Department of microbiology microbial food technology group a diploma in quality assurance in microbiology diploma



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History


The 19th-century English physiologist Sydney Ringer developed salt solutions containing the chlorides of sodium, potassium, calcium and magnesium suitable for maintaining the beating of an isolated animal heart outside of the body.[1] 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.[3] Ross Granville Harrison, working at Johns Hopkins Medical School and then at Yale University, published results of his experiments from 1907–1910, establishing the methodology of tissue culture.[4]

Cell culture techniques were advanced significantly in the 1940s and 1950s to support research in virology. Growing viruses in cell cultures allowed preparation of purified viruses for the manufacture of vaccines. The injectable polio vaccine developed by Jonas Salk was one of the first products mass-produced using cell culture techniques. This vaccine was made possible by the cell culture research of John Franklin Enders, Thomas Huckle Weller, and Frederick Chapman Robbins, who were awarded a Nobel Prize for their discovery of a method of growing the virus in monkey kidney cell cultures.


Concepts in mammalian cell culture

Isolation of cells


Cells can be isolated from tissues for ex vivo culture in several ways. Cells can be easily purified from blood, however only the white cells are capable of growth in culture. 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. Alternatively, 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.

Cells that are cultured directly from a subject are known as primary cells. With the exception of some derived from tumors, most primary cell cultures have limited lifespan. After a certain number of population doublings (called the Hayflick limit) cells undergo the process of senescence and stop dividing, while generally retaining viability.

An established or immortalised cell line has acquired the ability to proliferate indefinitely either through random mutation or deliberate modification, such as artificial expression of the telomerase gene. There are numerous well established cell lines representative of particular cell types.

Maintaining cells in culture


Cells are grown and maintained at an appropriate temperature and gas mixture (typically, 37°C, 5% CO2 for mammalian cells) in a cell incubator. Culture conditions vary widely for each cell type, and variation of conditions for a particular cell type can result in different phenotypes being expressed.

Aside from temperature and gas mixture, the most commonly varied factor in culture systems is the growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrients. The growth factors used to supplement media are often derived from animal blood, such as calf serum. One complication of these blood-derived ingredients is the potential for contamination of the culture with viruses or prions, particularly in biotechnology medical applications. Current practice is to minimize or eliminate the use of these ingredients wherever possible, but this cannot always be accomplished. Alternative strategies involve sourcing the animal blood from countries with minimum BSE/TSE risk such as Australia and New Zealand, and using purified nutrient concentrates derived from serum in place of whole animal serum for cell culture.[5]



Plating density (number of cells per volume of culture medium) plays a critical role for some cell types. For example, a lower plating density makes granulosa cells exhibit estrogen production, while a higher plating density makes them appear as progesterone producing theca lutein cells.[6]

Cells can be grown in suspension or adherent cultures. Some cells naturally live in suspension, without being attached to a surface, such as cells that exist in the bloodstream. There are also cell lines that have been modified to be able to survive in suspension cultures so that they can be grown to a higher density than adherent conditions would allow. Adherent cells require a surface, such as tissue culture plastic or microcarrier, which may be coated with extracellular matrix components to increase adhesion properties and provide other signals needed for growth and differentiation. Most cells derived from solid tissues are adherent. Another type of adherent culture is organotypic culture which involves growing cells in a three-dimensional environment as opposed to two-dimensional culture dishes. This 3D culture system is biochemically and physiologically more similar to in vivo tissue, but is technically challenging to maintain because of many factors (e.g. diffusion).


Cell line cross-contamination


Cell line cross-contamination can be a problem for scientists working with cultured cells. Studies suggest that anywhere from 15–20% of the time, cells used in experiments have been misidentified or contaminated with another cell line.[7][8][9] Problems with cell line cross contamination have even been detected in lines from the NCI-60 panel, which are used routinely for drug-screening studies.[10][11] Major cell line repositories including the American Type Culture Collection (ATCC) and the German Collection of Microorganisms and Cell Cultures (DSMZ) have received cell line submissions from researchers that were misidentified by the researcher.[10][12] Such contamination poses a problem for the quality of research produced using cell culture lines, and the major repositories are now authenticating all cell line submissions.[13] ATCC uses short tandem repeat (STR) DNA fingerprinting to authenticate its cell lines.[14]

To address this problem of cell line cross-contamination, researchers are encouraged to authenticate their cell lines at an early passage to establish the identity of the cell line. Authentication should be repeated before freezing cell line stocks, every two months during active culturing and before any publication of research data generated using the cell lines. There are many methods for identifying cell lines including isoenzyme analysis, human lymphocyte antigen (HLA) typing, Chromosomal analysis, Karyotyping, Morphology and STR analysis.[14]

One significant cell-line cross contaminant is the immortal HeLa cell line.

