MICROBIAL CELL CULTIVATIONS
- Microorganisms are used in the production of pharmaceuticals,
industrial chemicals, enzymes, recombinant DNA technologies,
▪ All living organisms, classified as animal kingdom, plant
kingdom, (multicellular) and protist (Unicellular), (Haekel,1866).
▪ Protist, simple biological organism including algae, protozoa,
fungi, and bacteria.
▪ Unit structure: prokaryotes and eukaryotes.
▪ Prokaryotic cell: bacteria and blue-green algae, has two internal
regions: cytoplasm and nuclear region (nucleoplasm)
- is surrounded with a cell wall and a cell membrane.
▪ Cell wall, protects the cell from external influences.
▪ Cell membrane (cytoplasmic membrane), a selective barrier
between the cell interior & the external environment
- serves as the surface onto which other cell substances attached
▪ Cytoplasm has grainy dark spots (ribosomes: contains protein &
ribonucleic acid (RNA)
▪ Ribosome is the site for protein
▪ Nuclear region contains DNA (genetic
information that determines the
production of proteins & other cellular
▪ DNA fragments & low molecular
weight proteins – largest molecules to
cross the cell membrane.
▪ Eukaryotic cell: plants, animals, protozoa, fungi, and algae.
- has internal unit membrane systems that segregate the functional
components of the cell.
- 1,000 to 10,000 times larger/complex than prokaryotic cells.
◦ Nucleus - surrounded by a double membrane with pores 40 - 70μm
wide, containing chromosomes.
- controls hereditary properties and all vital activities of the cell.
◦ Chromosomes - long, threadlike bodies found in the nuclei of cells,
which contain the genes arranged in nucleoproteins.
◦ Cytoplasm contains ribosome (large numbers of granules,
concentrated along the rough surface of the endoplasmic reticulum).
◦ Endoplasmic reticulum - an irregular network of interconnected
◦ Mitochondria - contain the electron transport enzymes that utilize
oxygen in the process of energy generation.
◦ Vacuole & lysosome - organelles that serve to isolate various
chemical reactions in a cell.
Microbial Nomenclature: binomial system
word: species name
Bacillus: a small rod
Lactobacillus: a small milk rod
Micrococcus: a small grainClostridium. a small spindle
Pasteurella: after Louis Pasteur, Latinized
Salmonella: after Daniel E. Salmon, Latinized
Saccharomyces: sugar fungus
Bacteria - unicellular microscopic organisms
- 1,500 known species occurring in all natural environments.
- typical cell diameter: 0.5 to 1μm.
◦ Shapes: cocci: spherical or ovoid
bacilli: cylindrical or rod shaped
spirilla: helically coiled
◦ Growth Pattern: binary fission, asexual reproductive process.
- involves cell elongation, cell wall
invagination, distribution of nuclear
material, transverse cell wall
formation, distribution of cellular
material into two cells, and
separation into two new cells.
◦ Nutritional Req’ts: actively
growing bacterial cells are 90%
- phototrophs: organisms that capable of employing radiant energy.
- chemotrophs: organisms that obtain energy for their activities and
self-synthesis from chemical reactions that can occur in the dark.
- autotrophs: organisms that use CO2 or CO3` as a carbon source.
- heterotrophs: organisms which cannot use CO2 but glucose or
amino acids as a sole carbon source.
◦ Physical Conditions: temperature, gaseous environment, pH
- the rate of microbial growth is temperature dependent.
- Types of bacteria with respect to temperature: psychrophiles (-7 to
35°C: opt. temp, 20-30°C), mesophiles (7 to 45°C: opt. temp, 30-
40°C), or thermophiles (40 to 75°C: opt. temp, 45-60°C)
- Types of bacteria with respect to O2: Aerobic bacteria (presence of
free oxygen), anaerobic bacteria (absence of free oxygen),
Facultatively anaerobic bacteria (either absence or presence of free
oxygen), Microaerophilic bacteria (presence of minute quantities of
- optimum pH for bacterial growth: 6.5 - 7.5
◦ Gram Reaction: Gram staining, differential staining technique that
separates bacteria into gram-positive (purple) & gram-negative (red).
▪ Fungi - plants devoid of chlorophyll & are unable to synthesize
their own food. (yeast & molds)
- Chlorophyll, a complex molecule which adsorbs visible light
◦ Yeasts: are unicellular organisms with spherical shape, 1-5 μm in
width and 5-30 μm in length. (budding, its growth pattern)
- Saccharomyces cerevisiae, used in the manufacture of wine & beer
and in the leavening of bread.
◦ Molds, a filamentous fungi (single cell ─ hyphae ─ mycelium)
- Imp. Classes: Aspergillus and Penicillium
▪ Culture Media: cultivation, growth of microbial population in
◦ Pure culture - culture that contains only one kind of
◦ Mixed culture - contains more than one kind of microorganism.
Steps for cultivating microorganisms:
(a) culture medium preparation, (b) sterilization, (c) inoculation
◦ Types of culture media:
- natural media (empirical/complex), used on the basis of
- synthetic media (chemically defined), consist of dilute, reproducible
solutions of chemically pure, in/organic compounds.
◦ Sterilization: moist heat (steam under pressure) in an autoclave
- @15 psi & 121°C (15-20 mins for liquid media).
◦ Inoculation - seeding of a culture vessel with the microbial material
ANIMAL CELL CULTIVATIONS
- mammalian cell culture technique employed to produce human
growth hormones, interferon, plasminogen activator, viral vaccines,
and monoclonal antibodies, extracted from human cadavers.
