EXPERIMENT 1: Preparation of animal cell culture media

Objective: To prepare media for culture of mammalian cells

Materials required: Reagents:

• Dulbecco's modified Eagle's medium (DMEM)

• Fetal bovine serum (FBS)
• Penicillin-Streptomycin solution (Antibiotic)

Apparatus/Instruments:

• Laminar Air flow hood

• CO2 incubator
• Micropipette

Theory: Cell culture is one of the major techniques in all biology

experiments. Basic environmental requirements for cell to grow
optimally are:

1. Controlled temperature

2. Appropriate growth medium for providing nutrients to the cells and

incubator that maintain correct pH and osmolarity

The most important and crucial step in all culture is selecting

appropriate growth medium for the in vitro cultivation. Cell culture
media generally comprise of an appropriate source of energy and
compounds which regulate and promote cell growth.

A typical culture medium that is like DMEM is composed of a

complement of amino acids, vitamins, inorganic salts, high glucose
content, L-glutamine, sodium pyruvate as a source of growth factors,
hormones and attachment factors. In addition to nutrients the medium
also helps to maintain pH and osmolarity.

There are two types of media:

1. Natural / complex media: This media consists only of naturally

occurring bodily fluids. It is used in a wide range of animal cell culture.
However, exact composition of the media is not known.

2. Artificial / synthetic media: this media is prepared by adding

nutrients, vitamins, salts, O2 and CO2 gas phases, serum, protein,
carbohydrates and cofactors. The exact composition of each
component is known.

Buffer system is used in bicarbonate / carbonate system at pH 7.2 to

7.4.

Phenol red is used as an indicator. If it turns yellow then the media is

acidic where as when it turns purple the media is basic.
Procedure:

1. DMEM, FBS and antibiotic solution mix were available

commercially and were provided to us.
2. The final concentration of FBS would be 10%.
3. In a conical tube, 5ml commercially available FBS and 1 ml
antibiotic solution were added and volume made upto 50 ml with
serum free DMEM (commercially available).
4. The media was labelled as complete media and was stored at 4
deg C until use.

Observations:

Inference:

Precautions:

1) The pH of the media should be maintained.

2) Sterilized equipments to be used while preparing the media.
3) The place where the media is prepared should be properly made
aseptic by autoclaving equipments and keeping the air free of
microorganisms by laminar air flow.
EXPERIMENT 2: Animal cell culture process (Adherent cells)

Objective: To culture the adherent animal cells under the aseptic

conditions and observe them under the microscope

Theory: Cell culture is the process where animal cells are cultured ‘in-
vitro’ (within artificially controlled environment) to promote growth. A
specific medium is added to allow the growth by providing all the
essential energy, amino acids, growth factors (supplied by FBS) required
for optimum growth of the cell. The buffer system in the media
maintenance physiological pH

Materials required:

• Dulbecco's Modified Eagle's Medium (DMEM), along with fetal

bovine serum (FBS) and other reagents (complete media).
• Laboratory equipments including
o Coated Petridish
o Micropipettes
o Microfuge tubes.
• Inverted microscope
• CO2 incubator
• Laminar air flow cabinet
• Animal cell (NIH-313 mouse embryo fibroblasts cells)
Procedure:

1) The NIH-313 cells were already maintained in coated Petriplate.

2) The adhered cells were dislodged from the petriplate by adding
0.25% Trypsin solution. Trypsin solution disrupts the association
of cells to the plate surface.
3) The dislodged cells were transferred to a fresh sterile centrifuge
tube. The cells were harvested by centrifugation at 3000 rpm for 5
minutes at room temperature.
4) The supernatant was discarded and the cell pellet was suspended
in fresh complete medium (DMEM + 10% FBS) by brief vortexing
5) 3 ml of complete media were added to each of 60 mm coated
petridish.
6) Cell suspension from step (4) was added to each of four coated
petridish (step 5) to a final cell number of 10⁴ cells.
7) In order to maintain the cell growth successfully, a cell incubator
is maintained at temperature 37⁰ C, relative humidity of 95% and
5% CO2 to keep the cells in desirable state and allow proliferation.
8) Plates were incubated in CO2 incubator and observed under
inverted microscope.

Observations: cells observed immediately after seeding were round

and floating. After 24 hours of seeding, the petridishes were taken out
from the incubator and we observed the cells under the inverted
microscope. The cells proliferated and had taken the
shapes/morphology and found to be adhered to the surface. There was
no contamination noticed.
Inference: The conditions must be ideal for the particular cell which is
being cultured in order for the cell culture to be successful. A cell
incubator is thus maintained at temperature 37⁰C, relative humidity of
95% and 5% CO2 to keep the cells in desirable state and allow for
proliferation

Precautions:

1) The process of cell culture should be carried out in an

environment free from any contamination thus it is carried out
inside the tissue culture hood / laminar air flow cabinet
2) We should wear gloves and then wash our hands with 70%
ethanol
3) The incubator should be maintained and ideal condition for cell
culture to be successful.

EXPERIMENT 3: Enumeration of cell culture (Adherent /

suspension ) using Haemocytometer

Objective: To count the number of cells in a cell culture using

Haemocytometer
Theory: Haemocytometer is a counting chamber device originally
designed for counting blood cells. It consists of a thick glass microscope
slide with rectangular indentation that creates a chamber. It is divided
into 9 major squares of 1 mm x 1 mm in size. The four corner squares
are further subdivided into four x four grids.

Each cell in the corner square has volume = 1 mm (length) x 1 mm

(breadth) x 0.1 mm (height) = 0.1 mm³ =10-4ml

Let the average cell count in each of the sets of 16 corner squares be
‘N’

Then in 10-4ml volume ---> ‘N’ no. of cells present

Therefore, in 1ml volume, no. of cells present ---> N x 10⁴

Now, if the dilution factor is ‘D’, then

No. of cells present = ‘N’ x ‘D’ x 10⁴/ml

Materials required:

• Haemocytometer with glass slide.

• 0.5 ml of cell suspension in an Eppendorf tube
• Micropipette
• 70% ethanol
• Microscope
Procedure:

1) The Haemocytometer was cleaned with 70% ethanol and

coverslip was affixed to it
2) The cell suspension was diluted 10 fold before counting
3) Using a micropipette hundred µL of diluted cell suspension was
taken
4) The chambers underneath the coverslip were gently filled,
allowing the cell suspension to be drawn out by capillary action.
5) Using a microscope the grid lines of the haemocytometer was
focus with the 10x objective
6) The number of cells present in each of the four sets of 16 corner
squares was counted.

Observation:

Number of cells in quadrant 1 (Q1) =

Number of cells in quadrant 2 (Q2) =

Number of cells in quadrant 3 (Q3) =

Number of cells in quadrant 4 (Q4) =

Inference:

Total number of cells counted =

Therefore, average number of cells present =

Therefore, number of cells present in 1 ml =

Now the dilution factor =

Hence, number of cells present =

Precautions:

1. The cell culture must be done with utmost care and ideal condition
must be provided for proliferation.

2. Transfer of cell to Haemocytometer should be done carefully to

ensure homogeneous distribution of cells.

3. Counting should preferably done in all four corner squares and then
the average number should be considered.