Tissue Culture LAB 3 TISSUE CULTURE CONTAMINATION T

Tissue Culture LAB# 3 TISSUE CULTURE CONTAMINATION T. A Roba Attar

Introduction • Contamination of cell cultures is easily the most common problem encountered in cell culture laboratories, sometimes with very serious consequences. • Cell culture contaminants can be divided into two main categories, chemical contaminants such as impurities in media, sera, and water, plasticizers, and detergents, and biological contaminants such as bacteria, molds, yeasts, viruses, mycoplasma, as well as cross contamination by other cell lines. • While it is impossible to eliminate contamination entirely, it is possible to reduce its frequency and seriousness by gaining a thorough understanding of their sources and by following good aseptic technique.

Source of contamination å The potential routes to contamination are summarized in the table, including failure in the sterilization procedures for glassware and pipettes, turbulence and particulates (dust and spores) in the air, in room poorly maintained incubators and refrigerators.

Types of microbial contamination å Bacteria, yeast, fungi, molds, and mycoplasma all a pear as contaminations in the tissue culture. å If protozoology is carried on in the same laboratory, some protozoa can infect cell line. å Usually, the species or type of infection is not important, unless it becomes a frequent occurrence.

Bacteria • Bacteria are a large and ubiquitous group of unicellular microorganisms. • They are typically a few micrometers in diameters, and can have a variety of shapes, ranging from spheres to rods and spirals. • Because of their ubiquity, size, and fast growth rates, bacteria, along with yeasts and molds, are the most commonly encountered biological contaminants in cell culture. • Bacterial contamination is easily detected by visual inspection of the culture within a few days of it becoming infected.

Yeast • Yeasts are unicellular eukaryotic microorganisms in the kingdom of Fungi, ranging in size from a few micrometers (typically) up to 40 micrometers (rarely).

Molds • Molds are eukaryotic microorganisms in the kingdom of Fungi that grow as multicellular filaments called hyphae. • Spores of many mold species can survive extremely harsh and inhospitable environments in their dormant stage, only to become activated when they encounter suitable growth conditions.

Viruses • Viruses are microscopic infectious agents that take over the host cells machinery to reproduce. • Their extremely small size makes them very difficult to detect in culture, and to remove them from reagents used in cell culture laboratories. • Because most viruses have very stringent requirements for their host, they usually do not adversely effect cell cultures from species other than their host. However, using virally infected cell cultures can present a serious health hazard to the laboratory personnel, especially if human or primate cells are cultured in the laboratory.

Mycoplasma • Mycoplasma are simple bacteria that lack a cell wall, and they are considered the smallest self-replicating organism. • Because of their extremely small size (typically less than one micrometer), mycoplasma are very difficult to detect until they achieve extremely high densities and cause the cell culture to deteriorate; until then, there are often no visible signs of infection. • Some slow growing mycoplasma may persists in culture without causing cell death, but they can alter the behavior and metabolism of the host cells in the culture.

Visible microbial contamination 1. Bacteria: infected cultures usually appear cloudy (i. e. , turbid), sometimes with a thin film on the surface. • Sudden drops in the p. H of the culture medium is also frequently encountered. • Under a low- power microscope, the bacteria appear as tiny, moving granules between the cells and spaces between cells appear granular and may shimmer with bacterial contamination. and observation under a highpower microscope can resolve the shapes of individual bacteria.

CONT……. 2. yeast: Like bacterial contamination, cultures contaminated with yeasts become turbid, especially if the contamination is in an advanced stage. • There is very little change in the p. H of the culture contaminated by yeasts until the contamination becomes heavy, at which stage the p. H usually increases. • Yeast appear as separate round or ovoid particles that may bud off smaller particles.

