Unit 3: Nutrition, Cultivation and Maintenance

I. Orientation

Microbial growth depends on obtaining nutrients, converting them into cellular material and energy, and maintaining viable cells under suitable environmental conditions. Cultivation therefore involves more than providing a medium: a microorganism must be isolated from a mixed population, purified into a genetically and physiologically consistent culture, and preserved without losing viability or important characteristics.

• Nutrient requirement: Cells need carbon, energy, nitrogen, minerals, water, and often growth factors such as vitamins or amino acids.
• Cultivation principle: A culture develops when physical and chemical conditions support metabolism and cell division.
• Isolation principle: Individual cells or colonies must be separated from a mixed sample.
• Purity convention: A pure culture contains one microbial species or strain, whereas a mixed culture contains two or more types.
• Maintenance principle: Preservation slows death and genetic change while retaining viability and characteristic properties.

II. Nutritional Categories of Microorganisms — Energy, carbon, and electron sources

Microorganisms are classified nutritionally according to how they obtain energy, carbon, and reducing power. These categories predict their habitats, metabolic pathways, and suitable culture media.

A. Nutritional categories of microorganisms

Nutritional categories of microorganisms are defined by combining the source of energy with the source of carbon and electrons.

• Energy source:
  • Phototrophs use light energy; cyanobacteria use chlorophyll-based photosynthesis.
  • Chemotrophs obtain energy by oxidizing chemical substances.
• Carbon source:
  • Autotrophs use inorganic carbon, mainly carbon dioxide, to synthesize cell material.
  • Heterotrophs obtain organic carbon from compounds such as glucose, acetate, or amino acids.
• Electron or hydrogen source:
  • Lithotrophs oxidize inorganic electron donors, including H₂, NH₃, H₂S, Fe²⁺, or H₂S₂O₃.
  • Organotrophs obtain electrons from organic compounds.
• Major combined categories:
  • Photoautotrophs: light energy, CO₂ carbon, inorganic electron donors; examples include cyanobacteria and purple sulfur bacteria.
  • Photoheterotrophs: light energy but organic carbon; examples include some purple nonsulfur bacteria.
  • Chemoautotrophs, or chemolithoautotrophs: energy and electrons from inorganic chemicals, with CO₂ as carbon; nitrifying bacteria are examples.
  • Chemoheterotrophs, or chemoorganoheterotrophs: energy, electrons, and carbon from organic compounds; fungi, protozoa, and many pathogens belong here.
• Special nutritional relationships:
  • Saprophytes digest dead organic matter extracellularly and absorb soluble products.
  • Parasites obtain nutrients from living hosts and may be obligate or facultative.
  • Symbionts exchange nutrients with another organism; mutualism benefits both partners.
• Growth-factor dependence: Some organisms are prototrophs, synthesizing all essential metabolites, while auxotrophs require a supplied factor such as biotin, tryptophan, or hemin.

B. Applications and limitations

Nutritional classification guides medium design, but organisms may change metabolism according to available substrates and environmental conditions.

• Medium selection: A photosynthetic organism requires light and inorganic nutrients, whereas Escherichia coli can grow on a glucose-based organic medium.
• Metabolic flexibility: Facultative anaerobes use respiration when oxygen is available but may ferment or respire anaerobically when oxygen is absent.
• Limitation of categories: A single label does not describe temperature, pH, oxygen requirement, or salt tolerance; these must also be controlled during cultivation.

III. Methods of Isolation of Microorganisms — Separating organisms from mixed samples

Isolation methods reduce the number of cells distributed in each region of a solid medium until individual cells form spatially separate colonies. A colony is treated as a clone only when it arose from one cell or a genetically equivalent cell group.

A. Methods of isolation of microorganisms

Methods of isolation of microorganisms use dilution, physical separation, selective growth, or environmental sampling to obtain distinct colonies.

• Streak-plate method:
  • A sterile loop is charged with sample and progressively streaked over successive sectors of agar.
  • Each sector mechanically dilutes the inoculum; isolated colonies usually appear in the final streaked area.
• Spread-plate method:
  • A measured sample, commonly 0.1 mL, is distributed across the agar surface with a sterile spreader.
  • Surface colonies can be counted and selected; excessive inoculum produces confluent growth.
• Pour-plate method:
  • A diluted sample is mixed with molten agar cooled to approximately 45–50°C and allowed to solidify.
  • Colonies form both within and on the agar; heat-sensitive organisms may be injured.
• Serial dilution:
  • The original sample is diluted stepwise, often tenfold, to lower cell density before plating.
  • If 0.1 mL of a 10⁻⁶ dilution produces 85 colonies, the estimated count is:

CFU/mL = colonies × dilution factor / volume plated
       = 85 × 10⁶ / 0.1
       = 8.5 × 10⁸ CFU/mL

• CFU means colony-forming units; it does not necessarily equal the number of individual cells.
  • Enrichment culture: A medium and condition favor a desired organism, such as cellulose as the sole carbon source for cellulose degraders.
  • Selective medium: Inhibitory agents suppress unwanted organisms; bile salts can inhibit many Gram-positive bacteria while allowing some Gram-negative enteric bacteria.
  • Differential medium: Indicators reveal biochemical differences; lactose fermentation on MacConkey agar produces colored colonies in fermenting bacteria.
  • Single-cell isolation: Micromanipulation, fluorescence-activated cell sorting, or limiting dilution can isolate cells that do not grow well in mixed populations.

