Unit 3: Nutrition, Cultivation and Maintenance - Subjective Questions

Microbial nutrition is the process by which microorganisms obtain and utilize substances required for energy production, growth, cell formation, and reproduction.

The major nutritional requirements include:

• Carbon: Needed for synthesis of cellular components. It may be obtained from organic compounds or carbon dioxide.
• Hydrogen and oxygen: Required in water, organic molecules, and oxidation-reduction reactions.
• Nitrogen: Essential for proteins, nucleic acids, enzymes, and some cell wall components.
• Sulfur: Present in amino acids such as cysteine and methionine and in certain coenzymes.
• Phosphorus: Required for nucleic acids, phospholipids, ATP, and phosphorylated metabolites.
• Minerals: Elements such as potassium, magnesium, calcium, and iron support enzyme activity, osmotic balance, and cellular structures.
• Growth factors: Organic compounds such as vitamins, amino acids, purines, or pyrimidines that some microorganisms cannot synthesize.
• Water: Acts as a solvent and is essential for transport, chemical reactions, and maintenance of cell structure.

The exact nutritional requirements vary according to the species and its metabolic capabilities.

Microorganisms can be classified nutritionally using three criteria:

• Energy source:

  • Phototrophs obtain energy from light.
  • Chemotrophs obtain energy by oxidizing chemical compounds.

• Source of electrons or hydrogen:

  • Lithotrophs use reduced inorganic substances such as hydrogen, ammonia, hydrogen sulfide, or ferrous iron.
  • Organotrophs use organic compounds as electron or hydrogen donors.

• Carbon source:

  • Autotrophs use carbon dioxide as their principal carbon source.
  • Heterotrophs obtain carbon from preformed organic compounds.

These categories may be combined. For example:

• Photoautotrophs: Use light for energy and carbon dioxide as the carbon source. Examples include cyanobacteria and algae.
• Photoheterotrophs: Use light for energy but require organic compounds as carbon sources.
• Chemoautotrophs or chemolithoautotrophs: Obtain energy by oxidizing inorganic compounds and use carbon dioxide as carbon source. Examples include nitrifying bacteria.
• Chemoheterotrophs or chemoorganoheterotrophs: Oxidize organic compounds for both energy and carbon. Most fungi, protozoa, and many bacteria belong to this group.

Autotrophs convert inorganic carbon into organic matter through photosynthesis or chemosynthesis. Heterotrophs obtain carbon and energy from organic materials such as sugars, proteins, and lipids. Some microorganisms are mixotrophic, meaning they can switch between autotrophic and heterotrophic nutrition depending on environmental conditions.

Culture media are nutrient preparations used to grow microorganisms in the laboratory. They may be classified as follows:

• Simple or basal media: Support the growth of non-fastidious organisms. Examples include nutrient broth and nutrient agar.
• Complex media: Contain ingredients such as peptones, yeast extract, or meat extract whose exact chemical composition is not completely known.
• Defined or synthetic media: Prepared from pure chemicals in known concentrations. They are useful for studying nutritional requirements and metabolism.
• Enriched media: Basal media supplemented with blood, serum, egg yolk, or other nutrients to support fastidious organisms. Blood agar is an example.
• Enrichment media: Liquid media that favor the multiplication of a desired organism present in a mixed population.
• Selective media: Contain substances that inhibit unwanted organisms and permit the growth of a particular group.
• Differential or indicator media: Contain indicators that reveal biochemical differences between microorganisms, often through color changes.
• Reducing media: Contain substances that remove oxygen and support anaerobic organisms.
• Transport media: Preserve microorganisms in a viable condition during transfer without permitting significant multiplication.

The choice of medium depends on the organism being studied and the purpose of cultivation.

Microbial growth is strongly influenced by environmental conditions:

• Temperature: Each microorganism has a minimum, optimum, and maximum growth temperature. Psychrophiles grow at low temperatures, mesophiles prefer moderate temperatures, thermophiles grow at high temperatures, and hyperthermophiles grow at very high temperatures. Temperature affects enzyme activity, membrane fluidity, and protein stability.
• pH: Microorganisms have an optimum pH for growth. Acidophiles prefer acidic conditions, neutrophiles grow near neutrality, and alkaliphiles prefer alkaline conditions. Extreme pH values alter protein structure, membrane function, and nutrient transport.
• Oxygen: Obligate aerobes require oxygen, obligate anaerobes are harmed by it, facultative anaerobes grow with or without oxygen, aerotolerant anaerobes do not use oxygen but tolerate it, and microaerophiles require low oxygen concentrations.
• Osmotic pressure: High concentrations of salts or sugars remove water from cells and may cause plasmolysis. Halophiles require high salt concentrations, whereas halotolerant organisms tolerate salt but do not require it.

