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Saturday, July 19, 2008


Cell Counting using Haemocytometer Hemocytometer or Haemocytometer is a device used to count cells.Cell Counting using haemocytometer is a technique used to count cells in a given sample. A known volume of cell culture is counted using a haemocytometer and the total number of cells is calculated. Haemocytometer is a device that was originally used to count blood cells. But now it is also used to count many other types of cells. It consists of a thick glass microscope slide with a rectangular indentation that creates a chamber. This chamber is engraved with a laser-etched grid of perpendicular lines. The simplest dimension for us to use is the 1mm2 area when a special cover slip is placed over the haemocytometer.

Haemocytometer Calculation

The volume of the numbered squares is 0.1 mm3 (0.1 µl) and the average number of cells in one square must be multiplied by 10^4 and by a factor to correct for the dilution (if any) to calculate cells/ml.

Number of cells/ml = Average number of cells/grid x Dilution factor x 10^4.

Trypan blue is a vital stain used to selectively color dead tissues or cells blue. It is a diazo dye.
Live cells or tissues with intact cell membranes are not colored  Since cells are very selective in the compounds that pass through the membrane, in a viable cell Trypan blue is not absorbed; however, it traverses the membrane in a dead cell. Hence, dead cells are shown as a distinctive blue color under a microscope. Since live cells are excluded from staining, this staining method is also described as a Dye Exclusion Method.

Procedure for calculating cell count and % viability of  BHK-21 cell line

  1. 100uL of the BHK-21 cell suspension was aseptically transferred into an eppendorf tube inside laminar air flow unit.
  2. Mix the cell suspension with 100 uL of Trypan Blue.
  3. Fill the haemocytometer chamber with the cell suspension and observe under phase contrast microscope in 10X magnification.
  4. Count the cells in each of the four grids bordered by triple lines on any two side of the square.
  5. the percentage of vialbility can be calculated using the formula
% viability = (No of viable cells / Total no of cells) * 100


No of Viable cells
No of non viable cells

Cell Concentration 

Number of cells/ml = Average number of cells/grid x Dilution factor x 10^4.

= 24.75 * 2 * 10^4
= 4.9*10^5

% viability = (No of viable cells / Total no of cells) * 100

% viability = (24.75/ 27.75) * 100 = 89.18%

Animal Cell Culture

Animal cell culture (ACC) is the process of culture of animal cells outside the tissue (ex vivo) from which they were obtained. The process of ACC is carried out under strict laboratory conditions of asepsis, sterility and controlled environment involving temperature, gases and pressure. It should mimic the in vivo environment successfully such that the cells are capable of survival and proliferation in a controlled manner.

Culturable Cells:
Theoretically, cells of any type can be cultured upon procurement in a viable state from any organ or tissue. However, not all types of cells are capable of survival in such an artificial environment because of many reasons on which the artificial environment may fail to mimic the biochemical parameters of the source environment. Some good examples include the absence of Growth Regulators, cell-to-cell signal molecules, etc. Under optimal conditions of maintenance, the cell culture established can be sub-until a of specific cell type is obtained. This can repeatedly sub-cultured to maintain as a cell-line. As a matter of fact, cell lines from cancerous tissues have also been established. The presence of excess growth regulators or other factors may often render the cells to undergo rapid uncontrolled proliferation resulting in a cancerous state. Good examples of established cell lines are Hela, BHK,Vero and CHO etc.

Important Terms:
Confluent – crowding of cell substrate such that there is no space for cells to grow.

Passage/subculture – the transfer of cell with or without dilution, from one culture vessel to another.

Passage Number – the number of times the cells in the culture have been subcultured or passaged.

Anchorage dependent: cells or cultures derived from them, which will grow, survive, or maintain  function only when attached to a surface such as glass or plastic.

Suspension culture: a type of culture in which cells, or aggregates of cells, multiply while suspended in a liquid medium.

