So after knowing the basic concept as well as the applications of upstream and down stream

processes Id like to discuss about the stages of the upstream process in detail.
The Upstream process is the first step of bioprocess from early cell isolation and
cultivation, to cell banking and culture development of the cells until final harvest where the
desired quantity is reached. Since this is the early stage of bio-processing, the quality of the
product is of critical importance. Sustainability of this procedure must also be considered.

Upstream processing is usually divided into the following stages:

1. Substrate Preparation
2. Media Preparation
3. Cell Culture

I. Substrate Preparation
What is a substrate?
 The substances with which the enzymes react to are called substrates. And in biochemistry, we
can define a substrate as a substance the enzyme acts upon.
 Substrate also plays an important role in determining the growth of microorganisms, thereby
increasing the product yield. Substrate is chosen in such a way that it should provide physical
support as well as nutrients to the growing culture.
 One example is in the fermentation of yeast, the substrate the yeast acts upon is sugar to
produce ethanol and carbon dioxide.

The types of substrates that are used for ethanol production are the following:
(a) Starch containing substrate
If yeast strains are to be used, the starch must be hydrolyzed as yeast does not contain
amylases. After hydrolysis, it is supplemented with celluloses of microbial origin so as to obtain
reducing sugars. About 1 ton of starch required 1 litre of amylases and 3.5 litre of glucoamylases.
(b) Juice from sugarcane or molasses or sugar beet,
(c) Waste products from wood or processed wood - lignocellulosic biomass
(d) Corn Meal
(e) Sulphite waste-liquor, a waste left after production of paper, also contains hexose as well as
pentose sugar. The former can be microbially easily converted.

Substrate Preparation
On industrial scale, ethanol is produced by the fermentation of molasses. Molasses is the mother
liquor left after the crystallization of sugarcane juice. It is a dark colored viscous liquid. Molasses
contains about 60% fermentable sugar.
1) Dilution of molasses
Molasses is first diluted with water in 1:5 (molasses: water) ratio by volume addition of water.
2) Ammonium sulphate.
If nitrogen content of molasses is less, it is fortified with ammonium sulphate to provide adequate
supply of nitrogen to yeast. (extra nutrients added to it)
3) Addition of sulfuric acid
Fortified solution of molasses is then acidified with small quantity of sulfuric acid. Addition of
acid favours the growth of yeast but unfavours the growth of useless bacteria.
4) Fermentation
The resulting solution is received in a large tank and yeast is added to it at 35°C and kept for
2 to 3 days. During this period, enzymes zymase which are present in yeast, convert sugar into
ethyl alcohol.

II. Media Preparation

Using specific recipes required for each bioreactor stage of scale up from inoculum to
harvest. Cell culture medium is critical to cell growth, metabolism, and protein expression. It
provides for optimum pH, osmolality, and nutrients in an environment that is essential for cell
survival, growth, and expression of proteins and/or metabolites and drug-substance modalities of
interest.

Classification of Culture Media:

1. BASAL MEDIA. Basal media are those that may be used for growth (culture) of bacteria that do
not need enrichment of the media. They contain amino acids, glucose, and ions (calcium,
magnesium, potassium, sodium, and phosphate) essential for cell survival and growth
Examples: Nutrient broth, nutrient agar and peptone water. Staphylococcus grow in these media.
2. ENRICHED MEDIA. The media are enriched usually by adding blood, serum or egg. Enriched
media contain the nutrients required to support the growth of a wide variety of organisms,
including some of the more fastidious ones. Examples: Enriched media are blood agar and
Lowenstein-Jensen media. Streptococci grow in blood agar media.
3. SELECTIVE MEDIA. These media favour the growth of a particular bacterium by inhibiting the
growth of undesired bacteria and allowing growth of desirable bacteria. For example, if a
microorganism is resistant to a certain antibiotic, such as ampicillin or tetracycline, then that
antibiotic can be added to the medium in order to prevent other cells, which do not possess the
resistance, from growing. Examples: tellurite media (Tellurite inhibits the growth of most of the
throat organisms except diphtheria bacilli).
5. INDICATOR (DIFFERENTIAL) MEDIA. An indicator is included in the medium.
Differential/indicator media distinguish one microorganism type from another growing on the
same media. This type of media uses the biochemical characteristics of a microorganism growing
in the presence of specific nutrients or indicators (such as neutral red, phenol red, eosin y, or
methylene blue) added to the medium to visibly indicate the defining characteristics of a
microorganism. A particular organism causes change in the indicator, e.g. blood, neutral red,
tellurite. Examples: Blood agar and MacConkey agar are indicator media. (are used to distinguish
one microorganism type from another growing on the same media.)
6. TRANSPORT MEDIA. These media are used when specie-men cannot be cultured soon after
collection. Transport media for microorganisms are essentially buffer solutions containing
carbohydrates, peptones and other nutrients (excluding growth factors) designed to preserve the
viability of bacteria during transport without allowing them to multiply. The main purpose of using
the transport medium is to keep the sample as close as possible to its original state.
Examples: Cary-Blair medium, Amies medium, Stuart medium.
7. STORAGE MEDIA. Media used for storing the bacteria for a long period of time. Examples:
Egg saline medium, chalk cooked meat broth

