Contamination Chapter 19

Sources of Contamination Failure in sterilization procedures for solutions, glasswares and pipettes Turbulence and particulates (dust and spores) in the air in the room Poorly maintained incubators and refrigerators Faulty laminar-flow hoods Importation of contaminated cell lines or biopsies Lapses in sterile technique Generally the above ones except the last one is rare to happen. The last one is most significant. If the skill and level of care of the operator is high and the atmosphere is clean, free of dust and sill, contamination as a result of manipulation will be rare. A laminar hood should not be busy and all equipment brought into work space should be wiped down. Humid incubators needs to be cleaned if there are any spills and regularly checked by engineers. Your cell lines need to be screened for contamination. Non-tested material should be tested quarantine areas

Types of Microbial Contamination Bacteria, yeast, fungi, molds, mycoplasmas and protozoa – contaminants Important to note the kind of contaminant, how it was detected, location where culture was last handled and operator’s name Frequent occurrence – identify its origin Rapidly growing ones – easily detected and discarded Difficulties – cryptic contaminants

Monitoring for Contamination Table 19.1 – home work Check for contamination by eye + microscope Suspicious – clear hood, observe under phase contrast microscope, discard confirmed cultures and used pipettes, swab with 70% alcohol and do not use the hood till next day Record the nature of contamination

Monitoring for Contamination New contamination – discard the culture, the medium and trypsin bottles (used to feed it) into disinfectant – outside tissue culture area New and widespread contamination – discard all media, stock solutions and trypsin

Monitoring for Contamination If same kind of contamination – check stock solutions – by incubation alone or in nutrient broth By plating out the solution on nutrient agar Proves negative and contamination still persists then incubate 100 ml of solution, filter it through 0.2 µm filter and plate out on nutrient agar with an uninoculated control

Monitoring for Contamination If contamination is widespread, multispecific and repeated, check for temperatures of ovens, autoclaves, duration of sterilization cycle etc Packaging and storage practices Aseptic room and laminar flow hood filters Never decontaminate cultures unless irreplaceable

Visible microbial contamination Characteristic features of microbial contamination: Sudden change in pH – decreases with bacterial infection, little change with yeast infection and increase with fungal contamination Cloudiness in medium – slight film or scum on surface or spots on growth surface Cloudiness dissipates when media is moved

Visible microbial contamination Characteristic features of microbial contamination: Under low-power microscope – spaces between cells will appear granular and may shimmer with bacterial contamination Yeasts appear as separate round or ovoid particles Fungi produce thin filamentous mycelia and denser clumps of spores

Visible microbial contamination Characteristic features of microbial contamination: Under high-power microscopy – resolves individual bacteria – rods and cocci Under 1000x bacterial slides can observed – - Microbial infection may be confused with media constituents Shimmering is due to the mobility of bacteria. Microbial infection can be observed and distinguished by shaking bottle and seeing their different shapes

Mycoplasma Detection of mycoplasma – fluorescent staining, PCR, ELISA assay, immunostaining, autoradiography or microbiological assay Fluorescent staining of DNA by Hoescht 33258 Reveals infections as fine particulate or filamentous staining over cytoplasm Nuclei of cultured cells are brightly stained – acts as positive control Low levels of contamination with micrococci can also be detected. DNA in mycoplasma will pick up the stain.

Mycoplasma Can or might not alter cell behavior and metabolism + alter medium composition Continuous cell lines – grow slowly and do not destroy host cells Take thymidine from medium and infected cultures show abnormal labeling Immunological studies – attempts to raise antibodies can give rise to antimycoplasma antibodies Do different periodic assays to detect contamination of all cell cultures

PCR for mycoplasmas Direct detection of mycoplasmas (FIG – 19.2) Several published primer sequences 16S rDNA sequences – target sequences PCR or RT-PCR – band size = 502 to 520 bp 16S rDNA or an RT-PCR with cDNA of 16S rDNA

Alternative Methods for Detecting Mycoplasma Biochemical: detects mycoplasma-specific enzymes as arginine deaminase or nucleoside phosphorylase

Alternative Methods for Detecting Mycoplasma Microbiological culture – Cultures are seeded into mycoplasma broth – grown for 6 days Plated out onto special nutrient agar Colonies form in 8 days – 200µm – fried egg morphology Fried egg – dense centre with lighter periphery. Much slower and difficult to perform than fluorescence

Alternative Methods for Detecting Mycoplasma Molecular hybridization: Molecular probes specific to mycoplasmal DNA can be used in Southern blot analysis to detect infections by conventional molecular hybridization techniques

Alternative Methods for Detecting Mycoplasma 3H thymidine incorporation: Autoradiography with 3H thymidine Culture is incubated overnight with 4KBq/ml of 3H thymidine Autoradiograph is prepared Grains over cytoplasm – indicate contamination No nuclear labeling because of trapping of thymidine at cell surface by mycoplasma

Viral contamination Incoming cell lines Serum in media Trypsin – sources of contamination Collect products - animal free from known viral contamination ELISA assays and PCR Viruses removed by manufacturers

Eradication of Contamination Bacteria, Fungi, Yeasts, Mycoplasma and Virus Eliminate – discard culture and medium and reagents used Decontamination should be attempted in extreme situations, under quarantine and with expert supervision Unsuccessful – cultures and reagents should be autoclaved Treating a culture will be unsuccessful or may lead to development of an antibiotic-resistant microorganisms

Eradication of Mycoplasma Kanamycin, Gentamycin, Tylosin, Polyanethol sulfonate, 5-bromouracil in combination with Hoechst 33258 and UV light

Persistent Contamination Deterioration in aseptic technique Increased spore count in atmosphere Poorly maintained incubators Contaminated cold room or refrigerator Faulty sterilizing oven or autoclave Sterilization cycle

Persistent Contamination Favors development of chronic contamination Inhibit growth – no death Undetected but appear – changes in conditions Should be cultured in antibiotic-free conditions for some time Cryptic contaminations will persist, hard to determine their origin and impossible to eliminate them

Homework Cross contamination

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