Isolation and Cultivation of Microorganisms

Culture media

Isolation technique

Preservation techniques

Culture collection

Definition

Cultivation

Nutrients

Pure culture or Axenic culture

Culture medium

Uses of culture medium

Types of culture media

contains a single species of microorganisms where its population arise from a single cell

increasing population of microorganisms by providing the nutritional and physical requirements

extracellular substance that used the cells for building protoplasm and energy generation

nutrient material for growth and cultivation of microorganisms

Uses

for growth and maintenance of microbial cultures

favor the production of particular compounds (to reproduce)

study microbial action on some constituents of the medium (microbial action on different medium)

Liquid (broth)

Semi-solid

Solid

no solidifying agent

0.1 - 0.5% solidifying agent (stab inoculation test)

1.5 - 2.0% solidifying agent

Solidifying agent

Agar

Gelatin

complex polysaccharide that is extracted from the red algae (seaweed)

melts at boiling point and solidifies at room temp

has no nutritional value

protein derived from collagen of skin and bone of animals (pigs)

some bacteria able to liquify gelatin at room temp

According to chemical composition

Synthetic

Complex

components are chemically defined

components are not chemically defined

According to principal function, purpose or application

General purpose

Differential

Selective

Enrichment

Assay

can support almost all types of species

Nutrient Agar (NA)

Potato Dextrose Agar (PDA)

Aspergillus flavus

Penicillium chrysogenum

distinguishes one type of bacteria from another with special reagents like pH indicators or dyes

Eosin Methylene Blue Agar (EMBA) - gram positive selector and selective in microorganisms

contains lactose fermentors (lactic acid) where acidic methylene blue and eosin, which will make the E. coli react, will produce black in color

Spirit blue agar

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A. left negative lipase (lipids); B. right positive lipase (lipids)

Thiosulfate citrate bile salts agar

made of sucrose where it is highly alkene medium

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allows the growth of specific type of microorganisms

selective agents

salts

dyes

antibiotics

Bacillus Cereus Agar

increase the number of microorganisms with unusual physiological characteristics with special nutrients

Blood agar

prescribed composition used for assay of vitamins, amino acids and antibiotics

used to determine qualitative/ quantitative production of such a compound by an organism

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Triple Sugar Iron Medium

Antibiotic sensitivity testing Using Disk Diffusion Assay

Fermentation media

color red means fermentation

Plating

Enrichment culture

Single cell isolation technique

Serial dilution

Membrane filter technique

Based on consistency

forms colonies (a macroscopically visible (surface or subsurface) growth or cluster of microorganisms on a solid medium)

Spread plating

Pour plating

Streak plating

agar first then incolumum with loopful of size

0.1 ml or less inoculum with agar first before the inoculum. only shows surface colonies

1 ml inoculum used where sample is directly inputed before agar. agar should not be too hot preferably at 45 degrees celcius. incubate and surface and subsurface colonies will appear.

combination of nutrient an physical condition where it is used to isolate unusual physiological types of microorganisms which are present in small numbers and grow slowly

used to thin out microbial population and is used if the desired microorganisms is present at a higher level than other microorganisms

dilution = (aliquot or the volume of the sample / volume of the container + volume of the sample) (past dilution)

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

Limiting dilution

Manual isolation

Microfluidics

Immunopanning

Laser microdissection (LMD)

Fluorescence activated cell sorting (FACS)

Magnet - activated cell-sorting (MACS)

for samples with higher volume but with low population

sterile membrane filters are used have a pore size that retains microorganisms

Steps in preparing pure culture

Verify the purity (microscope, restreak on agar medium, physiological and biochemical test, and gram staining)

Make stock cultures

Transfer desired colony to a slant or stab

Isolation

Objective

retain the viability of the stock culture for long period of time while maintaining its purity

Culture preservation methods

Freeze drying (lyophilization)

Overlaying cultures with mineral oil

Periodic transfer to fresh media

Freezing with liquid nitrogen

Drying

Considerations

Time intervals of transfers

Proper medium

Proper storage temperature

Aim: Limit the availability of O2 that helps reduce the metabolic rate

Advantage

simple

enables one to remove some growth under the oil and inoculate it in a fresh medium and still preserve the initial culture

Disadvantages

viability of microorganisms varies with species

rapid drying in frozen state (-20 degrees celcius)

Advantages

long term survival

less opportunity for changes in characteristics since it is freeze

small storage containers

drying using -196 celsius degrees

Consideration

liquid-nitrogen refs

Cryoprotective agent (glycerol)

grown on sterile paper disk saturated with nutrients and air dried and stored aseptically drying temperature (45 degrees celcius)

limitation: spore- and cyst- formers

procedure

drops of bacterials suspension in gelatin are placed on sterile plastic petri plates and dried off over phosphorus pentoxide under vacuum

Bacteria in small ampoules are dried from liquid state using vacuum pump, desiccant, and water bath

organisms are dried over calcium chloride in vacuum and stored in ref

definition

organizations which maintain authentic pure cultures of microorganisms

provide "type" strains to microbiologist throughout the world

Examples

National Collection of Type Cultures (NCTC) - London

Japan Collection of Microorganisms

American Type Culture Collection (ATCC) - Maryland

Philippine National Collection of Microorganism