Growth medium or culture medium is a gel or liquid designed to support the growth of
microorganisms or cells. There are different types of media for growing different types of
organisms or cells. One commonly used type of media is nutrient broth or agar. Some organisms,
termed fastidious organisms, require more specialized types of media. Media preparation is
usually carried out in tanks, carboys, bottles or bags to which the media is introduced. Similar to
humans, cells also require a proper nutrition to function and ultimately produce the protein product.
Therefore, media is predominantly made up of various components: carbohydrate (glucose),
nitrogen (amino acids), fats (lipids) and trace amounts of salt.
Media components are most often in powder form which is introduced to a high purity
grade of water for injection (WFI). Prior to introducing the cells to culture it is very important the
media is homogeneous and thoroughly mixed. After doing this step, the mixed solution is brought
from a bag or carboy towards the bioreactor.

Upstream Processing Areas

Dispensing room
 Materials needed by the biomanufacturing process to make product are weighed and measured
in a traceable, controlled, and sanitary area prior to their use. Optimally, raw materials are
received into the manufacturing facility in packaging which is suitable for cGMP processing
areas (e.g., glass or plastic with no cardboard or other fiber shedding materials). In cases where
larger containers of bulk raw material are received, the exact amount required for the
production batch will need to be removed from the bulk, packaged appropriately, and then
transferred to the point of use. The dispensing room is the location where raw materials for use
in the production process are weighed or measured.
 In some biomanufacturing facilities, the dispensing process can be electronic. One operator
can perform the weighing using a scale that provides electronic verification and recording of
the data. However, in many companies a manual system is still used. An operator and a
verifier perform the weighing then record the data on a paper document.
 There are certain performance characteristics required of the dispensary room and dispensary
booths (also called containment booths). The air cleanliness classification of the dispensing
room is Class 100,000 (EMA Grade C, the European classification) which is achieved using
HEPA (High Efficiency Particulate Air = 0.3 micrometer) filtration of the air supply to the
room.
 Clean rooms are classified according to the cleanliness level of the air inside the controlled
environment. The clean room class is the level of cleanliness the room complies with,
according to the quantity and size of particles per cubic meters of air.
 The key component is the High Efficiency Particulate Air (HEPA) filter that is used to trap
particles that are 0.3 micron and larger in size. “Class 100,000" denote the number of particles
of size 0.5 µm or larger permitted per cubic foot of air. ISO 8
 The HVAC (Heating, ventilation, and air conditioning system) also maintains the room at a
positive pressure with respect to the surrounding rooms and corridors (generally +0.10 inches
water column (wc), positive pressure). The positive pressure keeps the airflow in the room
blowing outward into adjacent rooms and airlocks when doors are opened. The room pressures
are continuously monitored by the building automation system so that data is always available
to demonstrate that the dispensary room is meeting the requirements needed to maintain a
controlled state. Additionally, the pressures are tied to alarms so that personnel will be notified
when the room is not meeting requirements.
 Positive Pressure: This means that the air pressure inside your cleanroom is greater than the
pressure outside of it. This is achieved by pumping clean, filtered air into the cleanroom,
generally through the ceiling. Positive pressure is used in cleanrooms where the priority is
keeping any possible germs or contaminants out of the cleanroom. In the event that there was a
leak, or a door opened, clean air would be forced out of the cleanroom, rather than unfiltered
air being allowed into the cleanroom. Negative pressure rooms, also called isolation rooms,
are a type of hospital room that keeps patients with infectious illnesses, or patients who are
susceptible to infections from others, away from other patients, visitors, and healthcare staff.
This means that when the door is opened, potentially contaminated air or other dangerous
particles from inside the room will not flow outside into non-contaminated areas.

