Microbiology: Freeze-Dried Cultures

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Questions and Answers

What is a drawback of using freeze-dried cultures?

They are quick to open and revitalize.

They require minimal sub-culturing.

They maintain typical characteristics easily.

They are tedious to open and revitalize. (correct)

How many sub-cultures may be necessary before cells regain their typical characteristics?

Several sub-cultures may be needed. (correct)

Only one sub-culture is needed.

None, they regain immediately.

Only sub-culturing is not needed.

What is the primary challenge associated with revitalizing freeze-dried cultures?

They can be tedious to open and revitalize. (correct)

They produce higher yields than fresh cultures.

They require complex nutrient media.

They instantly return to their characteristics.

Which of the following statements is true regarding freeze-dried cultures?

They may need several attempts to successfully revitalize. Signup and view all the answers

In the context of freeze-dried cultures, what is meant by 'revitalize'?

To restore the culture to its active state. Signup and view all the answers

Under which condition can the time of subculture be extended to one year?

If slops are covered with sterile medicinal grade mineral oil Signup and view all the answers

What is the recommended storage temperature for culture grown in broth?

-20 oC Signup and view all the answers

What should be added to cultures before freezing to preserve them?

10-20% glycerol Signup and view all the answers

If slops are not covered with mineral oil, what is the maximum duration for subculture?

Six months Signup and view all the answers

Which of the following is NOT mentioned as a way to extend the time of subculture?

Using a vacuum seal Signup and view all the answers

Which biological systems can be preserved using this universally applicable technique?

Fungi, bacteriophage, viruses, algae, yeasts, animal and plant cells Signup and view all the answers

Which of the following is NOT included in the biological systems that can be preserved by this technique?

Non-biological materials Signup and view all the answers

What is the main characteristic of the preservation method described?

It is applicable to a wide variety of biological systems. Signup and view all the answers

Which statement about the preservation method mentioned is true?

It effectively preserves a wide range of cells and biological systems. Signup and view all the answers

Why is this preservation technique considered universally applicable?

It has been successfully used for a variety of biological entities. Signup and view all the answers

What should be done if the samples fail any one of the tests?

The entire batch should be destroyed. Signup and view all the answers

Which method is NOT mentioned for improving the yield of a product?

Enhancing enzyme activity. Signup and view all the answers

Which of the following is considered a mutagenic agent?

Nitrous acid Signup and view all the answers

What is one of the physical conditions that can be improved to increase yield?

Temperature control Signup and view all the answers

What is the role of mutations in improving industrial micro-organisms?

To improve the efficiency of product yield. Signup and view all the answers

What is one method mentioned for increasing cell permeability?

Surfactants Signup and view all the answers

Which of the following is a result of increased cell permeability?

Corruption of selective permeability Signup and view all the answers

What type of chemical structure is mentioned in relation to increasing cell permeability?

Beta-lactams Signup and view all the answers

What mechanism is potentially overcome by increasing cell permeability?

Negative feedback mechanism Signup and view all the answers

What type of substance is likely to enhance the selective permeability of a cell?

Detergents Signup and view all the answers

What is the purpose of washing and re-suspending the cells in a medium containing homoserine?

To promote the proliferation of the desired auxotroph strain Signup and view all the answers

Why must the medium used for the culture lack penicillin?

To ensure that only auxotrophic strains thrive Signup and view all the answers

What is the effect of UV treatment on the cells before they are added to the medium?

It induces genetic mutations, creating new auxotrophic strains Signup and view all the answers

What defines an auxotrophic strain in this context?

A strain that requires specific nutrients not found in minimal medium Signup and view all the answers

What role does homoserine play in the described experiment?

It is a nutrient that auxotrophic strains need for growth Signup and view all the answers

Study Notes

Chapter II: Isolation, Preservation, and Improvement of Industrial Microorganisms

Industrial microorganisms are originally isolated from nature, but their quality is improved via genetic manipulation.
Genetic manipulation methods include mutagenesis and selection, nuclear fusion, protoplast fusion (for fungi, streptomycetes, and actinomycetes), and recombinant DNA technology.

Characteristics of Industrial Organisms

Nutritional Characteristics: The organism must be able to grow in inexpensive or pre-defined media.
Optimum Temperature: The ideal temperature for maximum productivity is above 40°C.
Availability in Pure Culture: The organism must be available in pure culture.
Genetic Stability: The organism should be genetically stable, but amenable to genetic manipulation.
High Productivity: The organism must have high productivity of desired metabolites or products.
Ease of Handling: The organism should be easy to handle (e.g., produce spores for easy inoculation and separation from the product for recovery).
Rapid Growth: The organism should grow rapidly and produce products quickly in large-scale cultures.
Non-harmful: The organism should not be harmful to humans, plants, or animals.

Sources of Microbes

Microbes are obtained from culture collections such as ATCC (American Type Culture Collection).
Standard organisms (from culture collections) are mainly used for assays, training, and educational purposes, not for production.
These organisms usually offer low yields of the desired product.

