Handbook of Microbiological Media, Fourth Edition part 128 pps

Mycoplasma Broth 1265
Basal Medium:
Composition
per 700.0mL:
Sorbitol 50.0g
Beef heart, solids from infusion 16.2g
Peptone 3.26g
NaCl 1.62g
Fructose 1.0g
Glucose 1.0g
Sucrose 1.0g
Pancreatic digest of casein 1.0g
Preparation of Basal Medium: Add components to distilled/de-
ionized water and bring volume to 700.0mL. Mix thoroughly. Adjust
pH to 7.5–7.8. Autoclave for 15 min at 15 psi pressure–121°C. Cool to
50°C.
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 Bak-
er’s yeast to 100.0mL of distilled/deionized water. Autoclave for 90
min at 15 psi pressure–121°C. Allow to stand. Remove supernatant so-
lution. Adjust pH to 6.6–6.8.
Preparation of Medium: Filter sterilize horse serum and fresh
yeast extract solution. Aseptically add to cooled, sterile basal medium.
Mix thoroughly. Aseptically distribute into sterile tubes or flasks.
Use: For the cultivation and maintenance of Mycoplasma mycoides,
Spiroplasma apis, Spiroplasma citri, and Spiroplasma melliferum.
Mycoplasma Broth
Composition per 950.0mL:

Glucose 1.0g
Nicotinamide adenine dinucleotide 0.1g
PPLO broth without Crystal Violet 680.0mL
Swine serum (56°C, 30 min) 150.0mL
Fresh yeast extract solution 100.0mL
Phenol Red (0.1% w/v solution) 20.0mL
pH 7.8 ± 0.2 at 25°C
PPLO Broth without Crystal Violet:
Composition
per 680.0mL:
Beef heart, solids from infusion 11.3g
Peptone 2.28g
NaCl 1.13g
Source: PPLO broth without Crystal Violet is available as a premixed
powder from BD Diagnostic Systems.
Preparation of PPLO Broth without Crystal Violet: Add
components to distilled/deionized water and bring volume to 680.0mL.
Autoclave for 15 min at 15 psi pressure–121°C. Cool to 56°C.
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 Bak-
er’s yeast to 100.0mL of distilled/deionized water. Autoclave for 90
min at 15 psi pressure–121°C. Allow to stand. Remove supernatant so-
lution. Adjust pH to 6.6–6.8.
Preparation of Medium: Mix glucose, nicotinamide adenine dinu-
cleotide, swine serum, fresh yeast extract solution, and Phenol Red.
Mix thoroughly. Heat to 56°C. Add to cooled, sterile PPLO broth with-
out Crystal Violet. Mix thoroughly. Aseptically distribute into sterile

tubes or flasks.
Use: For the cultivation and maintenance of Mycoplasma anseris and
Mycoplasma lipofaciens.
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 25°C
Hartley’s Digest Broth:
Composition
per 10.0L:
Ox heart 3,000.0g
Pancreatin 50.0g
Na
2
CO
3
, anhydrous (0.8% solution) 5.0L
HCl, concentrated 80.0mL
pH 7.5 ± 0.2 at 25°C
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 80°C. Add the 5.0L of Na
2
CO

3
solution. Cool to 45°C.
Add pancreatin and maintain at 45°C for 4 hr while stirring. Add the
HCl and steam at 100°C 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 pressure–121°C.
Pig Serum:
Composition
per 100.0mL:
Pig serum 100.0mL
Preparation of Pig Serum: Adjust pH of pig serum to 4.4 with
sterile 1N HCl. Do not let pH go below 4.2. Let serum stand at 4°C for
18-20 hr. Adjust pH to 7.0 with sterile 1N NaOH. Centrifuge at 9000
rpm for 20 min. Discard pellet. Filter supernatant solution through a
0.2μm membrane. Store at −70°C.
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 Bak-
er’s yeast to 100.0mL of distilled/deionized water. Autoclave for 90
min at 15 psi pressure–121°C. Allow to stand. Remove supernatant so-
lution. Adjust pH to 6.6–6.8.
Enzymatic Hydrolysate of Lactalbumin:
Composition
per 100.0mL:
Enzymatic hydrolysate of lactalbumin 5.0g
Preparation of Enzymatic Hydrolysate of Lactalbumin: Add
enzymatic hydrolysate of lactalbumin to 100.0mL of phosphate buff-

ered saline, 1X, pH 7.0.
Phosphate Buffered Saline Solution, 1X:
Composition
per liter:
NaCl 8.0g
Na
2
HPO
4
·7H
2
O 2.16g
KCl 0.2g
KH
2
PO
4
0.2g
© 2010 by Taylor and Francis Group, LLC
1266 Mycoplasma Broth Base
MgCl
2
·6H
2
O 0.1g
CaCl
2
0.1g
Hanks’ Balanced Salt Solution, 10X:
Composition

per liter:
NaCl 80.0g
Glucose 10.0g
KCl 4.0g
CaCl
2
1.4g
MgCl
2
·6H
2
O 1.0g
MgSO
4
·7H
2
O 1.0g
Na
2
HPO
4
·7H
2
O 0.9g
KH
2
PO
4
0.6g
Preparation of Hanks’ Balanced Salt Solution, 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 di-
gest broth, pig serum, enzymatic hydrolysate of lactalbumin, and fresh
yeast extract solution. Mix thoroughly. Add 36.0mL of distilled/deion-
ized water. Adjust pH to 7.4 with 1N NaOH. Filter sterilize through a
0.2μm membrane. Store at 4°C 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 25°C
Source: This medium is available as a premixed powder from BD Di-
agnostic 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–121°C.
Use: Used as a basal medium that should be enriched for the isolation and
cultivation of Mycoplasma species.
Mycoplasma Broth Base, Frey with Horse Serum
Composition per 1100.0mL:
Pancreatic digest of casein 7.5g
Papaic digest of soybean meal 2.5g