Cell Growth & Propagation :
Monolayer Cultures:

Cell Culture in T-Flask - Image Source

1. Anchorage dependent cultures:

Advantages: -Easy to change medium and wash cells before adding fresh medium.
- Microscopic evaluations
- Homogenous distribution

Disadvantages: – Scale up is difficult and expensive
- Require more space than suspension cultures
Properties: – Confluency
- Pretreated surfaces for growth

2. Suspension cultures:

Advantages: Trypsin treatment not required, so subculture is quicker and less traumatic for the cells.
- Scale up is easier

Disadvantages: Prolonged use of same vessel can lead to contamination
Properties: Untreated vessels used, minimise cost
- kept in suspension mechanically

Culture on Microcarriers :
· bead like structures used (90-250µm dia) which provide a large surface area to cells made from cellulose,sephadex.
· cells attach and spread on beads, grow to a confluent monolayer.
· held in medium and stirred constantly
· combination of suspension and adherent technique.

Suspension Culture- Image Source

Cell Growth in Media

The proliferation of anchorage dependent cells can only occur after adhesion to a suitable culture surface. Extracellular surface molecules are found naturally in some media supplements and are also secreted by cells themselves. ECM (extracellular matrix) molecules are adsorbed onto the substrate surface and then cells bind to the matrix through cell surface receptors.

Components of cell culture media :
· water
· inorganic salts
· trace elements
· sugars
· amino acids
· vitamins
· fatty acids & lipids
· hormones & growth factors
· carriers  ECM proteins
· antibiotics

Serum is an important component of most media formulations because it:
Ø is a universal growth supplement; complex mixture of components and absolutely essential for cell growth & proliferation.
Ø increases viscosity of medium thereby protecting cells from shear.
Ø buffers the cell culture environment
Ø binds and neutralises toxins( heavy metal ions and endotoxins)

Wednesday, July 16, 2008

40 L Fermenter

Ø Sampling
1. 2 hrs subsequent to inoculum transfer, the sterile bottle was clamped at the sampling port and the sampling line was steam sterilized for 10minutes.
1. Sample was collected by first draining off a few ml media.
2. pH and the O.D. (at 600nm) were checked and the specific growth rate (µ) was calculated.
Ø Dosing
1. Peristaltic pump was calibrated.
2. 400 ml of autoclaved Dextrose solution was transferred to a sterile transfer bottle in laminar airflow.
3. The 400 ml was transferred to the vessel in equal amounts in 4 hours at six-pulses/ hour.
4. 16.66 ml of the solution was transferred to the vessel at a ten-minute interval over the 4 hours.
5. A sample of the media was taken every one hour.
6. OD was checked at 600nm and the specific growth rate (µ) was calculated.
Ø Harvesting & CIP
1. On the third day of fermentation, the fermentation process was shut down and the biomass was harvested into the harvest vessel by pressurizing the vessel.
2. The dosing bottle of dextrose and antifoam were unclamped.
3. CIP skid was connected to the fermenter.
4. 9 liters (30% of working volume) of soft water was filled in the mixing vessel in the C.I.P station.
5. Appropriate valves were opened such that water entered the fermenter vessel through the spray ball and was agitated for 5 minutes. Water was then drained.
6. Washing with 1% alkali (NaOH) was done
- Concentrated alkali was prepared in the alkali bottle in the CIP station
- 1 liter of this concentrated alkali was pumped into the vessel and mixed with 8 liters of water and was heated up to 50°C and supplied to the fermenter vessel through the spray ball.
- The spray ball supply was stopped and alkali supplied to the transfer lines, one at a time for 5 minutes followed by air line alkali wash. The alkali was recirculated.
- Alkali was drained using drain 2 valve and it was ensured that the steam traps were open so as to drain off any remnants.
7. Rinsing with purified water was done using recirculation
8. Acid wash (0.5% HNO3) was performed in the same manner as the alkali wash.
9. Rinsing with purified water was done as before.
10. W.F.I. rinse without recirculation.
- The first two rinses were for the transfer and air lines followed by the third rinse for the fermenter vessel.
- Contents drained using the drain 1 and drain 2 valves of the CIP skid.


· To carryout FSIP of 40 L Fermenter
· To perform fermentation of yeast culture in batch mode
· To study the effect of dosing on the growth of yeast
· Cleaning in place and ESIP

Sterilization in place of any equipment can be done conveniently thereby eliminating the need of subsequent aseptic connections or shut down of the whole process. Typically SIP uses saturated steam at 15 psi for at least 30 minutes. In Microbial fermentation, FSIP is performed. The whole sterilization is done after charging the media into the vessel. On the other hand, in animal cell culture ESIP is carried out where the sterilization is done before charging the vessel with media. Before ESIP, integrity of vessels and filters is checked.