CIP/SIP Systems
 Vessels used during the biomanufacturing process and all associated piping/hoses must be free
of any foreign substances prior to use. Foreign substances include cell debris, medium,
cleaning chemicals, and even the target protein from a prior batch. As most bioreactors are
multi-use (and may be multi-product), any substances inadvertently left behind can
contaminate the next batch. Product left behind from the previous run could encourage
microbial growth. Clean in Place (CIP) and Steam in Place (SIP) are validated cleaning and
sterilization procedures that ensure the bioreactor is safe for use.
 CIP involves automatic cleaning of processing equipment, vessels, piping, and in-line devices
with minimal manual setup and shutdown and little or no operator intervention during
cleaning. Sprayballs are used to clean the inner surfaces of the tank during CIP. Sprayballs are
located within a vessel and have precisely located holes that ensure the cleaning solutions
contact the entire interior surface of the tank.
 Chemicals used in CIP are usually strong base solutions (such as potassium hydroxide) that are
applied over all surfaces using the spray balls, followed by rinsing and the application of a
strong acid such as phosphoric acid. These substances are all rinsed away by a final spray of
high purity grade water so that no substances remain.
1. CAUSTIC
Also known as caustic soda, sodium hydroxide or NaOH. This is an alkali with a very high pH
that is typically used in a concentration range of 0.5-2.0%. Concentrations as high as 4% may be
used for highly soiled surfaces.
Application:
Typically used as the main detergent in most CIP wash cycles
Softens fats, making them easier to remove
Non-foaming formulation can help reduce pump cavitation and increase efficiency.
2. ACID
Nitric acid is the most commonly used wash for scale removal and pH stabilization after a
caustic wash. At a typical concentration of 0.5%, it can be used effectively at lower temperatures
than caustic solutions, requiring less heating.
(Phosphoric acid is sometimes used but is somewhat less common.)
Application:
Used by dairies regularly to remove milk scale or “milk stone”
Excellent for brightening up discolored stainless steel by removing calcified mineral stains
Must be used with caution because they can attack some elastomers in the system like gaskets or
valve seats causing premature degradation or failure.
3. STERILIZER
Sterilizing a system means completely eliminating all living microorganisms. Sterilization can be
done using chemicals but it is usually done with high pressure steam (approx. 250° F for 30
minutes).
 The SIP process, also referred to as 'Steam-In-Place', is an extension of the CIP process by an
additional sterilisation, without any necessity for disassembling the plant and the measuring
equipment1. The sterilisation of hygiene-critical processes takes place at the end of the actual
CIP process.

III. Cell Culture

Cell culture is the process by which cells are grown under controlled conditions, generally
outside their natural environment. All living things grow by cellular division and same applies to
cell culture, the cells divide and continue to multiply once in a suitable environment. The correct
environment consists of a growth medium as a food source and cell culture vessels for controlling
gases and temperature. During the growth stage, the medium will turn an opaque colour as the cells
density increases. The growth rate is monitored accurately to determine how well cells are growing
over particular cycles; this is carried out by taking samples and precisely counting the cells.

Bacterial growth curve

 In higher organism growth refers as increase in size and volume of organism but in bacteria
growth refers as increase in number. When fresh liquid medium is inoculated with a given
number of bacteria and incubated for sufficient period of time, it gives a characteristic growth
pattern of bacteria. If the bacterial population is measured periodically and log of number of
viable bacteria is plotted in a graph against time, it gives a characteristic growth curve which is
known as growth curve or growth cycle. The growth curve is hyperbolic due to exponential
bacterial growth pattern.
The growth curve has following phases
 Lag phase
 Log phase or exponential phase
 Stationary phase
 Death phase or decline phase

1. Lag phase: 
 When bacteria is inoculated into new fresh media, it do not divide immediately. Bacteria takes
some time to adjust to the new environment. The time period in which bacteria is
metabolically active but do not divide is called as lag phase. Size of bacteria increase
continuously so the bacteria have largest size at the end of lag phase. In this phase,
microorganism tries to adopt in new environment. It is the phase of adjustment necessary for
the synthesis of enzymes and co-enzymes for physiological activities. If the culture organism
is taken from old culture, the duration will be longer but if the culture is fresh, duration is
short. Similarly if the culture media is different from the previous culture then duration is long
because bacteria takes some more time to adjust in the new media. At the end of lag phase,
bacteria become fully prepared for cell division.

2. Log phase or exponential phase: 

 During this phase bacteria divides continuously at constant rate and the number of bacteria
increase exponentially. In this phase all bacteria are in their rapid stage of cell division and
show balanced growth. Due to rapid cell division, bacteria have smallest size in this phase.
Bacterial population is nearly uniform in terms of their metabolic activities, chemical
composition of cell and other physiological characteristics. Generation time is shortest during
log phase and is strongly dependent upon growth factors present in the medium. This phase
lasts for several hour depending on the type of organism, conditions of growth and density of
organism.

3. Stationary phase: 
 The bacteria growth reaches a state during which there is no net increase in bacterial
population. This is called as stationary phase. In this phase a constant bacterial population is
maintained by balance between cell division and cell death. In some bacteria, complete
cessation of cell division occurs hence there is no net increase or decrease in number of
bacteria. Stationary phase is induced by- increased bacterial cell density, depletion of nutrition
in media and accumulation of toxic secondary metabolic wastes. Production of antibiotics such
as Penicillin, streptomycin etc and enzymes by certain bacteria occur during stationary phase
of their growth. 