KCl 0.4g
MgSO
4
0.2g
Na
2
PO
4
1.6g
KH
2
PO
4
0.1g
Horse serum 100.0mL
pH 7.7 ± 0.2 at 25°C
Source: This medium is available as a premixed powder from BD Di-
agnostic Systems.
Preparation of Medium: Add components, except horse serum, to
distilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pres-
sure–121°C. Cool to 50°C. Add sterile, heat-inactivated horse serum.
Mix thoroughly. Aseptically distribute into sterile tubes.
Use: For the cultivation of avian mycoplasmas.
Mycoplasma Broth Base, Frey with Horse Serum
Composition per 1100.0mL:
Pancreatic digest 7.5g
Yeast extract 5.0g
NaCl 5.0g
Papaic digest of soybean meal 2.5g

Na
2
HPO
4
1.6g
KCl 0.4g
MgSO
4
·7H
2
O 0.2g
KH
2
PO
4
0.1g
Horse serum 100.0mL
pH 7.7 ± 0.2 at 25°C
Preparation of Medium: Add components, except horse serum, to
distilled/deionized water and bring volume to 1.0L. Mix thoroughly.
Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pres-
sure–121°C. Cool to 50°C. Add sterile, heat-inactivated horse serum.
Mix thoroughly. Aseptically distribute into sterile tubes.
Use: For the cultivation of avian mycoplasmas.
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 25°C
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 Bak-
er’s yeast to 100.0mL of distilled/deionized water. Autoclave for 90
min at 15 psi pressure–121°C. Allow to stand. Remove supernatant so-
lution. Adjust pH to 6.6–6.8.
Preparation of Medium: Add components, except horse serum
and fresh yeast extract solution, to distilled/deionized water and bring
volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Dis-
tribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–
121°C. Cool to 50°C. To each 75.0mL of cooled, sterile basal medium,
add 20.0mL of sterile horse serum and 5.0mL of fresh yeast extract so-
lution. Mix thoroughly. Aseptically distribute into sterile tubes.
Use: For the isolation and cultivation of Mycoplasma 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 25°C
Source: This medium is available as a premixed powder from Oxoid
Unipath.

© 2010 by Taylor and Francis Group, LLC
Mycoplasma HiVeg Broth Base with Crystal Violet and Tellurite 1267
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 thor-
oughly. Filter sterilize.
Caution: Thallous acetate is a poison.
Preparation of Medium: Add components, except Mycoplasma
supplement G, to distilled/deionized water and bring volume to 1.0L.
Mix thoroughly. Gently heat and bring to boiling. Distribute into flasks
in 80.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C.
Cool to 50°C. Aseptically add 20.0mL of sterile Mycoplasma supple-
ment 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 25°C
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 thorough-
ly. Filter sterilize.
Caution: Thallous acetate is a poison.
Preparation of Medium: Add components, except Mycoplasma
supplement P, to distilled/deionized water and bring volume to 1.0L.
Mix thoroughly. Gently heat and bring to boiling. Distribute into bot-
tles in 1.0mL volumes. Autoclave for 15 min at 15 psi pressure–121°C.
Cool to room temperature. Aseptically add 2.0mL of sterile Mycoplas-
ma supplement P to each bottle.
Use: For the cultivation of Mycoplasma species.
Mycoplasma Broth with 10% Swine Serum
Composition per liter:
Pancreatic digest of casein 5.6g
NaCl 4.0g
Yeast extract 2.6g
Beef extract 2.4g
Beef heart, solids from infusion 1.6g
Swine serum, heat inactivated 100.0mL

Fresh yeast extract solution 100.0mL
pH 7.8 ± 0.2 at 25°C
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 Bak-
er’s yeast to 100.0mL of distilled/deionized water. Autoclave for 90
min at 15 psi pressure–121°C. Allow to stand. Remove supernatant so-
lution. Adjust pH to 6.6–6.8.
Preparation of Medium: Add components, except swine serum and
fresh yeast extract solution, to distilled/deionized water and bring volume
to 800.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave
for 15 min at 15 psi pressure–121°C. Cool to 50°C. Add sterile swine se-
rum and fresh yeast extract solution. Mix thoroughly. Aseptically distrib-
ute into sterile tubes.
Use: For the cultivation and maintenance of Mycoplasma columbinum
and Mycoplasma columborale.
Mycoplasma HiVeg Agar Base with
Horse Serum and Yeast Extract
(PPLO HiVeg Agar Base)
Composition per liter:
Agar 15.0g
Plant peptone 10.0g
Plant infusion 6.0g
NaCl 5.0g
Horse serum 260.0mL
Fresh yeast extract solution 65.0mL
pH 7.8 ± 0.2 at 25°C
Source: This medium, without horse serum and yeast extract solution,

