WO2018175681A1

WO2018175681A1 - Recovery of stressed bacteria

Info

Publication number: WO2018175681A1
Application number: PCT/US2018/023696
Authority: WO
Inventors: Yaowei Kang; Claire PELLIGRA; Jessica Mann
Original assignee: Novozymes Bioag A/S
Priority date: 2017-03-22
Filing date: 2018-03-22
Publication date: 2018-09-27

Abstract

Methods are described for recovering certain unculturable bacteria into a culturable form. In some examples, the unculturable bacteria have been exposed to at least one stress. In some examples, a sample containing bacteria is cultured using a medium containing humic acid, a salt thereof, an analog thereof, or peat, and the number of culturable bacteria from the sample are enumerated. This number of bacteria is greater than the number enumerated using the same medium that does not contain humic acid, a salt thereof, an analog thereof, or peat. The increased number of bacteria from the humic acid-containing medium generally represent recovery of bacteria that are unculturable in absence of humic acid or related substances.

Description

RECOVERY OF STRESSED BACTERIA

Background

[0001] Historically, bacteria have been considered viable if they could be cultured in the laboratory and nonviable if they could not be cultured. Now, however, it's accepted that some bacteria can exist in viable states that are not culturable (Pinto, D. et al., 2015, Thirty Years of Viable but Nonculturable State Research. Critical Reviews in Microbiology, 41, 61- 76). These bacteria may be called nonculturable or unculturable. The unculturable bacterial states may be caused by various environmental stresses on the bacteria, like lack of nutrients (e.g., starvation), temperatures too high or low to be permissive for growth,

sunlight/ultraviolet light, low oxygen availability, sulfur dioxide or certain other chemicals or substances, low redox potential, high saline concentration, desiccation, rehydration, heavy metal exposure, non-optimal pH, and others.

Summary

[0002] In some situations, it may be useful to recover or resuscitate bacteria from unculturable states and to culture them. For example, colony-forming unit (CFU) assays are often used to enumerate the number of culturable bacteria in a sample. However, if the sample contains unculturable bacteria, which generally will not form colonies in CFU assays, the number of viable bacteria in the sample will be underestimated using this assay.

Therefore, methods for culturing unculturable bacteria may yield improved methods for determining viable bacteria.

[0003] We have found that some bacteria, thought to have been rendered unculturable by stress conditions, may be cultured using culture medium that contains humic acid, salts thereof, analogs thereof, or peat. In some examples, Gram-negative bacteria subjected to desiccation conditions by coating the bacteria onto a seed, and then subjected to rehydration conditions by dissolving the seed coat in an aqueous solution, can be cultured after plating the bacteria on nutrient medium that contains humic acid or related substances.

[0004] We have shown that bacteria subjected to certain stresses have a higher efficiency of plating on agar plates containing humic acid, than on identical plates that do not contain humic acid. These bacteria, when not stressed, have similar efficiencies of plating on both plates containing humic acid and on plates that do not. For bacteria that are stressed by coating onto seeds, CFU counts obtained using culture medium that contains humic acid, salts, analogs, or peat, gives better estimates of the number of viable bacteria on the seeds, than do CFU counts using similar medium that does not contain humic acid or related substances.

Detailed Description

Definitions

[0005] The following includes definitions of selected terms that may be used throughout the disclosure and in the claims. The definitions include various examples and/or forms of components that fall within the scope of a term and that may be used for implementation. The examples are not intended to be limiting. Both singular and plural forms of terms fall within the definitions.

[0006] As used herein, "agar" means a gelatinous substance, generally derived from seaweed, and used in culture media to provide media that is solid or semisolid in consistency. In some examples, agar concentrations of about 0.5-1.5% (weight/volume) in media may be used for microbial culture plates. Herein, agar is considered a type of gelling agent.

[0007] As used herein, "about" means ± 10% with respect to the stated value or parameter.

[0008] As used herein, "agrochemical" means chemicals used in agriculture like, for example, chemicals used as acaricides, fungicides, gastropodicide, herbicides, insecticides, miticides, and the like.

[0009] As used herein, an "analog" of a first substance (e.g., humic acid) refers to a second substance that is structurally similar to the first substance, but with some differences. An analog may be synthetic.

[0010] As used herein, an "assay" means a test to determine something.

[0011] As used herein, "bacteria" means prokaryotic organisms that have peptidoglycan in their cell walls, and have lipids in their membranes, where the lipids contain fatty acids.

[0012] As used herein, "capable" refers to the ability or capacity to do or achieve a specific thing. [0013] As used herein, "colony" means a visible cluster of bacteria, generally on the surface of a solid or semisolid medium (e.g., medium containing agar), and probably originating from division of a single cell. A colony formed by bacteria may be called a "bacterial colony" or "colony-forming unit" (CFU).

[0014] As used herein, "contain" means to have or hold. In some examples, when a medium contains or is containing something (e.g., humic acid), the something is within or part of the medium.

[0015] As used herein, "count" when used as a verb, means to tally or total. "Counting" is an act to tally or total.

[0016] As used herein, "desiccate" means to reduce or remove the moisture from something. "Desiccation" refers to an act to reduce or remove moisture from something.

[0017] As used herein, "determine" means to establish or find out. "Determining" is an act to establish or find out. Something that has been established or found out may be said to be "determined."

[0018] As used herein, "dilution," when used as a noun, refers to a liquid that contains a reduced concentration of a thing as compared to the liquid when undiluted. "Diluting" is an act to create a dilution.

[0019] As used herein, "elute" means to remove by washing or dissolving.

[0020] As used herein, "enumerate" means to establish the number of something.

[0021] As used herein, "expose" means to subject a thing to something. "Exposure" is an act to expose.

[0022] As used herein, "gelling agent" refers to substances that are added to liquid to cause the liquid to become solid or semisolid in consistency. A variety of these substances exist. Example gelling agents may include agar, agarose, alginic acid, carrageenan, gelatin, gellan gum, guar gum, xanthan gum, and the like.

[0023] As used herein, "Gram-negative" refers to bacteria that, in a Gram staining reaction, lose the crystal violet stain and take the color of the counterstain. [0024] As used herein, "high," with reference to temperature, for example, means greater than a temperature that can sustain growth or, possibly, survival.

[0025] As used herein, "humic acid" refers to a principal component of humic substances (fulvic acid and humin are other principal components of humic substances) that is soluble in dilute alkali but which becomes insoluble as the pH becomes acidic. Substances "related to" humic acid may include salts of humic acid, humic acid analogs, synthetic humic acids, and may also include peat.

[0026] As used herein, "hydrate" means to absorb water. "Hydration" is an act to cause something to absorb water.

[0027] As used herein, "lack" means to be without.

[0028] As used herein, "long-term storage" generally refers to bacteria stored for a period of time, generally more than 1 -month. An example of long-term stored bacteria are bacteria stored in a liquid formulation in a bladder.

[0029] As used herein, "low," with reference to temperature or oxygen levels, for example, means less than required for growth or, possibly, survival.