4. Death phase or decline phase: 

 In this phase, number of bacteria decrease continuously and exponentially. It is just inverse of
log phase. But the death rate is slower than growth rate. Death phase is brought about by
 various reasons, such as depletion of nutrition and accumulation of toxic wastes. Not all
bacteria die at same rate, some die faster and some are more resistant and remain viable for
longer time. Eg. Spore forming bacteria

Throughout this cycle the cells process through a number of generations, with the doubling from
the original seeding cell concentration constituting one generation. Depending on the organism,
the growth cycle can vary broadly. Figure 10-3 illustrates the phases of the growth cycle of a
cell culture.

Culture used to inoculate a fermentation satisfies the following criteria:

1. must be in a healthy, active state thus minimizing the length of the lag phase in the subsequent
fermentation.
2. must be available in sufficiently large volumes to provide an inoculum of optimum size.
3. must be in a suitable morphological form. Since shape affects critical biological functions,
including nutrient acquisition, motility, dispersion, stress resistance and interactions with other
organisms.
4. must be free of contamination. Biological contamination is caused due to the presence of living
organisms in the culture. Such organisms include easily identifiable bacteria, yeast, and molds or
hard to detect viruses, protozoa, and mycoplasmas.
5. must retain its product-forming capabilities.

Cell Culture Basic Equipments

• Cell culture hood (i.e., laminar-flow hood or biosafety cabinet):
• Incubator (humid CO2 incubator recommended)
• Water bath
• Centrifuge
• Refrigerator and freezer (–20°C)
• Cell counter (e.g., Countess® Automated Cell Counter or hemacytometer)
• Inverted microscope
• Liquid nitrogen (N2) freezer or cryostorage container
• Sterilizer (i.e., autoclave)

1. Cell Culture Hood

The major requirement of a cell culture laboratory is the need to maintain an aseptic work
area that is restricted to cell culture work. Although a separate tissue culture room is preferred, a
designated cell culture area within a larger laboratory can still be used fort sterile handling,
incubation, and storage of cell cultures, reagents, and media. The simplest and most economical
way to provide aseptic conditions is to use a cell culture hood (i.e., biosafety cabinet).

Figure 2.1. The basic layout of a cell culture hood for right-handed workers. Left-handed workers may switch the
positions of the items laid out on the work surface.
A cell culture hood should be large enough to be used by one person at a time, be easily cleanable
inside and outside, have adequate lighting, and be comfortable to use without requiring awkward
positions. Keep the work space in the cell culture hood clean and uncluttered, and keep everything
in direct line of sight. Disinfect each item placed in the cell culture hood by spraying them with
70% ethanol and wiping clean. The arrangement of items within the cell culture hood usually
adheres to the following right-handed convention, which can be modified to include additional
items used in specific applications.
• A wide, clear work space in the center with your cell culture vessels
• Pipettor in the front right, where it can be reached easily
• Reagents and media in the rear right to allow easy pipetting
• Tube rack in the rear middle holding additional reagents
• Small container in the rear left to hold liquid waste
2. Incubator
The purpose of the incubator is to provide the appropriate environment for cell growth. The
incubator should be large enough for your laboratory needs, have forcedair circulation, and should
have temperature control to within ±0.2°C. Stainless steel incubators allow easy cleaning and
provide corrosion protection, especially if humid air is required for incubation. Although the
requirement for aseptic conditions in a cell culture incubator is not as stringent as that in a cell
culture hood, frequent cleaning of the incubator is essential to avoid contamination of cell cultures.
Types of Incubators
There are two basic types of incubators, dry incubators and humid CO2 incubators. Dry incubators
are more economical, but require the cell cultures to be incubated in sealed flasks to prevent
evaporation. Placing a water dish in a dry incubator can provide some humidity, but they do not
allow precise control of atmospheric conditions in the incubator. Humid CO2 incubators are
expensive, but allow superior control of culture conditions. They can be used to incubate cells
cultured in Petri dishes or multi-well plates, which require a controlled atmosphere of high
humidity and increased CO2 tension.

3. Inverted Microscope
 The inverted Microscope has a wide stage that favors it to view specimens in glass tubes and
Petri plates and therefore, it is commonly used to study live cells, by viewing the cells from the
bottom of the cell culture apparatus.

4. Centrifuge
 This apparatus is found in most laboratories from academic to clinical to research and used to
purify cells, subcellular organelles, viruses, proteins, and nucleic acids.

5. Water Bath
 Some protocols require prewarming a liquid medium before innoculating. For example, in this
protocol for E. coli competent cell preparation. As you know, cells are placed in incubator that
has 37 oC with 5% CO2. If you do not pre-heat your media to 37oC (same temperature with
the incubator), you will create stress to cells and possibly affect their 'health'. So it is important
to prevent creating further stress.

