Mycoplasma Agar Base

(PPLO Agar Base)

Composition per 1300.0mL:

Agar .............................................................. ...14.0g

Pancreatic digest of casein................................. .7.0g

NaCl................................................................. ..5.0g

Beef extract...................................................... ...3.0g

Yeast extract..................................................... ..3.0g

Beef heart, solids from infusion........................ ..2.0g

Horse serum............................................ ...260.0mL

Fresh yeast extract solution........................ ...65.0mL

pH 7.8 ± 0.2 at 25oC

Source: This medium is available as a premixed powder from  BD Diagnostic Systems.

Fresh Yeast Extract Solution:

Composition per 100.0mL:

Baker’s yeast, live, pressed, starch-free......... ...25.0g

Preparation of Fresh Yeast Extract Solution: Add the live Baker’s yeast to 100.0mL of distilled/ deionized water. Autoclave for 90 min at 15 psi pres-
sure-121oC. Allow to stand. Remove supernatant so-
lution. Adjust pH to 6.6-6.8.

Preparation of Medium: Add components, ex-
cept horse serum and fresh yeast extract, to distilled/
deionized water and bring volume to 1.0L. Mix thor-
oughly. Gently heat and bring to boiling. Distribute
into tubes or flasks. Autoclave for 15 min at 15 psi
pressure-121oC. Cool to 50oC. To each 75.0mL of
cooled, sterile basal medium, add 20.0mL of sterile
horse serum and 5.0mL of special yeast extract. Mix
thoroughly. Pour into sterile Petri dishes or distribute
into sterile tubes.

Use: For the preparation of media for the cultivation of Mycoplasma.


Mycoplasma Agar

with Increased Selectivity

Composition per 1300.0mL:

Agar .............................................................. ...14.0g

Pancreatic digest of casein................................. .7.0g

NaCl................................................................. ..5.0g

Beef extract...................................................... ...3.0g

Yeast extract...................................................... ..3.0g

Beef heart, solids from infusion........................ ..2.0g

Thallous acetate ................................................ ..0.7g

Penicillin................................................... ...70,000U

Horse serum............................................. ... 260.0mL

Fresh yeast extract solution ........................ .. 65.0mL

pH 7.8 ± 0.2 at 25oC

Caution: Thallous acetate is a poison.

Fresh Yeast Extract Solution:

Composition per 100.0mL:

Baker’s yeast, live, pressed, starch-free......... ...25.0g

Preparation of Fresh Yeast Extract Solution: Add the live Baker’s yeast to 100.0mL of distilled/ deionized water. Autoclave for 90 min at 15 psi pres-
sure-121oC. Allow to stand. Remove supernatant so-
lution. Adjust pH to 6.6-6.8.

Preparation of Medium: Add components—ex-
cept horse serum, special yeast extract, thallous ace-
tate, and penicillin—to distilled/deionized water and
bring volume to 1.0L. Mix thoroughly. Gently heat
and bring to boiling. Distribute into tubes or flasks.
Autoclave for 15 min at 15 psi pressure-121oC. Cool
to 50oC. To each of 70.0mL of cooled, sterile basal
medium, add 20.0mL of sterile horse serum, 10.0mL
of special yeast extract, 50.0mg of thallous acetate,
and 5000U of penicillin. Mix thoroughly. Pour into
sterile Petri dishes or distribute into sterile tubes.

Use: For the preparation of media for the cultivation of Mycoplasma species.


Mycoplasma Agar with Supplement G

Composition per liter:

Agar .............................................................. ...10.0g

Bacteriological peptone ................................. ...10.0g

Beef extract...................................................... .10.0g

NaCl.................................................................. ..5.0g

Special mineral supplement............................... ..0.5g

Mycoplasma supplement G..................... . 250.0mL

pH 7.8 ± 0.2 at 25oC

Source: This medium is available as a premixed powder from Oxoid Unipath.

