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Secretariat : c/o IFAH, rue Defacqz, 1 - B - 1000 Bruxelles (Belgium) - Tel. +32-2-543.75.72, Fax +32-2-543.75.85

e-mail: sec@vichsec.org – Internet:www.vichsec.org

VICH GL34 (BIOLOGICALS: MYCOPLASMA)

November 2011

For consultation at Step 4 - Draft 2

TESTING FOR THE DETECTION OF

MYCOPLASMA CONTAMINATION

Recommended for Consultation

at Step 4 of the VICH Process

in November 2011

by the VICH Steering Committee

THIS GUIDELINE HAS BEEN DEVELOPED BY THE APPROPRIATE VICH EXPERT WORKING GROUP 

AND IS SUBJECT TO CONSULTATION BY THE PARTIES, IN ACCORDANCE WITH THE VICH PROCESS.

AT STEP 7 OF THE PROCESS THE FINAL DRAFT WILL BE RECOMMENDED FOR ADOPTION TO THE 

REGULATORY BODIES OF THE EUROPEAN UNION, JAPAN AND USA.
1. INTRODUCTION

1.1. Objective of the guideline

This VICH (International Cooperation on Harmonization of Technical Requirements for 

Registration of Veterinary Medicinal Products) guideline is intended to facilitate the 

harmonized licensing of new products for veterinary use. It is important that biological 

products for veterinary use are free of contamination with Mycoplasmas to help assure 

consistency of production and final product safety. Mycoplasma contaminants may be 

introduced into cell culture and in ovo origin biological products through the master 

seeds, the master cell seed (stock), starting materials of animal origin, and in 

processing of biological materials during passage and product assembly. Therefore it 

is necessary to demonstrate through testing that Mycoplasmas are not present, within 

the limits of the test, in the final product, working seeds and cells and harvests, and 

starting materials such as the master seed, master cell seed, and ingredients of animal 

origin. This guideline establishes stages of manufacture to be tested and test 

procedures to detect the presence of Mycoplasma contamination. It will provide a 

unified standard that will facilitate the mutual acceptance of test data by the relevant 

regulatory authorities. Methods proven equivalent to the guideline method by 

scientifically accepted criteria could also be acceptable.

1.2. Background

The present methods for testing for Mycoplasma contamination are described in the 

Japanese “Minimum requirements of biological products for animal use (2002)”, the 

European Pharmacopoeia (7

th Edition, 2011, 2.6.7), and the United States Code of 

Federal Regulations, Title 9, 113.28. These requirements are all similar in that they 

require testing for Mycoplasma contamination using a broth and agar technique. The 

requirements do however differ in the specifics of these broth and agar tests as well as 

other alternative test methods that are required or approved for use in detecting 

Mycoplasma contamination.

1.3. Scope of guideline

This guideline describes the manner in which tests conducted to detect the presence 

of Mycoplasma contamination in cell culture and in ovo origin biological products for 

veterinary use shall be done to assure the absence of Mycoplasma contamination. 

Tests on master seeds, master cell seeds (stocks), working seeds and cells, 

ingredients of animal origin, harvests and live final vaccine and harvests for killed 

products are included. Bacterial products which grow in the mycoplasma test media 

and products for which mycoplasma contamination risk has been addressed through a 
validated mycoplasma inactivation procedure will be considered outside this guideline. 

The absence of mycoplasma contamination in eggs used for production is controlled 

by appropriate testing of the flock, which is not covered by this guideline.

1.4. Test Methods 

The guideline describes two test methods: 1) expansion in broth culture and detection 

by colony formation on nutrient agar plates; and 2) expansion in cell culture and 

characteristic fluorescent staining of DNA (a technique capable of detecting noncultivatable strains). 