[0030] As used herein, "medium," with reference to a culture medium for a bacterium, refers to compositions for supporting growth of bacteria. Example growth medium may include liquid media (e.g., broths) or solid/semisolid media (e.g., agar-containing media).

[0031] As used herein, "nutrients" means substances that support growth or maintenance of life.

[0032] As used herein, "peat" generally refers to partially decomposed vegetable/plant matter.

[0033] As used herein, "plating" refers to applying a sample, bacteria from a sample, or dilution of the sample or bacteria, to solid or semisolid bacterial culture medium (e.g., agar- containing medium). "Plated" refers to something that has been applied to solid or semisolid bacterial culture medium.

[0034] As used herein, "prior" means before. [0035] As used herein, "recover" means to culture a bacterium that is unculturable under another set of circumstances.

[0036] As used herein, "render" means to cause a thing to be or become something.

[0037] As used herein, "represent" means constitutes or regarded as.

[0038] As used herein, "salt" refers to an ionic form of a substance.

[0039] As used herein, "sample" refers to a representative part of a whole.

[0040] As used herein, "seed coat" refers to a layer of something (e.g., bacteria) on the surface of a seed. "Coating," when used as a verb, is an act to make a seed coat. A simple seed coat may be made by exposing seeds to bacteria and water or buffer, then allowing the water to dry, leaving the bacteria on the seeds. Other seed coats may contain various chemicals and/or other ingredients, along with the bacteria, and possibly additional microbes.

[0041] As used herein, "soil" generally refers to a mixture of organic matter, minerals, gases, liquids, microbes, and the like, present in the upper layer of the earth.

[0042] As used herein, "soluble" means able to be dissolved (e.g., in water).

"Solubilizing" is an act to dissolve something.

[0043] As used herein, "stress" means conditions that are not favorable to growth or survival.

[0044] As used herein, "synthetic" refers to something that is synthesized, rather than naturally occurring. A synthetic substance may be an analog.

[0045] As used herein, "unculturable," when referring to a bacterium, means unable to be cultured, using current technologies (i.e., technologies prior to this disclosure; e.g., without humic acid), and generally refers to a certain set of growth conditions (e.g., the medium does not contain humic acid). A bacterium that is considered unculturable may eventually be cultured, for example, when technologies are improved. In some examples, a bacterium cultured using the methods disclosed herein may not have been cultured previously. [0046] As used herein, "use" means to employ or put into service. "Using" is an act to employ or put into service. Something that has been employed or put into service may be said to be "used."

[0047] As used herein, "viable" means alive, surviving or living. Unculturable bacteria and stress

[0048] Generally, herein, unculturable bacteria are bacteria that were culturable at one time, under a specific set of conditions, but at a later time became unculturable (but retained viability) under those same conditions. In some examples, the culturable bacteria became unculturable because of exposure to various stresses.

[0049] In some examples, a method of determining whether unculturable bacteria exist in a bacterial population may be to show that some bacteria within a population of culturable bacteria becomes unculturable over time, without losing viability. In some examples, as described herein, it may be possible to show that bacteria that are unculturable under one set of conditions can be cultured (i.e., recovered) under another set of conditions (e.g., herein, when humic acid is present).

[0050] Unculturable bacteria may be formed in various ways. In some examples, unfavorable environmental conditions, or stress conditions, may cause culturable bacteria to enter into an unculturable state. A number of these conditions are listed in the Background section of this application. Other conditions, not listed herein, that cause culturable bacteria to become unculturable, likely exist and, it may even be that things other than stress conditions can cause bacteria to enter an unculturable state. Unculturable bacteria may exist and may be recovered from samples from soil, water, air, materials in the environment, from the surface of animals, from inside animals, from plants or plant-associated material, and the like.

[0051] For induction of bacteria into an unculturable state using stress, the amount of stress applied to the bacteria may have to be considered. For example, too much stress applied to bacteria (e.g., type of stress, time and/or intensity of the stress) may cause the bacteria to become nonviable and, therefore, not recoverable. Too little stress may fail to place bacteria into an unculturable state at all. There likely is an amount of each different type of stress that places the maximum number of bacteria in a population into an unculturable state. This amount of stress may have to be empirically determined. The percentage of bacteria within a population that have entered into an unculturable state may affect the ability of that population to demonstrate recovery (e.g., if fewer bacteria in a population are in an unculturable state, assays that detect recovery of unculturable bacteria to a culturable state, even if robust, may not detect recovery). Hence, even with an assay that can efficiently detect recovery of unculturable bacteria (e.g., humic acid in the medium), there need to be unculturable bacteria in the population in order for the assay to give positive results.

[0052] Many different types of bacteria are known to be capable of entering/exi sting in an unculturable state. Nonlimiting examples of unculturable bacteria that exist in an unculturable state may include γ-proteobacteria, β-proteobacteria, a-proteobacteria, ε- proteobacteria, bacteroidetes, acinobacteria, or firmicutes. In some examples, the bacteria capable of entering/exi sting in an unculturable state include Gram-negative bacteria.

[0053] Nonlimiting examples of unculturable bacteria that exist in an unculturable state may be from the genera Acetobacter, Acinetobacter, Aeromonas, Agrobacterium,

Alcaligenes, Arcobacter, Bifidobacterium, Bradyrhizobium, Burkholderia, Campylobacter, Citrobacter, Cytophaga, Enter obacter, Enter ococcus, Erwinia, Escherichia, Francisella, Helicobacter, Klebsiella, Lactobacillus, Legionella, Listeria, Oenococcus, Paracoccus, Pasteurella, Pseudomonas, Ralstonia, Ramlibacter, Rhizobium, Rhodococcus, Salmonella, Serratia, Shigella, Sinorhizobium, Vibrio, Xanthomonas, and Yersinia.

[0054] Nonlimiting examples of unculturable bacteria that exist in an unculturable state may be Acetobacter aceti, Acinetobacter calcoaceticus, Aeromonas hydrophilia, Aeromonas salmonicida, Agrobacterium tumifaciens, Alcaligenes eutrophus, Arcobacter butzleri, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacteriumanimalis, Bradyrhizobium japonicum, Bradyrhizobium elkaii, Burkholderia cepacia, Burkholderia pseudomallei, Campylobacter coli, Campylobacter jejuni, Campylobacter lari, Citrobacter freundii, Cytophaga allerginae, Enterobacter aerogenes, Enterobacter cloacae, Enter obacter agglomerans, Enterococcus faecalis, Enterococcus hirae, Enterococcus faecium, Erwinia amylovora, Escherichia coli, Francisella tularensis, Helicobacter pylori, Klebsiella aerogenes, Klebsiella pneumoniae, Klebsiella planticola, Lactobacillus plantarum,

Lactobacillus lindneri, Lactobacillus paracollinoides, Lactobacillus lactus, Legionella pneumophila, Listeria monocyhtogenes, Oenococcus oeni, Paracoccus pantotrophus, Pasteurella piscicida, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae, Ralstonia solanacearum, Ramlibacter sp., Rhizobium leguminosarum, Rhizobium meliloti, Rhodococcus rhodochrous, Salmonella enteritidis, Salmonella enterica, Serratia marcescens, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Sinorhizobium meliloti, Vibrio alginolyticus, Vibrio anguillarum, Vibrio campbellii, Vibrio cholera, Vibrio fischeri, Vibrio harveyi, Vibrio mimicus, Vibrio natriegens, Vibrio parahaemolyticus, Vibrio proteolytica, Vibrio shiloi, Vibrio vulnificus, Xanthomonas axonopodis, Xanthomonas campestris, Yersinia pestis and Yersinia entomophaga.

