chamber measures cells in small volume of liquid

  • Optical density at 600 nm proportional to number of cells present

• Viable count:

  • Soil shaken as for total count then a 4X or 10X dilution series prepared. Appropriate dilutions may then be pipeted onto culture media, incubated and colonies counted

• MPN count for specific organisms, ie denitrifiers/ Rhizobium. Dilution series as above then inoculated onto indicator medium

(17 of 21) Characterizing Soil Microorganisms

• Approach varies with type of organism

• Colony characteristics

• Spore production and fruiting structure, life cycles

• Cell characteristics

  • but bacteria relatively undifferentiated

• Cell chemical characteristics

  • Energy sources and/or amino acids utilized

  • End products of fermentation

  • Tolerance of antibiotics, heavy metals, salts

• Cell wall compositional differences

  • FAME

  • Serology

• Disease or infective ability DNA or RNA based differentiation

(18 of 21) Fatty Acid Methyl Ester (FAME) Analysis

• Cells grown in standard culture medium

• Membrane fatty acids extracted with saponifying agent

• Methylated and extracted

• Analyzed by gas chromatography, against a set of standard fatty acids

• Multivariate analysis of the fatty acids present, and their amount used to cluster organisms

• By comparison to standard strains can be used to identify isolates

• Community FAME profiles developed to show how soil populations change with treatment

• Permits study of change in membrane structure with stress: i.e. role of 19:0 cyclopropane in rigidity of the membrane; membrane structure in thermophiles

(19 of 21) Antigen-Antibody Interactions

	An antigen is any molecule recognized as foreign by the body, and inducing the production of antibodies Bacterial cells have many antigens, including the cell wall LPS, flagella proteins, and unique cell enzymes Antibodies are soluble proteins produced in the presence of an antigen, and which react with that antigen An antiserum is antibody-containing blood from which RBC, etc have been removed

(20 of 21) Molecular Techniques Used in Phylogenetic and Biodiversity Studies

• DNA melting point and hybridization determination

• Restriction fragment length polymorphism coupled to the use of specific probes

• Polymerase chain reaction (PCR) methods using random or specific primers 16S rRNA sequence analysis

(21 of 21) Exercises

• I want to determine the ~ number of organisms in soil that can utilize plant cell wall pectin as an energy source.How?

• I have an organism that I think will be useful as an inoculant for compost. What could I do to:

  a) monitor fluctuations in the numbers of this organism at different stages of the composting process?

  b) to evaluate its contribution to compostingNB. Remember that the compost will contain many different organisms

© 2001 Peter Graham and Deborah Allan
Web Site Design by Jenni Swenson