Mycoplasma Supplement G:

Composition per 20.0mL:

Thallous acetate .......................................... ...25.0mg

Horse serum................................................ .. 20.0mL

Yeast extract (25% solution)........................ . 10.0mL

Penicillin................................................... ...20,000U

Preparation of Mycoplasma Supplement G: Add components to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Filter sterilize. Caution: Thallous acetate is a poison.

Preparation of Medium: Add components, ex-

cept Mycoplasma supplement G, to distilled/deion-
ized  water  and  bring  volume  to 1.0L.  Mix

thoroughly. Gently heat and bring to boiling. Distrib-
ute into flasks in 80.0mL volumes. Autoclave for 15
min at 15 psi pressure-121oC. Cool to 50oC. Asepti-
cally add 20.0mL of sterile Mycoplasma supplement
G to each 80.0mL of basal medium. Mix thoroughly.

Use: For the growth of Mycoplasma species.


Mycoplasma Agar with Supplement P

Composition per liter:

Agar .............................................................. ...10.0g

Bacteriological peptone ................................. ...10.0g

Beef extract...................................................... .10.0g

NaCl................................................................. ..5.0g

Special mineral supplement............................... ..0.5g

Mycoplasma supplement P .................... .250.0mL

pH 7.8 ± 0.2 at 25oC

Source: This medium is available as a premixed powder from Oxoid Unipath.

Mycoplasma Supplement P:

Composition per 20.0mL:

Glucose ........................................................... ...0.3g

Mycoplasma broth base ................................ .0.145g

Thallous acetate ............................................. ..8.0mg

Phenol Red..................................................... .1.2mg

Methylene Blue chloride................................. .0.3mg

Penicillin................................................... ...12,000U

Horse serum.................................................. .6.0mL

Yeast extract (25% solution)........................ ...3.0mL

Preparation of Mycoplasma Supplement P: Add components to distilled/deionized water and bring volume to 20.0mL. Mix thoroughly. Filter sterilize. Caution: Thallous acetate is a poison.

Fresh Yeast Extract Solution:

Composition per 100.0mL:

Baker’s yeast, live, pressed, starch-free......... ...25.0g

Preparation of Fresh Yeast Extract Solution: Add the live Baker’s yeast to 100.0mL of distilled/ deionized water. Autoclave for 90 min at 15 psi pres-
sure-121oC. Allow to stand. Remove supernatant so-
lution. Adjust pH to 6.6-6.8.

Preparation of Medium: Add components, ex-
cept Mycoplasma supplement P, to distilled/deion-
ized  water  and  bring  volume  to 1.0L.  Mix

thoroughly. Gently heat and bring to boiling. Distrib-
ute into bottles in 1.0mL volumes. Autoclave for 15 min at 15 psi pressure-121oC. Cool to room temper-
ature. Aseptically add 2.0mL of sterile Mycoplasma supplement P to each bottle.

Use: For the growth of Mycoplasma species.


Mycoplasma Broth

(ATCC Medium 555)

Composition per 103.0mL:

Hartley’s digest broth................................. ... 30.0mL

Pig serum .................................................. ... 20.0mL

Enzymatic hydrolysate of lactalbumin ... ... 10.0mL

Hanks’ balanced salt solution, 10X ............... . 4.0mL

Fresh yeast extract solution ........................... . 2.0mL

Phenol Red (0.25% solution)........................ .. 1.0mL

pH 7.4 ± 0.2 at 25oC

Hartley’s Digest Broth:

Composition per 10.0L:

Ox heart ..................................................... .3,000.0g

Pancreatin ...................................................... ...50.0g

Na2CO3 , anhydrous (0.8% solution)............... .. 5.0L

HCl, concentrated ....................................... .. 80.0mL

pH 7.5 ± 0.2 at 25oC

Preparation of Hartley’s Digest Broth: Finely
mince the ox heart. Add the meat to 5.0L of distilled/
deionized water. Gently heat and bring to 80oC. Add
the 5.0L of Na2CO3  solution. Cool to 45oC. Add pan-
creatin and maintain at 45oC for 4 hr while stirring.
Add the HCl and steam at 100oC for 30 min. Cool to
room temperature. Adjust pH to 8.0 with 1N NaOH.
Gently heat and bring to boiling. Continue boiling for
25 min. Filter while hot. Cool to room temperature.
Adjust pH to 7.5. Autoclave for 15 min at 15 psi pres-
sure-121oC.