A third test methodology, nucleic acid amplification (NAT), is acknowledged, but is not 

included in this guideline. The use of validated NAT techniques is currently approved 

or under consideration by regulatory authorities for more rapid detection confirmation, 

and strain identification. Appropriately validated NAT techniques may be used as an 

alternative to the broth/agar culture method and/or the indicator cell culture method

provided the NAT test is shown to be at least equivalent in detection limit to the test 

methods in this guideline. A sample that tests positive by NAT may be directly 

considered unsuitable for use. If confirmation of the presence of living mycoplasma in 

the material under test is needed the broth/agar culture method or the indicator cell 

culture method should be used. Evaluation of NAT method use is encouraged in 

parallel testing to further develop, compare, and refine the technique for possible 

inclusion in future versions of this guideline. 

2. GUIDELINE FOR TESTING FOR MYCOPLASMA 

CONTAMINATION 

2.1. General test procedures for detecting Mycoplasma contamination

The culture method using broth and agar is the fundamental method of Mycoplasma 

detection. A solid and liquid media culture method shall be used to test harvests or 

final batches of vaccine, and ingredients of animal origin. Master seed, master cell 

seed (stock), and working seed and cell lots shall be tested using both a solid and 

liquid media culture method and an indicator cell culture method with DNA stain. 

Should either method result in a positive test for mycoplasma the sample is considered 

positive and is unsuitable for use.

Material Broth & Agar Culture DNA Stain

Master Seed & Master Cell Seed Required Required

Working Seed & Working Cell Seed Required Required

Ingredient of Animal Origin1,2 Required

Harvest When testing required3

Final Product When testing required3

1 Excluding eggs

2 Unless a validated mycoplasma inactivation procedure has been applied

3 The competent authorities require testing of different combinations of harvests and final 

product. 

2.2. Culture test system validation 

The culture method should be carried out to validate the detection limit of a 

laboratory’s mycoplasma detection method. A sufficient number of both solid and 

liquid media shall be used to insure the growth of a low level of the following 5 strains 

of mycoplasmas. 
Acholeplasma laidlawii

Mycoplasma hyorhinis

Mycoplasma orale

Mycoplasma synoviae

Mycoplasma fermentans

The species were selected to reflect a range (within a practical number) of antibiotic 

sensitivity (to detect inhibition of mycoplasma growth in the assay), fastidiousness, 

rapidity of growth, likelihood of being a contaminant, and pathogenicity in avian or 

mammalian target species. Acholeplasma laidlawii is a common cell culture 

contaminant of animal and possibly environmental origin. Mycoplasma hyorhinis is 

fastidious, a common cell culture contaminant of animal origin, and a mammalian 

pathogen. Mycoplasma orale is antibiotic sensitive and is a common cell culture 

contaminant of human origin. Mycoplasma synoviae is fastidious (having a 

nicotinamide-adenine-dinucleotide [DPN, NAD] and cysteine requirement) and is an 

avian pathogen. Mycoplasma fermentans is a slow-growing organism and a common 

cell culture contaminant of human origin.

References of the strains used to validate the laboratory mycoplasma contamination 

culture test system should be of low passage level (15 or less), and identified relative 

to type culture isolates, (see Appendix 3.2 for further information on reference strains). 

The reference strains used to validate the culture test system will be appropriate to the 

products tested (see table). Validation for M. synoviae is required when materials of 

avian origin are used at any stage in development and production. Validation for M. 

hyorhinis and A. laidlawii is required when materials of mammalian origin are used at 

any stage in development and production. Validation for M. orale is required when an 

antibiotic has been used at any stage in development and production. Reference 

Preparations shall be used to validate each production lot of broth and agar. At least 

one reference strain must be used as a control with each test. 

