Exercise 1_ Pure Culture Techniques

Question 1

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?

Answer 1

Microorganisms

Question 2

What are the several environmental factors that determine the type and number of microorganisms in a natural habitat?

Answer 2

Nutrients Temperature pH Amount of available water Atmospheric gasses Light pressure and other organisms

Question 3

What is the imperative requirement in order to study a specific role of a particular microorganism in its environment?

Answer 3

Isolate the particular microorganism in pure culture

Question 4

a population of cells or multicellular organisms that are derived from the growth of a single organism consisting of the same species or strain.

Answer 4

Pure culture

Question 5

Several ways employed in isolating pure culture from mixed populations of microorganisms that discretely form well-isolated colonies on solid media?

Answer 5

Streak plating Pour plating spread plating

Question 6

Isolation techniques employed in isolating pure culture from mixed populations of microorganisms that are not cultivated well on solid media?

Answer 6

Serial dilution method Single cell isolation enrichment method

Question 7

Success of enrichment, isolation, and cultivation of microorganisms is critically dependent on what?

Answer 7

Choice of suitable culture medium Incubation conditions

Question 8

After isolating a pure culture, what is the next important thing to do with the culture?

Answer 8

Maintain its viability and purity for an extended period of time

Question 9

the process of maintaining viability and purity of a pure culture microorganism for an extended period of time

Answer 9

Preservation/conservation

Question 10

Importance of preservation of microorganism

Answer 10

For future studies as reference for standardized assays and tests for taxonomic purposes as valuable stock for biotechnological applications

Question 11

Skills or techniques that are vital in laboratory manipulations involving microorganisms to prevent contamination and maintain pure culture.

Answer 11

Aseptic techniques

Question 12

the process of transferring a pure culture to liquid or agar media for the continuance of growth and viability.

Answer 12

Serial sub-culturing/serial subculture

Question 13

Disadvantages of repeated sub-culturing

Answer 13

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)

Question 14

Several methods of microorganism preservation

Answer 14

storage at low temperature freeze-drying (lyophilization) drying and storage in distilled water

Question 15

Approximate volume of medium per big plate?

Answer 15

15-20mL

Question 16

Approximate volume of medium per big tube (slant)?

Answer 16

8mL

Question 17

Approximate volume of medium per big tube (stab/broth)?

Answer 17

10mL

Question 18

Approximate volume of medium per small tube (slant)?

Answer 18

3mL

Question 19

Approximate volume of medium per small tube (stab/broth)?

Answer 19

5mL

Question 20

Approximate volume of liquid when using Erlenmeyer flasks?

Answer 20

not more than 60% of its total capacity

Question 21

How would you prepare 17 big plates of compounded nutrient agar? NB = 8g/1000ml

Answer 21

weigh 2.72g of NB and 5.1g of agar, place in a 750ml E-flask and dissolve in 340mL dH2O. Mix well

Question 22

How would you prepare 13 big tubes of compounded nutrient agar slant? NB = 8g/1000ml

Answer 22

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.

Question 23

How would you prepare 15 big tubes of compounded nutrient agar stab? NB = 8g/1000ml

Answer 23

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.

Question 24

How would you prepare 18 big tubes of Nutrient broth? NB = 8g/1000ml

Answer 24

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.

Question 25

How would you prepare 13 small tubes of compounded nutrient agar slant? NB = 8g/1000ml

Answer 25

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.

Question 26

How would you prepare 18 small tubes of Nutrient broth? NB = 8g/1000ml

Answer 26

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.

Question 27

Why is melted agar cooled to 45-47 degree Celsius before pouring into plates and before slanting?

Answer 27

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.

Question 28

Why cultures for further use kept in the refrigerator?

Answer 28

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.

Question 29

Why is important to avoid cutting into the agar surface during inoculation?

Answer 29

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.

Question 30

Why are mineral oil and glycerol sterilized for three consecutive days?

Answer 30

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.

Question 31

What are the characteristics of a good cryoprotective agent?

Answer 31

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).

Question 32

How do mineral oil and glycerol preserve the culture?

Answer 32

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.