is available as a premixed powder from HiMedia.
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 Bak-
er’s yeast to 100.0mL of distilled/deionized water. Autoclave for 90
min at 15 psi pressure–121°C. Allow to stand. Remove supernatant so-
lution. Adjust pH to 6.6–6.8.
Preparation of Medium: Add components, except horse serum
and fresh yeast extract solution, to distilled/deionized water and bring
volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Dis-
tribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–
121°C. Cool to 50°C. To each 75.0mL of cooled, sterile basal medium,
add 20.0mL of sterile horse serum and 5.0mL of special yeast extract
solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into
sterile tubes.
Use: For the preparation of media for the cultivation of Mycoplasma.
Mycoplasma HiVeg Broth Base
with Crystal Violet and Tellurite
(PPLO HiVeg Broth Base with CV)
Composition per liter:
Plant peptone 10.0g
Plant infusion 6.0g
NaCl 5.0g
Crystal Violet 0.01g
© 2010 by Taylor and Francis Group, LLC
1268 Mycoplasma HiVeg Broth Base without Crystal Violet and with Ascitic Fluid
Chapman tellurite solution 2.85mL
Ascitic fluid 250.0mL

pH 7.8 ± 0.2 at 25°C
Source: This medium, without tellurite, is available as a premixed
powder from HiMedia.
Chapman Tellurite Solution:
Composition per 100.0mL:
K
2
TeO
3
1.0g
Preparation of Chapman Tellurite Solution: Add K
2
TeO
3
to
distilled/deionized water and bring volume to 100.0mL. Mix thorough-
ly. Filter sterilize.
Caution: Potassium tellurite is toxic.
Preparation of Medium: Add components, except ascitic fluid and
Chapman tellurite solution, to distilled/deionized water and bring vol-
ume to 747.15mL. Mix thoroughly. Autoclave for 15 min at 15 psi
pressure–121°C. Cool to less than 37°C. Aseptically add sterile ascitic
fluid and 2.85mL of Chapman tellurite solution. Mix thoroughly. Asep-
tically distribute into sterile tubes or flasks.
Use: For the isolation of Mycoplasma species from clinical specimens.
Mycoplasma HiVeg Broth Base
without Crystal Violet and with Ascitic Fluid
(PPLO HiVeg Broth Base without CV)
Composition per liter:
Plant peptone 10.0g

Plant infusion 6.0g
NaCl 5.0g
Ascitic fluid 250.0mL
pH 7.8 ± 0.2 at 25°C
Source: This medium, without ascitic fluid, is available as a premixed
powder from HiMedia.
Preparation of Medium: Add components, except ascitic fluid, to
distilled/deionized water and bring volume to 750.0mL. Mix thorough-
ly. Autoclave for 15 min at 15 psi pressure–121°C. Cool to less than
37°C. Aseptically add sterile ascitic fluid. If desired, 0.5g of thallium
acetate or 100,000U of penicillin may be added for a more selective
medium. Mix thoroughly. Aseptically distribute into sterile tubes or
flasks.
Use: For the enrichment of pleuro-pneumonia-like organisms (PPLOs)
and Mycoplasma species from clinical specimens.
Mycoplasma Horse Serum Broth
(ATCC Medium 1959)
Mycoplasma broth base 660.0mL
Horse serum 200.0mL
Fresh 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.
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 Bak-
er’s yeast to 100.0mL of distilled/deionized water. Autoclave for 90
min at 15 psi pressure–121°C. 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 dis-
tilled/deionized water. Mix thoroughly. Filter sterilize.
Arginine Solution:
Composition
per 10.0mL:
L-Arginine 4.2g
Preparation of Arginine Solution: Add arginine to 10.0mL of
distilled/deionized water. Mix thoroughly. 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–121°C. Cool to 25°C. Asepti-
cally add 5.0mL sterile arginine solution, 100.0mL sterile fresh yeast
extract solution, 10.0mL sterile glucose solution, and 200.0mL filter
sterilized horse serum. Mix thoroughly. Aseptically distribute into ster-

ile tubes or flasks.
Use: For the preparation of media for the cultivation of Mycoplasma
spp.
Mycoplasma Liquid Medium
Composition per 1004.0mL:
Arginine 1.0g
Glucose 1.0g
L-Cysteine·HCl·H
2
O 1.0g
Mycoplasma broth base 850.0mL
Horse serum, not inactivated 100.0mL
Fresh yeast extract (25% solution) 50.0mL
Phenol Red (1.0% solution) 2.0mL
DNA calf thymus solution 2.0mL
pH 7.8 ± 0.2 at 25°C
Mycoplasma Broth Base:
Composition
per 850.0mL:
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 850.0mL. Mix thorough-
ly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi
pressure–121°C. Cool to 50°C.
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 Bak-
er’s yeast to 100.0mL of distilled/deionized water. Autoclave for 90
© 2010 by Taylor and Francis Group, LLC
Mycoplasma Medium, Revised 1269
min at 15 psi pressure–121°C. Allow to stand. Remove supernatant so-
lution. Adjust pH to 6.6–6.8.
DNA Calf Thymus Solution:
Composition
per 10.0mL:
DNA calf thymus 1.0g
Preparation of DNA Calf Thymus Solution: Add DNA calf
thymus to distilled/deionized water and bring volume to 10.0mL. Mix
thoroughly. Filter sterilize.
Preparation of Medium: Combine components, except Mycoplasma
broth base and DNA calf thymus solution, and mix thoroughly. Filter ster-
ilize through a 0.2μm membrane. Add sterile solution to 850.0mL of
cooled, sterile Mycoplasma broth base. Aseptically add 2.0mL of sterile
DNA calf thymus solution. Mix thoroughly. Aseptically distribute into
sterile tubes or flasks.
Use: For the cultivation and maintenance of Mycoplasma lipophilum
and Mycoplasma species.
Mycoplasma Medium
Composition per liter:
Heart infusion broth 25.0g
Mucin, bacteriological grade 5.0g
Agar, purified (optional) 7.0g
Hemoglobin 2.0g
Turkey serum, sterile inactivated 100.0mL