Microorganisms grow best when these factors remain within their physiological limits.

Isolation is the process of obtaining a microorganism as a separate, uncontaminated culture from a mixed sample. The general procedure is:

1. Collection of the sample: Obtain the sample aseptically from soil, water, food, clinical material, or another source.
2. Preparation of a suspension: Mix the sample in a sterile diluent to disperse the microorganisms.
3. Serial dilution: Prepare successive dilutions when the sample contains a large number of organisms.
4. Inoculation: Transfer the sample to a suitable solid or liquid culture medium using an aseptic technique.
5. Incubation: Incubate the inoculated medium at an appropriate temperature, pH, oxygen level, and time.
6. Recognition of colonies: Observe colony size, shape, margin, elevation, color, texture, and hemolysis where applicable.
7. Selection of a colony: Choose a well-isolated colony that appears representative of the desired organism.
8. Subculturing: Transfer the selected colony to a fresh sterile medium.
9. Verification of purity: Examine the culture microscopically and perform appropriate biochemical or molecular tests.

A culture is considered pure only when it contains a single type of microorganism.

The streak plate method is a mechanical method used to isolate individual colonies on the surface of solid culture media.

Principle: A loop carrying a mixed microbial population is progressively diluted by streaking it over different areas of an agar plate. Individual cells become separated and develop into distinct colonies.

Procedure:

1. Label a sterile agar plate.
2. Sterilize and cool the inoculating loop.
3. Pick up a small amount of the sample.
4. Streak the first section of the agar surface.
5. Sterilize and cool the loop again.
6. Drag the loop from the previous section into a new section and streak it repeatedly.
7. Continue the process through several sections.
8. Incubate the plate in an inverted position.
9. Select a well-isolated colony for subculture.

Advantages:

• Simple and inexpensive.
• Requires no special equipment.
• Useful for obtaining pure cultures and observing colony morphology.

Limitations:

• Not suitable for accurate viable cell counts.
• Requires practice to obtain proper dilution.
• Overloading the loop or streaking incorrectly may prevent colony separation.
• Some organisms may not grow on the selected medium.

In both methods, serial dilution is commonly used to obtain countable plates. The viable count is generally expressed as colony-forming units per milliliter, calculated using the dilution factor and the volume inoculated.

Serial dilution is the stepwise dilution of a microbial suspension by transferring a measured volume into successive tubes containing sterile diluent. It reduces the number of cells to a level that permits separate colonies to develop on an agar plate.

Procedure:

1. Add a known volume of sterile diluent to several labeled tubes.
2. Transfer a measured volume of the original sample into the first tube and mix thoroughly.
3. Transfer the same measured volume from the first tube to the second tube.
4. Repeat the process through the required number of tubes.
5. Inoculate a measured volume from selected dilutions onto suitable agar plates.
6. Incubate the plates and count plates containing a suitable number of colonies.

For a tenfold dilution series, the dilution factor after tubes is:

The viable count is calculated as:

Only distinct colonies on plates within the accepted countable range should be used. Each colony is considered to have arisen from one viable cell or a group of cells, so the result is reported as colony-forming units rather than exact cell numbers.

Enrichment culture is a technique that increases the proportion of a desired microorganism in a mixed population by providing conditions favorable to its growth and unfavorable to competing organisms.

Steps involved:

• Select a medium containing a specific nutrient or energy source required by the target organism.
• Adjust environmental conditions such as pH, temperature, salinity, oxygen concentration, or incubation time.
• Inoculate the mixed sample into the enrichment medium.
• Incubate under the selected conditions.
• Transfer a portion to fresh enrichment medium several times if necessary.
• Plate the enriched culture on a suitable solid medium.
• Select isolated colonies and verify their identity and purity.

Importance:

• Helps isolate organisms present in very small numbers.
• Selects organisms with special metabolic properties.
• Supports the isolation of nitrogen fixers, cellulose degraders, hydrocarbon degraders, halophiles, and anaerobes.
• Facilitates the study of organisms that require unusual nutrients or environmental conditions.

Enrichment does not itself produce a pure culture; it only increases the relative abundance of the desired organism. Purification by subculturing is still required.