Humic acids, salts, and analogs

[0055] Soil organic matter may be classified as a humic substance or a non-humic substance. Humic substances are composed of altered or transformed components of plants, animals, microbes, and the like (e.g., decomposed organic matter). Non-humic substances include unaltered remains (e.g., not decomposed) of plants, animals, microbes, and the like. Humic substances are generally thought to include a humic acid component, a fulvic acid component, and a humin component. The humic acid component, and substances that may contain all or part of the humic acid component, is disclosed herein as capable of increasing the efficiency of plating of unculturable bacteria from samples.

[0056] These three components of humic substances - humic acid, fulvic acid, and humin - are defined, in part, based on their aqueous solubilities at different pH values. The humic acid component, for example, is generally water soluble at alkaline pH, but becomes less soluble under acidic conditions. In some examples, humic acid may be defined as the fraction of humic substances that are water insoluble at pH 2, but are increasingly soluble at higher pH values. The fulvic acid component is generally soluble in water at all pH values. The humin component is generally insoluble at all pH values.

[0057] Chemically, humic acid is a complex mixture of weak aliphatic and aromatic organic acids, often containing phenolic and carboxylic substituents. Humic acids may be called polydisperse because of their variable chemical features. The molecular sizes of humic acids may range, in some examples, from approximately about 10,000 to about 100,000 daltons. Humic acids may readily form salts with inorganic trace mineral elements. Both humic acids and salts thereof can be used and may be active in the methods disclosed herein.

[0058] Humic substances, and therefore humic acid, may be components of soil (e.g., humus), peat, lignite, coal, lake and stream sediments, seawater, and shale (e.g., Leonardite). Humic acid may be obtained or extracted from certain of these substances (e.g., convenient sources may be humus rich soil, peat moss, compost) using various methods. Humic acid may also be obtained from systems set up to facilitate degradation of organic materials (e.g., plant material) so that humic acid is produced. Humic acid may also be formed by polymerization of substances like polyphenols. Some of these methods are described in, for example, US Patent No. 5,854,032. Other methods for extracting or producing humic acids may be used. Humic acids can also be purchased commercially (e.g., Sigma-Aldrich No. 53680; Alfa Aesar No. 41747). The above-mentioned substances - like peat, lignite, coal, sediments, seawater, shale, and the like - are also within the scope of materials that increase plating efficiency of unculturable bacteria.

[0059] Salts of humic acid are within the scope of materials that can increase the efficiency of plating or recovery of unculturable bacteria from samples. In some examples, formation of salts of humic acid depends on the ability of carboxyl and/or hydroxyl groups therein to dissociate their hydrogen ions and bind to positive cations (e.g., metal cations like iron, copper, zinc, calcium, manganese, magnesium, and the like). Salts of humic acid can be purchased commercially (Sigma-Aldrich No. H16752).

[0060] Humic acid analogs and synthetic humic acids (a humic acid analog may also be synthetic) also exist and are within the scope of materials that may increase the efficiency of plating of unculturable bacteria. In some examples, certain quinones, one being

anthraquinone-2, 6-disulfonate (AQDS), are considered analogs of humic acid. Synthetic humic acids can be made by methods known in the art (e.g., V. A. Litvin, R. L. Galagan. "Synthesis and Properties of Synthetic Analogs of Natural Humic Acids." Russian Journal of Applied Chemistry 85, no. 2, 2012).

[0061] Humic acid may be fractionated and some of the fractions may be successfully used in the methods disclosed herein. In some examples of fractionating, humic acid is added to an aqueous solution of 0.1 M ammonium bicarbonate at a slightly basic pH. Insoluble material is removed from the mixture. The remaining solution is passed through a filter that retains molecules larger than 5,000 molecular weight on the filter, while molecules smaller than 5,000 molecular weight pass through the filter. The material retained on the filter may be shown to possess the activity of increasing the efficiency of plating of unculturable bacteria. Other methods of fractionating humic acid may be used. [0062] Herein, humic acid, salts thereof, analogs thereof, and peat, may include leonardite humic acids, lignite humic acids, peat humic acids or water-extracted humic acids. In some examples, humic acid, salts thereof, analogs thereof, and peat, may include ammonium humate, boron humate, potassium humate and/or sodium humate. In some examples, one or more of ammonium humate, boron humate, potassium humate and sodium humate is/are excluded. Nonlimiting examples of humic acids that may be useful various examples may include MDL Number MFCD00147177 (CAS Number 1415-93-6), MDL Number MFCD00135560 (CAS Number 68131-04-4), MDL Number MFCS22495372 (CAS Number 68514-28-3), CAS Number 93924-35-7, and CAS Number 308067-45-0.

Recovery of unculturable bacteria

[0063] Unculturable bacteria were generally once culturable under a specific set of conditions, but became unculturable at a later time under those same conditions. In some examples, application of stress to the culturable bacteria results in the bacteria becoming unculturable. Recovery of unculturable bacteria may occur when the unculturable bacteria become culturable later. In some examples, it may be possible to treat or stimulate the bacteria to regain culturability under the specific conditions under which they were previously unculturable. In some examples, it may be possible to culture the unculturable bacteria by changing the specific conditions. Herein, are disclosed methods for culturing unculturable bacteria by changing the conditions - by adding to the bacterial culture medium, humic acid, salts thereof, analogs thereof, or peat. In some examples, humic acid may be added without salts, analogs, or peat. In some examples, salts of humic acid may be added, without humic acid, analogs, or peat. In some examples, analogs of humic acid may be added, without humic acid, salts thereof, or peat. In some examples, peat may be added, without humic acid, salts thereof, or analogs thereof.

[0064] In some examples, the humic acids and/or related substances may be added to any bacterial medium. In some examples, the growth media may include YEM, R2A, TSA, LB, NA, ISP2, Jensen's, and the like. Media used for culturing bacteria may be liquid, semisolid or solid. Semisolid or solid medium may be made, in some examples, by adding a gelling agent to a liquid medium. A common gelling agent is agar. However, a number of other gelling agents exist and may be used. Examples include agarose, alginic acid, carrageenan, gelatin, gellan gum, guar gum, xanthan gum, and others. Generally, bacteria plated on a semisolid or solid medium may divide and form colonies after a time when the medium is placed in an environment conducive to growth of bacteria (e.g., 2-5 days of incubation at 30°C in an ambient atmosphere). However, these conditions (e.g., days of incubation, temperature, atmosphere) may vary and optimal conditions may be empirically determined.