pH 7.8 ± 0.2 at 25°C
Preparation of Medium: Add components, except turkey serum and
agar, to 850.0mL distilled water. Adjust pH to 7.8. Heat mixture at 93°–
95°C for 30 min in a water bath. Restore to original volume. Add 0.5% di-
atomaceous earth. Mix thoroughly. Filter through Whatman GFA (glass fi-
ber paper) in Buchner filter. Clarify using 0.45μm Millipore filter. Add
15% inactivated turkey serum. Sterilize using S3 (0.1μm) Seitz filter. Use
positive pressure. For solid medium
: Prepare 42.5mL of double-strength
broth and 42.5mL of distilled water containing 0.7g of purified agar. Ster-
ilize the solutions separately and combine aseptically at 56°C with 15.0mL
of sterile inactivated turkey serum for a final volume of 150.0mL.
Use: For the cultivation and maintenance of Mycoplasma hyosyn-
oviae.
Mycoplasma Medium
(CIP Medium 89)
(DSMZ Medium 1080)
Composition per liter:
Pancreatic digest of casein 7.0g
NaCl 5.0g
Beef extract 3.0g
Yeast extract 3.0g
Beef heart, infusion from (solids) 2.0g
Selective supplement solution 210.0mL
Yeast extract solution 100.0mL
Phenol Red solution 20.0mL
Yeast Extract Solution:
Composition per 100.0mL:
Yeast extract 25.0g
Preparation of Yeast Extract Solution: Add yeast extract to dis-

tilled/deionized water and bring volume to 100.0mL. Mix thoroughly.
Adjust pH to 7.0. Autoclave for 15 min at 15 psi pressure–121°C.
Phenol Red Solution:
Composition per 100.0mL:
Phenol Red 1.0g
Preparation of Phenol Red Solution: Add Phenol Red to dis-
tilled/deionized water and bring volume to 100.0mL. Mix thoroughly.
Adjust pH to 7.0. Autoclave for 15 min at 15 psi pressure–121°C.
Selective Supplement Solution:
Composition
per 210.0mL:
Ampicillin 1.0g
Horse serum 200.0mL
Arginine solution 10.0mL
Arginine Solution:
Composition per 100.0mL:
L-Arginine 50.0g
Preparation of Arginine Solution: Add L-arginine to distilled/de-
ionized water and bring volume to 100.0mL. Mix thoroughly. Adjust
pH to 7.0.
Preparation of Selective Supplement Solution: Add ampicillin
to 10.0mL arginine solution. Add horse serum. Mix thoroughly. Filter
sterilize.
Preparation of Medium: Add components, except yeast extract,
Phenol Red, and selective supplement solutions, to distilled/deionized
water and bring volume to 670.0mL. Mix thoroughly. Adjust pH to 7.2.
Distribute into tubes or flasks. Gently heat while stirring and bring to
boiling. Autoclave for 15 min at 15 psi pressure–121°C. Cool to room
temperature. Aseptically add yeast extract, Phenol Red, and selective
supplement solutions. Mix thoroughly. Aseptically distribute into tubes

or flasks.
Use: For the cultivation of Mycoplasma spp.
Mycoplasma Medium, Revised
Composition per 1030.0mL:
Noble agar 10.0g
Distilled water 360.0mL
Heart infusion broth 300.0mL
Pig serum, heat inactivated 200.0mL
Enzymatic hydrolysate of lactalbumin 100.0mL
Hanks’ balanced salt solution, 10X 40.0mL
Fresh yeast extract solution 20.0mL
Phenol Red (0.25% solution) 10.0mL
Heart Infusion Broth:
Composition
per liter:
Beef heart, infusion from 500.0g
Tryptose 10.0g
NaCl 5.0g
Preparation of Heart Infusion Broth: Add components to dis-
tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gen-
tly heat and bring to boiling.
Enzymatic Hydrolysate of Lactalbumin:
Composition
per 100.0mL:
Enzymatic hydrolysate of lactalbumin 5.0g
Phosphate buffered saline, 1X, pH7.0 100.0mL
Preparation of Enzymatic Hydrolysate of Lactalbumin: Add
enzymatic hydrolysate of lactalbumin to 100.0mL of phosphate buff-
ered saline, 1X, pH 7.0.
© 2010 by Taylor and Francis Group, LLC

1270 Mycoplasma pneumoniae Isolation Medium
Phosphate Buffered Saline Solution, 1X:
Composition
per liter:
NaCl 8.0g
Na
2
HPO
4
·7H
2
O 2.16
KCl 0.2g
KH
2
PO
4
0.2g
MgCl
2
·6H
2
O 0.1g
CaCl
2
0.1g
Preparation of Phosphate 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:
Na
2
Cl 80.0g
Glucose 10.0g
KCl 4.0g
CaCl
2
1.4g
MgCl
2
·6H
2
O 1.0g
MgSO
4
·7H
2
O 1.0g
Na
2
HPO
4
·7H
2
O 0.9g
KH
2
PO