6. Refrigerators
 For small cell culture laboratories, a domestic refrigerator (preferably one without a
autodefrost freezer) is an adequate and inexpensive piece of equipment for storing reagents
and media at 2–8°C. For larger laboratories, a cold room restricted to cell culture is more
appropriate. Make sure that the refrigerator or the cold room is cleaned regularly to avoid
contamination.
Freezers
 Most cell culture reagents can be stored at –5°C to –20°C; therefore an ultradeep freezer (i.e.,
a –80°C freezer) is optional for storing most reagents. A domestic freezer is a cheaper
alternative to a laboratory freezer. While most reagents can withstand temperature oscillations
 in an autodefrost (i.e., self-thawing) freezer, some reagents such as antibiotics and enzymes
should be stored in a freezer that does not autodefrost.

7. Liquid Nitrogen
 Systems for long-term storage of frozen cells provide a stable, low-temperature environment to
optimize the life span of the samples. Typically, lower storage temperatures enable a longer
viable storage period. Most cells need to be maintained at temperatures of –130°C or below in
order to completely halt the chemical reactions responsible for cellular degradation.

Stage Course of the fermentation

Before the culture can be used as inoculum for a large scale fermentation, it must go through
several preparation steps:
1. Inoculation.
The inoculum stage involves the thawing of a frozen vial of cells (ampoule). In brewing,
inoculation is when a yeast starter culture is introduced (pitched) into a wort, must or wash. By
inoculating with a specific yeast strain(s), that yeast will be able to grow more quickly than wild
yeasts or bacteria. The result is that the preferred yeast characteristics will dominate over any wild
yeasts that could have infected the wort/must/wash. The inoculum stage is comparable to the
childhood stage in the production lifecycle.
The cells are given specific conditions that promote the multiplication of those cells. Once
thawed, the cells are added to prepared media and the inoculum phase of the process begins.
Depending on the type of cells, this may take a few hours or a few weeks to reach the required
volume and cell density volume expansion. Bacteria can also be used in inoculations. An example
of this is the use of muck or dunder in Rum or Lactobacillus in whiskey.
There are two common approaches to inoculating plates for viable counts: the pour plate
and the spread plate methods which I’ll be discussing later on.
2. Inoculum build-up/activation: 1-2 shake flask cultures (100-500 mL)
Inoculum build up is the preparation of the seed culture in amounts sufficient to be used in
the large fermenter vessel. This involves growing the microorganisms obtained from the pure stock
culture in several consecutive flask cultures. Inoculum built up in a number of stages usually 2-3
stages in a shake or spinner flasks to produce sufficient biomass to inoculate to the production
stage fermenter.
3. Prefermenter: 1-3 prefermenter cultures
Culture that has satisfied all criteria for a good inoculum must be transferred to shake flasks
for culture activation. Before it can be used in a production fermenter, the activated inoculum must
be gradually upscaled in prefermenters. This process cuts down the time required for the growth of
microorganisms in the fermenter, thereby increasing the rate of productivity. Then the seed culture
obtained through this process is used to inoculate the fermentation medium.

4. Final fermenter
After all steps are done without contamination, the inoculum is ready to be introduced in a
final fermenter.
 Innoculation: Introduction of the cells to media to initiate cell expansion.
 Cell expansion: Increasing the amount of cells by scaling up the process.
 Cell Bank: Cell banking systems are set up to assure that a uniform population of cells is
preserved, their integrity is maintained, and a sufficient supply of material is introduced. Cell
banking refers to the collection, processing, and storage of specific cell lines, especially stem
cells, with the capacity to provide life-saving biopharmaceutical products. Cell banking offers
a reproducible way to manufacture high-quality, safe, and efficient biological products that
have undergone proper characterization. Processes involved in cell banking are safe, non-
invasive and risk free.

Inoculum preservation
There are two common approaches to inoculating plates for viable counts: the pour plate
and the spread plate methods. Although the final inoculation procedure differs between these two
methods, they both start with a serial dilution of the culture.
Serial Dilution
The serial dilution of a culture is an important first step before proceeding to either the pour plate
or spread plate method. The goal of the serial dilution process is to obtain plates with (Colony-
forming unit) CFUs in the range of 30–300, and the process usually involves several dilutions in
multiples of 10 to simplify calculation. The number of serial dilutions is chosen according to a
preliminary estimate of the culture density. Figure 6 illustrates the serial dilution method.

Figure 6. Serial dilution involves diluting a fixed volume of cells mixed with dilution solution
using the previous dilution as an inoculum. The result is dilution of the original culture by an
exponentially growing factor.
A fixed volume of the original culture, 1.0 ml, is added to and thoroughly mixed with the first
dilution tube solution, which contains 9.0 ml of sterile broth. Sterile conditions require the
complete absence of microorganisms including bacteria, fungus, and their spores. This step
represents a dilution factor of 10, or 1:10, compared with the original culture. From this first
dilution, the same volume, 1.0 ml, is withdrawn and mixed with a fresh tube of 9.0 ml of dilution
solution. The dilution factor is now 1:100 compared with the original culture. This process
continues until a series of dilutions is produced that will bracket the desired cell concentration for
accurate counting.
From each tube, a sample is plated on solid medium using either the pour plate method
(Figure 7) or the spread plate method (Figure 8). The plates are incubated until colonies appear.
Two to three plates are usually prepared from each dilution and the numbers of colonies counted
on each plate are averaged. In all cases, thorough mixing of samples with the dilution medium (to
ensure the cell distribution in the tube is random) is paramount to obtaining reliable results.