Selective media contain substances that inhibit the growth of some microorganisms while allowing the growth of others. Selective agents may include bile salts, dyes, antibiotics, high salt concentrations, or specific chemicals.

Differential media contain indicators or substrates that allow different microorganisms growing on the same medium to be distinguished by visible changes such as color, precipitate formation, or hemolysis.

Some media are both selective and differential. For example, MacConkey agar inhibits many Gram-positive bacteria and differentiates Gram-negative enteric bacteria according to lactose fermentation.

Purification is the process of obtaining a culture containing only one species or strain of microorganism.

Purification procedure:

1. Examine the original culture and select a well-isolated colony.
2. Transfer the colony aseptically to a fresh sterile medium.
3. Incubate under suitable conditions.
4. Repeat streaking or subculturing if mixed colony types are observed.
5. Maintain the final culture on an appropriate medium.

Methods commonly used:

• Streaking for isolated colonies.
• Repeated dilution and plating.
• Picking a single colony from a selective or differential medium.
• Micromanipulation or flow sorting for special applications.
• Antibiotic or nutritional selection when appropriate.

Confirmation of purity:

• All colonies should have similar morphology.
• A stained microscopic preparation should show uniform cell morphology and staining reaction.
• The culture should produce consistent biochemical reactions.
• Growth on different media should be uniform.
• Molecular tests, such as species-specific PCR or sequencing, may be used when necessary.

A culture should be handled aseptically throughout the process to prevent contamination. A single colony does not always guarantee purity, so microscopic and biochemical confirmation is important.

Aseptic technique refers to procedures used to prevent the introduction of unwanted microorganisms into cultures, media, equipment, or the working environment.

Important practices include:

• Sterilizing media, glassware, instruments, and work surfaces.
• Washing and disinfecting hands before and after handling cultures.
• Using sterile pipettes, loops, and tips.
• Flaming or otherwise sterilizing the mouth of culture vessels when appropriate.
• Keeping containers open for the shortest possible time.
• Avoiding contact between sterile materials and nonsterile surfaces.
• Working near a flame or within a biological safety cabinet when suitable.
• Correctly labeling and sealing cultures.
• Disposing of contaminated materials safely.

Aseptic technique is important because it:

• Prevents contamination of pure cultures.
• Ensures reliable experimental results.
• Protects laboratory personnel and the environment.
• Prevents the spread of pathogens.
• Maintains the characteristic properties of the organism being studied.

Without aseptic technique, it becomes difficult to determine whether observed growth or biochemical activity is caused by the intended microorganism.

The single-cell isolation method aims to separate one microbial cell from all other cells so that its descendants form a clonal culture.

Principle: A single cell is physically separated from a mixed population using a micromanipulator, capillary pipette, cell sorter, or another specialized device. The isolated cell is transferred to a sterile nutrient medium and allowed to multiply.

General procedure:

1. Prepare a dilute suspension of the microorganism.
2. Observe individual cells using microscopy or a cell-sorting instrument.
3. Pick or sort one cell into a sterile drop or culture vessel.
4. Transfer the cell to suitable growth medium.
5. Incubate under optimal conditions.
6. Confirm the purity and identity of the resulting culture.

Applications:

• Isolation of organisms that occur in very low numbers.
• Recovery of particular cells from a mixed population.
• Establishment of clonal microbial strains.
• Study of cell-to-cell variation and mutation.
• Isolation of organisms that cannot be separated easily by colony morphology.

The method is precise but requires specialized equipment, skilled personnel, and careful control of contamination.

Microbial preservation aims to maintain cultures in a viable, pure, and genetically stable condition for future use. Major methods include:

• Periodic subculturing: The organism is transferred regularly to fresh medium. It is simple but laborious and may allow contamination, mutation, or loss of characteristics.
• Refrigeration: Cultures are stored at low temperatures, commonly around , to slow metabolism. It is suitable for short-term storage of many organisms.
• Deep freezing: Cultures are stored at temperatures such as or , usually with a cryoprotectant such as glycerol.
• Lyophilization: Also called freeze-drying. The culture is frozen and water is removed under vacuum. The dried preparation can remain stable for long periods.
• Mineral oil overlay: A sterile layer of mineral oil reduces evaporation and oxygen exposure in cultures maintained on agar slants.
• Cryopreservation in liquid nitrogen: Storage at approximately greatly reduces cellular metabolism and is useful for long-term preservation.
• Desiccation: Removal of water can preserve some resistant microorganisms, spores, and viruses.

The method selected depends on the organism, desired storage period, available facilities, and need to preserve genetic and physiological properties.

