Sterile Cell Culture Technique
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1 Contamination
2 Sterile Cell Culture Technique
3 Contamination Contamination is one of the most common problems in cell culture laboratories. It is the case in which the culture medium in which the cells are cultured is invaded by a number of undesirable viable or inanimate factors. Contamination damages the cells as well as the results of the study.
4 Contamination Contamination problems can be divided into 3 classes; - Minor problems: Contamination of several plates occurs in the incubator. - Serious problems: The frequency of contamination increases, where a whole experiment or whole plate is contaminated. - Large-scale problems: These are situations in which the source of contamination detected risks all of the previous and planned experiments.
5 Contamination Loss of time, money and labor, irreparable damages on cells, effect of experimental results, stress on researcher, loss of valuable products (such as special cell lines ...)
6 Contamination Sources
7 Contamination Sources Processes in the Laboratory Welded; Movement of particles - Sneezing, coughing, or rapid movement of the air in the laboratory - Careless pipetting, vortexing and centrifugation Solution or mixing of materials used Sloppy autoclaving Working with multiple cells at the same time 7
8 Sources of Contamination Welded Equipment in the Laboratory; Incubators, refrigerators, water baths, etc. that are not regularly cleaned and maintained. Non-sterile media and used plastic materials Dirty clothing or lab coat Use of feeder cells 8
9 Sources of Contamination Sources of contamination can be examined under two headings; Chemical Contaminants - Chemical agents that will adversely affect the life of the cells and the results of the experiments in the cell culture environment Biological Contaminants - Biological agents that will adversely affect the life of the cells and the results of the experiments in the cell culture environment 9
10 Sources of Chemical Contamination Media; content or water-derived Serum; differences in serum content and growth factors and hormone content as they are obtained from living organisms.
11 Sources of Biological Contamination Bacteria Fungus and yeast - Bacteria, fungi and yeast contamination are the most common contaminants, as they are present in large quantities in the air and can easily multiply in the appropriate environment. Mycoplasma Virus Intercellular cross contamination 11
12 Sources of Biological Contamination Contact of materials used with non-sterile surfaces The drop of air particles into the media during culture, transport and incubation of the cells.
13 Sources of Biological Contamination Bacteria, Mushrooms and Yeasts are found almost everywhere and are very suitable for growing and growing medium. They may occur in a 2-3 day period, especially in the absence of anbio_ks (as can be seen under the microscope, as well as changes in the color, pH and clarity of the nutrient medium). An_bio_ks that are used in very high amounts can lead to the formation of an_bio-resistant organisms. 13
14 Sources of Biological Contamination 14
15 Sources of Biological Contamination Fungus 15
16 Sources of Biological Contamination 16
17 Sources of Biological Contamination Virus Due to their small size, the most difficult to detect in a cell culture environment is contamination. Being small also makes it difficult for them to move away from the environment they are in. Virus contamination is primarily a threat to the researcher. Extra care should be taken when working with cells at risk of virus contamination (primary culture, cells from other species). 17
18 Sources of Biological Contamination Mycoplasma Mycoplasmas are the smallest organisms that can spontaneously divide and multiply. It is estimated that 10-15% of currently existing cells are contaminated with mycoplasma. While the presence of mycoplasma prevents the healthy growth of the cells, there are many problems including chromosomal abnormalities in the cells. It is an important threat for cell culture studies as they are usually not detected visually and are very common. 18
19 Sources of Biological Contamination 19
20 Sources of Biological Contamination Cross-contamination Cells that are aggressive (shorter than half-life) are potential sources of cross-contamination. Cross-contamination is particularly caused by worker error. Since it directly affects the results of the experiments, the cells should be regularly monitored for characterization. 20
21 Sources of Biological Contamination 21
22 How to Avoid Contamination? Use of asep_k technique while working Regular use of an_biyo_k in the feeding places, Good training of the laboratory personnel, Aliquoting the materials used (divided into small amounts), 22
23 How to avoid contamination? Take care to work in laminar cabinets at every stage of the work, expose the laminar cabinet to UV for 30 minutes before use and start to use at least 5 minutes after opening the laminar cabinet. Regular cleaning of the laboratory 23
24 How can it be protected from contamination? Careful monitoring of cell morphology to prevent cross-contamination, Working with only one cell hau at the same time, Carefully marking used petri and flask 24
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