2. slide 2
What Contaminations Can You See Under
A Microscope?
Bacteria
small black specks
pH change/ cloudy media
mistaken for cell debris
definite movement
Fungus
Filamentous Strands
web-like mesh
3. slide 3
What Contaminations Can You See Under
A Microscope?
Yeast
oval/round in shape
smaller than cells
appear as bright beads
reflect light
form branched chains
Mycoplasma
INVISIBLE!
5. slide 5
Mycoplasma
Smallest, simplest prokaryotes
Size ranges from 0.2 to 0.8 µΜ
Many species cannot be removed by
filtration
Cannot be visualized, even at high
concentrations
Lacks Rigid Cell Wall
Not affected by traditional antibiotics
used in cell culture
Limited Biosynthetic capabilities
Utilize nutrients from “host”
6. slide 6
Adverse Effects of Mycoplasma
Contamination
DNA fragmentation associated with mycoplasmal nucleases
Induction of Apoptosis
Alters Gene Expression in contaminated cells
Affects Cell Metabolism
Changes in Signal Transduction
7. slide 7
Mycoplasma - Effects
Inhibition of cell growth
Inhibition of cell metabolism
Disruption of nucleic acid synthesis
Chromosomal aberrations
Changes in cell membrane antigenicity
Alteration of DNA transfection
efficiency
Increased sensitivity
to inducers of apoptosis
DNA fragmentation due to
Mycoplasmal nucleases
NOT apoptosis
CELL DEATH
Production of viruses
compromised
8. slide 8
Most Common Species
Mycoplasma orale (human)
20-40%
Mycoplasma hyorhinis (swine)
10-40%
Mycoplasma arginini (bovine)
20-30%
Mycoplasma fermentans (human)
10-20%
Mycoplasma hominis (human)
10-20%
Acholeplasma laidlawii (bovine)
5-20%
10. slide 10
Sources
Cross contamination from infected cultures
Laboratory Personnel
Culture Reagents (i.e. bovine serum)
Original isolate tissue (<1%)
11. slide 11
Continuous Monitoring
Monitoring of U937 cells for the presence of mycoplasma
0
0.2
0.4
0.6
0.8
1
1.2
1.4
week1 week 2 week3 week 4 week 5 week 6
MycoAlert®ratio
12. slide 12
Summary
Mycoplasma are a real problem in cell culture
Changes in gene expression
Changes in cell functioncytotoxicity
Mycoplasma contamination is often invisible
Infection from Laboratory personnel and reagents requires
routine monitoring
Mycoplasma testing of new cells is essential
14. slide 14
MycoAlert® for Mycoplasma detection
The assay detects the activity of two enzymes found in
mycoplasma and other mollicutes
Enzymes are associated with energy generation pathways that
result in ATP synthesis
The enzymes are found in all 6 of the main mycoplasma cell
culture contaminants and the majority of mollicute species
Being an enzyme assay, MycoAlert® only detects live
mycoplasma
The enzymes are not found in eukaryotic cells
17. slide 17
Assay Sensitivity
HepG2 cells were infected with mycoplasma at the concentration listed, the cells
were then cultured for 4 hours before being tested with a range of techniques
A. Laidlawii Culture Fluorescence PCR MycoAlert®
0 Negative Negative Negative Negative
20 Positive Positive Negative Positive
200 Positive Positive Positive Positive
2000 Positive Positive Positive Positive
20000 Positive Positive Positive Positive
200000 Positive Positive Negative Positive
0.1
10.0
1.0
100.0
CFU/ml
Mycoalert®ratio
0 20 200 2000 20000 200000
Samples provided, enumerated and tested by the European Collection of Cell Cultures (ECACC) with the exception of the MycoAlert® testing which was performed by Lonza
18. slide 18
Effect of Common Media Components
10
0.1
1
RPMI
Penicillin/
Streptomycin
10% DMSO
Trypsin/
EDTA
Serum 20%
Non Essential
Amino Acids
Glutamate
Sodium
Pyruvate
EMEM
Iscove’s
Negative
Mycoplasma positive
M. faucium added to create the positive samples
MycoAlert®Ratio
20. slide 20
Best Practices
Receive cells
Quarantine & test for mycoplasma
NEGATIVE
Put cells in normal
culture
Prepare cryo
samples
Monitor frequently
Use in experimental
protocols
POSITIVE
Destroy cells
Inform source
Quarantine cells
Treat with MycoZap™
Inform source
Monitor daily for
mycoplasma
Keep in quarantine until
infection eradicated for
4 weeks
21. slide 21
Best Practices
We strongly recommend to discard contaminated cultures
It is not known if all cell functions return upon elimination
In cases where fresh stocks cannot be obtained one should
consider treatment of the cells…
22. slide 22
Treatments
Standard Antibiotics- most prominent
Time-consuming (prolonged periods of >6 weeks needed)
Usually the success rates are relatively low…
Low antibiotic concentration
Heat-instability of the antibiotic
Early termination of the treatment
Formation of resistant mycoplasma
Recontamination possible
MycoZap™ Reagent
Fast (clear contamination in little as 4 days)
Highly reliable and definite elimination of mycoplasma
23. slide 23
MycoZap™ Reagent