4
0.6g
Preparation of Hanks’ Balanced Salt Solution, 10X: Add
components to distilled/deionized water and bring volume to 1.0L. Mix
thoroughly.
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 Bak-
er’s yeast to 100.0mL of distilled/deionized water. Autoclave for 90
min at 15 psi pressure–121°C. Allow to stand. Remove supernatant so-
lution. Adjust pH to 6.6–6.8.
Preparation of Medium: Add agar and heart infusion broth to dis-
tilled/deionized water and bring volume to 660.0mL. Mix thoroughly. Ad-
just pH to 7.4. Autoclave for 15 min at 15 psi pressure–121°C. Aseptically
add pig serum, enzymatic hydrolysate of lactalbumin, Hanks’ balanced
salt solution, 10X, fresh yeast extract solution, and Phenol Red solution.
Mix thoroughly. Aseptically distribute into sterile tubes.
Use: For the cultivation and maintenance of Mycoplasma dispar,
Mycoplasma flocculare, and Mycoplasma hyopneumoniae.
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 25°C

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 dis-
tilled/deionized water. Mix thoroughly. Gently heat and bring to boil-
ing. Filter through Whatman #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–121°C. Store at −20°C.
Preparation of Medium: Add components, except yeast extract so-
lution and α-gamma horse serum, 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–121°C. Cool to 45°–
50°C. 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 Mycoplasma 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 25°C
Preparation of Medium: Add components, except agarose, to dis-
tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Ad-
just pH to 7.3 with 1N NaOH. Add agarose. Mix thoroughly. Gently
heat and bring to boiling. Distribute into tubes or flasks. Autoclave for
15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes or leave
in tubes.
Use: For the isolation and cultivation of human mycoplasmas and ure-

aplasmas.
Mycorrhiza Medium
Composition per liter:
Agar 15.0g
Glucose 4.0g
Ammonium tartrate 1.0g
Malt extract 1.0g
KH
2
PO
4
0.2g
MgSO
4
·7H
2
O 0.1g
CaCl
2
·2H
2
O 26.0mg
NaCl 20.0mg
Inositol 10.0mg
ZnSO
4
·7H
2
O 0.88mg
MnSO

4
·4H
2
O 0.81mg
FeCl
3
·6H
2
O 0.8mg
Nicotinamide 100.0μg
p-Aminobenzoic acid 100.0μg
Pantothenic acid 100.0μg
Pyridoxine 100.0μg
Thiamine 100.0μg
Biotin 25.0μg
Riboflavin 25.0μg
Preparation of Medium: Add components to distilled/deionized wa-
ter and bring volume to 1.0L. Mix thoroughly. Gently heat until boiling.
Distribute into tubes or flasks. Autoclave for 10 min at 15 psi pressure–
121°C. Pour into sterile Petri dishes or leave in tubes.
Use: For the cultivation and maintenance of Thelephora terrestris.
Mycosel™ Agar
(Cycloheximide Chloramphenicol Agar)
Composition per liter:
Agar 15.5g
Papaic digest of soybean meal 10.0g
Glucose 10.0g
© 2010 by Taylor and Francis Group, LLC
MYX Agar 1271
Cycloheximide 0.4g

Chloramphenicol 0.05g
pH 6.9 ± 0.2 at 25°C
Caution: Cycloheximide is toxic. Avoid skin contact or aerosol for-
mation and inhalation.
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Gently heat while
stirring and bring to boiling. Autoclave for 15 min at 14 psi pressure–
118°C. Avoid overheating. Pour into sterile Petri dishes or distribute
into sterile tubes.
Use: For the selective isolation of pathogenic fungi from specimens
with other fungi and bacteria.
MYCT Medium
(DSMZ Medium 972)
Composition per liter:
KH
2
PO
4
13.6g
Cyclomaltoheptaose (ß-cyclodextrin) 7.0g
(NH
4
)
2
SO
4
4.0g
Yeast extract 3.0g
Casein hydrolysate 3.0g
Tween™ 80 1.0g

MgCl
2
0.2g
Sodium citrate 0.25g
FeSO
4
·7H
2
O 25.0mg
MnSO
4
·4H
2
O 25.0mg
pH 6.5 ± 0.2 at 25°C
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes
or flasks. Autoclave for 15 min at 15 psi pressure–121°C.
Use: For the cultivation of Lactobacillus sp.
Mykorrhiza Agar
Composition per liter:
Agar 15.0g
Malt extract 8.0g
Glucose 7.0g
Casein hydrolysate 1.0g
Yeast extract 1.0g
Asparagine 0.5g
KH
2
PO

4
0.5g
MgSO
4
·7H
2
O 0.5g
Preparation of Medium: Add components 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–121°C. Pour into sterile Petri dishes or leave in tubes.
Use: For the cultivation and maintenance of Klebsiella pneumoniae.
MYP Agar
See: Mannitol Yolk Polymyxin Agar
MYP Agar Base, HiVeg
with Egg Yolk and Polymyxin B
(Phenol Red Egg Yolk Polymyxin Agar Base, HiVeg)
Composition per liter:
Agar 15.0g
D-Mannitol 10.0g
Plant peptone 10.0g
NaCl 10.0g
Plant extract No. 1 1.0g
Phenol Red 0.025g
Egg yolk emulsion, 20% 10.0mL
Polymyxin B solution 1.0mL
pH 7.1 ± 0.2 at 25°C
Source: This medium, without egg yolk and polymyxin B, is avail-
able as a premixed powder from HiMedia.
Egg Yolk Emulsion, 20%:

Composition per 100.0mL:
Chicken egg yolks 11
Whole chicken egg 1
NaCl (0.9% solution) 80.0mL
Preparation of Egg Yolk Emulsion, 20%: Soak eggs with 1:100
dilution of saturated mercuric chloride solution for 1 min. Crack eggs
and separate yolks from whites. Mix egg yolks with 1 chicken egg.
Measure 20.0mL of egg yolk emulsion and add to 80.0mL of 0.9%
NaCl solution. Mix thoroughly. Filter sterilize. Warm to 45°–50°C.
Polymyxin B Solution:
Composition
per 1.0mL:
Polymyxin B 1.0mg
Preparation of Polymyxin B Solution: Add polymyxin B to dis-
tilled/deionized water and bring volume to 1.0mL. Mix thoroughly. Fil-
ter sterilize.
Preparation of Medium: Add components—except egg yolk emul-
sion, 20%, and polymyxin B solution—to distilled/deionized water and
bring volume to 100.0mL. Mix thoroughly. Gently heat and bring to boil-
ing. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.
Aseptically add 10.0mL of sterile egg yolk emulsion, 20%, and 1.0mL
of sterile polymyxin B solution. Mix thoroughly. Pour into sterile Petri
dishes.
Mysorens Medium
Composition per liter:
Peptone 10.0g
Meat extract 10.0g
Yeast extract 5.0g
NaCl 3.0g
Preparation of Medium: Add components to distilled/deionized

water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes
or flasks. Autoclave for 15 min at 15 psi pressure–121°C.
Use: For the cultivation and maintenance of Arthrobacter mysorens.
MYX Agar
(DSMZ Medium 729)
Composition per liter:
Na
2
-glutamate 5.0g
Yeast extract 1.0g
MgSO
4
·7H
2
O 1.0g
Glucose solution 10.0mL
pH 7.2 ± 0.2 at 25°C
Glucose Solution:
Composition
per10.0mL:
Glucose 2.0g
Preparation of Glucose Solution: Add glucose to distilled/deion-
ized water and bring volume to 10.0mL. Mix thoroughly. Filter steril-
ize.
© 2010 by Taylor and Francis Group, LLC
1272 Myxobacteria Medium
Preparation of Medium: Add components, except glucose solution,
to distilled/deionized water and bring volume to 990.0mL. Mix thor-
oughly. Adjust pH to 7.2. Aseptically add 10.0mL glucose solution. Mix
thoroughly. Aseptically distribute into sterile tubes or bottles.

Use: For the cultivation of Taxeobacter spp.
Myxobacteria Medium
Composition per liter:
Agar 15.0g
Skim milk powder 5.0g
Yeast extract 0.5g
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Do not adjust pH.
Gently heat and bring to boiling. Distribute into tubes or flasks. Auto-
clave for 15 min at 15 psi pressure–121°C.
Use: For the cultivation and maintenance of Archangium primige-
nium, Chondrococcus macrosporus, and Myxococcus coralloides.
Myxococcus flavescens Medium
Composition per liter:
Agar 15.0g
Soluble starch 5.0g
Casitone 2.5g
Galactose 1.0g
Raffinose 1.0g
Sucrose 1.0g
Yeast extract 1.0g
MgSO
4
·7H
2
O 0.5g
K
2
HPO
4

0.25g
pH 6.0 ± 0.2 at 25°C
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Adjust pH to 6.0.
Gently heat and bring to boiling. Distribute into tubes or flasks. Auto-
clave for 15 min at 15 psi pressure–121°C. Pour into sterile Petri dishes
or leave in tubes.
Use: For the cultivation and maintenance of Myxococcus flavescens.
Myxococcus Medium
Composition per liter:
Agar 12.0g
Pancreatic digest of casein 1.0g
Meat extract 1.0g
Glucose solution 50.0mL
pH 7.2 ± 0.2 at 25°C
Glucose Solution:
Composition
per 50.0mL:
Glucose 1.0g
Preparation of Glucose Solution: Add glucose to distilled/deion-
ized water and bring volume to 50.0mL. Mix thoroughly. Autoclave for
15 min at 15 psi pressure–121°C. Cool to 25°C.
Preparation of Medium: Add components, except glucose solu-
tion, to distilled/deionized water and bring volume to 950.0mL. Mix
thoroughly. Adjust pH to 7.2. Gently heat and bring to boiling. Auto-
clave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C. Asepti-
cally add sterile glucose solution. Mix thoroughly. Pour into sterile
Petri dishes or distribute into sterile tubes or bottles. Allow tubes or
bottles to cool in a slanted position.
Use: For the cultivation of Myxococcus species.

Myxococcus xanthus Medium
Composition per liter:
Agar 20.0g
Pancratic digest of casein 10.0g
MgSO
4
·7H
2
O 0.5g
K
2
HPO
4
0.148g
KH
2
PO
4
0.017g
pH 7.6 ± 0.2 at 25°C
Preparation of Medium: Add components 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–121°C. Pour into sterile Petri dishes or leave in tubes.
Use: For the cultivation and maintenance of Myxococcus xanthus.
N plus C Medium
Composition per liter:
Pancreatic digest of casein 10.0g
Glucose 10.0g
Citric acid·H

2
O 4.04g
KH
2
PO
4
2.0g
Yeast extract 1.5g
CaCl
2
·2H
2
O 0.6g
MgSO
4
·7H
2
O 0.6g
FeCl
2
·4H
2
O 0.06g
ZnSO
4
·7H
2
O 0.034g
Hemin solution 1.0mL
pH 4.6 ± 0.2 at 25°C