Pre-inoculum of Pichia pastoris culture, conical flask, TSB media powder, dextrose powder, antifoam, measuring cylinder, brown paper, spatula, cotton, rubber bands, pipette, test tubes, test tube stand, beaker, tissue, cuvette, distilled water, WFI

40 liter Fermenter and other utilities, HMI (Human machine interface), weighing machine, pH meter, autoclave, laminar air flow, shaker cum incubator, spectrophotometer, media preparation vessel, harvest vessel, CIP skid.

Ø Pre- fermentation steps
1. Prior to the first day of the fermentation process in the 40l Fermenter, the Pichia Pastoris culture inoculum was setup by inoculating 5l Fermenter with volume 3l of TSB media of pH 5.0..
2. 250 ml of 25% of antifoam (silicone oil) was prepared and autoclaved.
3. 2N NaOH (250ml) was prepared and autoclaved.
4. 20 liter of TSB media pH 5.0 prepared in a 3l concentrated form by dissolving 600g of TSB in 3 liter W.F.I. and autoclaved.
Ø Thermo siphon sterilization
1. All the utility lines were checked.
2. The mechanical seal and the thermo siphon pot were sterilized by passing steam for 20 minutes. The corresponding steam traps were opened.
3. Chilled water supply was started after closing the steam supply valve so that the condensate forms in the pot. Once the condensate was formed, the corresponding valves were opened such that the condensate reached the mechanical seal.
4. The probes (pH and DO) were attached. The calibration of pH probe was done using buffer of pH 7.0 and 4.0.
5. The 4 filters (pre-filter, surface, sparger, vent) were fixed into the respective slots and the corresponding housings were placed and tightened using TC clamps.
6. 17 L of W.F.I. from the media preparation room was transferred into the Fermenter vessel.
7. 3 liter of xxxxxx(autoclaved)xxxxxx concentrated media was poured.
Ø Perform the Pressure Hold Test
1. Adjust 2 bar pressure using the PRV and wait till the pre-filter pressure gauge attains 2 bar. Pressure hold test was performed in the surface and sparger air filter by opening the respective lines and isolating the filters. The pressure hold test was carried out from the surface filter to the vessel and then from the vessel to the exhaust filter.
2. The agitation was started at 200 RPM and the vent drain valve was opened.
Ø Draining of the jacket
1. The drain and jacket inlets were opened and closed after 2-3 min.
Ø Heating step
1. Steam was passed to the jacket and the corresponding steam trap was opened.
2. The exhaust and the 3 steam traps on the exhaust line were opened.
3. When the temperature of the vessel reached 70°C the needle valve on the exhaust line was opened for 2 minutes to remove the air traps.
4. All transfer lines and their respective steam traps were opened to remove air trap
Ø Sterilization step
1. When the temperature reached 121°C, steam was supplied to the vessel also by opening the respective valve. Steam valves and the drain valves of the surface and sparger air filters were opened to sterilize them. The sampling and the harvest port lines were also sterilized. Sterilization process was continued for 20 minutes and when the temperature shooted up, it was regulated by supplying and cutting off the steam supply into the vessel through the surface and sparger air filters.
2. Now, supply only jacket steam for 10 minutes. Close the sparger line to the Fermenter and surface line.
Ø DRying of filter
1. The surface and sparger air lines and their corresponding drain were opened for 10 minutes.

Ø Completion of sterilization
1. After total sterilization time of 30 min, the transfer lines were closed. The steam valves were also closed. The surface air valve was opened to allow air to pass through the vessel.
Ø Cooling of the fermenter
1. This was done by supplying chilled water through the open loop first. It was noted that the vessel was pressurized during this process so as to avoid collapsing of the vessel due to the sudden temperature change. When the temperature dropped down to 60ºC, the chilled water was supplied to the jacket using the closed loop.
Ø Setting of parameters
1. Aeration - 0.5 vvm (10 LPM).
2. Before agitation was started the thermo siphon was set at 2 bar pressure.
3. Temperature loop was started.
4. Condenser was switched on so that evaporation loss due to aeration is minimised.
5. Heat exchanger on the vent was switched on so that the exhaust filter does not choke.
6. Back pressure of 0.2 bar was maintained.
7. Antifoam was added into the fermenter.
8. DO probe was calibrated. The present amount of oxygen was treated as 100%.
Ø Inoculation
1. Temperature inside the vessel was brought down to a set point of 300C which was the optimum for yeast culture. The transfer ports were connected to their respective positions and were sterilized by passing steam. The inoculum was added to the vessel using a peristaltic pump and the process was started.

