1. Rapid Mycoplasma Testing
Lonza BioResearch
Rapid Mycoplasma Testing
June 2012

2. Content Mycoplasma – An Introduction Mycoplasma Testing for Research
Other tools
So what are we taking about in the next 45 min? I will give a brief introduction on what Mycoplasma are and what impact they have on cells. Then I’m going to present one method of testing for Mycoplasma contamination: for research purposes

3. What Contaminations Can Be Seen in the Microscope?
Bacteria
Small black specks
pH change
Often mistaken as cell debris
Definite movement
Yeast
Oval/round in shape
Smaller than cells
Reflect light (“bright beads”)
Formed branched chains
Fungus
Filamentous strands
Web-like mesh
Mycoplasma
Invisible
Only visible via electron microscopy
So onto familiar ground. I am sure I am not the only one amongst us who has pulled a flask out of the incubator after the weekend and realised that the carefully planned week has just gone up in smoke. When we look down the microscope what can we see.
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 which goes yellow 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.
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.
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 as filamentous strands associated with the cells. This can sometimes be quite difficult to spot. As the infection progresses, this web of strands gets larger and more intermeshed.
Mycoplasma
There is only a electron micrograph and 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”. In my experience cells infected with mycoplasma invariably look absolutely great. They increase their grow rate , they look great under the microscope but, unfortunately, this does not last. Changes start to occur 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 usually 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.

4. What Are Mycoplasma? Smallest, simplest prokaryotes
Size ranges from 0.2 to 0.8 µm
Many species cannot be removed by filtration
Cannot be visualized even at very high concentrations
Lack of rigid cell wall
Not affected by traditional antibiotics used in cell culture
Limited biosynthetic capabilities
Utilize nutrients from “hosts”
So what are Mycoplasma?
They 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 like penicillin 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)

5. Effects of Mycoplasma on Cells
Increased sensitivity
to inducers of apoptosis
Inhibition of cell growth
Alteration of DNA transfection efficiency
Chromosomal aberrations
Inhibition of cell metabolism
Disruption of nucleic acid synthesis
Production of viruses
compromised
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 based proliferation assays, also known as MTT 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.
It has been quoted that a mycoplasma infection affects every known process in the cell. This slide shows examples of processes which are affected by mycoplasma infections.
( Read out the labels…)
DNA fragmentation due to
Mycoplasmal nucleases
NOT apoptosis
Changes in cell membrane antigenicity
CELL DEATH

6. Mycoplasma Impair Transfection Efficiency
HeLa cells infected with
Mycoplasma fermantans
HeLa cells uninfected
Program I-013
no DNA
no program
+ DNA
Here, an example for the impact of Mycoplasma infection on the overall status of the cells: Transfection efficiency can be significantly reduced demonstrated by the following experiment: Hela cells transfected with a GFP expression construct using the electroporation based Nucleofector technology show an expression of GFP twice as high as Mycoplasma-infected Hela’s do.
In addition to decreasing transfection efficiency, Mycoplasma contamination can:
induce chromosomal abnormalities
alter antigenicity of surface proteins (Mycoplasma lives on cell membrane, and can cover up or hide surface proteins)
can compete with your cells for nutrients in tissue culture media
Program I-013
+ DNA
57.7% GFP+
23.0% GFP+
Preliminary data kindly provided by customer

7. Mycoplasma – Types & Frequency
This pie chart shows the 6 most common species which account for 95% of all cell culture infections. As can be seen we have 3 human, 2 bovine and one swine species. The bovine and swine species can be introduced into cell cultures by means of contaminated serum and trypsin respectively.
The human species, as you may imagine, get into the cultures via laboratory personnel.
I am sure you can hazard a guess as to how the human ones get into the cultures – Aaachoo ( excuse me….) just in case you were struggling…