Simulated phase contrast images of 293 cells in adherent culture that is contaminated with yeast. The contaminating yeast cells appear as ovoid particles, budding off smaller particles as they replicate

CONT……. 3. Molds: Similar to yeast contamination, the p. H of the culture remains stable in the initial stages of contamination, then rapidly increases as the culture become more heavily infected and becomes turbid. • Under microscopy, the mycelia usually appear as thin, wisp-like filaments, and sometimes as denser clumps of spores. • With some toxic infection some deterioration of the cells will be apparent.

fungi

Un-Visible microbial contamination • Viral infection of cell cultures can be detected by electron microscopy, immunostaining with a panel of antibodies, ELISA assays, or PCR with appropriate viral primers. • Chronic mycoplasma infections might manifest themselves with decreased rate of cell proliferation, reduced saturation density, and agglutination in suspension cultures; however, the only assured way of detecting mycoplasma contamination is by testing the cultures periodically using fluorescent staining (e. g. , Hoechst 33258), ELISA, PCR, immunostaining, autoradiography, or microbiological assays.

Cell lines are initially tested by at least two different assays: the classical broth-agar microbiological culture method and by a polymerase chain reaction (PCR) assay

Photomicrographs of mycoplasma-free cultured cells (panel A) and cells infected with mycoplasma (panels B and C). The cultures were tested using the Myco. Fluor. TM Mycoplasma Detection Kit, following the kit protocols. In fixed cells, the Myco. Fluor. TM reagent has access to the cell nuclei, which are intensely stained with the reagent, but the absence of fluorescent extranuclear objects indicates that the culture is free from mycoplasma contamination (panel A).

Monitoring for contamination 1. Check for contamination by eye and with a microscope at each handling of a culture. 2. If it is suspected, but not obvious that a culture is contaminated but the fact cannot be confirmed in situ, clear the hood or bench of everything except your suspected culture and one can of Pasteur pipettes. Because of the potential risk to other cultures, this is best done after all your other culture work is finished. Remove a sample from the culture and place it on a microscope slide (Kovaslides are convenient for this, as they do not require a cover slip). Check the culture with a microscope, preferably by phase contrast.

pasteur pipettes kovaslide

CONT……. If it is confirmed that the culture is contamination, discard the pipettes, swab the hood or bench with 70% alcohol containing a phenolic disinfectant and do not use the hood or bench until the next day. 3. record the nature of the contamination. 4. If the contamination is new and not widespread, discard the culture, the bottle with medium used to feed it, and any other bottle (e. g. , trypsin) that has been used in conjunction with the culture. Discard all these into disinfectant, preferably in a fume hood and outside the tissue culture area.

CONT……. 5. If the contamination is new and widespread (i. e. . , in at least two different cultures), discard all media, stock solutions, trypsin , etc…. . 6. If the same kind of contamination has occurred before, check stock solutions for contamination: a) by incubation media alone or nutrient broth or b) by plating out the solution on nutrient agar c) if (a) and (b) prove negative , but contamination is still suspected , incubate 100 ml of solution , filter on through a 0. 2 m filter and place out filter on nutrient agar.

CONT……. 7. If the contamination is widespread, multispecific and repeated, check the laboratory’s sterilization procedures (e. g. , the temperatures of oven and autoclaves, particularly in the center of the load, the durations of sterilization cycle, the packaging, the storage practices (e. g. , unsealed glassware should be resterilized every 24 hr), and the integrity of the aseptic room and the filters on the laminar-flow hood filters).

Cross-contamination å A number of cell strains have evolved with very short doubling times and high plating efficiencies. å Although these properties make such cell lines valuable experimental material, they also make them potentially hazardous for cross-infection other cell lines. Such Hela cell line.

To avoid cross-contamination 1. Obtain cell lines from a reputable cell bank that has performed appropriate characterization of the cells. 2. Do not have media bottles or culture flasks with more than one cell line open simultaneously. 3. Handle rapidly growing lines, such as He. La, on their own and after other cultures. 4. Never use the same pipette for different cell lines. 5. Never use the same bottle of medium, trypsin, etc for different cell lines.

CONT……. 6. Do not put a pipette back into a bottle of medium, trypsin, etc. , after it has been in a culture flask containing cells. 7. Add medium and any other reagents to the flask first and then add the cells last. 8. Check the characteristics of the culture regularly and suspect any sudden change in morphology growth rate, etc. cross-contamination or its absence may be confirmed by DNA fingerprinting, karyotype or isoenzyme analysis.