Talk:SOMS Honours Research Techniques - Tissue Culture
- 1 JoVE Videos
- 2 Bacteria
- 3 Mycoplasma
- 4 Fungi
- 5 Introduction
- 6 Practical Methods Video
- 7 Practical Methods
- 8 Cell Lines
- 9 Primary Cells
- Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord
- Organotypic Hippocampal Slice Cultures
- The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury
- Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice
- Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons
- Preparation of Dissociated Mouse Cortical Neuron Cultures
- Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons
- Derivation of Human Embryonic Stem Cells by Immunosurgery
- Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)
- Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay
- Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation
- Cardiac Myocytes
- Actinomyces, Bacillus, Enterococcus, Escherichia coli, Fusobacterium, Klebsiella, Lactobacillus, Micrococcus, Mycobacterium, Peptostreptococcus, Pseudomonas, Porphyromonas, Prevotella, Salmonella, Serratia, Staphylococcus, Streptococcus
- Standard antibiotics commonly used in cell culture (e.g. Pen/Strep) do not affect mycoplasmas.
- two methods to get rid of mycoplasma contamination:
- Mycoplasma-EX - a combination of non-antibiotic and antibiotic reagents.
- BIOMYC 1-3 - three highly effective antibiotics.
Text modified from http://www.unclineberger.org/tcf/Fungus.asp
- Fungi or molds produce thin filamentous mycelia and sometimes denser clumps of spores.
- They are easy to observe under a low power microscope and can even be seen without magnification in advanced stages of contamination.
- They can appear whiteish, yellowish, or black in culture and when in advanced mycelial growth stages look like large fuzzy patches in the dish or flask.
Characteristics in mammalian cell culture
- In early stages of contamination, fungi do not typically cause pH changes in the medium
- do not have significant toxic effects on mammalian cells.
- grow mostly in the medium unattached to the cells or growth vessel, but can become attached to either.
- spores that give rise to the mycelia formation are often hard to detect in cultures
Cell cultures can often be cured of fungus contamination when detected early by treatment with certain antibiotics (actually antimycotics).
Typical routes of infection in cultures:
- Fungus and mold spores are ubiquitous in the environment and generally infect cultures via an airborne route.
- Heating and air-conditioning systems are notorious for having high concentrations of spores.
- seasonal changes of fall and spring usually result in an increase in this type of contamination in cultures as heating or A/C systems are switched on or off.
- particularly in the spring, the higher bioburden in the air from pollen particles can carry fungal spores into air handling systems and into labs on the clothes of lab personnel.
- The two most common antimycotic agents used in cell culture that are effective against fungi or molds are Amphotericin B (Fungizone) and mycostatin (Nystatin).
- routinely used antibiotics such as penicillin/streptomycin (pen/strep), gentamicin, and kanamycin are NOT effective against fungi or molds.
- used in media at final working concentrations between 0.25ug/ml and 2.5ug/ml.
- typically very toxic in cell culture systems and should be used conservatively.
- used at final working concentrations between 100U/ml and 250U/ml.
- a colloidal suspension rather than a solution and should be mixed thoroughly before it is added to cell culture media.
- nystatin is in medium and viewed under a microscope, it will appear as small crystal-like particles.