8. Mycoplasma – Prevalence & Sources
15-35 % of continuous cell lines
5% of early passage cell cultures
1% primary cell cultures
Sources
Cross-contamination from infected cultures
Laboratory personnel
Culture reagents (e.g. bovine serum)
Original isolate tissue (<1%)
This is the scary slide. A conservative estimate states that between 15-35% of all continuous cell lines are contaminated with mycoplasma. This has even been reported to be as high as 85% in some countries. We also have incidences of 5% in early passage cultures and 1% of primary cells. The importance of knowing what you are dealing with is paramount.
So where does it come from.
The primary source is normally cross contamination from infected cultures. So the sooner you find out about your contamination, the lower is the risk of cross contamination.

9. The Speed of An Infection
MycoAlert™ Ratio
So how soon can you find out if you have a problem.
This does depend a little on the mycoplasma species you have been lucky enough to get. This graph shows an infection ( a deliberate one!) of some suspension cells. The cells were cultured as normal after infection and a sample of the culture supernatant taken at the time periods shown and assayed to mycoplasma. As you can see, nothing much happened for the first 48hbut then overnight the infection suddenly took off in both species and in less than 3days they were showing a significant contamination. Time for the bin for these babies…

10. Content Mycoplasma – An Introduction Mycoplasma Testing for Research
Other tools
So what are we taking about in the next 45 min? I will give a brief introduction on what Mycoplasma are and what impact they have on cells. Then I’m going to present two methods of testing for Mycoplasma contamination: one for research purposes, one for product release in industry.

11. Mycoplasma – Classical Detection Methods
Non-luminescence methods
Agar culture test (gold standard)
2 – 3 weeks
Often done externally
PCR methods
4-5 hours
Species detection depends on primer set
Also detects dead mycoplasma
Hoechst stain
Time-consuming, poor indicator
Experience required
So how do you go about mycoplasma testing
Well there are the good old ways…
Agar culture. take a sample of your cells and send them off to a specialised lab who will culture them for you. They normally get a result but it takes 2to 4 weeks
Or you could look for mycoplasma using PCR or Hoechst staining. Both these methods involve the use of DNA either amplifying it or staining it. They are much quicker than culture but still require some specialist skills and interpretation. As anyone who has done immunofluorescent staining will know it is usual to get some stained debris on the slide no matter how careful you are and in the case of the Hoescht staining you are looking for little blue dots. You have to do a fair number of these stains before you can be confident, your blue dots are the right ones. This can also be quite subjective: one person may see them another not
And PCR… well most of the time this is a routine procedure ( as easy as riding a bike) until it goes wrong. Anything that upsets the delicate balance in that tube can lead to empty wells on a gel

12. MycoAlert™ Mycoplasma Detection Kit
Unique 20 min bioluminescent assay for mycoplasma detection in cell cultures
Mechanism: 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, the MycoAlert™ Kit only detects viable mollicutes
The enzymes are not found in eukaryotic cells
A much easier way to determine Mycoplasma infection is the MycoAlert Detection assay. This assay is based on bioluminescence and gives you a result within 20 min.
How does the assay work? It detects the activity of two enzymes, specifically found in mycoplasma and other Mollicutes. Both enzymes are associated with ATP synthesis. The enzymes are found in all 6 of the main mycoplasma cell culture contaminants which- as a reminder- account for 95% of all mycoplasma infections. As the assay is enzyme based, the MycoAlert™ Kit only detects viable Mycoplasma.

