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
A. left negative lipase (lipids); B. right positive lipase (lipids)
Thiosulfate citrate bile salts agar
made of sucrose where it is highly alkene medium
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