Figure 7. In the pour plate method of cell counting, the sample is mixed in liquid warm agar (45–
50 °C) poured into a sterile Petri dish and further mixed by swirling. This process is repeated for
each serial dilution prepared. The resulting colonies are counted and provide an estimate of the
number of cells in the original volume sampled.

Figure 8. In the spread plate method of cell counting, the sample is poured onto solid agar and then
spread using a sterile spreader. This process is repeated for each serial dilution prepared. The
resulting colonies are counted and provide an estimate of the number of cells in the original
volume samples.
Contamination
Contamination of cell cultures is easily the most common problem encountered in cell culture
laboratories, sometimes with very serious consequences. While it is impossible to eliminate
contamination entirely, it is possible to reduce its frequency and seriousness by gaining a thorough
understanding of their sources and by following good aseptic technique.
During Cell Culture preventive measures must be taken to ensure that contaminating organisms do
not get into the product. Any time a sample is taken from a bioreactor or whenever a container or
vessel is opened, contamination is possible. During inoculum, aseptic technique is critical to
prevent contamination of the culture. Preventive measures include ensuring proper cleaning of all
materials, proper gowning (e.g., gloves, face masks, hair nets, etc.), and using proper aseptic
techniques in the BSC. Figure 10-7 demonstrates bacterial growth from a single human hair.

Sterilization
Sterilization is essential for preventing the contamination with any undesired microorganisms. Air
is sterilized by membrane filtration while the medium is usually heat sterilized.
 Air sterilization is the physical removal of microorganisms from the air by filters of
appropriate retention efficiency. The selection of filters for air sterilization must account for
the size range of the contaminants to be excluded. Filtration of air is accomplished using high-
efficiency particulate air (HEPA) filters designed to remove organisms larger than 0.3 μm
from isolation rooms, operating rooms, and biological safety cabinets.
Any nutrient component which is heat labile is filter-sterilized and later added to the sterilized
medium. The fermenter may be sterilized together with the medium or separately.
 Filtration is the preferred method of sterilizing heat sensitive liquid and gases without
exposure to denaturing heat. Rather than destroying contaminating microorganisms, it simply
removes them. It is the method of choice for sterilizing antibiotic solutions, toxic chemicals,
radioisotopes, vaccines, and carbohydrates, which are all heat-sensitive. The liquid or gas is
passed through a filter, a device with pores too small for the passage of microorganisms, but
large enough to allow the passage of the liquid or gas. These filters are made of different
materials; Working Mechanism of Filtration Sterilization
 Filters work by physically trapping particles larger than the pore size and by retaining
somewhat smaller particles via electrostatic attraction of the particles to the filters. Besides
porosity, other factors also influence the efficiency of filtration, they are:
 electric charge of the filter
 electric charge carried by the organisms
 nature of the fluid being filtered
 Filtration of liquids is accomplished either by pulling the solution through a cellulose acetate
or cellulose nitrate membrane with a vacuum (i.e, by applying negative pressure in the filter
paper) or by forcing the solution through filter paper by imposing positive pressure above the
fluid.
There are two reliable methods used to sterilize microbial culture media:
1. autoclave
2. Dry heat oven
 Always sterilize any reagents, media, or solutions prepared in the laboratory using the
appropriate sterilization procedure (e.g., autoclave, sterile filter). Of all the methods available
for sterilization (killing or removal of all microorganisms, including bacterial spores), moist
heat in the form of saturated steam under pressure is the most widely used and the most
dependable method. Moist heat has better penetrating power than dry heat and, at a given
temperature, produces a faster reduction in the number of living organisms. Steam sterilization
is nontoxic, inexpensive, rapidly microbicidal, and sporicidal. It rapidly heats and penetrates
fabrics. Moist heat sterilization using autoclave is commonly used for the sterilization of
biohazardous trash, heat, and moisture resistant materials such as aqueous preparation (culture
media). This method is also used for the sterilization of surgical dressings and medical
devices. The recommendation for sterilization in an autoclave is 15 minutes at 121°C (200
kPa). The temperature should be used to control and monitor the process; the pressure is
mainly used to obtain the required steam temperature. When using an autoclave, use the “wet”
 setting for sterilizing liquids (flasks, bottles, culture tubes, etc), and use the “dry” setting when
sterilizing empty containers, stoppers, etc.
 Dry heat sterilization (killing or removal of all microorganisms, including bacterial spores)
technique requires a longer exposure time (1.5 to 3 hours) and higher temperatures than moist
heat sterilization. Various available methods of dry heat sterilization are; hot air oven,
incineration, flaming (wire loop), etc. Dry heat ovens are used to sterilize items that might be
damaged by moist heat or that are impenetrable to moist heat (e.g., powders, petroleum
products, sharp instruments). The most common time-temperature relationships for
sterilization with hot air sterilizers are:
 170°C (340°F) for 30 minutes,
 160°C (320°F) for 60 minutes, and
 150°C (300°F) for 150 minutes or longer depending up the volume.
Advantages of dry heat sterilization
 A dry heat cabinet is easy to install and has relatively low operating costs;
 It penetrates materials
 It is nontoxic and does not harm the environment;
 And it is noncorrosive for metal and sharp instruments.
Disadvantages for dry heat sterilization
 Time consuming method because of slow rate of heat penetration and microbial killing.
 High temperatures are not suitable for most materials e.g. plastic and rubber items cannot be
dry-heat sterilized because temperatures used (160–170°C) are too high for these materials.
 The time and temperature required will vary for different substances and overexposure may
ruin some substances.