Manipulation of cultured cells


As cells generally continue to divide in culture, they generally grow to fill the available area or volume. This can generate several issues:

  • Nutrient depletion in the growth media

  • Accumulation of apoptotic/necrotic (dead) cells.

  • Cell-to-cell contact can stimulate cell cycle arrest, causing cells to stop dividing known as contact inhibition or senescence.

  • Cell-to-cell contact can stimulate cellular differentiation.

Among the common manipulations carried out on culture cells are media changes, passaging cells, and transfecting cells. These are generally performed using tissue culture methods that rely on sterile technique. Sterile technique aims to avoid contamination with bacteria, yeast, or other cell lines. Manipulations are typically carried out in a biosafety hood or laminar flow cabinet to exclude contaminating micro-organisms. Antibiotics (e.g. penicillin and streptomycin) and antifungals (e.g. Amphotericin B) can also be added to the growth media.

As cells undergo metabolic processes, acid is produced and the pH decreases. Often, a pH indicator is added to the medium in order to measure nutrient depletion.


Media changes


In the case of adherent cultures, the media can be removed directly by aspiration and replaced.

Passaging cells


Passaging (also known as subculture or splitting cells) involves transferring a small number of cells into a new vessel. Cells can be cultured for a longer time if they are split regularly, as it avoids the senescence associated with prolonged high cell density. Suspension cultures are easily passaged with a small amount of culture containing a few cells diluted in a larger volume of fresh media. For adherent cultures, cells first need to be detached; this is commonly done with a mixture of trypsin-EDTA, however other enzyme mixes are now available for this purpose. A small number of detached cells can then be used to seed a new culture.

Transfection and transduction


Another common method for manipulating cells involves the introduction of foreign DNA by transfection. This is often performed to cause cells to express a protein of interest. More recently, the transfection of RNAi constructs have been realized as a convenient mechanism for suppressing the expression of a particular gene/protein. DNA can also be inserted into cells using viruses, in methods referred to as transduction, infection or transformation. Viruses, as parasitic agents, are well suited to introducing DNA into cells, as this is a part of their normal course of reproduction.

[edit] Established human cell lines


One of the earliest human cell lines, descended from Henrietta Lacks, who died of the cancer that those cells originated from, the cultured HeLa cells shown here have been stained with Hoechst turning their nuclei blue.

Cell lines that originate with humans have been somewhat controversial in bioethics, as they may outlive their parent organism and later be used in the discovery of lucrative medical treatments. In the pioneering decision in this area, the Supreme Court of California held in Moore v. Regents of the University of California that human patients have no property rights in cell lines derived from organs removed with their consent.

A SELECTION OF MEDIA COMMONLY USED IN FOOD MICROBIOLOGY:

MEDIUM USE

1. Plate count agar Aerobic mesophilic count

2. Mac Conkey broth MPN of coliforms in water

3. Brilliant Green/Lactose/ Bile broth MPN of coliforms in food

4. Violet red/ bile/Glucose agar Enumeration of Enterobacteriaceae.

5. Crystal violet /Azide / Blood agar Enumeration of faecal Streptococci.

6. Baird- Parker agar Enumeration of S. aureus

7. Vassiliadis broth Selection enrichment of Salmonella.

8. Thiosulfate / bile/ citrate/ Sucrose agar Isolation of Vibrios

9. Rose Bengal/ Chloramphenicol agar Enumeration of moulds and yeasts

10. Mac Conkey agar E. coli .



4.Explain the Enumeration methods in detail.

Enumeration methods:-

Plate counts-

  • It has already been suggested that to count microorganisms in a food sample by direct microscopy has a limited sensitivity because of the very small sample size in the field of view at the magnification needed to see microorganisms, especially bacteria.

  • In a normal routine laboratory the most sensitive methods of detecting the presence of a viable bacterium is to allow it to amplify itself to form a visible colony.

  • This forms the basis of the traditional pour plate and spread plate and most probable number counts.


5.Explain the Alternative methods in detail.
Alternative methods-

    • .Cultural methods are relatively labour intensive and require time for adequate growth to occur.

    • . Many food microbiologists also consider that the traditional enumeration methods are not only too slow but lead to an over dependence on the significance of numbers of colony forming units.

    • . A number of methods have been developed which aim to give answer of redox to as “Rapid methods”.

Dye- reduction test:-

      • A group of tests which have been used for some time in the dairy industry dependent on the response of a number of redox dye to the presence of metabolically active microorganisms.

      • They are relatively simple and rapid to carry out at low cost.

      • The redox dyes are able to take up electrons from an active biological system and this results in a change of colour.

Dye Reduction Tests: Methylene Blue and Resazurin.
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