Lyophilization, or freeze-drying, is a long-term preservation method in which a microbial suspension is frozen and water is removed by sublimation under reduced pressure.

Procedure:

1. Grow the microorganism in a suitable medium.
2. Harvest the cells or spores during an appropriate growth stage.
3. Suspend them in a protective medium containing substances such as skim milk, serum, or sugars.
4. Dispense the suspension into sterile vials or ampoules.
5. Freeze the preparation rapidly.
6. Apply a vacuum so that ice changes directly into vapor.
7. Seal the dried vials under vacuum or inert gas.
8. Store them under recommended conditions.

Advantages:

• Suitable for long-term storage.
• Requires little space.
• Cultures can often be transported easily.
• Metabolic activity and genetic change are greatly reduced.
• Properly sealed preparations may remain viable for many years.

Limitations:

• Some microorganisms are damaged during freezing or drying.
• The process requires specialized equipment.
• Rehydration must be performed carefully.
• Moisture or oxygen entering the container may reduce viability.

A small sample is rehydrated and cultured when the organism is required.

Cryopreservation is the storage of microbial cultures at very low temperatures, commonly in mechanical freezers or liquid nitrogen. At these temperatures, cellular metabolism and chemical reactions are reduced to extremely low levels.

Procedure:

• Grow a healthy culture under suitable conditions.
• Mix the cells with a sterile cryoprotective agent.
• Dispense the preparation into labeled cryovials.
• Cool the culture at a controlled rate when appropriate.
• Store at approximately or in liquid nitrogen at about .
• Thaw rapidly and transfer a small portion to fresh medium when needed.

Cryoprotective agents: Glycerol and dimethyl sulfoxide are commonly used. They reduce ice-crystal formation and limit damage to cell membranes during freezing and thawing. Sugars and proteins may also provide protection.

Advantages:

• Maintains viability for long periods.
• Reduces genetic and physiological changes.
• Requires little handling after storage.
• Suitable for many bacteria, fungi, and cell-associated microorganisms.

Precautions: Repeated freeze-thaw cycles should be avoided, vials must be accurately labeled, and backup stocks should be maintained to prevent loss of valuable cultures.

Short-term methods are convenient for organisms used frequently, but they may lead to contamination, drying, and genetic changes. Long-term methods are preferred for valuable strains because they minimize metabolism and the number of cell divisions. A laboratory commonly maintains an actively used working culture along with a secure long-term master stock.

Anaerobic cultivation involves growing microorganisms in the absence of oxygen. It is necessary for obligate anaerobes and useful for studying organisms with oxygen-sensitive enzymes.

Methods used:

• Reducing media: Media containing substances such as thioglycollate or cysteine remove dissolved oxygen.
• Anaerobic jars: Inoculated plates are placed in a sealed jar, and oxygen is removed chemically or replaced with gases such as nitrogen, hydrogen, or carbon dioxide.
• Gas-generating systems: Commercial sachets produce hydrogen and carbon dioxide; a catalyst converts residual oxygen into water.
• Anaerobic chambers: The entire manipulation is performed in an oxygen-free atmosphere.
• Deep agar inoculation: The organism is inoculated deep into agar, where oxygen concentration is low.
• Sealed tubes and roll tubes: Cultures are protected from atmospheric oxygen by suitable sealing and gas replacement.

Indicators such as resazurin or methylene blue may be included to show whether oxygen is present. Media, instruments, and containers must be reduced or pre-reduced, and exposure to air should be minimized during inoculation and examination.

Fastidious microorganisms have complex nutritional or environmental requirements and cannot grow on ordinary basal media.

Nutritional requirements may include:

• Specific amino acids, vitamins, purines, or pyrimidines.
• Blood, serum, or tissue extracts.
• Special carbon or energy sources.
• Reduced oxygen tension or increased carbon dioxide.
• Strict temperature, pH, or osmotic conditions.

Cultivation methods:

• Use enriched media, such as blood agar or chocolate agar.
• Add appropriate growth factors or specific supplements.
• Employ enrichment culture to increase the number of target organisms.
• Use selective conditions to suppress competing microorganisms.
• Incubate in a controlled atmosphere, such as increased carbon dioxide or reduced oxygen.
• Provide suitable temperature, moisture, and incubation time.
• Use living cells or tissue culture when the organism is obligately intracellular.

Successful cultivation requires knowledge of the organism's natural habitat and metabolism. Failure to provide even one essential factor may result in no visible growth, although the organism remains viable.