Role of pH in cell culture

To achieve optimal cell culture conditions and maximize product yield, pH must be controlled
during the processing. pH can affect growth and is referred to as a “critical process parameter.”
pH will not remain stable for a long period of time in an actively growing culture. The media
generally contains bicarbonate, which when combined with the 5% CO2 in the incubator air
makes a buffering system to control the pH of the culture (Figure 10-11)

Figure 10-11.

In the bioreactor pH is measured by an in-line pH probe and controlled by an associated pH

transmitter via the bioreactor controls system. Figure 10-12 depicts an in-line pH transmitter
and probe. The probe is inserted into the bioreactor and is connected via a cable.
 Figure 10-12. Combination Dissolved Oxygen (DO) and pH probe transmitter
The optimal pH is between 6.8 to 7.4
At the incubator: Culture media contains bicarbonate which, when combined with CO2
infused into the culture incubator makes a buffering system to control pH.
At the bioreactor: pH is measured bu inline probe and bioreactor control system.
pH of the media decreases during cell growth as cell metabolize glucose in the presence of O2
Alkaline solutions such as NaCO3 and NaOH are added to increase the pH.

Optimal pH increases enzyme rate of reaction while less than optimal pH decreases it. Increasing
temperature also increases enzyme rate of reaction, until things get too hot, then the enzyme
denatures and ceases to function. Denaturing an enzyme essentially destroys it.

Maintaining and Monitoring Culture

Cell counts are important for monitoring cell health and proliferation rate, assessing
immortalization or transformation, seeding cells for subsequent experiments, transfection or
infection, and preparing for cell-based assays.

Yeasts are an economically important organism used for ethanol production, in the beverage and
alternative fuels industries as well as a leavening agent in the baking industry. Concentration and
viability determinations are routinely performed for quality control purposes in yeast production,
fermentation processes, and fungicides research to monitor proliferation of pathogenic yeasts.
1. Manual Cell Counting with Hemocytometer

A device used for determining the number of cells per unit volume of a suspension is called a
counting chamber. The most widely used type of chamber is called a hemocytometer, since it was
originally designed for performing blood cell counts. Place the hemacytometer under a
microscope with a typical magnification of 100.

2. Automated cell counters were designed to be a faster, easier, automated alternative to manual
counting. They use the same principles of operation as hemocytometers; they perform multiple
counts of cells within a known area and average out the results. They also can discern live cells
from dead cells using dye exclusion methods (such as trypan blue). Automated cell counters may
either operate as standalone devices or require a connection to a computer. In addition to a cell
count, most counters also provide statistical information on cell size
1. Osmotic shock or osmotic stress is physiologic dysfunction caused by a sudden
change in the solute concentration around a cell, which causes a rapid change in the
movement of water across its cell membrane.
In this technology, cells are first exposed to either high or low salt
concentration. Then the conditions are quickly changed to opposite conditions which
leads to osmotic pressure and cell lysis (figure 6). The reason for that is that water
quickly flows from low salt concentration conditions towards conditions with high
salt concentration. Thus, if the cells are first exposed to high salt concentration
solution, water flows into cell after exposure to low salt concentration. As a result,
pressure in cell increases and cell explodes (Stanbury et al. 2016). Conversely, if cell
are exposed to high salt concentration (~1 molar solution) after exposure to low
concentration, water flows out of the cell which leads to cell disruption.
Through the process of osmosis, water can be moved into the cell causing its
volume to increase to the point that it bursts. The method however, can only work
with animal cells and protozoa, since they do not have cell walls.