Pig Serum:

Composition per 100.0mL:

Pig serum .................................................. . 100.0mL

Preparation of Pig Serum: Adjust pH of pig se-
rum to 4.4 with sterile 1N HCl. Do not let pH go be-
low 4.2. Let serum stand at 4oC for 18-20h. Adjust
pH to 7.0 with sterile 1N NaOH. Centrifuge at 9000
rpm for 20 min. Discard pellet. Filter supernatant so-
lution through a 0.2ตm membrane. Store at −70oC.

Enzymatic Hydrolysate of Lactalbumin: Composition per 100.0mL:

Enzymatic hydrolysate of lactalbumin ............. ...5.0g

Preparation of Enzymatic Hydrolysate of
Lactalbumin: Add enzymatic hydrolysate of lac-
talbumin to 100.0mL of phosphate buffered saline,
1X, pH 7.0.

Phosphate Buffered Saline Solution, 1X: Composition per liter:

NaCl.................................................................. ..8.0g

Na2HPO4*7H2O............................................... .2.16g

KCl..................................................................... .0.2g

KH2PO4........................................................... ...0.2g

MgCl2*6H2O .................................................... .0.1g

CaCl2................................................................. .0.1g

Preparation of Buffered Saline Solution, 1X:

Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Hanks’ Balanced Salt Solution, 10X: Composition per liter:

NaCl.............................................................. ...80.0g

Glucose ........................................................... .10.0g

KCl.................................................................... .4.0g

CaCl2................................................................. .1.4g

MgCl2*6H2O .................................................... .1.0g

MgSO4*7H2O.................................................. ...1.0g

Na2HPO4*7H2O.............................................. ...0.9g

KH2PO4........................................................... ...0.6g

Preparation of Hanks’ Balanced Salt Solu-
tion, 10X: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly.
Preparation of Medium: Combine components
in the following order: Hanks’ balanced salt solution,
10X, Phenol Red, Hartley’s digest broth, pig serum,
enzymatic hydrolysate of lactalbumin, and fresh
yeast extract solution. Mix thoroughly. Add 36.0mL
of distilled/deionized water. Adjust pH to 7.4 with 1N
NaOH. Filter sterilize through a 0.2ตm membrane.
Store at 4oC for up to 3 weeks.

Use: For the cultivation of Mycoplasma species.


Mycoplasma Broth Base

(PPLO Broth Base

without Crystal Violet)

Composition per liter:

Pancreatic digest of casein................................. .7.0g

NaCl................................................................. ..5.0g

Beef extract...................................................... ...3.0g

Yeast extract..................................................... ..3.0g

Beef heart, solids from infusion........................ ..2.0g

pH 7.8 ± 0.2 at 25oC

Source: This medium is available as a premixed powder from  BD Diagnostic Systems.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Gently heat and bring to boiling. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure-121oC.

Use: Used as a basal medium that should be enriched
for the isolation and cultivation of Mycoplasma species.

Mycoplasma Broth, Supplemented Composition per liter:

Pancreatic digest of casein................................. .7.0g

NaCl.................................................................. ..5.0g

Beef extract...................................................... ...3.0g

Yeast extract...................................................... ..3.0g

Beef heart, solids from infusion........................ ..2.0g

Horse serum............................................. ... 260.0mL

Fresh yeast extract solution ........................ .. 65.0mL

pH 7.8 ± 0.2 at 25oC

Fresh Yeast Extract Solution:

Composition per 100.0mL:

Baker’s yeast, live, pressed, starch-free......... ...25.0g

Preparation of Fresh Yeast Extract Solution: Add the live Baker’s yeast to 100.0mL of distilled/ deionized water. Autoclave for 90 min at 15 psi pres-
sure-121oC. Allow to stand. Remove supernatant so-
lution. Adjust pH to 6.6-6.8.