13. The MycoAlert™ Reaction
Specific mollicute substrate
Photinus pyralis
(The Fire Fly)
Mycoplasma enzymes
ATP
Luciferase
+ Luciferin + O2
LIGHT
+ Oxyluciferin + AMP + PPi + CO2
Luciferase reaction:
Very sensitive (detection of 50 attomols ATP)
Wide dynamic range (six orders of magnitude)
Amount of light is proportional to the ATP present
Compatible with 96-well and 384-well formats
A little bit more background on the Biochemistry of the assay: A mollicute specific substrate is turned into ATP by the two Mycoplasma specific enzymes. The amount of generated ATP is determined by the luciferase-luciferin reaction. This reaction is one of the most efficient reactions in nature with approximately one photon of light being produced from each molecule of ATP. If all other factors are kept constant then the emitted light intensity is linearly related to the ATP concentration and is measured using a luminometer or beta counter. The assay is conducted at ambient temperature (18°C-22°C), the optimal temperature for luciferase. Bioluminescence is now the most widely used method for the assay of ATP due to its very high sensitivity, wide dynamic range, and ease of use.
NB 50 attomols of ATP standard is roughly equivalent to 10 mammalian cells. With cells there is 5 orders of magnitude.

14. MycoAlert™ Protocol 10 min 5 min B/A < 0.9 = negative
ReadB
MycoAlert™ Substrate
10 min
Specific Substrate for Mollicutes
Read A
MycoAlert™ Reagent
5 min
Lysis, Luciferase, Luciferin
Here we have a quick schematic showing the process and how straight forward it is: You have your cleared culture supernatent sample in the tube and add the MycoAlert reagent which contains the lysis agent to break open the mycoplasma and the luciferase/luciferin. The mix is incubated at RT for 5mins to allow the lysis to occur and then a reading is taken in the luminometer –read A. This gives us the background level of ATP present in the mix. We then add the specific mycoplasma substrates and wait for 10mins. If there are mycoplasma there, they make ATP – not the Mycoplasma of course- but the specific enzymes release by the lysis step! We can see this when we take a reading B and see an increase in light levels. If there aren’t any mycoplasma present, the luciferase luciferin reaction with the background ATP will show a reduction due to the ATPase action of this combination and the light levels will decrease.
When you take reading B and divide it by reading A there are 3 out comes:
If the ratio is below 0.9 the cell culture is uninfected. A value above 1,2 means the culture is infected, ratios between 0.9 and 1.2 mean that you need to put your cells in quarantine.
Read these
Sample
100 µl
B/A < 0.9 = negative
B/A > 1.2 = positive
0.9 < B/A < 1.2 = quarantine

15. MycoAlert™ Kit – Assay Kinetics
Time (minutes) 1 10
100
1000
10000 2 4 6 8 12 14 16
RLUs
Reading A +
MycoAlert™ Substrate added
Reading B
MycoAlert™ Reagent added
Clean sample
Infected sample*
This graph shows a time course of the MycoAlert reaction with a positive and negative sample. K562 cells were deliberately infected with Mycoplasma Hyorhinis and assayed against a clean sample of these cells from another laboratory. As you can see the blue line from the clean sample shows a gradual decrease in the relative light units over the incubation periods. This data produces a ratio of les than 1.
in the infected sample, the mycoplasma generate ATP when given the MycoAlert substrate and this is shown by a significant increase in the relative light units seen. The ratio of reading B over reading A is bout significantly greater than 1 and showing a positive for infection.
*Infected sample = supernatant from K562 cell culture infected for 72 hours with M. hyorhinis