for Mycoplasma Elimination
Minimal toxic effect on host cells
Optimized reagent mix- combination of antibiotic and antimetabolic
agents
Reagent 1 is a detergent
Integrates into the mycoplasma cell wall and
disintegrates the mycoplasma particle
Pretreatment will reduce the mycoplasma titer drastically
Reagent 2 is an antibiotic
Mycoplasma particles which survived this treatment by
hiding in cell clusters or cell crinkles will be destroyed
subsequently by the antibiotic
25. slide 25
MycoZap™ Summary
Easy to use
Simply add Reagent to your culture
Universal
One protocol to eliminate all mollicutes
Complete
All required reagents are in one kit, one protocol
Effective clean up
Combination of antibiotic and antimetabolic agents- disrupts
mycoplasma
Minimum toxic effect on host cells
Allows you to rescue priceless or high value cell line from
destruction and enables lab to continue using cell line and add
value to their investment
Hello everyone
My name is ……. and I am the Life Science specialist for the Cell Discovery Division of Lonza Bioscience.
I would like to talk to you today about mycoplasma. This is something every cell biologist has heard about
There seem to be three camps of people when you start to discuss mycoplasma, those who are aware and test regularly – we salute you! Those who are aware , know they ought to test but somehow never quite get round to it and those who definitely know that their lab is clean even though they have never tested and therefore can ignore any issues.
Hopefully this webinar will demystify the whole mycoplasma issue, raise awareness of the issues and offer convenient and really easy solutions to ensure that the fear – whilst we can’t guarantee to make it disappear will definitely become more manageable.
Mycoplasma –Background (What is it, what effects it can have), Detection techniques, Our Solution for detection. Best Practices for Clean up.
Some questions to ask before doing presentation or demo
What would be the consequences of an infected cell line to you and your lab?
How would the loss of a cell line adversely affect your lab and your research?
What routine testing can you keep in house to ensure you are mycoplasma free?
Bacterial infections of media ( especially media containing no antibiotics) can be quite common. The infection is normally quite rapid and can be seen by a pH change in the media as well as the media becoming cloudy. When observed under light microscopy the bacteria can be seen as small black specks which when closely observed are under motion. Observation is necessary for low lying infections as bacteria can often be mistaken as cell debris. Cell debris will only undergo Brownian motion ( a slight trembling type motion) whereas bacteria will show a defined ( sometimes quite rapid) activity.
Fungus is one of the easier contaminations to spot when established as it is usually floating on the surface of the culture media. When observed down the microscope it appears in the early stages of infection it appears as filamentous
strands associated with the cells but can be quite difficult to spot. As the infection progresses this web of strands gets larger and more intermeshed.
Yeast infections usually produce oval/round shaped organisms which are quite easy to spot as they reflect the light well so appear quite bright under light microscopy. They are characterised by their tendency to appear as groups of “buds” in the early stages. If left to progress they form branched chains rather like beads. They are distinctly smaller than cells.
Mycoplasma
There is no picture in this section as mycoplasmal infections cannot be seen under light microscopy like bacteria, fungus and yeast. They do not produce any black moving specks or bright beads. They do not even affect the pH of the media or its clarity. The most disturbing aspect of a mycoplasma infection is that it is virtually invisible.
Usually the first indication that cells are infected with mycoplasma is the niggling doubt that the cells are not “acting quite right”. This may be noted by a difference in the population doubling rate or the response to a normal drug dosing regime. Unfortunately by the time these effects are seen the culture is invariably heavily infected and the best course of action is to discard it immediately before other precious stocks are also infected.
This electron micrograph shows mycoplasma attached to the cell. From this picture it becomes more apparent why these infections are so detrimental to the cell. The mycoplasma are almost like parasites requiring the cell to work much harder just to stay alive. They deplete essential nutrients from the media making life in the flask a struggle.