Hemin Solution:
Composition
per 100.0mL:
NaOH 1.0g
Hemin 250.0mg
Preparation of Hemin Solution: Add components to distilled/de-
ionized water and bring volume to 100.0mL. Mix thoroughly. Auto-
clave for 15 min at 15 psi pressure–121°C.
Preparation of Medium: Add components, except hemin solution, to
distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Ad-
just pH to 4.6. Distribute into tubes or flasks. Autoclave for 15 min at 15
psi pressure–121°C. Prior to inoculation, add 0.1mL of hemin solution per
100.0mL of medium.
Use: For the cultivation and maintenance of Physarum polycephalum.
N DeVogel Medium
(Vogel N Medium)
Composition per liter:
Sucrose 15.0g
KH
2
PO
4
5.0g
Trisodium citrate·2H
2
O 3.0g
NH
4
NO
3

2.0g
MgSO
4
·7H
2
O 0.2g
CaCl
2
·H
2
O solution 20.0mL
Biotin solution 5.0mL
Trace elements solution 5.0mL
CaCl
2
·H
2
O Solution:
Composition
per 20.0mL:
CaCl
2
·H
2
O 0.1g
© 2010 by Taylor and Francis Group, LLC
NAM Medium 1273
Preparation of CaCl
2
·H

2
O Solution: Add CaCl
2
·H
2
O to dis-
tilled/deionized water and bring volume to 20.0mL. Mix thoroughly.
Biotin Solution:
Composition
per 100.0mL:
Biotin 5.0mg
Preparation of Biotin Solution: Add biotin to 50% ethanol and
bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Store at 5°C.
Trace Elements Solution:
Composition
per 100.0mL:
Citric acid·H
2
O 5.0g
ZnSO
4
·7H
2
O 5.0g
Fe(NH
4
)
2
(SO
4

)
2
·6H
2
O 1.0g
CuSO
4
·5H
2
O 0.25g
H
3
BO
3
, anhydrous 0.05g
MnSO
4
·H
2
O 0.05g
Na
2
MoO
4
·2H
2
O 0.05g
Preparation of Trace Elements Solutions: Add components
successively to distilled/deionized water and bring volume to
100.0mL. Mix thoroughly after addition of each component. Filter

sterilize. Add 2–3.0mL of chloroform as a preservative. Store at 25°C.
Preparation of Medium: Add components, except biotin solution
and trace elements solution, to distilled/deionized water and bring vol-
ume to 990.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pres-
sure–121°C. Aseptically add 5.0mL of sterile biotin solution and
5.0mL of sterile trace elements solution. Mix thoroughly. Aseptically
distribute into sterile tubes or flasks.
Use: For the cultivation and maintenance of Neurospora crassa.
NAG Medium
(DSMZ Medium 366)
Composition per liter:
Agar 15.0g
Glucose 10.0g
Peptone 5.0g
Meat extract 3.0g
pH 7.0 ± 0.2 at 25°C
Preparation of Medium: Add components 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–121°C. Do not overheat. Pour into sterile Petri dishes or
leave in tubes.
Use: For the isolation and cultivation of Xanthomonas fragariae.
Nakayama Glucose Agar
Composition per 1001.0mL:
Yeast extract 15.0g
Agar 10.0g
Glucose 10.0g
Peptone 10.0g
Solution A 10.0mL
Solution B 10.0mL

Solution C 1.0mL
pH 6.8 ± 0.2 at 25°C
Solution A:
Composition
per 100.0mL:
K
2
HPO
4
0.5g
KH
2
PO
4
0.5g
Preparation of Solution A: Add components to distilled/deionized
water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15
min at 15 psi pressure–121°C.
Solution B:
Composition
per 100.0mL:
MgSO
4
·7H
2
O 3.0g
MnSO
4
·5H
2

O 0.1g
NaCl 0.1g
CuSO
4
·5H
2
O 0.01g
Preparation of Solution B: Add components to distilled/deionized
water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15
min at 15 psi pressure–121°C.
Solution C:
Composition
per 100.0mL:
Trisodium citrate 2.0g
FeSO
4
·7H
2
O 0.1g
Preparation of Solution C: Add components to distilled/deionized
water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15
min at 15 psi pressure–121°C.
Preparation of Medium: Add components, except solution A, so-
lution B, and solution C, to distilled/deionized water and bring volume
to 980.0mL. Mix thoroughly. Gently heat and bring to boiling. Auto-
clave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C. Asepti-
cally add 10.0mL of sterile solution A, 10.0mL of sterile solution B,
and 1.0mL of sterile solution C. Mix thoroughly. Pour into sterile Petri
dishes or distribute into sterile tubes.
Use: For the cultivation and maintenance of Bacillus laevolacticus.

NAM Medium
Composition per liter:
Pancreatic digest of casein 15.0g
Agar 15.0g
Papaic digest of soybean meal 5.0g
NaCl 5.0g
Sheep blood, defibrinated 50.0mL
Hemin solution 10.0mL
N-Acetyl muramic acid (NAM) solution 1.0mL
pH 7.3 ± 0.2 at 25°C
Hemin Solution:
Composition
per 100.0mL:
Hemin .0.050g
NaOH (1N solution) 1.0mL
Preparation of Hemin Solution: Add hemin to NaOH solution.
Mix thoroughly. Adjust volume to 100.0mL with distilled/deionized wa-
ter. Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.
N-Acetyl Muramic Acid (NAM) Solution:
Composition
per 10.0mL:
N-Acetyl muramic acid 100.0mg
Preparation of N-Acetyl Muramic Acid (NAM) Solution:
Add N-acetyl muramic acid to distilled/deionized water and bring vol-
ume to 10.0mL. Filter sterilize.
Preparation of Medium: Add components, except sheep blood,
hemin solution, and N-acetyl muramic acid (NAM) solution, to dis-
tilled/deionized water and bring volume to 49.0mL. Mix thoroughly.
Autoclave for 15 min at 15 psi pressure–121°C. Cool to 50°–55°C.
Aseptically add 50.0mL of sterile sheep blood, 10.0mL of sterile hemin