[0065] Different forms of humic acid may require different concentrations within media to produce increased efficiency of plating of bacteria isolated from samples, as compared to media that lacks the humic acids (e.g., the optimal concentration of humic acid may not be the same as the optimal concentration of a salt of humic acid). In some examples, concentrations of any of the various humic acid forms above 0% (weight/volume) may be used. In some examples, humic acid forms may be used at concentrations above 0% and less than about 5% (e.g., 0.25, 0.50, 1.50, 2.00, 2.50%). In some examples, humic acid forms may be used at concentrations above 0% and less than about 0.25% (e.g., 0.10, 0.15, 0.20, 0.25%). In some examples, a concentration of humic acid used in the medium is not 0.1% or is above 0.1%. In some examples, concentrations of humic acid between about 0-5% or 0.05- 2.00%) may be used. In some examples, a concentration of a salt of humic acid below about 0.25%) may be used. In some examples, a concentration of peat of about 0.5% may be used.

[0066] Many different types of bacteria may be recoverable using the methods disclosed herein. Nonlimiting examples of these bacteria may include γ-proteobacteria, β- proteobacteria, a-proteobacteria, ε-proteobacteria, bacteroidetes, acinobacteria, or firmicutes, In some examples, the bacteria capable of being recovered using the methods disclosed herein include Gram-negative bacteria.

[0067] Nonlimiting examples of bacteria recoverable using the methods disclosed herein may be from the genera Acetobacter, Acinetobacter, Aeromonas, Agrobacterium,

[0068] Nonlimiting examples of bacteria recoverable using the methods disclosed herein may be Acetobacter aceti, Acinetobacter calcoaceticus, Aeromonas hydrophilia, Aeromonas salmonicida, Agrobacterium tumifaciens, Alcaligenes eutrophus, Arcobacter butzleri, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacteriumanimalis, Bradyrhizobium japonicum, Bradyrhizobium elkaii, Burkholderia cepacia, Burkholderia pseudomallei, Campylobacter coli, Campylobacter jejuni, Campylobacter lari, Citrobacter freundii, Cytophaga allerginae, Enterobacter aerogenes, Enterobacter cloacae, Enter obacter agglomerans, Enterococcus faecalis, Enterococcus hirae, Enterococcus faecium, Erwinia amylovora, Escherichia coli, Francisella tularensis, Helicobacter pylori, Klebsiella aerogenes, Klebsiella pneumoniae, Klebsiella planticola, Lactobacillus plantarum,

[0069] In some examples, the bacteria cultured using the methods disclosed herein may not be from the order Actinomycetales (e.g., microbes from this order may be excluded). In some examples, the bacteria cultured using the methods disclosed herein may not be from the phyla Acidobacteria and Verrucomicrobia (e.g., microbes from one or both of these phyla may be excluded). In some examples, the excluded Acidobacteria may belong to subdivision 1 only. In some examples, the excluded Verrucomicrobia may belong to subdivision 4 only.

[0070] In some examples, the unculturable bacteria that have been recovered are enumerated. The enumerated bacteria may be counted directly. The enumerated bacteria may be calculated. In some examples of calculating unculturable bacteria, total bacteria may be enumerated (total = culturable + previously unculturable) and the number of culturable bacteria may be subtracted from the total. In some examples, a sample or dilution thereof may be cultured on agar-containing nutrient media. One of the media may contain humic acid, a salt thereof, an analog thereof, or peat. The other of the media may not contain humic acid or related substances. In the case where unculturable bacteria have been recovered on the medium containing humic acid, the bacteria enumerated on that medium are generally greater than the number of bacteria enumerated on the same medium not containing humic acid. Subtraction of the latter from the former (number on humic acid medium minus number on medium not containing humic acid) yields an estimate of the number of unculturable bacteria in the sample.

[0071] In some examples, the bacteria enumerated or counted using medium containing humic acid, salts thereof, analogs thereof, or peat, are greater than the number of bacteria enumerated using the same medium without humic acid and/or related substances. In some examples, the number of bacteria in presence of humic acid and/or related substances may be at least about 1.2-, 1.4-, 1.6-, 1.8-, 2.0-, 2.2-, 2.4-, 2.6-, 2.8-, 3.0-, 3.2-, 3.4-, 3.6-, 3.8-, 4.0-, 4.2-, 4.4-, 4.6-, 4.8-, 5.0-, 5.2-, 5.4-, 5.6-, 5.8-, 6.0-, 6.2-, 6.4-, 6.6-, 6.8-, 7.0-, 7.2-, 7.4-, 7.6-, 7.8-, 8.0-, 8.5-, 9.0-, 10-, 20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, or 100-fold greater than the number of bacteria obtained using medium without humic acid. Recovery of unculturable bacteria using the methods disclosed here may not be complete. In other words, the number of unculturable bacteria recovered using the methods may not be all of the unculturable bacteria within a population. Perhaps a fraction of the total unculturable bacteria in a bacterial population are recovered using the disclosed methods.

Example embodiments of the invention

[0072] 1. A method, comprising, consisting essentially of, or consisting of:

[0073] recovering unculturable bacteria from a sample using a medium containing humic acid, a salt thereof, an analog thereof, or peat; and

[0074] enumerating the bacteria recovered from the sample.

[0075] 2. The method of embodiment 1, where the unculturable bacteria from the sample have been exposed to at least one stress.

[0076] 3. The method of any one of embodiments 1 or 2, where the unculturable bacteria from the sample have been exposed to at least one stress, including lack of nutrients, high or low temperatures, ultraviolet light, low oxygen, desiccation or hydration, long-term storage, or exposure to agrochemicals.

[0077] 4. The method of any one of embodiments 1-3, where the unculturable bacteria from the sample have been exposed to at least one stress, including desiccation or hydration. [0078] 5. The method of any one of embodiments 1-4, where the unculturable bacteria from the sample have been exposed to an amount of stress capable of rendering bacteria unculturable.

[0079] 6. The method of any one of embodiments 1-5, where the sample includes a seed coat.

[0080] 7. The method of any one of embodiments 1-6, where the bacteria recovered from the sample are eluted from the seed coat prior to the recovering.

[0081] 8. The method of any one of embodiments 1-7, where the recovering step uses a CFU assay.

[0082] 9. The method of any one of embodiments 1-8, where the medium includes a gelling agent.

[0083] 10. The method of any one of embodiments 1-9, where the medium includes agar.

[0084] 11. The method of any one of embodiments 1-10, where the enumerating step includes counting colonies that form on the medium after about 2-5 days of incubation at about 30°C, and the medium includes agar.

[0085] 12. The method of any one of embodiments 1-11, where a concentration of the humic acid in the medium is greater than 0% and less than about 5% (weight/volume).

[0086] 13. The method of embodiment 12, where the humic acid includes Sigma- Aldrich No. 53680 or Alfa Aesar No. 41747.

