1. Contamination
Chapter 19

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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

11. 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.

12. 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

13. 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

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

15. 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

17. 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

18. 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

19. 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

20. 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

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

22. 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

23. 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

24. Homework
Cross contamination

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