Required Reference Organisms by: product type; test method, and presence of 

antibiotics

Vaccine type

Antibiotic content

Test Method

A.

laidlawii

M.

orale

M. 

hyorhinis

M.

synoviae

M. 

fermentans

Avian in ovo origin 

vaccine

Without Antibiotics 

Broth/Agar Method

X X

Avian in ovo origin 

vaccine

With Antibiotics

Broth/Agar Method

X X X

Avian cell culture origin 

vaccine

Without Antibiotics

Broth/Agar Method

X X X

Avian cell culture origin 

vaccine 

With Antibiotics

Broth/Agar Method

X X X X
Mammalian cell culture 

origin vaccine 

Without Antibiotics 

Broth/Agar Method

X X X

Mammalian cell culture 

origin vaccine With 

Antibiotics

Broth/Agar Method

X X X X

Vaccine Without 

Antibiotics

DNA Staining Method X X

Vaccine With Antibiotics

DNA Staining Method

X X

2.3. Culture Method

2.3.1. Incubation conditions

Incubate the broth culture medium or media in tightly stoppered containers in 

air. Incubate all agar plates under microaerophilic conditions (nitrogen 

containing 5-10% CO2). For the solid medium or media, maintain an 

atmosphere of adequate humidity to prevent desiccation of the agar surface.

2.3.2. Nutritive properties of a new batch of medium

Each new lot (batch) of medium must be tested for the nutritive properties using 

references specified above in Section 2.2. Each testing laboratory must 

determine the inoculum for each of their references that will contain a low level 

(not more than 100 CFU). Inoculate the solid medium with a low level (not 

more than 100 CFU) per 60 mm plate and per 100 ml container of broth 

medium. Use at least one agar plate and broth container for each reference. 

Incubate the agar and broth media and make subcultures from the broth onto 

agar at the specified intervals. The agar medium batch complies with the test 

for nutritive properties if for all the references specified, growth obtained does 

not differ by a factor greater than 5 from the value calculated with respect to the 

inoculum. The broth complies if Mycoplasma growth on those agar plates 

subcultured from the broth is achieved for each reference specified. Media 

formulations found effective are included in Appendix 3.1 of this guideline.

2.3.3. Inhibitory substances

Carry out the test for nutritive properties in the presence and absence of the 

material to be tested at the time of prelicense and whenever there is a change 

in the production method that may affect the detection of mycoplasmas. If 

growth of the references occurs more than one sub-culture sooner without the 

test material than with the test material, or if plates directly inoculated with the 

test material have less than one-fifth of the colonies of those directly inoculated 

without the test material, the test material contains inhibitory substances. 

These substances must be neutralized or their effect otherwise countered, e.g., 

through passage in substrates not containing inhibitors or dilution in a larger 

volume of medium, before the test for mycoplasma contamination is carried 

out. For the dilution technique, larger medium volumes may be used or the 

inoculum volume may be divided among multiple 100 ml flasks. The 
effectiveness of the neutralization or other process is confirmed by repeating 

the test for inhibitory substances after neutralization.

2.3.4. Test method

2.3.4.1. The amount of inoculum for each plate of solid medium is 0.2 ml of 

product to be examined. When an assay for mycoplasma concerns 

master and working seeds, master and working cells, and ingredients 

of animal origin a volume of not less than 10 ml of undiluted sample 

shall be tested in each liquid medium. The volume of final product to 

be tested in each liquid medium shall be as required by the regulatory 

authority issuing the marketing authorization. These are currently not 

less than 1 ml in Japan and the US and not less than 10 ml in the EU. 

Incubate the agar plates at 35oC to 38oC, microaerophilically, for 10-14 

days in an atmosphere of adequate humidity to prevent desiccation of 

the surface. Incubate the liquid media at 35o C to 38o C in tightly 

stoppered containers in air for 20-21 days. At the same time incubate 

an uninoculated 100 ml portion of each liquid medium and agar plates 

as a negative control. If any significant pH change occurs upon the 

addition of the product to be examined (this should be determined at 

the time of prelicense), the liquid medium shall be restored to its 

original pH value by the addition of a solution of either sodium 

hydroxide or hydrochloric acid. Between the 2nd and 4th day after 

inoculation, subculture each liquid culture by inoculating at least 1 

plate of each solid medium with 0.2 ml and incubate them at 35oC to 

38oC microaerophilically for 10-14 days. Repeat the procedure 

between the 6th and 8th day, again between the 13th and 15th day, and 

again between the 19th and 21st day of the test. Incubate those agar 

plates inoculated on day 19, 20, or 21 for 7 days. Observe the liquid 

medium or media every 2 or 3 days and if a color change occurs, 

subculture. Color change detection requires the addition of phenol red 

to the media.