Preparation of Medium: Add components, ex-
cept horse serum and fresh yeast extract, to distilled/
deionized water and bring volume to 1.0L. Mix thor-
oughly. Gently heat and bring to boiling. Distribute
into tubes or flasks. Autoclave for 15 min at 15 psi
pressure-121oC. Cool to 50oC. To each 75.0mL of
cooled, sterile basal medium, add 20.0mL of sterile
horse serum and 5.0mL of fresh yeast extract. Mix
thoroughly. Aseptically distribute into sterile tubes.

Use: For the isolation and cultivation of Myco-
plasma species.


Mycoplasma Broth with Supplement G

Composition per liter:

Bacteriological peptone ................................. ...10.0g

Beef extract...................................................... .10.0g

NaCl.................................................................. ..5.0g

Special mineral supplement, Oxoid Unipath......0.5g
Mycoplasma supplement G... ................. . 250.0mL

pH 7.8 ± 0.2 at 25oC

Source: This medium is available as a premixed powder from Oxoid Unipath.

Mycoplasma Supplement G:

Composition per 20.0mL:

Thallous acetate .......................................... ...25.0mg

Horse serum................................................ .. 20.0mL

Yeast extract (25% solution)........................ . 10.0mL

Penicillin................................................... ...20,000U

Preparation of Mycoplasma Supplement G:
Add components to distilled/deionized water and
bring volume to 20.0mL. Mix thoroughly. Filter ster-
ilize.

Caution: Thallous acetate is a poison.

Preparation of Medium: Add components, ex-
cept Mycoplasma supplement G, to distilled/deion-
ized  water  and  bring  volume  to 1.0L.  Mix

thoroughly. Gently heat and bring to boiling. Distrib-

ute into flasks in 80.0mL volumes. Autoclave for 15
min at 15 psi pressure-121oC. Cool to 50oC. Asepti-
cally add 20.0mL of sterile Mycoplasma supplement
G to each 80.0mL of basal medium. Mix thoroughly.

Use: For the growth of Mycoplasma species.

Mycoplasma Broth with Supplement P Composition per liter:

Bacteriological peptone ... ............................ ...10.0g

Beef extract... ................................................. .10.0g

NaCl... ............................................................ ..5.0g

Special mineral supplement, Oxoid Unipath......0.5g
Mycoplasma supplement P ... ................ .250.0mL

pH 7.8 ± 0.2 at 25oC

Source: This medium is available as a premixed powder from Oxoid Unipath.

Mycoplasma Supplement P:

Composition per 20.0mL:

Glucose ... ...................................................... ...0.3g

Mycoplasma broth base ... ........................... .0.145g

Thallous acetate ... ........................................ ..8.0mg

Phenol Red... ................................................ .1.2mg

Methylene Blue chloride... ............................ .0.3mg

Penicillin... .............................................. ...12,000U

Horse serum... ............................................. .6.0mL

Yeast extract (25% solution)... ................... ...3.0mL

Preparation of Mycoplasma Supplement P:
Add components to distilled/deionized water and
bring volume to 20.0mL. Mix thoroughly. Filter ster-
ilize.

Caution: Thallous acetate is a poison.

Preparation of Medium: Add components, ex-
cept Mycoplasma supplement P, to distilled/deion-
ized  water  and  bring  volume  to 1.0L.  Mix

thoroughly. Gently heat and bring to boiling. Distrib-
ute into bottles in 1.0mL volumes. Autoclave for 15 min at 15 psi pressure-121oC. Cool to room temper-
ature. Aseptically add 2.0mL of sterile Mycoplasma supplement P to each bottle.

Use: For the cultivation of Mycoplasma species.