[0087] 14. The method of any one of embodiments 1-11, where a concentration of the salt of humic acid in the medium is greater than 0% and less than about 0.25% (weight/volume).

[0088] 15. The method of embodiment 14, where the salt of humic acid includes Sigma- Aldrich No. HI 6752. [0089] 16. The method of any one of embodiments 1-15, where the enumerating step determines a number of bacteria greater than a number determined by using the medium that does not contain humic acid, a salt thereof, an analog thereof, or peat.

[0090] 17. The method of any one of embodiments 1-15, where a number of bacteria enumerated using medium containing humic acid, a salt thereof, an analog thereof, or peat, is greater than a number of bacteria enumerated using the same medium that does not contain humic acid, a salt thereof, an analog thereof, or peat, by at least about 1.2-, 1.4-, 1.6-, 1.8-, 2.0-, 2.2-, 2.4-, 2.6-, 2.8-, 3.0-, 3.2-, 3.4-, 3.6-, 3.8-, 4.0-, 4.2-, 4.4-, 4.6-, 4.8-, 5.0-, 5.2-, 5.4-, 5.6-, 5.8-, 6.0-, 6.2-, 6.4-, 6.6-, 6.8-, 7.0-, 7.2-, 7.4-, 7.6-, 7.8-, 8.0-, 8.5-, 9.0-, 10-, 20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, or 100-fold.

[0091] 18. The method of any one of embodiments 1-17, where the unculturable bacteria recovered from the sample comprise, consist essentially or, or consist of, Gram- negative bacteria.

[0092] 19. The method of any one of embodiments 1-17, where the unculturable bacteria recovered from the sample include Bradyrhizobium, Paracoccus, or Ramlibacter.

[0093] 20. The method of any one of embodiments 1-17, where the unculturable bacteria recovered from the sample include Bradyrhizobium japonicum, Bradyrhizobium elkanii, Paracoccus pantotrophus, or Ramlibacter sp.

[0094] 21. A method for determining viable bacteria in a sample, comprising, consisting essentially of, or consisting of:

[0095] plating the sample, or dilution thereof, on an agar-containing medium containing humic acid, a salt thereof, an analog thereof, or peat, such that bacterial colonies form on the medium; and

[0096] counting the bacterial colonies,

[0097] where a number of bacterial colonies formed on the medium containing humic acid, a salt thereof, an analog thereof, or peat, is at least about 1.2-, 1.4-, 1.6-, 1.8-, 2.0-, 2.2-, 2.4-, 2.6-, 2.8-, 3.0-, 3.2-, 3.4-, 3.6-, 3.8-, 4.0-, 4.2-, 4.4-, 4.6-, 4.8-, 5.0-, 5.2-, 5.4-, 5.6-, 5.8-, 6.0-, 6.2-, 6.4-, 6.6-, 6.8-, 7.0-, 7.2-, 7.4-, 7.6-, 7.8-, 8.0-, 8.5-, 9.0-, 10-, 20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, or 100-fold greater than a number of bacterial colonies formed when the sample or dilution is plated on the same medium that does not contain humic acid, a salt thereof, an analog thereof, or peat.

[0098] 22. The method of embodiment 21, where the sample includes a seed coat.

[0099] 23. The method of embodiment 22, where the seed coat includes at least one agrochemical.

[00100] 24. The method of embodiment 21, where the sample includes long-term stored bacteria.

[00101] 25. The method of any one of embodiments 21-24, where a concentration of the humic acid in the medium is greater than 0% and less than about 5% (weight/volume).

[00102] 26. The method of embodiment 25, where the humic acid includes Sigma- Aldrich No. 53680 or Alfa Aesar No. 41747.

[00103] 27. The method of any one of embodiments 1-26, where at least some of the bacterial colonies are formed by Gram-negative bacteria.

[00104] 28. The method of any one of embodiments 1-27, where at least some of the bacterial colonies are formed by Bradyrhizobium.

[00105] 29. A method for determining viable bacteria, comprising, consisting essentially of, or consisting of:

[00106] providing a seed that has a seed coat that contains bacteria;

[00107] solubilizing the seed coat;

[00108] plating the solubilized seed coat, bacteria therefrom, or a dilution thereof, on an agar-containing medium containing humic acid, a salt thereof, an analog thereof, or peat, such that bacterial colonies form on the medium; and

[00109] enumerating the bacterial colonies,

[00110] where a number of bacterial colonies formed on the medium containing humic acid, a salt thereof, an analog thereof, or peat, is at least is at least about 1.2-, 1.4-, 1.6- , 1.8-, 2.0-, 2.2-, 2.4-, 2.6-, 2.8-, 3.0-, 3.2-, 3.4-, 3.6-, 3.8-, 4.0-, 4.2-, 4.4-, 4.6-, 4.8-, 5.0-, 5.2- , 5.4-, 5.6-, 5.8-, 6.0-, 6.2-, 6.4-, 6.6-, 6.8-, 7.0-, 7.2-, 7.4-, 7.6-, 7.8-, 8.0-, 8.5-, 9.0-, 10-, 20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, or 100-fold greater than a number of bacterial colonies formed when the solubilized seed coat, bacteria therefrom, or dilution thereof, is plated on the same medium that does not contain humic acid, a salt thereof, an analog thereof, or peat,

[00111] where the number of bacterial colonies formed on the medium containing humic acid, a salt thereof, an analog thereof, or peat, that do not form on the medium that does not contain humic acid, a salt thereof, an analog thereof, or peat, represent unculturable bacteria.

[00112] 30. The method of embodiment 29, where the seed coat includes at least one agrochemical.

[00113] 31. The method of any one of embodiments 29 or 30, where the seed coat includes long-term stored bacteria.

[00114] 32. The method of any one of embodiments 29-31, where a concentration of the humic acid in the medium is greater than 0% and less than about 5% (weight/volume).

[00115] 33. The method of embodiment 32, where the humic acid includes Sigma- Aldrich No. 53680 or Alfa Aesar No. 41747.

[00116] 34. The method of any one of embodiments 29-33, where at least some of the bacteria forming colonies on the medium containing humic acid, a salt thereof, an analog thereof, or peat, include Gram-negative bacteria.

[00117] 35. The method of embodiment 34, where at least some of the Gram-negative bacteria include Bradyrhizobium.

Examples

[00118] The following examples are for illustrating various embodiments and are not to be construed as limitations.

Example 1. Improved efficiency of CFU formation of bacteria eluted from seeds with humic acid

[00119] Coating bacteria onto seeds results in decreased numbers of bacteria that form colonies in CFU assays when the bacteria are eluted from the seed coats. Bacteria coated onto seeds undergo desiccation and, when the seed coat is dissolved, the bacteria undergo rehydration. Desiccation and/or rehydration are stressors for the bacteria. Whether the stress events kill the bacteria (i.e., the bacteria are not viable), cause the bacteria to become unculturable but still viable, cause some combination of killing and unculturability, or something else, is unclear.