2.3.4.2. If the liquid medium or media shows bacterial or fungal contamination, 

repeat the test. If it is not possible to read at least one plate per 

inoculation day, the test must be repeated.

2.3.4.3. Include in the test, positive controls prepared by inoculating a low level 

(not more than 100 CFU) of at least one of the reference species onto 

the agar plates and into the broth medium or media. If the test is run 

on a routine basis, the control species should be rotated on a regular 

basis. This control shall be used in each test conducted with a 

medium that has been validated for nutritive properties using 

references determined by the types of products being tested as 

specified in Section 2.2 of this guideline.

2.3.5. Judgment of the culture method 

At the end of the incubation period, examine all the inoculated solid media 

microscopically for the presence of mycoplasma colonies. The product is 

negative for Mycoplasma contamination if the growth of typical Mycoplasma 

colonies has not occurred on any of the inoculated solid media. If growth of 

typical Mycoplasma colonies has occurred on any of the solid media, the test 

and sample tested are considered positive for Mycoplasma contamination. The 
test is invalid if the positive controls do not show growth of mycoplasma on at 

least one subculture plate or the negative controls are positive for mycoplasma 

contamination. If either of the controls is invalid the test must be repeated. If 

suspect colonies are observed, confirmation of mycoplasma contamination may 

be accomplished using an appropriate and validated method. 

2.4. Indicator cell culture method

Cell cultures are stained with a fluorescent dye that binds to DNA. Mycoplasmas are 

detected by their characteristic particulate or filamentous pattern of fluorescence on 

the cell surface, and if contamination is heavy, in the surrounding areas. Mitochondria 

in the cytoplasm may be stained, but may be differentiated from mycoplasma.

2.4.1. Validation of the indicator cell culture method

Using a VERO or other equivalent in efficiency indicator cell culture substrate, 

validate the procedure using an inoculum not more than 100 CFU or CFU-like 

micro-organisms of appropriate references of M. hyorhinis and M. orale. Both 

references must be positive when stained with the DNA stain at the end of the 

test.

If for viral, etc., suspensions the interpretation of results is affected by 

cytopathic effects, the virus may be neutralized using a specific antiserum that 

has no inhibitory effects on mycoplasmas, or an alternative cell culture 

substrate that does not allow the growth of the virus may be used. To 

demonstrate the absence of inhibitory effects of serum, carry out the positive 

control tests in the presence of neutralizing antiserum. Antiserum lots may be 

qualified once rather than at use.

2.4.2. Test method

2.4.2.1. Seed the indicator cell culture at a suitable density that will yield 

confluence of the cells after 3 days of growth (example: 2 X 104to 2 X 

105cells per ml, 4 X 103to 2.5 X 104cells/cm2) in a cell culture vessel 

of not less than 25 cm2. The indicator cell culture should be subcultured without antibiotic prior to use. Inoculate 1 ml of the sample to 

be examined into the cell culture vessel and incubate at 35o C to 38o C. 

2.4.2.2. After at least 3 days of incubation and the cells have grown to 

confluence, make a subculture onto cover slips in suitable containers 

or on some other surface (chambered slides) suitable for the test 

procedure. Seed the cells in the second subculture at a low density so 

that they reach only 50% confluence after 3-5 days of incubation. 

Complete confluence must be avoided because it impairs visualization 

of mycoplasmas after staining.

2.4.2.3. Remove medium from cover slips or chambered slides. Rinse the 

monolayer of indicator cells with phosphate buffered saline (PBS) and 

then fix with glacial acetic acid/methanol (1 to 3) or some other 

suitable fixing solution.

