Question

Please explain in details.

For cell culture medium,

1. Why cell need 5% CO2?

Humidity is typically >95%?

•Why use ethanol to sterilize equipment?

•Why use iodine and methylene blue to stain plant and human cell, respectively?

2)Describe in detail about how to prepare low, medium and high density cultures. Show steps by steps calculation.

Answer 1

1. Culturing of animal cell lines which are used to maintain the same environment that usually needed inside the body, as our blood contains carbon dioxide around 40mmHg which is equal to 5% of CO2, to maintain the cell culture medium as like blood we need to supply. In blood, it is in the form of bicarbonate, which acts as a buffer in resisting the pH change, avoid fluctuations caused by the gases, metabolites. ALL cells release carbon dioxide, Lactate that affects the pH. Like our body, we need to maintain cell lines in 5% carbon dioxide concentration.

95% of humidity is maintained to minimize the evaporation of culture media, incubators are used to maintain the humidify environment for the growth of cell lines.

Ethanol is used as a sterilizer to minimize the contamination, as we know the pure form of alcohol coagulates the proteins, dissolving lipids so we use 70 - 95% ethanol as surface sterilizer so that microorganisms like bacteria and viruses will die.

Staining is used to differentiate and marked the difference of the particular part of the cell, iodine is used to stain the plant materials, as we know plant materials are made of starch, due to its coiled structure iodine molecules hold within and give blue color which is used for identification of plant material

in case of human cells methylene blue is used for staining, these stain DNA /nuclei, so this is easy to identify the type of nucleus and type of cell, due to brilliant in color and easy identification this dye is so handy in carrying of experiments on human cells when visualizing under the microscope.

2. In cell culturing cell, density is calculated by the passage number, aseptically transferring of cell inoculum, taking of cells present in exponential or log phase, used for culturing. primary cultures are usually subcultures in 1:2 ratio.Population doubling level (PDL)

PDL = 3.32 (Xe - logXb) + S

Xb means the cell number at beginning of the incubation time

Xe is the cell number at the end of the incubation time

S is the starting of PD

so if we need low denstiy we will choose less innoculum relatively medium and high density of cell cuturing