Lecture 9

Question 1

list and describe potential sources of contaminations

Answer 1

equipment: room air, work surface, improper hood use, incubators
cells: storage and import
media/reagents: solutions, glassware and tools/instruments, culture flask, media
operator (person): manipulations, pipetting, dispensing, hand hair breath clothing

Question 2

list common cell culture contaminants

Answer 2

Bacteria
Fungi
Yeast
-these three can be easily seen via microscope
Mycoplasma
Viruses. very small and pass through filters
Cross contamination (other cell lines)

Question 3

describe indications for visible contaminations and the specific appearances of bacteria and yeast

Answer 3

sudden pH change. pH decreases with bacteria and yeast, it increases slightly with fungi
cloudy medium. yeast and bacteria especially, sometimes media precipitates or cell debris (this is fine)
bacterial contamination can be seen as flickering/shimmering under microscope
yeast looks like ovoid particles budding off smaller particles

Question 4

describe why mycoplasma contaminations are especially difficult for cell cultures

Answer 4

mycoplasma are the smallest free living organism. they are resistant to penicillin and are smaller than 1 micrometer, can only be seen clearly by staining or electron microscope. They are super hard to identify and super hard to get rid of
No visible sign of contamination (no turbidity, cytopathic effect, or pH change). Lack cell wall and have small size to pass through filters

Question 5

list potential sources for mycoplasma contaminations and describe adverse effects of mycoplasma on these sources

Answer 5

laboratory personnel; can infect humans and cause fertility issues and cancer (possibly)
media components; serum is very problematic! cannot autoclave it, only filter. filters don’t remove mycoplasma
contaminated incubator
contaminated reagents
contaminated virus pool
infected cell lines

Question 6

describe common tests used to detect mycoplasma contaminations

Answer 6

PCR: quick and high sensitivity. can give false positives as primers don’t detect ALL species. quantitation possible
DNA fluorescence stain: use Hoechst 33258 or DAPI which stain DNA. short assay with so so sensitivity. watch out for extranuclear fluorescence and interpret appropriately (not necessarily mycoplasma)
cultivation on agar: Gold standard! culture and grow mycoplasma (risky). very sensitive, takes a long time. fried egg look is mycoplasma
immunological assays
DNA hybridization
electron microscopy

Question 7

describe treatment and/or decontamination options for contaminated cultures and justify/explain decisions

Answer 7

Always dispose!! Autoclave or bleach. only in extreme situations would decontamination be attempted (irreplaceable culture). quarantine, use antibiotics or commercial product.

Question 8

describe adverse effects of mycoplasma on cells

Answer 8

alter metabolism, nucleic acid synthesis, and gene expression
change enzyme kinetics
alter membrane antigenicity
reduce cell fusion efficiency (reduce hybridoma)
cause chromosomal abnormalities (cause transformation and cancer)
deplete nutrients from media and interfere with growth rate
reduce tumorigenicity of some lymphoid cell lines
changes viral yields by nutrient depletion and reduced immune response

Question 9

when to test for mycoplasma

Answer 9

once a month standard
newly arrived cultures
prior to freezing
suspicious cultures (growth change, etc)

Question 10

general rules for detecting mycoplasma

Answer 10

need positive and negative controls
assay cultures 2-4 days after passage (mycoplasma conc will be high)
use antibiotic free medium
no proteolytic enzymes that can detach mycoplasma
use 2 different procedures