Module 3 Flashcards
- a culture which contains a single species of microorganism
- a population of cells arising from a single cell
Pure Culture (Axenic Culture)
- increasing the population of microorganisms by providing
their nutritional and physical requirements
Cultivation
- extracellular substances which provide the cell with materials for building protoplasm and for energy generation
Nutrients
- any nutrient material for growth and cultivation of microorganisms in the laboratory
Culture Medium
Uses of culture medium
- for growth and maintenance of microbial cultures
- to favor the production of particular compounds
- to study microbial action on some constituents of the medium
types of culture media according to physical state
Liquid (Broth) – no solidifying agent
Semi-solid – 0.1 - 0.5% solidifying agent
Solid – 1.5 – 2.0% solidifying agent
solidifying agent used
agar or gelatin
types of culture media according to chemical composition
synthetic, complex, dehydrated, and compounded
all components are chemically defined
Synthetic
not all components are chemically defined
examples are:
potato infusion (plant origin)
beef extract (animal origin)
yeast extract (microbial origin)
Complex
types of culture media according to function, purpose, or application
General Purpose, Differential , Selective, Enrichment, Assay
all the components are in one media bottle
dehydrated
from separated bottles then combined into one
compounded
- can support most or almost all types of species
e.g. Nutrient Agar (NA) - for bacteria, Potato Dextrose Agar (PDA) - for yeast/mold
General Purpose
- distinguishes one type of bacteria from another
- with special reagents like pH indicators or dyes
e.g. Eosin Methylene Blue Agar (EMBA) - kills gram positive bacteria
Differential
- allows the growth of a specific type of microorganism only
- with selective agents (ex. salts, dyes, antibiotics, etc.)
e.g. Bacillus Cereus Agar (BCA)
Selective
- used to increase the number of microorganisms with unusual physiological characteristics
- with special nutrients (ex. blood)
e.g. Blood Agar
Enrichment
- used to determine qualitative/ quantitative production of such a compound by an organism
- of prescribed composition used for assay of vitamins, amino acids and antibiotics
Assay
types of isolation techniques
Plating, Enrichment, Serial Dilution, Single-Cell Isolation Technique, Membrane Filter Technique
- a macroscopically visible (surface or subsurface)
growth or cluster of microorganisms on a solid medium
Colony
types of plating
streak, spread-plate, pour-plate
- isolation of specific types of microorganisms by a
combination of nutrient and physical conditions - used for the isolation of unusual physiological types of
microorganisms which are present in small numbers and
which grow slowly
Enrichment Culture
- used if the desired microorganism is present at a higher level than any other microorganism
Serial Dilution
- uses a micropipette or a microprobe to physically pick a
single cell and transfer it on an agar medium
Single-Cell Isolation Technique
- for samples with low population
- uses a sterile membrane filter having a pore size that retains microorganism
Membrane Filter Technique
Steps in Preparing Pure Cultures
- Isolation
- Transfer desired colony to a slant or stab
- Verify the purity
- microscope
- restreak on agar medium
- physiological and biochemical test
- Make stock cultures
Culture Preservation Methods
Periodic transfer to fresh media
Overlaying cultures with mineral oil
Freeze-drying (lyophilization)
Freezing with liquid nitrogen
Drying
considerations for Periodic transfer to fresh media
- time interval of transfers
- proper medium
- proper storage temperature
aim of Overlaying cultures with mineral oil
limit the availability of O2 to reduce metabolic rate
Advantages of Overlaying cultures with mineral oil
- Simple
- enables one to remove some growth under the oil and
inoculate it in a fresh medium and still preserve the initial
culture
disadvantages of Overlaying cultures with mineral oil
- viability of microorganisms varies with species
rapid drying in frozen state
Freeze-drying (lyophilization)
Advantages of Freeze-drying (lyophilization)
- long-term survival
- less opportunity for changes in the characteristics of culture
- small storage containers
Considerations of Freezing with liquid nitrogen
cryoprotective agent (glycerol)
liquid-nitrogen refs
Samples are grown on sterile paper disk saturated with nutrient, then the disks are allowed to air dry and stored aseptically drying temperature = 45oC
Drying
Limitation of Drying
for spore- and cyst-formers
steps in drying
- Drops of bacterial 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 the ref
- organizations which maintain authentic pure cultures of
microorganisms - provide ‘type’ strains to microbiologists throughout the
world
Culture Collections
examples of Culture Collections
American Type Culture Collection (ATCC) in (Maryland)
National Collection of Type Cultures (NCTC) in (London)
Japan Collection of Microorganisms
Philippine National Collection of Microorganisms
