Cell cultivation & measurements

• Cultivation  the growth of microbial

population in artificial environments
• The first stage in the screening for
microorganism of potential industrial
application is their isolation.
• Isolation (pure or mixed cultures)
followed by the assessment to de­
termine which carry out the desired
reaction or product.
• A pure culture a culture that
contains only one kind of
microorganism
• A mixed culture is one that contains
more than one kind of microorganism.
• important criteria in the choice of organism:
1. The nutritional characteristics of the organism.
Natural medium or a pre-determined one
2. The optimum conditions of the organism  pH, T, nutrisions, gas
3. The reaction of the organism with the equipment to be employed
4. The suitability of the organism to the type of process to be used.
5. The stability of the organism and its amenability to genetic manipulation.
6. The productivity of the organism; ability to convert substrate into product
and to give a high yield of product per unit time.
7. The ease of product recovery from the culture.

• Isolation method  enrichment culture

• Enrichment culture is a technique resulting in an increase in the number
of organism relative to the number of other type of original inoculum
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• To cultivate microorganisms, culture medium has to be prepared in one of
the commonly employed culture vessels: a test tube, a flask, a Petri dish,
or a fermenter.
• Inoculation is the seeding of a culture vessel with the microbial material
(inoculum).
• The inoculum is introduced with a metal wire or loop which is rapidly
sterilized just before its use by heating it in a flame.
• Transfers of liquid culture are often made by using a sterilized pipette.
• The inoculation is usually done in a laminar flow hood to minimize the risk
of contamination
• The necessary steps for cultivating microorganisms are:
1. Preparing a culture medium in which a microorganism can
grow best.
2. Sterilizing in order to eliminate all living organisms in the
vessel.
3. Inoculating the microorganism in the prepared medium.
 CELL GROWTH
• growth increase of all chemical components: increasing in size
and/ number
• Increase of mass might not really reflect growth because the cells
could be simply increasing their size
• balanced growth growth during which a doubling of the biomass is
accompanied by a doubling of all other properties (e.g. protein, DNA,
RNA)
• In an adequate medium to which they have become adapted,
inoculum are in a state of balanced growth
• Cell growth can be determined by measuring cell number, cell mass,
or cell activity.
 The measurement methods
• Cell number • Indirect methods
1. Microscopic count (2) 1. Nutrient consumption
2. viable plate count (2) 2. Product formation
3. Coulter counter 3. Cell components
4. Heat evolution
• Cell mass 5. Viscosity
1. Cell dry weight
2. turbidity
 Cell numbers
Microscopic Count
• measured under a microscope by counting cells placed in
special counting chambers.
• Very dense suspensions can be counted if they are diluted
appropriately
• Two types of chambers used (liquid samples):
1. hemocytometer. a blood cell counting chamber (> 3um).
2. Petroff-Hausser counting chamber, for use primarily with
bacteria.
hemacytometer
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Petroff-Hausser counting chamber
• The (+) are: • The disadvantages are:
1. Dead cells cannot usually be
1. Minimal equipment is
distinguished
required.
2. not suitable for cell suspensions
2. Results are obtained of low density.
rapidly. 3. Small cells are difficult to see
3. The morphological under the microscope and can
characteristics of the be missed when counting.
organisms can be 4. The actual counting procedure is
observed. tiresome and may cause
eyestrain.
5. not suitable for mycelium.

• A viable cell is defined as
one that is able to divide &
form a colony.
• Two methods: count-the
spread plate method and
pour plate method.
• The spread plate method: a
volume of no larger than 0.1
mL is spread over the agar
surface.
Viable plate count
• The pour plate method: the
sample is mixed with
melted agar and poured
into a sterile plate.
• The plate is incubated until
the colonies appear 
counted.
• It is important that the
number of colonies should
be neither too large nor too
small diluted
 Coulter counter
• not only the cell number, but the cell
size can be measured
• A typical Coulter counter has one or
more microchannels that separate
two chambers containing electrolyte
solutions. As fluid containing
particles or cells is drawn through
each microchannel, each particle
causes a brief change to the electrical
resistance of the liquid. The counter
detects these changes in electrical
resistance
• The disadvantage:
1. it cannot distinguish between cells
and impurity.
2. Difficult to use with organisms in
chains and is useless with mycelial
 Measurement of Cell Mass:
cell dry weight
• Cell Dry Weight: directly by • the most direct approach and
taking an aliquot of cell is probably the most reliable
suspension and centrifuging it and reproducible  for the
supernatant is discarded, quantitative of a cell mass
the cells are thoroughly • (-) time consuming and
washed with distilled water to relatively insensitive to small
eliminate all soluble matter.
changes of the cell mass.
• The suspension is
• can only be used with dense
recentrifuged and the settled
cell suspensions, and the cells
cells are dried in an oven and
must be completely free of all
weighed.
extraneous matter
 Turbidity
• Cell measured optically by
determining the amount of light
scattered by a suspension of
cells. • Such measurements are usually
• The technique is based on the made in a spectrophotometer,
fact that small particles scatter which reads in absorbency (A)
light proportionally, within units.
certain limits, to their
concentration.
• When a beam of light is passed • A calibration curve can be
through a suspension of obtained by measuring the
organisms, the reduction in the
amount of light transmitted as a absorbance of the samples with
consequence of scattering is known cell concentration
thus a measure of the cell • Usually at 600-700 nm.
density.
 The indirect methods
• based on the overall stoichiometry for growth and product
formation, which may be written in the general form:

• The change of the cell mass can be monitored indirectly by

measuring: nutrient consumption, product formation, cell
components, heat evolution, or other physical properties of
broth.
1. Nutrient consumption
• necessary to choose a nutrient which is not likely to be used to
synthesize a metabolic product.
• i.e. : Phosphate, sulfate, or magnesium
• When cell mass is the major product, the concentration of a carbon
source can be measured to estimate the cell mass

2. Product Formation:
• the product formed is growth associated.
• Some products are formed after cell mass reaches the stationary
phase of the growth cycle and, therefore, are not growth
associated.
• The evolution of carbon dioxide can be measured
3. Cell Components:
• For cultures undergoing balanced growth, the macro-
molecular cell components such as protein, RNA, and DNA can
be measured instead of cell mass.
• However, care is needed because the proportion of these
materials in a cell can change with time if the culture does not
undergo balanced growth
4. Heat Evolution
• Cell growth evolves heat
• The heat of combustion of organisms is fairly constant with a
typical value of 5 kcal/g.
• The amount of heat evolved depends on the efficiency of the
carbon energy utilization. Therefore, the measurement of the heat
of fermentation can be related indirectly to cell growth.
• However, the total heat accumulated in a fermentation system is
the combined effect of various sources of heat generation and
disappearance such as the heat from agitation and evaporation, the
heat dissipated to the surroundings through the fermenter walls,
and the sensible heat in the air stream.
• Therefore, to measure the growth by heat evolution, a complete
energy balance of a fermentation system has to be made, which
may not be an easy task
5. Viscosity
• Mycelial growth or polysaccharide formation increase the
viscosity of the fermentation broth.
• Therefore, the measurement of broth viscosity is useful in
many commercial fermentations.
• Frequently, the fermentation broth shows non-Newtonian
behavior, which makes the measurement of actual viscosity
very complicated.