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Unit 3 of 3 · B.Sc MLS Sem 3

Unit 3: Culture preservation

Applied Bacteriology notes · PTU syllabus (BMLS307-18)

3 min read3 topics8 exam questions
On this page
  1. Unit summary
  2. Why preserve cultures
  3. Preservation methods
  4. Lyophilisation
  5. Key terms
  6. Quick revision
  7. Important questions

Unit summary

Reference and clinical isolates must be kept alive and unchanged for years. This unit covers the rationale and methods of preserving microorganisms, with special reference to lyophilisation.

After this unit you can

  • Explain why cultures are preserved
  • Compare short- and long-term preservation methods
  • Describe lyophilisation
  • Revive preserved cultures safely

PTU syllabus topics

Basic concepts and rationale for preserving microbes, principle and procedures of preservation methods with special reference to lyophilization

ComparisonMethods of preserving cultures
How
Shelf life

Subculturing

Transfer to fresh media regularly

Weeks

Refrigeration

2–8 °C

Weeks to months

Glycerol stocks at −80 °C

Deep freeze

Years

Lyophilisation

Freeze-drying under vacuum

Many years

1

Topic 1

Why preserve cultures

  • Maintain reference and QC strains, keep isolates for outbreak investigation, research and teaching; preserve viability, purity and characteristics without genetic change.
2

Topic 2

Preservation methods

ComparisonPreservation methods
Method
Duration

Periodic subculture

Transfer to fresh media

Weeks; risk of mutation and contamination

Refrigeration (2–8 °C)

Slants or stabs

Weeks to months

Paraffin oil overlay

Reduces drying and oxygen

Months to years

Glycerol stocks frozen at −20 °C to −80 °C

10–20% glycerol protects cells

Years

Liquid nitrogen (−196 °C)

Cryopreservation

Decades

Drying on beads, soil or silica

Desiccation

Years

Lyophilisation (freeze-drying)

Freeze and remove water by sublimation

Decades

3

Topic 3

Lyophilisation

ProcessLyophilisation
  1. 1

    Grow a young culture

  2. 2

    Suspend in a protective medium (skimmed milk, serum or sucrose)

  3. 3

    Dispense into ampoules

  4. 4

    Freeze rapidly (−40 °C or lower)

  5. 5

    Dry under vacuum — ice sublimes

  6. 6

    Seal ampoules under vacuum

  7. 7

    Store in the cold and dark

  • Advantages: long viability, easy storage and transport at room temperature, small space. Revival: open ampoule safely (score and wrap), add broth, incubate and check purity.

Key terms

Lyophilisation
Freeze-drying under vacuum
Cryoprotectant
Agent such as glycerol protecting cells during freezing
Sublimation
Ice turning directly to vapour
Reference strain
Standard strain for quality control
Revival
Restoring a preserved culture to growth

Quick revision

  • Reasons for preservation.
  • Subculture, refrigeration, oil overlay, glycerol freezing, liquid nitrogen, drying.
  • Lyophilisation steps, advantages, revival.

Important exam questions

Practice questions written to the PTU exam pattern for this unit's syllabus: short answers (Section A style) and long answers (Sections B and C style).

Short-answer questions

  1. Q1.Why are cultures preserved?
  2. Q2.What is the role of glycerol in frozen stocks?
  3. Q3.Define lyophilisation.
  4. Q4.Name a protective medium used in freeze-drying.
  5. Q5.State one drawback of repeated subculture.
  6. Q6.How is a lyophilised culture revived?

Long-answer questions

  1. Q1.Describe the methods of preserving microorganisms.
  2. Q2.Describe lyophilisation in detail.

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