solution, and 1.0mL of sterile N-acetyl muramic acid (NAM) solution.
© 2010 by Taylor and Francis Group, LLC
1274 NANAT Agar
Mix thoroughly. Aseptically and anaerobically distribute into sterile
tubes under a gas phase of 80% N
2
+ 10% CO
2
+ 10% H
2
.
Use: For the cultivation of Bacteroides forsythus.
NAMn
See: Nutrient Agar with Manganese
NANAT Agar
(Nalidixic Acid Novobiocin Actidione Tellurite Agar)
Composition per liter:
Pancreatic digest of casein 17.0g
Agar 15.0g
NaCl 5.0g
Tween™ 80 5.0g
Papaic digest of soybean meal 3.0g
K
2
HPO
4
2.5g
Glucose 2.5g
Yeast extract 1.0g
Tellurite solution 10.0mL

Antibiotic solution 10.0mL
pH 7.2 ± 0.2 at 25°C
Tellurite Solution:
Composition per 100.0mL:
K
2
TeO
3
0.05g
Preparation of Tellurite Solution: Add K
2
TeO
3
to distilled/de-
ionized water and bring volume to 100.0mL. Mix thoroughly. Filter
sterilize.
Antibiotic Solution:
Composition
per 10.0mL:
Actidione (cycloheximide) 0.04g
Polymyxin B (optional) 0.03g
Novobiocin 0.025g
Nalidixic acid 0.02g
Preparation of Antibiotic Solution: Add components to distilled/
deionized water and bring volume to 10.0mL. Mix thoroughly. Filter
sterilize.
Caution: Potassium tellurite is toxic.
Caution: Cycloheximide is toxic. Avoid skin contact or aerosol for-
mation and inhalation.
Preparation of Medium: Add components, except tellurite solu-

tion and antibiotic solution, to distilled/deionized water and bring vol-
ume to 980.0mL. Mix thoroughly. Gently heat and bring to boiling.
Autoclave for 15 min at 15 psi pressure–121°C. Cool to 45°–50°C.
Aseptically add sterile tellurite solution and antibiotic solution. Mix
thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes.
Use: For the isolation and cultivation of Rhodococcus (Corynebacte-
rium) equi from animal feces, especially from horses and swine. The
addition of polymyxin B inhibits the growth of Pseudomonas aerugi-
nosa which may interfere with the isolation of Rhodococcus equi.
Nannocystis Agar
Composition per liter:
Agar 15.0g
CaCl
2
·2H
2
O 1.0g
pH 7.2 ± 0.2 at 25°C
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring
to boiling. Autoclave for 15 min at 15 psi pressure–121°C. Pour into
sterile Petri dishes. After the agar has solidified, overlay the surface
with 0.5mL of a suspension of dead (autoclaved) Escherichia coli cells.
Use: For the cultivation and maintenance of Nannocystis species.
Naphthalene Medium
Composition per liter:
NH
4
NO
3

2.5g
Na
2
HPO
4
·2H
2
O 1.0g
Naphthalene 0.64g
MgSO
4
·7H
2
O 0.5g
Fe(SO
4
)
3
·5H
2
O 0.01g
Co(NO
3
)
2
·6H
2
O 5.0mg
CaCl
2

·2H
2
O 1.0mg
KH
2
PO
4
0.5mg
MnSO
4
·2H
2
O 0.1mg
(NH
4
)
6
Mo
7
O
24
·4H
2
O 0.1mg
Preparation of Medium: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes
or flasks. Autoclave for 15 min at 15 psi pressure–121°C.
Use: For the cultivation of Pseudomonas alcaligenes.
Naphthalene Mineral Salts Medium
See: Medium for Hydrocarbon-Degrading Bacteria

Naphthalene Sulfonic Acid Medium
Composition per 1004.0mL:
Na
2
HPO
4
·2H
2
O 3.5g
KH
2
PO
4
1.0g
NH
4
Cl 0.31g
MgCl
2
·6H
2
O 0.1g
Ca(NO
3
)
2
·4H
2
O 0.05g
Solution A 100.0mL

Solution B 3.0mL
Trace elements solution SL-4 1.0mL
pH 7.0 ± 0.2 at 25°C
Solution A:
Composition
per liter:
Glucose 3.0g
Glycerol 3.0g
Sodium succinate 3.0g
Preparation of Solution A: Add components to distilled/deionized
water and bring volume to 1.0L. Mix thoroughly. Filter sterilize.
Solution B:
Composition
per liter:
Naphthalene sulfonic acid 2.3g
Preparation of Solution B: Add naphthalene sulfonic acid to dis-
tilled/deionized water and bring volume to 1.0L. Mix thoroughly. Filter
sterilize.
Trace Elements Solution SL-4:
Composition
per liter:
EDTA 0.5g
FeSO
4
·7H
2
O 0.2g
Trace elements solution SL-6 100.0mL
Preparation of Trace Elements Solution SL-4: Add components
to distilled/deionized water and bring volume to 1.0L. Mix thoroughly.

Filter sterilize.
© 2010 by Taylor and Francis Group, LLC