Microbial Contamination

Microbiological
contamination
refers to the
nonintended or
accidental
introduction of
infectious material
like bacteria,
yeast, mould,
fungi, virus,
prions, protozoa or

Types of Microbial C0ntamination

Bacteria: are

microorganisms with a
size of up to 5 m and
represent the most
important group of
pathogen.
Viruses: subcellular
biological objects with a
size of 20-200 nm. They
exist with and without
envelopes and can
cause serious infections .

Prions:

infectious protein
particles. They are
the smallest
pathogens, which
are below 5 nm in
size.
Fungi:Yeasts and
protozoa with up
to 200 m in
diameter are three

Detection of Microbial
Contamination
A

sudden change in pH.

Cloudiness in the medium.
Under a 10 objective, spaces between
cells will appear granular and may
shimmer with bacterial contamination.
Under a 100 objective, it may be
possible to resolve individual bacteria
and distinguish between rods and cocci.
Precipitates of media constituents.

Mycoplasma
A

genus of bacteria.
Lack a cell wall around their cell
membrane.
Unaffected by many common
antibiotics such that target cell wall
synthesis as penicillin or other betalactam antibiotics .

Detecting Mycoplasmal
Infections
Fluorescent

staining

PCR
ELISA assay
Immunostaining
Autoradiography

or
Microbiological assay.

FLUORESCENCE DETECTION
OF MYCOPLASMA
Seed indicator cells into Petri dishes
without using antibiotics.
Add 1.5 mL of medium.
Incubate the culture .
Remove the medium .
Rinse the monolayer with BSS-PR .
Add fresh BSS-PR .
Add pure flaxiative ,rinse.
Add more fixative and fix for 10 min.
Remove and discard the fixative.

Dry the monolayer completely.

Wash off the fixative with deionized
water and discard the wash.
Add Hoechst in BSS-PR .
Remove and discard the stain.
Rinse the monolayer.
Mount a coverslip in a drop of buffered
glycerol mountant.
Examine the monolayer by
epifluorescence .

Analysis For Fluorescence

Detection
Check

for extranuclear fluorescence.

Mycoplasmas give pinpoints over the
cytoplasm .
Sometimes a light, uniform staining
of the cytoplasm is observed
probably due to RNA.
If there is any doubt regarding the
interpretation of the fluorescence
test, it should be repeated .

DETECTION OF MYCOPLASMA BY PCR:

Collection

of samples, culture without any

antibiotics.
Collection of 1 mL of the supernatant medium
of adherent cells.
Centrifuge the supernatant medium.
Centrifuge the suspension again and wash
one more time with D-PBSA .
After centrifugation, resuspend the pellet in
D-PBSA.
After lysing the cells, extract and purify the
DNA by standard phenol/chloroform extraction.

Interpretation of Results For PCR

All samples containing

the internal control
DNA show a band at
986 bp.
Mycoplasma-positive
samples show a band
at 502 to 520 bp
Contaminations of
reagents are revealed
by a band in the
negative control
sample.

Cross Contamination
Bacteria

or other microrganisms are

unintentionally transrerred from one
subtance or object to another.
Makes harmful effect.
First discoverd by chromosome
analysis with HeLa cells .

Avoid crosscontamination
Obtain

cell lines from a reputable cell

bank.
Do not have culture flasks of more
than one cell line or media bottles
used with them, open
simultaneously.
Handle rapidly growing lines, such
as HeLa, on theirown and after other
cultures.
Never use the same pipette for

 Never

use the same bottle of

medium or trypsin.
Do not use unplugged pipettes, or
pipettors without plugged tips, for
routine maintenance.
Check the characteristics of the
culture regularly, and suspect any
sudden change in morphology,
growth rate.