[00120] To learn more about what happens to bacterial cells when they are coated onto, and then eluted from seeds, experiments were performed to examine this loss of CFUs in response to coating the bacteria onto seeds. Experiments were also performed to determine if addition of certain substances, here humic acid, to agar media used for the CFU assays, could increase the efficiency of CFU formation when the bacteria were eluted from the seeds.

[00121] In these studies, Bradyrhizobium elkanii strain 5019 was grown in two different production liquid media (#1 or #2) at 30°C with shaking at 200 rpm for 3 days. An aliquot of the cells that had been grown for 3 days was then diluted to an optical density at 600 nm of 0.5. Sixty soybean seeds were contacted with 1.0 ml of the diluted culture to coat the seeds with the bacteria, and the seeds were then allowed to dry under sterile conditions for 4 hours at room temperature. The coated seeds were stored in a covered sterile beaker at 30°C under ambient humidity until the seeds were treated as described below.

[00122] To examine the number of culturable bacteria recovered from the seed coats over time, the following experiment was performed. At 4 hours (at the end of the drying period), 24 hours, and 96 hours after contacting the soybean seeds with the bacteria, 5 seeds were soaked in 5 ml of sterile phosphate buffer, pH 7.2 (Weber Scientific, Hamilton, NJ) for 30 minutes. The phosphate buffer used to soak the seeds was serially diluted and then plated onto YEM (yeast mannitol extract) agar plates (10 g mannitol, 0.5 g yeast extract, 0.1 g sodium chloride, 0.5 g potassium phosphate dibasic anhydrous, 0.2 g magnesium sulfate heptahydrate, 12 g agar, and water to give a 1 liter volume, pH 6.8, with 1 ml of 0.003 g/ml vancomycin and 0.67 ml of 0.084 g/ml cycloheximide added). After plating the bacteria onto the YEM agar plates, the plates were incubated at 30°C until single colonies formed and could be counted. Colony counts were determined and used to calculate the approximate number of bacteria coated onto each seed. The determination for each time point was an average of colony counts from at least 8 agar plates.

[00123] The data in Table 1 show the average CFU per seed from seed coats at various time points (4, 24 and 96 hours) after bacteria were applied to seeds. The number of CFU from each of the two production media was normalized to the number of CFU obtained at 4 hours, which was given the value 1.

[00124] These data showed that the number of culturable bacteria obtained from seed coats using CFU assays (YEM plates) decreased over time. This experiment does not ascertain whether the decrease in cell number was due to cell death (i.e., nonviable cells), culturable cells becoming unculturable, or something else.

[00125] To determine whether the number of bacteria recovered from seed coats using CFU assays could be affected by addition of humic acid to the agar plates used for the CFU assays, the following experiment was performed.

[00126] Bacteria grown in the first liquid production medium, coated onto seeds, and eluted from the seeds at the 96-hour time point, as described above, were plated onto YEM agar plates, YEM agar plates containing 0.3% (weight/volume) humic acid, R2A plates (Fisher Scientific; catalog no. DF1826-07-3), or R2A plates containing 0.3% humic acid. For plates containing humic acid, humic acid (No. 53680 from Sigma-Aldrich, St. Louis, Missouri, USA, or No. 41747 from Alfa Aesar, Tewksbury, Massachusetts, USA) was added to the media before autoclaving to give a final concentration of 0.3%. After autoclaving, the media was swirled while pouring the plates to ensure humic acid was uniformly distributed throughout the media.

[00127] The average CFU per seed from seed coats at 96 hours after bacteria were applied to the seeds are shown in Table 2. Table 2. CFU of bacteria after 96 hours on seed when plated onto agar plates ± 0.3% humic acid

Agar plate composition CFU (relative to same medium without humic acid)¹

YEM 1.4 x 10⁴ (1.0)

YEM + humic acid 9.7 x 10⁴ (6.9)

R2A 3.4 x 10⁴ (1.0)

R2A + humic acid 7.3 x 10⁴ (2.2)

*Ιη parenthesis are shown the number of CFU from plates containing humic acid normalized to the number of CFU from plates containing the same medium (YEM or R2A) without humic acid. The number of CFU from plates not containing humic acid was given the value 1.

[00128] These data showed that the number of culturable bacteria recovered from the seed coats could be increased by addition of humic acid to the agar plates used for the CFU assays. These data indicated that, when culturability of bacteria is to be used as an estimate for viable bacteria eluted from seeds, a higher estimate of viable bacteria on the seeds can be obtained when humic acid is used in the culture media, as compared to using agar plates not containing humic acid. These data also indicate that there are likely unculturable bacteria in the seed coats. That is, the bacteria that form colonies on plates containing humic acid, but not on plates without humic acid, are viable on the seeds, but not culturable without humic acid.

Example 2. No improved efficiency of CFU formation of non-stressed cells with humic acid

[00129] The data shown in Example 1 indicate that there are viable but unculturable bacteria in seed coatings, and that at least some of the unculturable bacteria can be rescued using humic acid. These data do not indicate what caused the bacteria to be unculturable. One hypothesis we sought to test was that unculturability of the bacteria was caused by the seed coating process (e.g., desiccation of the bacteria) and/or the rehydration process (e.g., elution of the bacteria from the seed coat).

[00130] We knew from the experiments described in Example 1 that humic acid would rescue at least some unculturable bacteria from seed coats. If the seed coating/rehydration process caused the bacteria to become unculturable, there should be fewer unculturable bacteria in a bacterial population that had not been coated onto/rehydrated from seeds, and humic acid would be less efficient in rescuing cells. To test this hypothesis, we determined the effect of humic acid on colony-forming efficiency of bacteria grown in liquid culture, that had not been coated onto/rehydrated from seeds.

[00131] For these experiments, Bradyrhizobium elkanii strain 5019 was inoculated from a single colony on a YEM agar plate into production medium #1 and grown at 30°C with shaking at 200 rpm for 3 days. An aliquot of the culture was then serially diluted in phosphate buffer and plated on YEM agar plates, and on YEM agar plates containing 0.3% humic acid, as described in Example 1. After plating the bacteria onto the plates, the plates were incubated at 30°C until single colonies formed and could be counted. Colony counts were determined and used to calculate the approximate number of bacteria coated onto each seed. The determination for each time point was an average of colony counts from at least 3 agar plates.

[00132] The results from the experiment are shown in Table 3.

[00133] The data showed that any increase in efficiency of plating of the non-stressed cells on plates containing humic acid, as compared to plates not containing humic acid, was small (1.2 times more colonies on YEM with humic acid). In contrast, the increased plating efficiency of stressed cells, as shown in Table 2, was larger (6.9 times more colonies on YEM with humic acid). These data indicated that formation of the unculturable bacteria (i.e., bacteria that grew with humic acid, but not without humic acid) was related to the stress of seed coating and/or rehydration from seeds. Example 3. Rescue of dried bacterial preparations from coated seeds using humic acid

[00134] The data above indicated that, for bacteria grown in liquid media, and coated onto seeds, some of the bacteria became unculturable but capable of being rescued by humic acid. The studies below test whether bacteria grown by other methods (e.g., dry preparations of bacteria) behave similarly.

