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Isolation and Cultivation of

Microorganisms
Class MCB11

Completed

Created @July 5, 2020 12:40 PM

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Type Lecture

Pure Culture (Axenic Culture)

a culture which contains a single species of microorganism

a population of cells arising from a single cell

Isolation and Cultivation of Microorganisms 1

 Cultivation

increasing the population of microorganisms by providing their nutritional

and physical requirements

Nutrients

extracellular substances which provide the cell with materials for building
protoplasm
and for energy generation

Culture Medium

any nutrient material for growth and cultivation of microorganisms in the

laboratory (e.g. Agar)

Uses of Culture Media

1. for growth and maintenance of microbial cultures

2. to favor the production of particular compounds

3. to study microbial action on some constituents of the medium

Types of Culture Media

According to physical state

Liquid (Broth) – with no solidifying agent

Semi-solid – with 0.1 - 0.5% solidifying agent

Solid – with 1.5 – 2.0% solidifying agent (ex. agar or gelatin)

According to chemical composition

Isolation and Cultivation of Microorganisms 2

 Synthetic – all components are chemically defined

Complex – not all components are chemically defined

examples

potato infusion (of plant origin)

beef extract (of animal origin)

yeast extract (of microbial origin)

According to principal function, purpose or application

General Purpose – can support most or almost all types of species

e.g.

Nutrient Agar (NA),

Potato Dextrose Agar (PDA)

Differential – distinguishes one type of bacteria from another

with special reagents like pH indicators or dyes

e.g.

Eosin Methylene Blue Agar (EMBA),

Spirit Blue Agar,

Thiosulfate Citrate Bile Sucrose Agar (TCBSA),

CHROMagar

Selective – allows the growth of a specific type of microorganism only

with selective agents (ex. salts, dyes, antibiotics, etc.)

e.g.

Bacillus Cereus Agar (BCA)

Enrichment – used to increase the number of microorganisms with

unusual physiological characteristics

with special nutrients (ex. blood)

e.g.

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 Blood Agar.

Chocolate Agar

Assay – of prescribed composition used for assay of vitamins, amino

acids, and antibiotics

used to determine qualitative/quantitative production of such a

compound by an organism

e.g.

Triple Sugar Iron Agar (TSIA),

Mueller-Hinton Agar

Techniques

Plating

Colony - a macroscopically visible (surface or subsurface) growth or

cluster of microorganisms on a solid medium

Enrichment Culture

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 isolation of specific types of microorganisms by a combination of nutrient
and physical conditions (basically targeted)

used for the isolation of unusual physiological types of microorganisms

which are
present in small numbers and which grow slowly (those with specific
requirements to grow) (most sensitive to several factors)

Serial Dilution

Used if the desired microorganism is present at a higher level than any

other microorganism

uses a series of tubes with diluents

Single-Cell Isolation

Isolation and Cultivation of Microorganisms 5

 uses a micropipette or a microprobe to physically pick a single cell and
transfer it on an agar medium

Common methods used: FACS (fluorescence-activated cell sorting),

laser capture microdissection,

manual cell picking,

random seeding/dilution

microfluidics/lab- on-a-chip devices

Membrane Filter

Isolation and Cultivation of Microorganisms 6

 for samples with low population

uses a sterile membrane filter having a pore size that retains

microorganism

Basic Steps in preparing Pure Cultures

1. Isolation

2. Transfer desired colony to a slant or slab

3. Very the purity

Microscopic

restreak on agar medium

subject to different physiological/biochemical tests

4. If colony is pure; make stock cultures

Purity is maintained

Viability is retained

Culture Preservation Methods

Isolation and Cultivation of Microorganisms 7

 Objectives: to retain the viability of the stock culture for long period of time while
maintaining purity

Periodic Transfer to fresh media

Considerations:

time interval of transfers

proper medium

proper storage temp.

Overlaying cultures with mineral oil

Aim: limit oxygen (low oxygen = low metabolic rate)

Advantages:

Simple

easier to get sample from the oil coating

Disadvantages:

not for all microorganism (most need oxygen)

Freeze Drying (lyophilization)

employs rapid drying in frozen state

dry ice in alcohol (-70C)

Advantages:

long term survival

less interference = less change

small storage container

Freezing with liq. nitrogen

Temp (-196C)

Isolation and Cultivation of Microorganisms 8

 specimens are coated with cryoprotective agent (glycerol) in liq-nitrogen ref

Drying

For spore and cyst former

drying temp (45C)

Banking Microbes
Culture Collections

organizations which maintain authentic pure cultures of microorganisms

provide ‘type’ strains to microbiologists throughout the world

Examples

American Type Culture Collection (ATCC) (Maryland)

National Collection of Type Cultures (London)

Japanese Type Culture Collection

Philippine National Collection of Microorganisms (at BIOTECH-UPLB)

END (7/6/2020)

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