2.4.2.4. Remove the fixing solution and discard. Wash the fixing solution with 

sterile water and dry slides completely if they are to be stained more 

than one hour later.
2.4.2.5. Add a suitable fluorescent dye that binds to DNA such as bisbenzimide 

stain (Hoechst compound 33258, bisbenzimidazole, 5 ug/L) and allow 

to stain for a suitable time.

2.4.2.6. Remove the stain and rinse the monolayer with water. Mount the 

cover slips if applicable and examine the slides by fluorescence (for 

bisbenzimide stain use a 330 nm/380 nm excitation filter, LP 440 nm 

barrier filter) at 400 X magnification or greater.

2.4.2.7. Compare the microscopic appearance of the test cultures with that of 

the negative and reference controls, examining for extranuclear 

fluorescence. Mycoplasmas produce pinpoints or filaments over the 

indicator cell’s cytoplasm. They may also produce pinpoints and 

filaments in the intercellular spaces. Multiple microscopic fields as 

validated should be examined.

2.4.3. Judgment of the indicator cell culture method

The product being examined is negative for Mycoplasma contamination if there 

is no evidence of pinpoints or filaments of extranuclear fluorescence. If the 

slides inoculated with the product contain evidence of pinpoints or extranuclear 

fluorescence indicative of Mycoplasma the test and sample tested are 

considered positive for Mycoplasma contamination. The test is invalid if the 

positive controls do not show the presence of the appropriate extranuclear 

fluorescence of the reference organisms or the negative cell controls contain 

extranuclear fluorescence. If either of the controls is invalid the test must be 

repeated.
3. APPENDICES

3.1. Regional examples of suitable broth and agar formulations

9 CFR Mycoplasma Broth

Heart Infusion Broth 62.5 g

Proteose Peptone #3 25.0 g

Yeast Extract 12.5 ml

1 % Thallium Acetate 62.5 ml

1 % Tetrazolium Chloride 13.75 ml

Penicillin (100,000 units/ml) 12.5 ml 

Heat inactivated Horse Serum 250 ml

H2O 2425 ml

Mix all ingredients well and adjust pH to 7.9 with 10 Normal NaOH.

Filter sterilize through a 0.2 μ filter. Dispense into sterile test vessels.

Add DPN/L-Cysteine solution before use, 2 ml/100 ml of broth.

9 CFR Mycoplasma Agar

Heart Infusion Agar 25 g

Heart Infusion Broth 10 g

Proteose Peptone #3 10 g

1% Thallium Acetate 25 ml

H2O 995 ml

Heat Inactivated Horse Serum 126 ml

Yeast Extract 5 ml

Penicillin (100,000 units/ml) 5.2 ml

DPN/L-Cysteine 21 ml

Combine heart infusion agar, heart infusion broth, proteose peptone #3, Thallium acetate, 

and H2O.

Mix and bring to boil, then cool. Adjust the pH to 7.9 with 10 Normal NaOH.

Autoclave 20 min. at 121o C. Cool in water bath to 56o C.

Aseptically add: horse serum, yeast extract, Penicillin, and DPN/L-Cysteine. 

Dispense 12 ml into each 15 X 60 mm petri dish.

DPN/L-Cysteine solution

Nicotiamide-adenine-dinucleotide (DPN, NAD) 5 g

Q.S. with H2O to 500 ml

L-Cysteine 5 g

Q.S. with H2O to 500 ml

Mix each chemical separately until dissolved.

Mix the two solutions and filter sterilize.

Japanese Liquid Medium for Mycoplasma

Basal Medium

50 % w/v Bovine Cardiac Muscle Extract 100 ml

Meat Peptone 10 g
Sodium Chloride 5 g

Glucose 1 g

Sodium L-glutamate 0.1 g

L-arginine hydrochloride 1 g

H2O QS to 1000 ml

Filter sterilize through 0.22 μ membrane filter or sterilize at 121o C for 15 min.