List of antibiotics, the organisms they are effective against, and recommended working concentrations
- compiled by the Sigma-Aldrich Company can be found as a pdf document (129Kb, 3 pages)
Contamination Risks and Comments Select 'General Identification' from the menu above or click here for contaminant examples and characteristics in cell culture
During times of high concentrations of airborne particulates, such as in the spring during pollen season and during construction when dust and dirt is created, there is a statistically higher chance of having contamination due to airborne spores and other microorganisms associated with the dust and the airborne particulates. I thought it might be helpful for us to alert our users of this potential and make some recommendations about what can be done to try to reduce the risk. Have the air flow rate in your hood(s) checked. As HEPA filters load over time, the pressure drop will increase and the flow rate may be reduced to levels below the rate required for the stability of the laminar flow. If this happens, there is a higher likelihood that air turbulence within the cabinet will occur during a manipulation that could lead to contamination from an airborne source. The air flow rate should not be less than 80-85 feet per minute. UNC Health and Safety has an anemometer and can check this for you or the TCF can recommend a third party vendor. Reduce traffic in your cell culture rooms, especially near the hoods. Traffic causes air turbulence at the hood opening and can compromise the protective air barrier that is created by the downward flow of air inside the cabinet at the opening. Spore-carrying dust particles or other airborne contaminates could get into the working area and ultimately find their way into your media or cultures. Do not use flames (bunsen burners, etc.) under the hood. This will cause air turbulence over the flame and in the working area that could lead to spores from your lab coat, for example, being caught up in the turbulence and blown into your media bottle, flask, or, dish, etc. All routine cell culture manipulations can be done without flaming by using appropriate aseptic and sterile technique. We will be glad to offer specific suggestions and training regarding this if necessary. Make sure all interior hood surfaces are adequately disinfected, preferably by using chemical disinfectants such as 70% ethanol, dilute hypochlorite, or a dilute quaternary ammonium solution such as 1% benzalconium chloride. Freshness of the solutions and adequate contact time are important for these to work properly. Consider using TC flasks with vented filter caps instead of flasks that require loosened caps for gas exchange or dishes. These flasks tend to reduce airborne contamination and especially the spread of contamination from one vessel to another while in the incubator. These flasks are more costly but it may be worth the extra cost considering the higher risk at this time. At least you may consider using them for stock cultures or for extremely important cultures. If you experience chronic contamination problems, you may want to consider using an antibiotic (with an anti-mycotic component for fungal or yeast problems). Whereas I don't recommend them for routine use, sometimes they may be necessary in certain situations or in areas with unusually high concentrations of airborne particulates. We can make specific recommendations as to which antibiotics to use and at what concentrations. Please note that pen/strep offers no protection against fungus. It may be useful to monitor the airborne microbial levels in your cell culture rooms and even within your hoods (it should be 0 in hoods) by using Nutrient Agar and Sabouraud Dextrose Agar plates. Nutrient agar will give you information about airborne bacteria levels and Sabouraud dextrose agar will detect airborne fungi and spores. Put plates at various locations in the lab and in your hood. Take the lids off for a set period of time, 30 minutes for example. Collect the plates and incubate the nutrient agar plates for 3 to 5 days and the Sabouraud dextrose agar plates for 7 to 10 days. Remember not to incubate these plates in incubators with your cell cultures! Take a total plate count and note the locations that seem to be high. Depending on what you find, there may be steps you can take to reduce the level of airborne contaminants. I hope no one has serious problems, but if you do, please feel free to contact us at the TCF if we can be of help. We will be happy to come to your cell culture lab and make specific recommendations and otherwise try to help with particular problems. We can also make recommendations regarding cleaning, preventive maintenance schedules, and disinfectants for hoods, water baths, and incubators.
The previous laboratory introduced cell culture, cells and practical considerations. This laboratory will look at the more "hands on" techniques required to carry out tissue culture.
Practical Methods Video
- Journal of Visualized Experiments
- General Guide For Cryogenically Storing Animal Cell Cultures (Corning)
- This 10-page PDF Corning Technical Guide examines both the theoretical and practical aspects of cryogenic preservation and reviews key strategies for managing a cell repository.
- Mouse (Mus musculus) muscle cell line
- Original Reference Blau HM, et al. Plasticity of the differentiated state. Science 230: 758-766, 1985. PubMed: 2414846
- Blau Lab Protocol Blau Lab - C2 Mouse Myoblast Culture
- Rat (Rattus norvegicus), adrenal gland cancer
- Original Reference Greene LA, Tischler AS. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Natl. Acad. Sci. USA 73: 2424-2428, 1976. PubMed: 1065897
- Mouse, Swiss albino (Mus musculus)
- Original Reference Todaro GJ, Green H. Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J. Cell Biol. 17: 299-313, 1963. PubMed: 13985244
- Human (Homo sapiens) cervical adenocarcinoma
- HeLa cells were named for Henrietta Lacks, who died in 1952 from cervical adenocarcinoma and her physician Margaret Gey then began working with these cells used throughout the world for medical research.