Hypotonic environment means having a lower osmotic pressure than a particular

fluid, typically a body fluid or intracellular fluid. Which causes the cell to swell.
Disadvantages:
Osmotic shock is not commonly used method for cell disruption because of its
low efficiency.This is not always effective so it is used in combination with other
process. The efficient disruption would commonly require for example enzymatic pre-
treatment to weaken the cells. In addition, this technology requires addition of high
amounts of salts and water usage is high. Also product may be diluted which
increases downstream processing costs.

2. Sonication
Sonication is a class of physical disruption commonly used to break open cells. The
method uses pulsed, high frequency sound waves to agitate and lyse cells, bacteria,
spores and finely diced tissue. The sound waves are delivered using an apparatus with
a vibrating probe that is immersed in the liquid cell suspension. Mechanical energy
from the probe initiates the formation of microscopic vapor bubbles that form
momentarily and implode, causing shock waves to radiate through a sample. To
prevent excessive heating, ultrasonic treatment is applied in multiple short bursts to a
sample immersed in an ice bath. Sonication is best suited for volumes <100mL.

Sonication can be very effective in small scale work; however, upscaling is very poor.
It has high energy requirements and heat generation, as well as high health and safety
issues, due to noise. It is an expensive process. And can generate free radicals that
might interfere desired product.

3. Thermolysis
Used to disrupt the bonds within cell walls, and also to denature proteins. Thermolysis
has shown potential in becoming more common in large scale production. Breakage
of cells by subjecting them to heat is relatively easy and cheap. But this technique
can be used only for a very few heat-stable intracellular products.
 Periplasmic proteins in G(-) bacteria are released when the cells are heated up to
50ºC.
 Cytoplasmic proteins can be released from E.coli within 10min at 90 ºC.
 This method is very easy and economical for heat stable product.
 High heat inactivates cell by disrupting cell wall and release intracellular
products.
 Improved protein release has been obtained after short high temperature
shocks, than when at longer temperature exposures at lower values. Unfortunately, the
results are highly unreliable, as the protein solubility changes with temperature
fluctuations. And uncontrolled amount of heat can easily denature or damage target
proteins and subtances.
Disadvantage:
Cannot be used for heat labile substances
Spore forming bacteria are also resistant to this method.

4. High Pressure homogenizer

High pressure homogenization is a mechanical process that works to reduce
particle size or to lyse cells. The higher the amount of energy applied during the
homogenization process, the smaller the particle size or the more complete the cell
lysis. In a French press, or high pressure homogenization, the cell suspension is drawn
through a valve into a pump cylinder (figure 4). Then it is forced under pressure of up
to 1500 bar, through a narrow annular gap and discharge valve, where the pressure
drops to atmospheric. Cell disruption is achieved due to the sudden drop in pressure
upon the discharge, causing the cells to explode. This method is one of the most
widely known and used methods. It is mostly used for yeast cells. It is a vital unit in
the dairy production industry, for milk homogenization. Homogenisers can vary in
design and has a high amount of solids, up to 50% of the feed. Heat generation is also
high – 1.5ºC/1000 psi.
Has several possible applications in the food sector and pharmaceutical industry. It is
commonly used for small scale recovery of intracellular materials from bacterial and
plant cell.

5. Impingement
In this procedure, a stream of suspended cells at high velocity and pressure are forced
to hit either a stationary surface or a second stream of suspended cells (impinge
literally means to strike or hit). The cells are disrupted by the forces created at the
point of contact. Micro fluidizer is a device developed based on the principle of
impingement. It has been successfully used for breaking E. coli cells. The advantage
with impingement technique is that it can be effectively used for disrupting cells even
at a low concentration.

6. GRINDING WITH GLASS BEADS

Bead mills have been originally used in the paint industry, and have been adapted for
cell disruption in both small scale and large scale production. It is an efficient way
of disrupting different microbial cells as different designs have been developed. The
main principle requires a jacketed grinding chamber with a rotating shaft, running in
its center. Agitators are fitted with the shaft, and provide kinetic energy to the small
beads that are present in the chamber. That makes the beads collide with each other.
The choice of bead size and weight is greatly dependent on the type of cells. The
diameter can affect the efficiency of cell disruption in relation of the location of the
desired enzyme in the cell. The increased number of beads increases the degree of
disruption, due to the increased bead-to-bead interaction. The increased number of
beads, however, also affects the heating and power consumption. An optimal
condition for bead load is considered between 80 and 85% of the free volume. The
discs run at a speed of 1500-2250 rpm. Glass beads with a diameter greater than 0.5
mm are considered best for yeast cells, and diameter lesser than 0.5 mm is optimal for
bacterial cells.
Advantages:
It is very useful for small sized materials and it doesn’t release harmful aerosols.
Bead mill can be carried out in both batch and continuous fashion.
Commonly used for disruption of yeast cells and for grinding animal tissues.
Disadvantages:
It produces large amount of heat so thermolabile materials cannot be disrupted. Main
issues related to bead mills, are the high temperature rises with increase of bead
volume, poor scale-up, and most importantly, there is a high chance of contamination.