[00135] Two types of dry Bradyrhizobium preparations were used in these studies. One type of Bradyrhizobium was formulated as a peat-based product. The peat based product contained two strains of Bradyrhizobium elkanii. A second type of dry Bradyrhizobium used in these experiments was grown, not in liquid medium, but by solid-state fermentation (SSF) on solid supports. The SSF product contained a single strain of Bradyrhizobium elkanii. Both the peat-based and SSF products were substantially dry formulations of the bacteria.

[00136] The peat-based and SSF dry products were coated onto seeds by adding the products to wet seeds, shaking the seeds, and allowing the seeds to air dry for 4 hours. At 4 hours (the end of the drying period), at 4 weeks and at 9 weeks, 50 seeds were transferred to a 250 ml Erlenmeyer flask containing 50 ml of sterile phosphate buffer and the mixture was stirred with a magnetic stirrer for 15 min. The phosphate buffer was then serially diluted and plated onto YEM plates or YEM plates containing 0.3% humic acid. The plates were incubated at 30°C until single colonies formed and could be counted. Colony counts were determined and used to calculate the approximate number of bacteria coated onto each seed.

[00137] The data in Table 4 show the average CFU per seed from the seeds coated with the dry bacterial products. The number of CFU from each of the two cultures was normalized to a value of 1 at 4 hours.

4 weeks 1.87 x 10⁵ (1.0) 3.43 x 10⁵ (1.8)

9 weeks 6.40 x 10⁴ (1.0) 1.60 x 10⁵ (2.5)

SSF 4 hours 1.82 x 10⁶ (1.0) NA²

4 weeks 5.57 x 10⁵ (1.0) 7.43 x 10⁵ (1.3)

9 weeks 1.19 x 10⁵ (1.0) 2.60 x 10⁵ (2.2) Ιη parenthesis are shown the number of CFU on YEM + humic acid p ates normalized to the number of CFU on YEM plates (normalized number on YEM is 1).

2NA is not available.

[00138] The data showed that there was some increase in efficiency of plating of the dry bacterial formulations eluted from seeds, on media containing humic acid, as compared to media not containing humic acid (1.8 times more CFU of the peat-based product on humic acid plates after 4 weeks on seed; 1.3 times more CFU of the SSF product on humic acid plates after 4 weeks on seed). The increased efficiency of plating with humic acid was greater for bacteria that had been on the seeds for 9 weeks (2.5 times more CFU of the peat- based product; 2.2 times more CFU of the SSF product) as compared to bacteria that had been on the seeds for 4 weeks.

Example 4. Efficiency of CFU formation of bacteria in seeds coats containing agrochemicals

[00139] The following study was performed to test the effects of agrochemicals in combination with bacteria in seed coats on culturability of the bacteria, and on the efficiency of CFU formation of bacteria eluted from the seeds in media containing humic acid. In this study, Acceleron® (Monsanto, St. Louis, Missouri, USA) was used as an exemplary agrochemical.

[00140] To perform the experiments, 3 ml of a Bradyrhizobium japonicum strain mixture (2 strains of Bradyrhizobium elkanii and 1 strain of Bradyrhizobium japonicum), grown in a liquid production medium, was mixed with 2 ml of a liquid additive (extender) used with the Monsanto Optimize® product. Two seed coating mixtures were prepared. The first mixture contained the strain mixture and extender, as above, and also 5.3 ml of Acceleron®. The second, control mixture, contained the strain mixture and extender, as above, along with 5.3 ml of water. The two seed coating mixtures were separately mixed with 1 kg of soybean seeds in an inflated plastic bag and shaken for 1 minute. The bag was then opened and the seeds left to dry at room temperature in ambient humidity for 4 hours.

[00141] At various times points, (the 0 day time point was taken immediately after completion of the 4 hour period for drying of the seed coats, also 1 day, 4 days, 11 days), 50 seeds (with agrochemicals and, separately, without agrochemicals) were transferred to a 250 ml Erlenmeyer flask containing 50 ml of sterile phosphate buffer and the mixture was stirred with a magnetic stirrer for 15 min. The phosphate buffer was then serially diluted and plated onto YEM plates or YEM plates containing 0.3% humic acid.

[00142] The data in Table 5 show the average CFU per seed from the seeds used in this study, over a period of 1 1 days. The number of CFU per seed from the various samples were normalized to the number of CFU obtained from control seeds (no agrochemistry) on YEM plates that did not contain humic acid at 0 days.

[00143] Similar to bacteria coated onto seeds without agrochemistry (Example 1), the number of CFU obtained from bacteria coated onto seeds in combination with agrochemicals decreased the longer the bacteria were present in the seed coats. At least for the

concentration of Acceleron® used here, the rate of decrease of CFU on seeds with agrochemistry, as compared to seeds without agrochemistry, appeared similar. For both bacteria coated onto seeds with agrochemicals and without, humic acid increased the efficiency of CFU formation of bacteria eluted from the seeds.

Example 5. Recovery with and without humic acid of long-term stored bacteria

[00144] The ability of humic acid to increase the efficiency of plating of bacteria exposed to additional types of stress was investigated. In this study, bacteria that had been stored in liquid form long term in a polypropylene bladder were tested.

[00145] A mixture of two strains of Bradyrhizobium elkanii and one strain of

Bradyrhizobium japonicum, grown in liquid medium, and stored in liquid form in a bladder for 1 year at room temperature was used in these studies. An aliquot of the culture was serially diluted in phosphate buffer and plated on YEM agar plates, and on YEM agar plates containing 0.3% humic acid, as described in Example 1. After plating the bacteria onto the plates, the plates were incubated at 30°C until single colonies formed and could be counted. Colony counts were determined and used to calculate the approximate number of bacteria coated onto each seed. The data from the experiment are shown in Table 6.

[00146] The data showed that humic acid in the plating medium increased the number of CFU obtained from bacteria (1.7-fold) stored long-term in liquid form in a bladder.

Example 6. Recovery of heat-stressed Paracoccus with humic acid

[00147] The ability of humic acid to increase the efficiency of plating of bacteria exposed to additional types of stress was investigated. In this study, Paracoccus that had been exposed to heat were tested. [00148] A strain of Paracoccus pantotrophus was streaked onto a nutrient agar plate and mistakenly left in an automobile parked outdoors in North Carolina over a warm June weekend. The following Monday, an attempt was made to rescue culturable bacteria from the plate left in the automobile by dragging an inoculating loop across the surface of the plate, then inoculating a new plate containing TSA medium and a second new plate containing TSA plus 0.25% humic acid. After incubation of the two plates, we observed that there was greater bacterial growth on the TSA + humic acid plate than on the TSA plate that did not contain humic acid.