5 Litre Fermenter Autoclaving

* To autoclave the 5L - Fermenter.
* To inoculate the required volume of the shaker flask culture to the fermenter.
* To maintain the conditions required for the growth of Pichia pastoris in the fermenter.
* To determine the KLa of the process by dynamic gassing out method.

v Vessel made of Borosilicate Glass
v Control Panel
v DO probe
v 0.2 air filters (air-in, exhaust-out, addition bottles)
v pH probe
v Temperature probe
v Transmission lines ( Acid, Alkali & Antifoam)
v 2 Rushton turbine type impllers on a shaft mounted on a motor
v Baffle cage
v Condensor
v Sparger
v Rotameter
v Sampling line
v Chiller
v Peristaltic pumps – 3 for acid, alkali, antifoam dosing
v Inoculum transfer bottle + needle inoculation assembly + inoculum port
v Dosing bottles for above
v Silicon tubings for attachments

Working volume : 3 Litres
pH : 5.0
Temperature : 30.0oC
RPM : 200
Media: Tryptone Soya Broth [3Litres]


Prepared TSB media was added.
The lid of thefermenter was closed properly.
The pH probe was calibrated and inserted into the fermenter.
The jacket was filled with water.
KCl elctrolyte was added into the DO probe and it was inserted into the fermenter.
The condenser, acid, base and dummy ports were connected and the tubings were clamped properly.
Tubings to the sampling line and the harvest line were connected. The ends were covered with cotton dipped in alcohol and foil.
The motor shaft (dry mechanical seal portion protruding out of the vessel top plate), and DO probes were covered with cotton and foil.
After making sure that the whole assembly had been connected properly, a pressure hold test was conducted. For this, air was started in to the fermentor at a very low rate, with one end of the condensor connected to the fermenter, and the other end to a beaker containing water. As bubbles were formed, it was ensured that the connections were proper and there is no leakage.
To the inoculation bottle used for this fermenter, tubings and filter were connected.
The fermenter and all its accessories were kept in the autoclave at 121oC for 30 minutes.
After autoclaving the fermentor was removed from the autoclave and immediately filtered air was connected to the sparger and the air was started to break the vaccume build-up during the cooling and to cool the contents. Also, all the probes were connected, especially the DO probe which being a polarographic probe, needs at least six hours of polarization for proper results.
1. An aeration rate of 2 LPM was maintained in the fermenter.
2. XXXXXX(DO probe was calibrated. )XXXXXX
3. Agitator speed was set at 200 RPM, temperature at 30oC.
4. After aeration and agitation at set temperature was maintained for at least 30-60 minutes, DO probe was calibrated to 100% DO saturation.
5. Shaker flask culture was transferred to the inoculation bottle used for the fermenter under sterile conditions.
It was optimized that 600 units had to be transferred to the 5L fermenter. OD of shake flask was 7.4, so by formula:
Vol of seed transferred= 600 Units/ OD =600/7.4 = 81ml
6. Inoculation bottle was connected through the peristaltic pump to the fermenter. Connect the inoculation needle was connected onto the fermenter under sterile conditions and inoculum was tranferred.
7. The fermenter was run for 24 hours so that the culture attained the required growth before transferring to the next fermenter.
8. Care was taken to ensure that all the set conditions in the fermenter remained same throughout the run. If there was any change in the pH of the culture, acid/base was added as the case may be.

1. Aeration was stopped to the fermentor when the DO level reached 70%.

2. The DO concentration (CL) was allowed to come to a level just above the critical oxygen concentration level. Aeration was started again at this level.

3. Timer was started and the increase in DO level was noted over a specific interval of time till it reached maximum DO level, i.e. saturation oxygen concentration level.

4. Saturation oxygen concentration (C*) was noted with this medium.

5. ln ( C* - CL ) was calculated.