Mycoplasma Horse Serum Broth

(ATCC Medium 1959)

Mycoplasma broth base ... ..................... ...660.0mL

Horse serum... ....................................... ...200.0mL

Yeast extract solution... ............................ .100.0mL

Phenol Red (0.1%)... ................................. .20.0mL

Glucose solution ... .................................... .10.0mL

NaOH (1N solution)... .............................. ..6.25mL

Arginine solution ... .................................... ..5.0mL 

Mycoplasma Broth Base:

Composition per liter:

Pancreatic digest of casein................................. .7.0g

NaCl................................................................. ..5.0g

Beef extract...................................................... ...3.0g

Yeast extract..................................................... ..3.0g

Beef heart, solids from infusion........................ ..2.0g

Preparation of Mycoplasma Broth Base: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Yeast Extract Solution:

Composition per 100.0mL:

Baker’s yeast, live, pressed, starch-free......... ...25.0g

Preparation of Fresh Yeast Extract Solution:
Add the live Baker’s yeast to 100.0mL of distilled/
deionized water. Autoclave for 90 min at 15 psi pres-

sure-121oC. Allow to stand. Remove supernatant so-
lution. Adjust pH to 6.6-6.8.

Glucose Solution:

Composition per 10.0mL:

Glucose ........................................................... ...5.0g

Preparation of Glucose Solution: Add glucose to 10.0mL of distilled/deionized water. Mix thor-
oughly. Filter sterilize.

Arginine Solution:

Composition per 10.0mL:

L-Arginine........................................................ ..4.2g

Preparation of Arginine Solution: Add argin-
ine to 10.0mL of distilled/deionized water. Mix thor-
oughly. Filter sterilize.

Preparation of Medium: Combine 660.0mL
Mycoplasma broth base, 20.0mL Phenol Red, and
6.25mL 1N NaOH. Mix thoroughly. Autoclave for

15 min at 15 psi pressure-121oC. Cool to 25oC. Aseptically add 5.0mL sterile arginine solution,

100.0mL sterile yeast extract solution, 10.0mL ster-
ile glucose solution, and 200.0mL filter sterilized
horse serum. Mix thoroughly. Aseptically distribute

into sterile tubes or flasks.

Use: For the preparation of media for the cultivation of Mycoplasma spp.


Mycoplasma pneumoniae

Isolation Medium

Composition per 1200.0mL:

Beef heart for infusion.................................... ..50.0g

Peptone ........................................................... .10.0g

NaCl................................................................. ..5.0g

Water........................................................ .. 900.0mL

Yeast extract solution................................. .100.0mL

α-Gamma horse serum, unheated............. ...200.0mL

pH 7.6-7.8 at 25oC

Yeast Extract Solution:

Composition per 10.0mL:

Yeast, active dry Baker’s ............................. ..250.0g

Preparation of Yeast Extract Solution: Add yeast to 1.0L of distilled/deionized water. Mix thor-
oughly. Gently heat and bring to boiling. Filter through Whatman no. 2 filter paper. Adjust the pH of the filtrate to 8.0 with NaOH. Distribute into tubes in 10.0mL volumes. Autoclave for 15 min at 15 psi pressure-121oC. Store at −20oC.

Preparation of Medium: Add components, ex-
cept yeast extract solution and α-gamma horse se-
rum, to distilled/deionized water and bring volume to
990.0mL. Mix thoroughly. Gently heat and bring to
boiling. Autoclave for 15 min at 15 psi pressure-
121oC. Cool to 45o-50oC. Aseptically add sterile
yeast extract solution and α-gamma horse serum.
Mix thoroughly. Aseptically distribute into sterile
tubes.

Use: For the isolation and cultivation of Myco-
plasma pneumoniae.


Mycoplasmal Agar

Composition per liter:

Papaic digest of soy meal................................. .20.0g

Agarose........................................................... .10.0g

NaCl................................................................. ..5.0g

Phenol Red (2% solution).............................. .1.0mL

pH 7.3 ± 0.2 at 25oC

Preparation of Medium: Add components, ex-
cept agarose, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.3 with 1N NaOH. Add agarose. Mix thoroughly. Gen-
tly heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure-
121oC. Pour into sterile Petri dishes or leave in tubes. Use: For the isolation and cultivation of human mycoplasmas and ureaplasmas.