Mycoplasmas are from the family Mollicutes, which includes Acholeplasma, Ureaplasma and other species. However the term Mycoplasma is most often used as a ‘cover-all’. More than 180 species have been identified of which 20 distinct Mycoplasma and Acholeplasma species from human, bovine and swine have been isolated from cell culture.
There are 6 species that account for 95% of all mycoplasma infections, these are M.orale, M.arginii, M.fermentans, M.salivarum, M.hyorhinis and A.laidlawii.
Mycoplasmas are widespread in nature as parasites of humans, mammals, reptiles, insects and plants. They are the smallest and simplest prokaryotes, they lack a rigid cell wall and are surrounded by a single plasma membrane. The lack of cell wall means that they are resistant to a number of commonly used antibiotics and are therefore difficult to eradicate.
They are dependant on their hosts for many nutrients as their biosynthetic capabilities are limited.
(Mycoplasma photos are of M. Hyorhinis, M. Orale and M. Pnemoniae)
Mycoplasma have long been recognised as common contaminants of cell lines in continuous culture. Unlike the turbid growth that is commonly associated with bacterial and fungal contamination, the presence of mycoplasma can go undetected for months.
As the mycoplasma compete with the cell lines for the nutrients in culture media, one of the first signs is a slow down in cell proliferation and slight changes in cellular responses and gene expression. They can cleave DNA to give the appearance of apoptosis, and also induce gene expression – for example IL-2 and interferons.
Their metabolism can also cause aberrant results with tetrazolium assays, and so could mask any cytotoxic effects of compounds and cause shifts in IC50 values. This is the result of the ability of certain mycoplasma species to reduce the tetrazolium salts and cause an increase in coloured product.
Mycoplasma contamination can alter experimental outcomes significantly and can have serious consequences as virtually every cellular process can be altered by the infection
Mycoplasmas grow slowly and colonies may take up to 3 weeks to develop and are typically very small.
There are currently different Mycoplasma detection methods:
Culture:
Advantage: Properly done this offers the greatest sense of security
Disadvantage: Costly and can take up to a month for results.
Indirect tests measuring biochemical markers and other
characteristics associated with mycoplasmas
DNA Flourochrome staining
Advantages: These are typically faster than direct culture methods. Can detect the non-cultivable mycoplasmal strains that direct culture may miss.
Disadvantages: They do require a higher level of contamination 10 to the 4 organisms/ml for detection. Sometimes hard to read
PCR
Advantages: Rapid, 6 to 8 hours, and inexpensive. requires small amount of sample 200-400 ul.
Disadvantages: Subject to False Positives
When the presence of Mycoplasma is suspected, it is usually confirmed by one of these three methods: DNA staining (Hoechst or DAPI), culture or PCR.
PCR requires both skill and time, and is currently not accepted by the FDA. Hoechst or DAPI DNA staining is subjective, especially with low contamination, requiring expertise, also time to prepare samples. Culture of mycoplasma is definitive but difficult, so is usually sent off to a specialist lab. Both PCR and Hoecsht staining will pick up mycoplasmal DNA from non-viable organisms, and therefore gives false positives. Also not all PCR primers will detect all the relevant contaminating species, for example the Takara kit does not pick up Acholeplasma, and A.laidlawii is a common contaminant.
A comparison of detection technologies. A HepG2 cell line was infected with mycoplasma at a range of CFUs per ml. The cells were cultured for 4 hours post infected as per ECACC SOPs and then tested by culture, fluorescence, PCR and MycoAlert. These results do not show absolute detection limits due to the culture period employed by ECACC which will have allowed time for some degree of mycoplasma multiplication. ECACC have developed their own PCR system as they have continued problems with all commercial kits as is illustrated by the data they supplied above.
The effect of common media components that may be found in samples tested with MycoAlert were tested to see if they interfered with the assay. All sample were made up in RPMI media, positive samples had the addition of M. faucium mycoplasma. The results show that none of the components used interfered significantly with the assay, positives remained positive and negatives remained negative.
The use of DMSO showed a reduction in the positive ratio and a more negative negative ratio. DMSO is known to inhibit luciferase to a degree at high concentrations. It should be noted that the concentration of DMSO found in cell culture would normally be no greater than 1%, only concentrated samples for freezing in liquid Nitrogen would contain as high as 10% and we do not recommend testing such samples without expansion into media.
Trypsin/EDTA showed a reduced positive ratio, EDTA at high concentrations inhibits luciferase.
20% Serum showed a reduced positive ratio, serum darkens the sample and quenches the light emitted by the reaction to a degree.
