Cell CultivationsMICROBIAL CELL CULTIVATIONS- Microorganisms are used in the production of pharmaceuticals, industrial chemicals, enzymes, recombinant DNA technologies, etc.Microbial Cells▪ 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 synthesis.▪ Nuclear region contains DNA (genetic information that determines the production of proteins & other cellular substances/structures).▪ 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 membrane-delimited channels.◦ 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- 1st word: genus- 2nd word: species nameBacillus: a small rodLactobacillus: a small milk rodMicrococcus: a small grainClostridium. a small spindlePasteurella: after Louis Pasteur, LatinizedSalmonella: after Daniel E. Salmon, LatinizedSaccharomyces: 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 ovoidbacilli: cylindrical or rod shapedspirilla: 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%

water.- 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 free oxygen.)- 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 artificial environment.◦ Pure culture - culture that contains only one kind of microorganism◦ 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 experience- 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 (inoculum).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 beadsMonoclonal 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.Cell Fusion:- 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

secondary metabolites that have specific metabolic functions.Plant Cells◦ size : 20-40μm in diameter &100-200μm long- 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 plant).◦ 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 orsecondary plant substances.- occupies 90 percent of the volume of the cell for a mature plant cell- 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 irradiationCELL 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 suspensionCELL IMMOBILIZATION- encouraging production of high yields of secondary metabolites

- can protect cells & decrease problems related to shear forces.