B. Applications and limitations

Isolation strategy must match the organism’s physiology and the sample’s complexity.

• Environmental samples: Soil or water may require enrichment before plating because the target organism is present in very low numbers.
• Anaerobes: Specimens must be handled without oxygen and incubated in reducing media or an anaerobic chamber.
• Viable-but-nonculturable cells: A cell may remain metabolically active but fail to produce colonies under routine laboratory conditions.
• Contamination control: Sterile media, instruments, containers, and aseptic transfers prevent laboratory organisms from being mistaken for sample organisms.

IV. Purification Techniques — Obtaining a pure culture

Purification removes accompanying organisms from an isolated colony or culture. It is essential for reliable identification, biochemical testing, research, industrial production, and clinical interpretation.

A. Purification techniques

Purification techniques repeatedly separate cells and verify that the resulting culture has consistent characteristics.

• Colony selection and re-streaking:
  • A well-isolated colony is transferred to fresh sterile agar and streaked again.
  • Repeated subculturing reduces the chance that neighboring cells were carried over.
• Quadrant streak purification:
  • The loop is sterilized between streaking sectors, creating progressive dilution.
  • A colony selected from the third or fourth sector is more likely to be isolated than one from heavy primary growth.
• Selective and differential purification:
  • Selective agents suppress contaminants while allowing the target to grow.
  • Differential reactions help distinguish colonies, but appearance alone does not prove purity.
• Enrichment followed by plating:
  • A target population is increased under favorable conditions, then separated on solid medium.
  • This is useful when the desired organism is rare but can introduce selection bias.
• Physical purification:
  • Filtration can retain bacteria while allowing viruses or dissolved molecules to pass, depending on pore size.
  • Centrifugation separates cells according to sedimentation properties but generally does not distinguish closely related bacteria.
• Purity verification:
  • Colony morphology: Examine size, shape, margin, elevation, pigmentation, and texture.
  • Microscopy: A pure bacterial culture should show consistent cell morphology and Gram reaction.
  • Subculture: Growth from a single colony on nonselective medium should remain uniform.
  • Biochemical or molecular testing: A consistent biochemical profile, MALDI-TOF result, or sequence supports identity and purity.

B. Applications and limitations

Purification is a probability-based process that requires evidence from more than one observation.

• Mixed colonies: A colony may contain variants with different nutritional requirements even when it appears uniform.
• Satellite growth: Small colonies around a nutrient-releasing organism may be dependent rather than contaminants of the same type.
• Genetic instability: Repeated subculture can select mutants, plasmid loss, or altered expression of virulence and metabolic traits.
• Best practice: Preserve a verified stock and use working cultures for routine experiments, limiting the number of transfers.

V. Preservation Techniques — Maintaining viability and characteristics

Preservation techniques reduce metabolic activity or cellular damage so that microorganisms remain viable and genetically stable during storage. The method depends on the organism, storage duration, and intended use.

A. Preservation techniques

Preservation techniques range from short-term refrigeration to long-term cryogenic or desiccation-based storage.

• Refrigeration:
  • Cultures are commonly stored at approximately 2–8°C on agar slants or in suitable broth.
  • Low temperature slows metabolism but does not stop death; periodic subculture may be needed.
• Deep freezing:
  • Bacterial or fungal cultures may be stored at −20°C or preferably −80°C with a cryoprotectant.
  • Glycerol, often used at approximately 15–25% final concentration, reduces ice-crystal injury.
• Cryopreservation:
  • Liquid nitrogen storage near −196°C greatly slows chemical reactions and cellular aging.
  • Controlled cooling and appropriate cryoprotectants improve survival during freezing and thawing.
• Lyophilization, or freeze-drying:
  • The culture is frozen, water is removed under vacuum by sublimation, and the dry preparation is sealed.
  • Stabilizers such as skim milk, serum, or sugars protect membranes and proteins.
• Mineral-oil overlay:
  • Sterile mineral oil covers an agar culture, limiting oxygen transfer and drying.
  • It is useful for some fungi and bacteria but is less standardized than frozen or lyophilized stocks.
• Desiccation:
  • Drying on sterile soil, silica gel, paper, or other carriers can preserve resistant cells and spores.
  • It is unsuitable for many fragile, non-spore-forming organisms.
• Spore preservation:
  • Bacterial endospores and fungal spores tolerate drying better than vegetative cells.
  • Spore suspensions must still be protected from contamination and excessive heat.
• Quality control after storage:
  • Viability: Recover growth on appropriate medium.
  • Purity: Check colony morphology and microscopy.
  • Identity: Confirm expected biochemical, molecular, or phenotypic traits.
  • Stability: Compare preserved cultures with the original reference characteristics.

B. Applications and limitations

A preservation system is successful only if it maintains both living cells and their useful properties.

• Short-term versus long-term: Refrigeration suits temporary laboratory use, whereas −80°C storage, liquid nitrogen, or lyophilization is preferable for reference strains.
• Freeze–thaw damage: Repeated thawing and refreezing increases membrane injury; cultures should be divided into single-use aliquots.
• Organism-specific survival: Mycoplasmas, many protozoa, and some fastidious bacteria require specialized media and cryoprotectants.
• Documentation: Each stock should record organism name, strain designation, date, medium, preservation method, storage temperature, and passage history.
• Biosafety: Pathogenic cultures require secure containment, labeled containers, restricted access, and validated decontamination procedures during storage and recovery.