Cell?

Basic, structural and functional unit Term coined by Robert Hooke Classified into 2 types
Prokaryotes
E.g: Bacteria such as E.coli

Eukaryotes
Unicellular E.g. Yeast Multicellular all higher organisms

Cell culture
Method of multiplying microbes or cells in predetermined culture media under controlled laboratory conditions. Uses:
To determine the type of organism Primary diagnostic method Study the characteristics of microbes

Steps for preparing a Bacterial cell culture

Preparation of media
Addition of components Adjust pH Sterilization
By autoclaving at 121o C and 15 psi

Inoculation of microorganisms
Material used for inoculation is called inoculum

Pure culture
Contains a single type of bacteria

Components of media
Carbon source
Source of energy Building blocks of cells

Nitrogen source
Required for synthesis of aminoacids and nucleic acids

Trace elements Growth factors Water

Important for survival

Agar
Gel like substance Used in solid media for solidification

Not all the microorganisms in the environment can be grown in the laboratory

Types of media
Nutrient rich media
Contains all the required nutrients
Generally used E.g.,nutrient agar etc

Minimal media
Contains minimal nutrients
Used to observe specific function E.g. M63, M9 media

Enriched media
Contains special components with very high nutrition
Used for growth of fastidious microorganisms E.g. Blood agar

Selective media
Contains components that selects growth of specific microbes
Used for isolation of specific micro-organism E.g. Eosine Methylene Blue agar

Differential media
Contains component that distinguish different microbes
Used to differentiate two species of microbes E.g. Mackonkey agar

Methods of inoculation
Spread plate Pour plate Streak plate Slant culture
Sample is pietted onto the surface of agar plate Spread the sample evenly by the glass sreader After incubation typical surfce colonies are obtained

Spread plate method

Methods of inoculation
Pour plate Method
To prepare pure culture from a mixed culture Inoculum is serially diluted before inoculation

Methods of inoculation
Streak plate method
To obtain single colony from an existing pure culture

Methods of inoculation
Slant culture
Provides more surface area More aeration Microbes survive longer than liquid cultures

Bacterial Growth
Bacterial growth is the division of one piece of bacteria into two daughter cells in a process called binary fission

Phases of bacterial growth are

A. Lag phase B. Exponential or log phase C. Stationary phase D. Death phase

Bacterial Growth Curve

Four Phases of Bacterial Growth

Lag phase During lag phase, bacteria adapt themselves to growth conditions. It is the period where the individual bacteria are maturing and not yet able to divide. During the lag phase of the bacterial growth cycle, synthesis of RNA, enzymes and other molecules occurs Exponential phase (sometimes called the log phase or the logarithmic phase) is a period characterized by cell doubling. The number of new bacteria appearing per unit time is proportional to the present population. Exponential growth cannot continue indefinitely, however, because the medium is soon depleted of nutrients and enriched with wastes Stationary phase The "stationary phase" is due to a growthlimiting factor; this is mostly depletion of a nutrient, and the formation of inhibitory products such as organic acids. Death phase Bacteria run out of nutrients and die.

Factors Influencing Bacterial Growth

Temperature Bacteria have a minimum, optimum, and maximum temperature for growth and it can be divided into 4 groups based on their optimum growth temperature.
Psychrophiles are cold-loving bacteria. Their optimum growth temperature is between -5C and 15C. They are usually found in the Arctic and Antarctic regions. Mesophiles are bacteria that grow best at moderate temperatures. Their optimum growth temperature is between 25C and 45C. Most bacteria are mesophilic and include common soil bacteria and bacteria that live in and on the body. Thermophiles are heat-loving bacteria. Their optimum growth temperature is between 45C and 70C and are commonly found in hot springs and in compost heaps. Hyperthermophiles are bacteria that grow at very high temperatures. Their optimum growth temperature is between 70C and 110C. They are usually members of the Archae and are found growing near hydrothermal vents at great depths in the ocean.

