To learn how to make sterile microbiological agar plate media and pour agar plates.
List of contaminated cell lines Cell line cross-contamination can be a problem for scientists working with cultured cells. 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.
Many methods are used to identify cell lines, including isoenzyme analysis, human lymphocyte antigen HLA typing, chromosomal analysis, karyotyping, morphology and STR analysis. Other technical issues[ edit ] As cells generally continue to divide in culture, they generally grow to fill the available area or volume.
This can generate several issues: Cell-to-cell contact can stimulate cellular differentiation. Genetic and epigenetic alterations, with a natural selection of the altered cells potentially leading to overgrowth of abnormal, culture-adapted cells with decreased differentiation and increased proliferative capacity.
These are generally performed using tissue culture methods that rely on aseptic technique. Aseptic 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.
As cells undergo metabolic processes, acid is produced and the pH decreases. Often, a pH indicator is added to the medium to measure nutrient depletion. Media changes[ edit ] In the case of adherent cultures, the media can be removed directly by aspiration, and then is replaced.
Media changes in non-adherent cultures involve centrifuging the culture and resuspending the cells in fresh media. Passaging 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.
Some cell cultures, such as RAW cells are mechanically scraped from the surface of their vessel with rubber scrapers. Transfection and transduction[ edit ] Main articles: Transfection and Transformation genetics Another common method for manipulating cells involves the introduction of foreign DNA by transfection.
This is often performed to cause cells to express a gene of interest.
DNA can also be inserted into cells using viruses, in methods referred to as transductioninfection 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.A.
Write a lab report about the Aseptic Technique and Culturing Microbes LabPaq experiment and the microbial identification experiments in which you do the following: 1.
Discuss the importance of using proper aseptic technique . Aseptic Techniques. Before inoculation with the desired microorganisms, microbiological media and all materials coming into contact with it must be sterile.
During any subsequent handling of the bacterial cultures, unwanted or contaminant organisms must be excluded employing aseptic techniques. Microbiology Theory: Media Preparation. Categories; Defined media are media composed of pure ingredients in carefully measured concentrations dissolved in double distilled water i.e., the exact chemical composition of the medium is known.
Typically, they contain a simple sugar as the carbon and energy source, an inorganic nitrogen source. Lab Exercise 3: Media, incubation, and aseptic technique Know the laboratory equipment and culture media needed to develop and maintain pure cultures 6. Carry out aseptic technique for the removal and transfer of microorganisms for culturing.
7. Correctly sterilize and flame transfer instruments and tubes.
culture experiments, laboratory safety, aseptic technique, and microbial contamination of cell cultures, as well as providing basic methods for passaging, freezing, and thawing cultured cells.
Aseptic technique Michele Pearson, Leah Christine Silver, and William Jarvis Purpose Aseptic technique is employed to maximize and maintain asepsis, the absence of pathogenic organisms, in the clinical setting.
The goals of aseptic technique are to protect the patient from infection and to prevent the spread of pathogens.