Adjust the pH of the medium after sterilization to 7.2-7.4.

Additives for 77 ml of the Basal medium;

Horse Serum 10 ml

Inactivated Porcine Serum 5 ml

25 % w/v Fresh Yeast Extract 5 ml

1 % w/v β-NAD (oxidized) 1 ml

1 % w/v L-cysteine HCL (1 H2O) 1 ml

0.2 % w/v phenol red 1 ml

Previously filter sterilize the additives and aseptically add to the sterilized basal 

medium. The additives which can be sterilized by high pressure can be autoclaved. 

Penicillin G potassium, 500 units/ml of the medium, or Thallium acetate, 0.02 % w/v, 

can be added.

Japanese Agar Medium for Mycoplasma

Basal Medium 78 ml

Agar 1 g

Sterilize by autoclaving 121oC for 15 min. 

Additives:

Horse Serum 10 ml

Inactivated Porcine Serum 5 ml

25 % w/v fresh yeast extract 5 ml

1 % w/v β-NAD (oxidized) 1 ml

1 % w/v L-cysteine HCl (1 H2O) 1 ml

Penicillin G potassium, 500 units per ml of medium, or thallium acetate, 0.02 % w/v can be 

added.

Add the additives to basal/agar medium which has been liquefied by heating, and divide into 

sterile petri dishes, 45-55 mm. Cool and allow to solidify.

EP Hayflick media (Recommended media for the general detection of mycoplasmas)

Liquid Medium:

Beef Heart Infusion Broth (1) 90 ml

Horse Serum (unheated) 20 ml

Yeast Extract (250 g/L) 10 ml

Phenol Red (0.6 g/L solution) 5 ml

Penicillin (20,000 I.U. per ml) 0.25 ml

Deoxyribonucleic acid (2 g/L solution) 1.2 ml

Adjust to pH 7.8.

Solid Medium:
Prepare as described for the liquid medium above but replace beef heart infusion broth 

with beef heart infusion agar containing 15 g/L of agar.

EP Frey media (Recommended Media for the detection of M. synoviae)

Liquid Medium:

Beef Heart Infusion Broth (1) 90 ml

Essential Vitamins (2) 0.025 ml

Glucose monohydrate (500 g/L solution) 2 ml

Swine serum (inactivated at 56oC for 30 min.) 12 ml

β-Nicotinamide adenine dinucleotide (10 g/L solution) 1 ml 

Cysteine hydrochloride (10 g/L solution) 1 ml

Phenol Red (0.6 g/L solution) 5 ml

Penicillin (20,000 I.U. per ml) 0.25 ml

Mix the solutions of β-nicotinamide adenine dinucleotide and cysteine hydrochloride 

and after 10 minutes, add the other ingredients. Adjust pH to 7.8.

Solid Medium:

Beef Heart Infusion Broth (1) 90 ml

Ionagar (3) 1.4 g

Adjust pH to 7.8, and sterilize by autoclaving, then add:

Essential Vitamins (2) 0.025 ml

Glucose monohydrate (500 g/L solution) 2 ml

Swine serum (unheated) 12 ml

β-Nicotinamide adenine dinucleotide (10g/L solution) 1 ml

Cysteine hydrochloride (10 g/L solutions) 1 ml

Phenol Red (0.6 g/L solution) 5 ml

Penicillin (20,000 I.U. per ml) 0.25 ml

EP Friis media (Recommended Media for the Detection of Non-avian Mycoplasmas)

Liquid Medium:

Hank’s Balanced Salt Solution (modified) (4) 800 ml

H2O 67 ml

Brain Heart Infusion (5) 135 ml

PPLO Broth 248 ml

Yeast Extract (170 g/L) 60 ml

Bacitracin 250 mg

Meticillin 250 mg

Phenol Red (5 g/L) 4.5 ml

Horse Serum 165 ml

Swine Serum 165 ml

Adjust the pH to 7.40-7.45

Solid Medium:

Hank’s Balanced Salt Solution (modified) (4) 200 ml

DEAE-dextran 200 mg

Ionagar (3) 15.65 g

Mix well and sterilize by autoclaving. Cool to 100o C. Add this to 1740 ml of the liquid 

medium described above.
EP Media Sub parts

(1) Beef Heart Infusion Broth

Beef Heart (for preparation of the infusion) 500 g

Peptone 10 g

Sodium Chloride 5 g

H2O QS to 1000 ml

Sterilize by autoclaving.

(2) Essential Vitamins

Biotin 100 mg

Calcium pantothenate 100 mg

Choline chloride 100 mg

Folic acid 100 mg

i-Inositol 200 mg

Nicotinamide 100 mg

Pyridoxal hydrochloride 100 mg

Riboflavin 10 mg

Thiamine hydrochloride 100 mg

H2O QS to1000 ml

(3) Ionagar

A highly refined agar for use in microbiology and immunology, prepared by an ionexchange procedure which results in a product having superior purity, clarity, and gel 

strength.

It contains approximately:

H2O

12.2 %

Ash 1.5 %

Acid insoluble ash 0.2 %

Chlorine 0.0 %

Phosphate (calculated as P2O5) 0.3 %

Total Nitrogen 0.3 %

Copper 8 ppm

Iron 170 ppm

Calcium 0.28 %

Magnesium 0.32 %

(4) Hank’s Balanced Salt Solution (modified)

Sodium chloride 6.4 g

Potassium chloride 0.32 g

Magnesium sulphate heptahydrate 0.08 g

Magnesium chloride hexahydrate 0.08 g

Calcium chloride, anhydrous 0.112 g

Disodium hydrogen phosphate dihydrate 0.0596 g

Potassium dihydrogen phosphate, anhydrous 0.048 g

H2O QS to 800 ml

(5) Brain heart infusion

Calf brain infusion 200 g

Beef heart infusion 250 g

Proteose peptone 10 g

Glucose 2 g
Sodium chloride 5 g

Disodium hydrogen phosphate, anhydrous 2.5 g

H2O QS to 1000 ml

(6) PPLO broth

Beef heart infusion 50 g

Peptone 10 g

Sodium chloride 5 g

H2O QS to 1000 ml

Bisbenzimide stain solution for DNA Staining

Hoechst compound 33258 (bisbenzimidazole), 5 μg per liter of buffered aqueous 

solution.

Note: The solution should be protected from light.

3.2. Mycoplasma References

Standardization of testing between laboratories and between regions would be 

enhanced by use of references common within or between regions, This has been 

shown to be presently impractical due to the difficulty of producing consistent batches 

of lyophilized references and the shipping issues with frozen references. Therefore, 

regions or laboratories may use their own references, providing that they are of low 

passage level (15 or less), identified relative to type culture isolates, stable, and 

appropriately validated as suitable for use in the context of this guideline. It is strongly 

recommended to include in the validation of detection limit a comparison to the 

EDQM reference strains (described below) for international recognition. Regions or 

laboratories may produce their own validated references, or may acquire commonly

available and appropriately validated references, such as the following produced by 

the EDQM.

The 5 strains of Mycoplasma listed in Section 2.3 were isolated by laboratories of the 

European Union and donated to the European Directorate for the Quality of Medicines 

and HealthCare (EDQM). EDQM produced a sufficient quantity of these frozen 

references, and performed an intra-region EU validation/stability study (C. Milne, A. 

Daas. Establishment of European Pharmacopoeia Mycoplasma Reference Strains. 