[00149] Another study was performed. A TSA plate onto which Paracoccus pantotrophus had been streaked and the bacteria had grown was used. This plate will be called the master plate. At the beginning of the experiment (called 0 days), a sterile inoculating loop was used to scrape and transfer colonies from the master plate to sterile phosphate buffer, which was then diluted to an optical density at 600 nm of 2.0. The diluted phosphate buffer was then serially diluted, and plated onto TSA plates and onto TSA plates containing 0.25% humic acid. The plates were incubated at 30°C until single colonies formed. Colony counts were determined. These are the counts shown in Table 7 for 0 hours.

[00150] The master plate was covered with parafilm and incubated at 45°C inside a sealed plastic bag that also contained a wet towel. After 72 hours at 45°C, and again after 80 hours, colonies were scraped from the master plate into sterile phosphate buffer, which was diluted to an optical density at 600 nm of 2.0, and then serially diluted, and plated onto TSA plates and onto TSA plates containing 0.25% humic acid. The plates were incubated at 30°C until single colonies formed. Colony counts were determined for the 72- and 80-hour time points, and the results are shown in Table 7.

Ιη parenthesis are shown the number of CFU per seed from plates containing humic acid relative to the number of CFU on the same plates without humic acid at the same time point (CFU number without humic acid given value 1.0).

[00151] For each time point, the ratio of CFU counts on TSA containing humic acid versus

CFU counts on TSA without humic acid were examined. The data show that there was an increase in plating efficiency of heat-treated bacteria on plates containing humic acid as compared to plates not containing humic acid (1.4-fold increase at 72 hours, 1.5-fold increase at 80 hours). The increase at 0 hours on plates with humic acid (1.2-fold) was similar to that obtained for non-stressed bacteria in Example 2.

Example 7. Other bacteria

[00152] The data above indicate that humic acid can rescue and/or increase the plating efficiency of stressed Bradyrhizobium elkanii, Bradyrhizobium japonicum, and Paracoccus pantotrophus bacteria. These are Gram-negative bacteria. Additional studies (data not shown) indicated that a strain of Ramlibacter, also a Gram-negative organism, after coating onto soybean seeds and elution from the seeds at 4 hours (the end of the drying period), as in Example 1, using 0.25% humic acid, also showed an increase in efficiency of plating on nutrient agar plates containing humic acid as compared to plates not containing humic acid.

[00153] In contrast, similar seed coating and elution studies using Pseudomonas kilonesis (Gram-negative) and Paenibacillus sp. (Gram-positive) did not show increased efficiency of plating with 0.25% humic acid.

[00154] While example compositions, methods, and so on have been illustrated by description, and while the descriptions are in considerable detail, it is not the intention of the applicant to restrict or in any way limit the scope of the application. It is, of course, not possible to describe every conceivable combination of components or methodologies for purposes of describing the compositions, methods, and so on described herein. Additional advantages and modifications will readily appear to those skilled in the art. Therefore, the invention is not limited to the specific details and illustrative examples shown and described. Thus, this application is intended to embrace alterations, modifications, and variations that fall within the scope of the application. Furthermore, the preceding description is not meant to limit the scope of the invention. [00155] To the extent that the term "includes" or "including" is employed in the detailed description or the claims, it is intended to be inclusive in a manner similar to the term

"comprising" as that term is interpreted when employed as a transitional word in a claim. Furthermore, to the extent that the term "or" is employed in the detailed description or claims (e.g., A or B) it is intended to mean "A or B or both". When the applicant intends to indicate "only A or B but not both" then the term "only A or B but not both" will be employed. Thus, use of the term "or" herein is the inclusive, and not the exclusive use. See, Bryan A. Garner, A Dictionary of Modern Legal Usage 624 (2d. Ed. 1995). Also, to the extent that the terms "in" or "into" are used in the specification or the claims, it is intended to additionally mean "on" or "onto." Furthermore, to the extent the term "connect" is used in the specification or claims, it is intended to mean not only "directly connected to," but also "indirectly connected to" such as connected through another component or components.

Claims

CLAIMS We claim:

1. A method, comprising:

recovering unculturable bacteria from a sample using a medium containing humic acid, a salt thereof, an analog thereof, or peat; and

enumerating the bacteria recovered from the sample.

2. The method of claims 1, where the unculturable bacteria from the sample have been exposed to at least one stress, including lack of nutrients, high or low temperatures, ultraviolet light, low oxygen, desiccation or hydration, long-term storage, or exposure to agrochemicals.

3. The method of any one of claims 1-2, where the unculturable bacteria from the sample have been exposed to an amount of stress capable of rendering the bacteria unculturable.

4. The method of any one of claims 1-3, where the sample includes a seed coat and the bacteria are eluted from the seed coat prior to the recovering.

5. The method of any one of claims 1-4, where the recovering step uses a CFU assay.

6. The method of any one of claims 1-5, where a concentration of the humic acid in the medium is greater than 0% and less than about 5% (weight/volume).

7. The method of any one of claims 1-5, where a concentration of the salt of humic acid in the medium is greater than 0% and less than about 0.25% (weight/volume).

8. The method of any one of claims 1-7, where the enumerating step determines a number of bacteria greater than a number determined by using the medium that does not contain humic acid, a salt thereof, an analog thereof, or peat.

9. The method of any one of claims 1-8, where the unculturable bacteria recovered from the sample include Gram-negative bacteria.

10. A method for determining viable bacteria in a sample, comprising:

plating the sample, or dilution thereof, on an agar-containing medium containing humic acid, a salt thereof, an analog thereof, or peat, such that bacterial colonies form on the medium; and

counting the bacterial colonies,

where a number of bacterial colonies formed on the medium containing humic acid, a salt thereof, an analog thereof, or peat, is at least about 1.2-fold greater than a number of bacterial colonies formed when the sample or dilution is plated on the same medium that does not contain humic acid, a salt thereof, an analog thereof, or peat.

11. The method of claim 10, where the sample includes a seed coat that contains at least one agrochemical.

12. The method of any one of claims 10-11, where the sample includes long-term stored bacteria.

13. A method for determining viable bacteria, comprising:

providing a seed that has a seed coat that contains bacteria;

solubilizing the seed coat;

plating the solubilized seed coat, bacteria therefrom, or a dilution thereof, on an agar- containing medium containing humic acid, a salt thereof, an analog thereof, or peat, such that bacterial colonies form on the medium; and

enumerating the bacterial colonies,

where a number of bacterial colonies formed on the medium containing humic acid, a salt thereof, an analog thereof, or peat, is at least is at least about 1.2-fold greater than a number of bacterial colonies formed when the solubilized seed coat, bacteria therefrom, or dilution thereof, is plated on the same medium that does not contain humic acid, a salt thereof, an analog thereof, or peat, where the number of bacterial colonies formed on the medium containing humic acid, a salt thereof, an analog thereof, or peat, that do not form on the medium that does not contain humic acid, a salt thereof, an analog thereof, or peat, represent unculturable bacteria.

14. The method of claim 13, where a concentration of the humic acid in the medium is greater than 0% and less than about 5% (weight/volume).

15. The method of any one of claims 13-14, where at least some of the bacteria forming colonies on the medium containing humic acid, a salt thereof, an analog thereof, or peat, include Gram-negative bacteria.