Exercise 1_ Pure Culture Techniques Flashcards
are ubiquitously found in nature, from the deep in the earth’s crust, to the polar ice caps and oceans, to the bodies of plants and animals?
Microorganisms
What are the several environmental factors that determine the type and number of microorganisms in a natural habitat?
Nutrients Temperature pH Amount of available water Atmospheric gasses Light pressure and other organisms
What is the imperative requirement in order to study a specific role of a particular microorganism in its environment?
Isolate the particular microorganism in pure culture
a population of cells or multicellular organisms that are derived from the growth of a single organism consisting of the same species or strain.
Pure culture
Several ways employed in isolating pure culture from mixed populations of microorganisms that discretely form well-isolated colonies on solid media?
Streak plating Pour plating spread plating
Isolation techniques employed in isolating pure culture from mixed populations of microorganisms that are not cultivated well on solid media?
Serial dilution method Single cell isolation enrichment method
Success of enrichment, isolation, and cultivation of microorganisms is critically dependent on what?
Choice of suitable culture medium Incubation conditions
After isolating a pure culture, what is the next important thing to do with the culture?
Maintain its viability and purity for an extended period of time
the process of maintaining viability and purity of a pure culture microorganism for an extended period of time
Preservation/conservation
Importance of preservation of microorganism
For future studies as reference for standardized assays and tests for taxonomic purposes as valuable stock for biotechnological applications
Skills or techniques that are vital in laboratory manipulations involving microorganisms to prevent contamination and maintain pure culture.
Aseptic techniques
the process of transferring a pure culture to liquid or agar media for the continuance of growth and viability.
Serial sub-culturing/serial subculture
Disadvantages of repeated sub-culturing
prone to contamination time-consuming laborious (esp. when maintaining a large number of isolates higher probability of genetic changes through spontaneous mutation (since microbes are allowed to grow)
Several methods of microorganism preservation
storage at low temperature freeze-drying (lyophilization) drying and storage in distilled water
Approximate volume of medium per big plate?
15-20mL
Approximate volume of medium per big tube (slant)?
8mL
Approximate volume of medium per big tube (stab/broth)?
10mL
Approximate volume of medium per small tube (slant)?
3mL
Approximate volume of medium per small tube (stab/broth)?
5mL
Approximate volume of liquid when using Erlenmeyer flasks?
not more than 60% of its total capacity
How would you prepare 17 big plates of compounded nutrient agar? NB = 8g/1000ml
weigh 2.72g of NB and 5.1g of agar, place in a 750ml E-flask and dissolve in 340mL dH2O. Mix well
How would you prepare 13 big tubes of compounded nutrient agar slant? NB = 8g/1000ml
weigh 0.832g of NB and 1.56g of agar, place in a250mL E-flask and dissolve in 104mL dH2O. Cook in the microwave oven and transfer 8mL in each tube.
How would you prepare 15 big tubes of compounded nutrient agar stab? NB = 8g/1000ml
weigh 1.2g of NB and 2.25g of agar, place in a 250mL E-flask and dissolve in 150mL dH2O. Cook in the microwave oven and transfer 10mL in each tube.
How would you prepare 18 big tubes of Nutrient broth? NB = 8g/1000ml
weigh 1.44g of NB, place in a 300mL E-flask and dissolve in 180mL dH2O. Cook in the microwave oven and transfer 10mL in each tube.
How would you prepare 13 small tubes of compounded nutrient agar slant? NB = 8g/1000ml
weigh 0.312g of NB and 0.585g of agar, place in a 100mL E-flask and dissolve in 39mL dH2O. Cook in the microwave oven and transfer 3mL in each tube.
How would you prepare 18 small tubes of Nutrient broth? NB = 8g/1000ml
weigh 0.72g of NB, place in a 250mL E-flask and dissolve in 90mL dH2O. Cook in the microwave oven and transfer 5mL in each tube.
Why is melted agar cooled to 45-47 degree Celsius before pouring into plates and before slanting?
To minimize condensation on the plates or tubes which may interfere in the growth pattern of the culture and may also lead to contamination. Also for the ease of pouring and proper handling of hot media bottle.
Why cultures for further use kept in the refrigerator?
Low temperature slows down growth and multiplication of microorganism by changing the conformation of its lipid cell membrane from fluid to solid thereby limiting nutrient uptake resulting into slow metabolism and growth. it also helps prevent contamination.
Why is important to avoid cutting into the agar surface during inoculation?
To prevent confluent growth of overlapping colonies compromising the purity of the culture. It can also introduce contaminants and mutations to the culture due to partial change in medium condition in the cuttings.
Why are mineral oil and glycerol sterilized for three consecutive days?
Oils have high boiling point and thus requires consecutive heat sterilization to eliminate all contaminants that may be present including spores from the environment or endospores that may have formed during first and 2nd sterilization which are relatively more resistant to heat compared to vegetative or growing cells.
What are the characteristics of a good cryoprotective agent?
high solubility in water even at low temperatures, non- or minimally toxic, depress the freezing point of a solution, able to penetrate cell membranes easily, and able to bind either with electrolytes (to increase concentration in the freezing process) or with water molecules (to delay freezing).
How do mineral oil and glycerol preserve the culture?
Mineral oil ensures anaerobic conditions and prevents dehydration of the medium. This condition helps microorganisms or pure culture to remain in a dormant state and, therefore, the culture can be preserved form months to years (varies with species). Glycerol is used as a cryoprotectant as it disrupts the hydrogen bonding between water molecules, hence preventing the formation of ice crystals during freezing which can damage cells by dehydration through a localized increase in salt concentration leading to denaturation of proteins, and puncture cellular membranes.
