Cell Cultivations

Prokaryotic Cell

Eukaryotic Cell

Microbial Nomenclature

• Binomial system

Bacillus subtilisName of the genus Name of the species

Bacillus Micrococuss Clostrodium Salmonella

Bacteria

• Unicellular microscopic organisms.• Diameter of cell .• Binary Fission: (Asexual reproductive process)• Cell elongation• Invagination of the cell wall• Distribution of cellular material into two cells• Separation into two new cells

Oxygen Requirement

• Aerobic bacteria grow in the presence of free oxygen.• Anaerobic bacteria grow in the absence of free oxygen.• Facultatively anaerobic bacteria grow in either the absence or the

presence of free oxygen.• Microaerophilic bacteria grow in the presence of minute quantities of

free oxygen.

Spore Formation

• A method of surviving unfavorable conditions.• Spores are more resistant than normal

cells to heat, drying, radiation and chemicals.• Spores can come back to normal cells

under favorable conditions.• Spores of tetanus and anthrax, for

example, can survive in the soil for many years

Gram Reaction

• Method of differentiating bacterial species into two large groups• Gram-positive• Gram-negative

• Gram staining differentiates bacteria by the chemical and physical properties of their cell walls by detecting peptidoglycan.• In a Gram stain test, gram-positive bacteria retain the crystal violet

dye.• Counterstain (commonly safranin or fuchsine) added after the crystal

violet gives all Gram-negative bacteria a red or pink coloring.

Experimental Procedure

A Gram stain of mixed Staphylococcus aureus and Escherichia coli

1. Cover bacterial smear on a slide with crystal violet and let stand for 20 seconds and wash off the stain. All cells stain violet.

2. Cover the smear with Gram's iodine solution and let stand for 1 minute. Crystal violet will form a complex with iodine within cells. All cells will remain violet.

3. Flood the smear with 95 percent ethyl alcohol for 10 to 20 seconds and rinse the slide with water. Gram-positive cells will remain violet, but gram-negative cells will be colorless.

4. Cover the smear with safranin, a red counterstain, for 20 seconds, wash for a few seconds, and dry. Gram-positive cells will remain violet, but gram-negative cells will become red.

Fungi

• Ranges from single-celled yeast to multicellular mushrooms.• Yeasts and molds are industrially

important.

Yeast

• Yeasts are generally unicellular organisms• Their shape is spherical to ovoid. • Their size is 1 to 5 μm in width and from 5 to 30 μm in length. • The cell wall is quite thin in young cells but thickens with age.

Molds

• Molds are filamentous fungi.• A single reproductive cell or spore (conidia) is germinated to form a

long thread, hyphae, which branches repeatedly as it elongates to form a vegetative structure called a mycelium.• This consists of a multinucleate mass of cytoplasm within a rigid,

much-branched system of tubes. • The most important classes of molds industrially are Aspergillus and

Penicillium.

Oyster mushroom (Pleurotus ostreatus) growing on coffee grounds

Culture Media

• The growth of microbial population in artificial environments is called cultivation. • A culture that contains only one kind of microorganism is a pure

culture. • A mixed culture is one that contains more than one kind of

microorganism.• The necessary steps for cultivating microorganisms are:• Preparing a culture medium in which a microorganism can grow best.• Sterilizing in order to eliminate all living organisms in the vessel.• Inoculating the microorganism in the prepared medium

Growth Medium

• Natural or complex media usually contain peptones, beef extract, or yeast extract. • When a solid medium is desired, a solidifying agent such as gelatin or

agar may be incorporated into the medium.• Synthetic media consist of dilute, reproducible solutions of chemically

pure, known inorganic and/or organic compounds.• The medium may be as simple as inorganic ammonium salt plus

minerals and a sugar, or as complex as purified casein with added vitamins, minerals, and a sugar.

Penicillin Production

• Penicillin is a secondary metabolite of certain species of Penicillium and is produced when growth of the fungus is inhibited by stress. • It is not produced during active growth.

Typical medium consists of corn steep liquor (4 percent to 5 percent dry weight); an additional nitrogen source such as soy meal, yeast extract, whey; a carbon source such as lactose; and various buffers.

Inoculation: Lyophilized spores are grown in an agar slant culture, which is inoculated into a shake flask culture, followed by primary, secondary seed culture and large-scale fermenter in increasing volume.

The most common method of sterilization is by moist heat (steam under pressure) in an autoclaveDownstream processing: After removing the mold mycelium, the penicillin is separated from the broth by means of a two-stage continuous countercurrent extraction with amyl or butyl acetate.

Fermenter

• A stirred fermenter is employed in fed-batch mode by feeding glucose and nitrogen during cultivation. • Typical size of the vessel is 40,000 to 200,000 liters. • Oxygen is supplied by sparging air at a rate of 0.5 to 1.0 volumes of air

per fluid volume per min. • The pH is maintained at 6.5. • Glucose and nitrogen are fed continuously during the fermentation.• Penicillin is excreted into the medium.

Animal Cells

• Animal cells are eukaryotic cells. • Bound together by intercellular material to form tissue.• Tissue is customarily divided into four categories:• Epithelium

• Covering or lining of all free body surfaces, both external and internal• Connective

• Tissue that connects, supports, binds, or separates other tissues or organs• Muscle

• Muscle cells are responsible for movement in higher animals• Nerve

• Specialized cells to react to stimuli and to conduct impulses to various organs in the body

Animal Cell Cultivation

• Cells removed from animal tissue can be cultivated in nutritional medium outside the donor's body. • Cultured cells grow by increasing in number and size.• The mammalian cell culture technique can be employed to produce• Clinically important bio-chemicals such as human growth hormones• Viral vaccines• Monoclonal antibodies

Difficulties in cultivating mammalian cells• They are larger and more complex than most microorganisms.• Their growth rate is very slow compared to the microorganisms.• They are enclosed with a delicate plasma membrane without the tough

cell wall normally found in microorganisms or plant cells. • Their nutritional requirements are not fully defined yet, requiring

expensive blood serum for medium.• They are part of an organized tissue rather than an individual cellular

organism.• Most animal cells only grow when attached to a surface.