Pharmeuropa Bio 2006(1):57-72). Completion of further validation studies by the 

regulatory agencies and the industries in Japan, USA, and Canada confirmed that the 

strains are very suitable for use in the context of this guideline. (VICH Collaborative 

Study on the Ph. Eur. Mycoplasma Reference Strains: EDQM Report Compiling and 

Analysing the Data Set for the VICH Collaborative Study on the European 

Pharmacopoeia Mycoplasma Reference Strains, EDQM Administrator Representative, 

C. Milne, 2010.) The BQMEWG commends the Staff at the EDQM for their efforts and 

perseverance in producing and validating these very excellent references.

For DNA staining validation, the following strains may also prove useful:

M. hyorhinis -- ATCC 29052 

M. orale -- ATCC 23714
3.3. Glossary

Batch (lot, serial) of starting material of animal origin

The total quantity of homogenous material (e.g., cells, serum) identified by a unique 

serial number.

Cell-seed system

A system whereby successive final lots (batches) of a product are manufactured by 

culture in cells derived from the same master cell seed. A number of containers from 

the master cell seed are used to prepare a working cell seed. 

Cell lines

Cultures of cells >10 passages or subcultures from the tissue of origin and having a 

high capacity for multiplication in-vitro. 

Final product, batch, lot, or serial

A collection of closed, final containers or other final dosage units that are expected to 

be homogeneous and equivalent with respect to risk of contamination during filling or 

preparation of the final product. The dosage units are filled, or otherwise prepared, 

from the same final bulk product, freeze-dried together (if applicable) and closed in one 

continuous working session. They bear a distinctive number or code identifying the 

final lot (batch, serial). Where a final bulk product is filled and/or freeze-dried on 

several separate sessions, there results a related set of final lots (batches, serials) that 

are usually identified by the use of a common part in the distinctive number or code; 

these related final lots (batches, serials) are sometimes referred to as sub-batches, 

subserials, sub-lots or filling lots. For the purposes of mycoplasma testing, a single 

sub-batch may be considered representative of the batch.

Harvests

Material derived on one or more occasions from a single production culture inoculated 

with the same working seed lot (single harvest) or pooled material containing a single 

strain or type of micro-organism or antigen and derived from a number of eggs, cell 

culture containers, etc. that are processed at the same time (monovalent pooled

harvest).

Master cell seed (stock)

A collection of aliquots of cells (primary or cell line) of a single passage level for use in 

the preparation of the product, distributed into containers in a single operation, 

processed together and stored in such a manner as to ensure uniformity and stability 

and to prevent contamination. Master cell seed is usually stored at temperatures of -

70oC or lower.

Master seed 

A collection of closed containers of a culture of micro-organisms of a single passage 

level used for the production of all batches of a designated veterinary biological 

product, distributed from a single bulk into containers and processed together in a 

single operation in such a manner as to ensure uniformity and stability and to prevent 

contamination.

Microaerophilic condition

A nitrogen atmosphere containing 5-10% carbon dioxide and sufficient humidity to 

prevent drying of the agar plates. 
Passage

One transfer of cells or microorganisms followed by the normally used incubation 

period for the cell or microorganism concerned.

Primary cell cultures

Primary cell cultures are cultures of cells essentially unchanged from those in the 

animal tissues from which they have been prepared and being no more than 10 in-vitro

passages to the test level from the initial preparation from the animal tissue. The first 

in-vitro cultivation is regarded as the first passage of the cells

Seed-lot system:

A system in which successive batches of a product are derived from the same master 

seed virus. For routine production, a working seed virus may be prepared from the 

master seed virus.

Working cell seed (stock)

A collection of aliquots of cells derived from the master cell seed and at the passage 

level used in the preparation of production cell cultures. The working cell seed is 

distributed into containers, processed and stored as described for master cell seed. 

The term includes production cell seed. 

Working References

A passage of the Reference strains of Mycoplasma produced in the testing laboratory 

for use as controls to satisfy the reference requirements specified in this document.

Working seed

A collection of aliquots of a microorganism derived from the master seed virus and at 

the passage level used in the preparation of product. Working seed virus is distributed 

into containers and stored as described for master seed virus. The term includes 

production seed.