1.6 Aseptic Technique and Cell Culture

Question 1

What is aseptic technique?

Answer 1

Aseptic technique involves the sterilisation of equipment and culture media by heat or chemical means and subsequent exclusion of microbial contaminants.

Question 2

Examples of aseptic technique

Answer 2

work surfaces are disinfected before and after working with 70% ethanol or virkon.​
All equipment and culture media sterilised to exclude microbial contaminants – e.g. use of heat (dry in an oven), flame inoculating loops or steam (using an autoclave) or chemicals (such as bleach).​

Question 3

Purpose of aseptic technique

Answer 3

Aseptic technique eliminates unwanted microbial contaminants when culturing micro-organisms or cells

Question 4

How can a microbial culture be started?

Answer 4

Using an inoculum of microbial cells on an agar medium, or in a broth with suitable nutrients

Question 5

What is microbial culture?

Answer 5

A method of multiplying microorganisms by letting them reproduce in a culture medium under controlled laboratory conditions.

Question 6

What does cell culture require?

Answer 6

Cell culture requires environmental factors such as temperature and pH to be controlled and the growing cells must have the opportunity for gas exchange.

Question 7

What does the medium that animal cells are grown in contain?

Answer 7

Growth factors

Question 8

What are growth factors

Answer 8

Growth factors are proteins that promote cell growth and proliferation. Growth factors are essential for the culture of most animal cells.

Question 9

Where do growth factors come from?

Answer 9

Serum

Question 10

How many times can primary cell lines divide in culture

Answer 10

A limited number of times

Question 11

How many times can tumour cell lines divide in culture?

Answer 11

Tumour cells lines can perform unlimited divisions

Question 12

What does a typical culture media contain?

Answer 12

Water, salts, amino acids, vitamins and glucose.

Question 13

What does the plating out of a liquid microbial culture on solid media allow?

Answer 13

The number of colony-forming units to be counted and the density of cells in the culture estimated

Question 14

What type of dilution may be needed to achieve a suitable colony count?

Answer 14

Serial dilution

Question 15

Equation used to calculate the number of bacteria in a diluted cell culture

Answer 15

number of colonies x dilution of sample = number of bacteria/ml

Question 16

What is a haemocytometer?

Answer 16

Haemocytometers are specialised slides that have a counting chamber with a known volume of liquid.

Question 17

What are haemocytometers used for?

Answer 17

They are used to estimate cell numbers in a liquid culture

Question 18

How would you calculate the number of cells per cm^3 of culture if 15 cells were counted in a haemocytometer?

Answer 18

15 cells counted = 15 cells per 0.1 μl (or 1mm^3)​

15 × 10 = 150 cells per μl​

150 × 1000 = 150000 (1⋅5 × 10^5) cells per cm^3 of culture

Question 19

How are cells on a haemocytometer made visible?

Answer 19

They are stained

Question 20

How can living cells prevent the stain entering through the cell membrane?

Answer 20

Closing pores in the membrane​.
Actively transporting the stain out the cell​.
Breaking down the stains.

Question 21

What kind of cells can the stain be taken up by?

Answer 21

Non-viable cells (dead cells) take up the stain and can be visualised as the stain colour (usually blue).

Question 22

What is a viable cell count?

Answer 22

Number of live actively growing/dividing cells in a sample.

Question 23

What is a total cell count?

Answer 23

The number of dead and live cells

Question 24

What state are the cells that take up Trypan Blue dye?

Answer 24

Dead

Question 25

Equation for calculating percentage viability of cells

Answer 25

Live cell count/ total cell count = percentage viability