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

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

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

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

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

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

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

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

Viral contamination

• Incoming cell lines• Serum in media• Trypsin – sources of contamination• Collect products - animal free from known

viral contamination• ELISA assays and PCR

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

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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