Isolation from Nature

Isolation is commonly from soil due to its high microbial content.
Methods involve enrichment liquid cultures based on selecting microorganisms with the desired traits.
Enrichment cultures increase the number of a specific organism relative to others in the original sample.
Selective forces (e.g., adding specific substances to enrichment liquid cultures) can be used to isolate desired organisms.
Environmental conditions (e.g., air-drying soil) can be altered to favor the survival of specific organisms before culturing.

Preservation of Industrial Microorganisms

Industrial cultures need to be viable and free from contamination to maintain productivity.
To eliminate genetic change, cultures are stored at reduced temperature or dehydrated.

1. Storage at Reduced Temperature

Agar Slopes: Cultures grown on agar slopes can be stored in a refrigerator (5°C) and subcultured at approximately 6-month intervals. Storing slopes covered with sterile mineral oil extends the time to a whole year.
Broth: Cultures grown in broth can be stored at -20°C after adding glycerol (10-20%).
Liquid Nitrogen: This is a universal method reducing metabolic activity by storage at very low temperatures (-150°C to -196°C). This involves growing cultures to maximum stationary phase, resuspending cells in a cryoprotective agent (e.g., 10% glycerol), and freezing the suspension in sealed ampoules. Fungi, bacteriophages, viruses, and algae can also be stored this way. Though freezing and thawing causes some loss of viability, there is little loss during storage. This method is useful for long-term preservation of cells that are unable to survive drying. The equipment, however, is expensive and liquid nitrogen must be replenished regularly to prevent loss of the collection.

2. Storage in a Dehydrated Form

Drying: Dried soil cultures are widely used for culture preservation, mostly for fungi, actinomycetes, and spore-forming bacteria. Moist soil is inoculated with medium, incubated for several days for growth, allowed to dry at room temperature, and kept in a dry atmosphere or refrigerator.
Lyophilization (Freeze-Drying): This process involves freezing a culture, then drying it under vacuum to sublimate cell water. Cultures are grown to the maximum stationary phase, resuspended in protective media (e.g., milk, serum, sodium glutamate), transferred to ampoules, frozen, and subjected to vacuum until sublimation is complete. Ampoules are then sealed and stored in a refrigerator. Cultures can stay viable for 10 years or more. While very convenient and stable for service culture collections, freeze-dried cultures are tedious to open and revitalize and may require multiple subcultures. This is not suitable for animal or plant cells.

Quality Control of Preserved Stock Cultures

Samples from every batch are routinely checked for purity, viability, and productivity (at least 3% of ampoules are tested).
If even one sample fails quality control, the whole batch is discarded.

Increasing Yield of Products

Improving the quality of industrial microorganisms: Mutations can create desired characteristics.
Improving production conditions: Physical and nutritional conditions can be optimized.

Improvement the Quality of Industrial Microorganisms

Mutations: Mutagenic agents (UV/ionizing radiation, chemicals like nitrous acid, nitrosoguanidine) can cause mutations and lead to changed characteristics; some may be desired mutations.

Primary Metabolites

Essential for cell growth and associated with cell activities/energy production.
Production correlates with cell growth and concentration.
Examples: Amino acids, fatty acids, vitamins, and alcohol.

Secondary Metabolites

Not essential for cell growth or energy production.
Production depends on media components and occurs after growth.
Produced at high concentrations during idiophase.
Examples: Antibiotics and some vitamins.

Selection of Mutant Producing Improved (Higher) Level of Primary Metabolites

Glutamic acid and lysine are significant commercially available primary metabolites.
Glutamic acid (glutamate) is an excitatory neurotransmitter used by the brain as fuel to increase neuron firing, and is used in treatments for muscular dystrophy Parkinson's Disease, Schizophrenia, etc.
Corynebacterium glutamicum is the main organism for Glutamic acid production. Two conditions are integral: the lack of an enzyme needed to convert alpha-ketoglutaric acid to succinic acid, and an auxotroph nature due to a biotin deficiency. This results in higher glutamic acid production as well as increased release outside the cell due to disrupted membrane permeability.

Selection of Secondary Metabolite Producing Strains

Strains can be isolated from nature, and improved via mutations, creating strains producing significantly more of the desired secondary metabolites.
Autotoxic resistance is a key trait for production of secondary metabolites. This strains are resistant to relatively high concentrations of the antibiotic in which they are used to produce.

Techniques used to improve strain qualities

Mutations of already existing enzyme-encoding genes
Genetically engineering by inserting a cloned gene, such as for 6'N.Kanamycin-acetyl transferase, to achieve desired characteristics in cells.

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Description

This quiz explores the advantages and disadvantages of using freeze-dried cultures in microbiology. Participants will answer questions regarding sub-culturing, preservation techniques, and the optimal conditions for maintaining these cultures. Test your understanding of this important preservation method in microbial biology.