16. MycoAlert™ Kit – Species Testing
Result
Acholeplasma laidlawii
positive
Acholeplasma modicum
Acholeplasma morum
Mesoplasma entomophilum
Mesoplasma florum
Mycoplasma alkalescens
Mycoplasma arginini
Mycoplasma arthritidis
Mycoplasma bovirhinis
Mycoplasma bovis
Mycoplasma bovoculi
Mycoplasma buccale
Mycoplasma californicum
Species
Result
Mycoplasma canadense
positive
Mycoplasma cloacale
Mycoplasma conjunctivae
Mycoplasma equirhinis
Mycoplasma faucium
Mycoplasma fermentans
Mycoplasma gallinacium
Mycoplasma gallisepticum
Mycoplasma genitalium
Mycoplasma hominis
Mycoplasma hyopneumoniae
Mycoplasma hyorhinis
Mycoplasma hyosynoviae
Species
Result
Mycoplasma lipophilum
positive
Mycoplasma muris
Mycoplasma neurolyticum
Mycoplasma opalescens
Mycoplasma orale
Mycoplasma pirum
Mycoplasma pneumoniae
Mycoplasma primatum
Mycoplasma pulmonis
Mycoplasma salivarium
Mycoplasma spermatophilum
Mycoplasma synoviae
Spiroplasma citri
To date we have tested 44 species of mollicute. These include species originally isolated from human, bovine, porcine, ovine, canine, murine, avian insect and plant sources. In 5 cases, the mollicute could not be detected, these were a ureaplasma species. This result was expected as Ureaplasmas utilise a different energy generation system from that of all other mollicutes utilising different substrates to those found in MycoAlert.
Mollicute species obtained from the National Collection of Type Cultures UK 39 of 118 species in the collection tested to date

17. MycoAlert™ Results MycoAlert™ Assay compared to a PCR kit K562* U937*
0.1 1 10
100
K562*
U937*
HL60
JURKAT
CHO*
BJAB
COS7*
Ratio
That’s all very well, I hear you say, but it’s relatively new
You might be used to a PCR based Mycoplasma detection kit to monitor your cell cultures. So how does the MycoAlert Kit compare to PCR? This graph shows the answer to the question:
A set of cell lines in culture were found to contain a covert mycoplasma contamination subsequently identified as M. hyorhinis. The cell lines were determined to be mycoplasma positive or negative by use of the MycoAlert assay, the results were verified by use of a Mycoplasma PCR kit. (Stratagene)
MycoAlert™ 39 32
0.4 23
0.8 25
PCR + -
* Positive cell lines (infected with M. hyorhinis)

18. MycoAlert™ Kit – No Interference with Media Components
Influence of different media components on MycoAlert® Results:
The effect of common media components that may be found in samples were tested with MycoAlert 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.
But there are a few components that have a minor effect on the result:
DMSO: 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.

19. Instrumentation Cuvette/tube luminometers Single sample throughput
E.g. Lucetta™ Luminometer with tailor-made MycoAlert™ Mode
Plate-reading luminometers
Up to 96 samples per plate
Can be semi-automated if fitted with reagent injectors for high sample throughput
But some are not sensitive enough for MycoAlert® measurements
Scintillation counters can be used in luminescence mode
List of MycoAlert® compatible luminometers is available at:
And what equipment do you need?
We have, during the development of this assay, tested a number of machines ourselves and also had feedback from other end users. In the MycoAlert section on the Lonza website there is a list of suggested luminometers which also includes machines that we don’t recommend and this is worth checking out. As a rough guide though, we have found that multifunction machines do not have the sensitivity in their luminescent mode to be used for MycoAlert but some manufacturer’s have addressed this by adding an adjustable gain in luminescent mode which can help increase the sensitivity. The web link is shown at the end of this webinar.

20. Content Mycoplasma – An Introduction Mycoplasma Testing for Research
Other Tools
So what are we taking about in the next 45 min? I will give a brief introduction on what Mycoplasma are and what impact they have on cells. Then I’m going to present two methods of testing for Mycoplasma contamination: one for research purposes, one for product release in industry.

21. Summary – Lonza Products
Lonza offers a complete product portfolio for managing mycoplasma contaminations:
Detection:
MycoAlert™ Kit and MycoAlert™ Plus Kit for basic research
Lucetta™ Luminometer
MycoTOOL® PCR Kit for final industrial release testing
Elimination:
MycoZap™ Elimination Kit
Prevention:
Specific MycoZap™ Antibiotics
MycoZap™ Spray (EU only)

22. Support Tools
Please contact the Scientific Support Team for additional assistance or to request a MycoAlert™ Demo
Other helpful tools
(for luminometer list)

23. Thank you for your attention