Growth Media

• The serum in the medium is expensive.• The presence of many different proteins in

serum can also complicate the downstream separation processes.

Structure of Immunoglobulin

Cell Fusion

Antibody Assay

Apply a specific antigen solution to a microtitre plate capable of adsorbing antigens.Add the monoclonal antibody solution to be analyzed to the plate. The antibody will be bound to the antigen on the solid surface.Add a second monoclonal antibody conjugated with an enzyme.Add the substrate which will be converted to product by enzymatic reaction to produce a color change.

Plant Cell Cutivations

• Plant Cell cultivation is a technique of growing plant cells, tissues, seeds or other plant parts in a sterile environment on a nutrient medium.

Totipotency

• Ability of a cell or tissue or organ to grow and develop into a fully differentiated organism.

Growth Medium

Plant Growth Regulators

• Auxin: Stimulates Root Development• Cytokinin: Stimulates Shoot Development• Generally, the ratio of these two hormones can

determine plant development• Auxin ↓Cytokinin = Root Development• Cytokinin ↓Auxin = Shoot Development• Auxin = Cytokinin = Callus Development

Plant Tissue Culture

Callus Culture

Suspension Culture

Protoplast Culture

Root Culture

Embryo Culture

Callus Culture

• Tissues and cells cultured on an agar-gelled medium form an unorganized mass of cells called callus.• The cell aggregates do not

correspond with any particular tissue of the whole plant.• Cell proliferation was induced

by plant growth regulators in the solid nutrient medium.

Suspension Culture

• A suspension culture consists of single cells and small groups of cells suspended in a liquid medium.• Suspension cultures must be constantly

agitated at 100-250 rpm (revolutions per minute). • Suspension cultures grow much faster than

callus culture.

Protoplast Culture• A protoplast is a plant cell that had its cell wall

completely or partially removed.• Protoplasts will be grown in solid or liquid medium.

Embryo Culture

• Embryo cultures may be established from embryos removed from sterilized seeds, ovules, or fruits.• The embryos produced from cell culture technique, known as

somatic embryos, can be isolated and germinated to provide one plant per explant.• Embryo culture can be employed for the rapid production of

seedlings from seeds which have a protracted dormancy period.• The method has many potential advantages over traditional

propagation systems, such as fast turnaround, genetic uniformity, mass production, and propagation of disease-free plants.

Reading Assignment

5.3.4 Secondary Metabolite Production

Cell Growth Measurement

• To follow the course of growth, it is necessary to make quantitative measurements. • Cell growth can be determined by measuring • Cell number• Cell mass• Cell activity

Measurement of Cell Number

• Microscopic Counts: The number of cells in a population can be measured under a microscope by counting cells placed in special counting chambers.• There are two types of chambers used for counting cell number

in liquid samples.• Hemocytometer : a blood cell counting chamber for use with organisms of 3

µm in diameter or larger. • Petroff-Hausser counting chamber: for use primarily with bacteria.

Hemocytometer• A special square grid marked on the surface

of the glass slide.• A ridge on each side of the grid holds a

cover slip off of the grid.

Pros and Cons

• The desirable features of direct-counting methods are: • Minimal equipment is required. • Results are obtained rapidly. • The morphological characteristics of the organisms can be observed.

• The disadvantages are: • Dead cells cannot usually be distinguished from live cells. • The method is not suitable for cell suspensions of low density. • Small cells are difficult to see under the microscope and can be missed when

counting. • The actual counting procedure is tiresome and may cause considerable eyestrain. • It is not suitable for highly flocculating cells such as mycelium.

Viable Plate Count

• The sample is mixed with melted agar and poured into a sterile plate. • The plate is then incubated until the

colonies appear, and the number of colonies is counted. • It is important that the number of

colonies developing on the plates should be neither too large nor too small.• To obtain the appropriate number of cells

per unit volume, the sample has to be diluted.

Coulter Counter

• A Coulter counter is an apparatus for counting and sizing particles suspended in electrolytes. It is used for cells, bacteria, prokaryotic cells and virus particles.• A typical Coulter counter has one or more micro-

channels 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.

Measurement of Cell Mass

• Cell dry weight can be measured directly by taking a sample of cell suspension and centrifuging it.• The cells are thoroughly washed with distilled water to eliminate all

soluble matter. • The suspension is re-centrifuged and the settled cells are dried in an

oven and weighed. • This technique can only be used with dense cell suspensions.

Turbidity

• The cell mass can be measured optically by determining the amount of light scattered by a suspension of cells. • The technique is based on the fact that small particles scatter light

proportionally, within certain limits, to their concentration.• When a beam of light is passed through a suspension of organisms,

the reduction in the amount of light transmitted as a consequence of scattering is thus a measure of the cell density. • The measurements are usually made at a wavelength of 600 ~ 700

nm.

Indirect Methods

Cell Immobilization

• Immobilization provides conditions conducive to cell differentiation and cell-to-cell communication, thereby encouraging production of high yields of secondary metabolites.• Immobilization can protect cells

and thereby decrease problems related to shear forces.