- post-translational modifications: involve proteolytic cleavage,
subunit association, or a variety of additional reactions
(glycosylation, can protect proteins against proteolytic breakdown,
maintain structural stability, & alter antigenicity).
Animal Cells - are eukaryotic cells.
- bound together by intercellular material to form tissue.
◦ Tissue: epithelium, connective tissue, muscle, & nerve.
- Epithelial tissue forms the covering or lining of all free body
surfaces, both external and internal.
- Connective tissue, where cells are embedded in an extensive
intercellular matrix as either be solid, semisolid, & liquid.
- Muscle cells are elongate & bound together into sheets or bundles
by connective tissue.
- Muscles are responsible for most movement in higher animals
- Nerve cells, composed of a cell body (fibers, long thin extensions
& nucleus), transmit impulses very rapidly.
▪ Suspension Cells: are nonanchorage-dependent cells from blood or
lymph fluids that do not require a surface to grow on.
- lymphocytes, derived from the lymphoid tissue & spherical.
▪ Anchorage-Dependent Cells: require a wettable surface for
attachment and growth.(epithelial or fibroblast, widely used)
- microcarriers: microscopically small beads
Monoclonal Antibodies (identical antibody)
- can be analyzed by the solid-phase assay techniques (ELISA,
enzyme linked immunosorbent assays; RIA, radioimmuno assays).
- antibodies can bind to the antigens to eliminate foreign substances
◦ 5 classes of antibodies (immunoglobulin, Ig): G, A, M, D, and E
- Ig G, has a Y-shaped protein structure comprised of one pair of
heavy chain and light chain polypeptides, linked by disulfide bonds.
- antibody-secreting cells cannot be maintained in a culture medium.
- myeloma cells, malignant tumor cells of the immune system.
- hybridoma, the fused cell of B-Iymphocytes with myeloma cells.
PLANT CELL CULTIVATIONS
◦ Secondary metabolites - serve as a chemical interface between the
producing plants and their surrounding environment.
- The technical rationale for using plant tissue is based on the unique
biochemical totipotency (ability to generate or regenerate a whole
organism from a part) of plant cells.
◦ Primary metabolites, produced in larger quantities than the
◦ size : 20-40μm
- its structure
similar to that of typical eukaryotic cells
- features: rigid wall, large vacuole, and presence of chloroplasts.
- surrounded by a cell wall (determines many features of the
◦ middle lamella - outer layer of the cell wall (carbohydrate in
nature), contains heavy layer of pectin (serves as the glue to hold
one plant cell to an adjacent cell).
◦ cell membrane (cytoplasmic membrane) - inner layer of the wall,
thin & flexible & composed of protein and lipid.
- regulates the movement of substances into and out of the cell
◦ vacuole serves as a receptacle for waste metabolic products or
secondary plant substances.
- occupies 90 percent of the volume of the cell for a mature plant
- is surrounded by a plasma membrane.
- component: water, which contains dissolved solutes,
◦ Cytosol - the liquid around all of the floating structures between
the nucleus and cell membrane.
◦ Chloroplast - site of photosynthesis in the plant cell, w/c contains
the green pigment chlorophyll (responsible for trapping the light for
the production of carbohydrates)
◦ Nucleus - cell's control center and contains DNA for the protein
translation and transcription.
◦ Endoplasmic reticulum – is a network of tiny tubes that
interconnect the different parts of the cells.
◦ Ribosome - is concentrated on the surface of endoplasmic
reticulum, which involved in the synthesis of proteins
◦ Mitochondrion contains genetic material and enzymes important
for cell metabolism.
▪ Plant Tissue Culture - refer only to those of unorganized aggregates
of cells - Types: Unorganized growth & organized growth
- Unorganized growth, occurs frequently when pieces of whole
plants are cultured in vitro.(Callus cultures, amorphous cell
aggregates; Suspension cultures, cell aggregates growing dispersed
in liquid medium; Protoplast cultures, growth of protoplasts in solid
or liquid medium).
- Organized growth, occurs when organized plant parts are
transferred to culture medium with their structure preserved. (Root
cultures, root tips taken from plants; Embryo cultures, embryos
removed from sterilized seeds, ovules, or fruits)
-- somatic embryos, embryos produced from cell culture technique.
Secondary Metabolite – classified into 3 major categories: alkaloids
(crystalline, nitrogen-containing compds. which can be extracted by
use of acidic solutions), essential oils (mixtures of terpenoids), and
glycosides (phenolics, tannin, saponins, dye, etc.)
- Biotic elicitors are compounds or substances of microbial origin
- Abiotic elicitors are stress agents such as UV irradiation
CELL GROWTH MEASUREMENT
◦ Balanced growth - growth during which a doubling of the biomass
is accompanied by a doubling of all other measurable properties of
the population such as protein, DNA, RNA, and intracellular water.
- Types of chambers for counting cell number: hemocytometer, a
blood cell counting chamber with organisms of 3μm in diameter;
Petroff-Hausser counting chamber, for use primarily with bacteria.
Viable Plate Count:
◦ Viable cell - able to divide and form a colony.
◦ 2 ways of performing a plate count: spread plate method, a volume
of no larger than 0.1 mL is spread over the agar surface; pour plate
method, sample is mixed w/ melted agar & pour into a sterile plate.
Measurement of Cell Mass
- absorbency is defined as the logarithm of the ratio of the intensity
of light striking the suspension to that transmitted by the suspension
- encouraging production of high yields of secondary metabolites
- can protect cells & decrease problems related to shear forces.