6. A graph was plotted between ln (C* - CL) versus time and the slope of the graph was calculated.


To inoculate the shaker flasks with the preserved freeze culture of Pichia pastoris
1. The prepared media was autoclaved.
2. Inoculated the autoclaved and cooled shake flasks media with the provided pre-inoculum culture under sterile conditions. Usually 4-5 % of the shake flask volume was inoculated into it.
3. Incubated the inoculated flasks on a shaker with the RPM and Temperature set suitable for the growth of the P.pastoris. Here, Orbital Shaker was used, with the RPM set at 200, and temperature at 30 oC.
4. Allowed the culture to grow for 24hours, before inoculating to the next fermenter, so that P.pastoris has grown to a suitable level.


1. Media suitable for the growth of Pichia pastoris was prepared. For this culture Tryptone Soya Broth (TSB, 30g/L) media was used.
2. For Shaker Flask a total of 300ml TSB media was prepared, divided into 75 * 4 ml, taken in 500ml flask to provide adequate headspace for the growth of microbes.
3. For 5l Fermenter 3l of TSB media was prepared.
4. The pH of the media was adjusted to 5.0 as this is the optimal pH for growth of Pichia pastoris.
1. 25% Silicon antifoam was prepared to control the foam formation in the 5l fermenter.
Acid & Base preparation:
1. 2N HCl & 2N NaOH were prepared to adjust the pH.


Microbial fermentation is the enzymatic decomposition and utilization of foodstuffs, particularly carbohydrates, by microbes. It has always been an important part of our lives: foods can be spoiled by microbial fermentations, foods can be made by microbial fermentations, and muscle cells use fermentation to provide us with quick responses. Fermentation is often defined as a process where cells produce energy anaerobically, or without oxygen. In general, it involves the breaking down of complex organic substances into simpler ones. Microbial or animal cells obtain energy through glycolysis, splitting a sugar molecule, and removing electrons in the process. The electrons are then passed to an organic molecule such as pyruvic acid. This results in the formation of waste products that are excreted from the cell.
Microbial fermentation is any large-scale cultivation of microbes or other single cells, occurring with or without air. In the teaching lab or at the research bench, fermentation is often demonstrated in a test tube, flask, or bottle-in volumes from a few milliliters to two liters. At the production and manufacturing level, large vessels called fermenters or bioreactors are used. A bioreactor may hold several liters to several thousand liters. Bioreactors are equipped with aeration devices as well as stirrers, and pH and temperature controls. In order to get a product from fermentation, fermentation scientists develop media and test growth conditions. Then, a scale-up must be done to reproduce the process at a large volume. During production, technicians monitor temperature, pH, and growth in the bioreactors to ensure that conditions are optimum for cell growth and product. Bioreactors are used to make products such as insulin and human growth hormone from genetically engineered microorganisms as well as products from naturally-occurring cells, such as the food additive xanthan.
For one, they don't use toxic reagents or require the addition of intermediate reagents. Microbiologists are now looking for naturally occurring microbes that produce desired chemicals. In addition, they are now capable of engineering microbes to enhance production of these chemicals. In recent years, microbial fermentations have been revolutionized by the application of genetically-engineered organisms. Many fermentations use bacteria but a growing number involve culturing mammalian cells.

Bio - Pharmaceutical Production

Biopharmaceutical production include two main processes; upstream process (USP) and down stream process (DSP). The upstream part of the bioprocess refers to the first step in which bio-molecules are produced, usually by microorganisms (bacteria, yeast, fungi, actinomycetes or animal/plant cell lines) in fermentors of various configurations. When these microorganisms produce the product in maximum concentration they are harvested and transferred to the downstream process. DSP refers to the recovery and purification of biopharmaceutical products. The methodology adopted for downstream processing depends on the nature of the end-product, its concentration, stability and the degree of purification desired. It is necessary to ensure that the above mentioned processes are carried out aseptically. Hence sterilization and filtration plays an important role in biopharmaceutical production. Biopharmaceuticals are also widely produced using mammalian cell lines in bioreactors. Animal cell culture (ACC) is the process of culture of animal cells outside the host animal (ex vivo) from which they were obtained. The process of ACC is carried out under strict laboratory conditions of asepsis, sterility and controlled environment involving temperature, gases and pressure. It should mimic the in vivo environment successfully such that the cells are capable of survival, proliferation and production of the desired product in a controlled manner.