Factors Influencing Bacterial Growth

pH

Neutrophiles grow best at a pH range of 5 to 8

Acidophiles grow best at a pH below 5.5 Alkaliphiles grow best at a pH above 8.5

Aeration requirement of oxygen

Aerobes requires oxygen Anaerobes do not require oxygen
Two types
Facultative anaerobes Obligatory anaerobes

Organisms that grow in extreme conditions are called extremophiles

Applications
Clinical diagnosis Industries
Production of proteins and peptides Antibiotics

Research
Identify pathogenicity Study gene expression

Animal Cell Culture

Animal cell Culture is the general term for the removal of cells, tissues, or organs from an animal and their subsequent placement into an artificial environment conducive to growth. History The 19th-century English physiologist Sydney Ringer developed salt solutions suitable for maintaining the beating of an isolated animal heart outside of the body. In 1885, Wilhelm Roux removed a portion of the medullary plate of an embryonic chicken and maintained it in a warm saline solution for several days, establishing the principle of tissue culture. Ross Granville Harrison, published results of his experiments from 1907 to 1910, establishing the methodology of tissue culture. Cell culture techniques were advanced significantly in the 1940s and 1950s to support research in virology

Several ways for Isolation of cells

Cells can be easily purified from blood; however, only the white cells are capable of growth in culture. Primary culture Cells that are cultured directly from a subject are known as primary cells Explant culture Pieces of tissue can be placed in growth media, and the cells that grow out are available for culture. This method is known as explant culture. Enzymatic dissiciation Mononuclear cells can be released from soft tissues by enzymatic digestion with enzymes such as collagenase, trypsin, or pronase, which break down the extracellular matrix.

Enzymatic dissociation

Cell fusion technique

Using cell fusion techniques, it is also possible to obtain hybrid cells by fusing cells from two different parents. These may exhibit characteristics of either parent or both parents. This technique was used in 1975 to create cells capable of producing monoclonal antibodies.These hybrid cells (called Hybridomas) are formed by fusing two different but related cells. The first is a spleen-derived lymphocyte that is capable of producing the desired antibody. The second is a rapidly dividing myeloma cell (a type of cancer cell) that has the machinery for making antibodies but is not programmed to produce any antibody. The resulting hybridomas can produce large quantities of the desired antibody. These antibodies, called Monoclonal Antibodies due to their purity, have many important clinical, diagnostic, and industrial applications.

E.coli as a model organism

Basic environmental Requirements for Happy Cells

Controlled temperature
- for Cold Blooded Vertebrates 18 to 25C

- for mammalian cells 36 to 37C.

Good substrate for cell attachment Appropriate medium and incubator. Maintenance of correct pH and osmolality

How to Decide if Cultured Cells Are Happy

a) Determination of Morphology b) Determination of the Growth rate c) Plating Efficiency is a testing method where small numbers of cells (20 to 200) are placed in a culture vessel and the number of colonies they form is measured. d) Expression of Specialized Functions, by using biochemical or immunologicalassays and tests.

What is Cell Culture Used For?

Model Systems Cell cultures provide a good model system for studying 1) Basic cell biology and Biochemistry 2) The interactions between disease-causing agents and cells 3)The effects of drugs on cells Toxicity Testing Cultured cells are used to study the effects of new drugs, cosmetics and chemicals on survival and growth in a wide variety of cell types. Especially important are liver- and kidney-derived cell cultures

Cancer Research Cell culture can be used in studying: 1. The basic differences between normal and cancerous cells. 2. The mechanisms that cause the change in normal cells. 3. Suitable drugs and methods for selectively destroying types of cancer. Virology Viruses can be replicated in cell cultures (in place of animals) for use in vaccine production.e.g vaccines for polio, rabies,chicken pox, hepatitis B and measles. Genetic Engineering Cells have genetically engineered to produce proteins that have medicinal or commercial value. These include monoclonal antibodies,insulin,hormones, etc.

Gene Therapy Cells can be removed from a patient lacking a functional gene and the missing or damaged gene can then be replaced. The cells can be grown for a while in culture and then replaced into the patient. An alternative approach is to place the missing gene into a viral vector and theninfect the patient with the virus in the hope that the missing gene will then be expressed in the patients cells. Drug Screening and Development Cell-based assays are important for the pharmaceutical industry, not just for cytotoxicity testing but also for screening of compounds that may have potential use as drugs.