Medios de Cultivo

En la página http://www.cfsan.fda.gov/~ebam/bam-mi.html se encuentra la descripción de los medios de cultivo que se utilizan en el  Manual Online de Bacteriología Analítica (U.S. Food & Drug Administration, Center for Food Safety & Applied Nutrition). Se recomienda su visita para ver la composición a los medios de cultivo que se detallan. La página de Oxoid http://www.oxoid.com/uk/index  y la de Merk http://pb.merck.de/servlet/PB/menu/1001723/index.html y la de bioMérieux http://www.biomerieux.com/servlet/srt/bio/portail/home permitirán  así mismo encontrar otras descripciones para identificación de bacterias

 

Baird-Parker Medium, pH 7.0

 

http://www.cfsan.fda.gov/~ebam/m17.html

 

Tryptone

10 g

Beef extract

5 g

Yeast extract

1 g

Sodium pyruvate

10 g

Glycine

12 g

Lithium chloride.6H2O

5 g

Agar

20 g

Autoclave 15 min at 121°C. Final pH, 7.0 ± 0.2. If desired for immediate use, maintain melted medium at 48-50°C before adding enrichment. Otherwise, store solidified medium at 4 ± 1°C up to 1 month. Melt medium before use.

 

Brilliant Green Lactose Bile Broth

http://www.cfsan.fda.gov/~ebam/m25.html

Peptona 10 g

Lactosa 10 g

Oxgall   20 g

Verde brillante 0.0133 g

Distilled water 1 liter

Autoclave 15 min at 121°C. Final pH, 7.2 ± 0.1.

Dissolve peptone and lactose in 500 ml distilled water. Add 20 g dehydrated oxgall dissolved in 200 ml distilled water. The pH of this solution should be 7.0-7.5. Mix and add water to make 975 ml. Adjust pH to 7.4. Add 13.3 ml 0.1% aqueous brilliant green in distilled water. Add distilled water to make 1 liter.

Dispense into fermentation tubes, making certain that fluid level covers inverted vials.

 

Brilliant Green Lactose Bile Broth

 

http://www.cfsan.fda.gov/~ebam/m25.html

 

Peptone

10 g

Lactose

10 g

Oxgall

20 g

Brilliant green

0.0133 g

Distilled water

1 liter

 

 

 

 

 

Dissolve peptone and lactose in 500 ml distilled water. Add 20 g dehydrated oxgall dissolved in 200 ml distilled water. The pH of this solution should be 7.0-7.5. Mix and add water to make 975 ml. Adjust pH to 7.4. Add 13.3 ml 0.1% aqueous brilliant green in distilled water. Add distilled water to make 1 liter.

Dispense into fermentation tubes, making certain that fluid level covers inverted vials.

Autoclave 15 min at 121°C. Final pH, 7.2 ± 0.1.

 

Christensen's Urea Agar

 

http://www.cfsan.fda.gov/~ebam/m40.html

 

Base

 

Peptone

1 g

NaCl

5 g

 

 

Dextrose

1 g

KH2PO4

2 g

Phenol red
(6 ml of 1:500 solution)

0.012 g

Agar

15 g

Distilled water

900 ml

Dissolve all ingredients except urea in 900 ml water (basal medium).

For halophilic Vibrio spp., add extra 15 g NaCl (final NaCl concentration, 2%).

Autoclave 15 min at 121°C. Cool to 50-55°C

Urea concentrate

Urea

20 g

Distilled water

100 ml

 

Dissolve urea in 100 ml water.


Filter-sterilize; add aseptically to cooled basal medium. Mix. Final pH, 6.8 ± 0.1. Dispense to sterile tubes or petri dishes. Slant tubes for 2 cm butt and 3 cm slant.

 

EC Broth

 

http://www.cfsan.fda.gov/~ebam/m49.html

 

Trypticase or tryptose   20 g

Bile salts No. 3 1.5 g

Lactose  5 g

K2HPO4 4 g

KH2PO4 1.5 g

NaCl 5 g

Distilled water 1 liter

Distribute 8 ml portions to 16 x 150 mm test tubes containing inverted 10 x 75 mm fermentation tubes. Autoclave 15 min at 121°C. Final pH, 6.9 ± 0.2.

 

Hektoen Enteric (HE) Agar

 

http://www.cfsan.fda.gov/~ebam/m61.html

 

 

Peptone

12 g

NaCl

5 g

Yeast extract

3 g

Sodium thiosulfate

5 g

Bile salts No. 3

9 g

Ferric ammonium citrate

1.5 g

Lactose

12 g

Bromthymol blue

0.065 g

Sucrose

12 g

Acid fuchsin

0.1 g

Salicin

2 g

Agar

14.0 g

Distilled water

1 liter

 

Heat to boiling with frequent agitation to dissolve. Boil no longer than 1 min. Do not overheat. Cool in water bath. Pour 20 ml portions into sterile 15 x 100 mm petri dishes. Let dry 2 h with lids partially removed. Final pH, 7.5 ± 0.2. Do not store more than 1 day.

 

Hugh-Leifson Glucose Broth (HLGB)

 

http://www.cfsan.fda.gov/~ebam/m63.html

 

  

 

Peptone

2 g

Yeast extract

0.5 g

NaCl

30 g

Dextrose

10 g

Bromcresol purple

0.015 g

Agar

3 g

Distilled water

1 liter

 

 

 

Heat with agitation to dissolve agar. Adjust pH to 7.4 ± 0.2. Autoclave 15 min at 121°C.

 

 

 

 

Indole Médium

 

http://www.cfsan.fda.gov/~ebam/m64.html

 

 

L-Tryptophan

1 g

NaCl

1 g

K2HPO4

3.13 g

KH2PO4

0.27 g

Distilled water

200 ml

 

 

 

 

 

Dissolve ingredients. Dispense 1 ml portions to 13 x 100 mm screw-cap tubes. Autoclave 15 min at 121°C. Final pH, 7.2 ± 0.2.

 

Lauryl Tryptose (LST) Broth

http://www.cfsan.fda.gov/~ebam/m76.html

Tryptose or trypticase 20 g

Lactose 5 g

K2HPO4 2.75 g

KH2PO4 2.75 g

NaCl 5 g

Sodium lauryl sulfate 0.1 g

Distilled water 1 liter

Dispense 10 ml portions into 20 x 150 mm tubes containing inverted 10 x 75 mm fermentation tubes. Autoclave 15 min at 121°C. Final pH, 6.8 ± 0.2

Levine's Eosin-Methylene Blue (L-EMB) Agar

http://www.cfsan.fda.gov/~ebam/m80.html

Peptone10 g

Lactose 10 g

K2HPO4 2 g

Agar 15 g

Eosin Y 0.4 g

Methylene blue 0.065 g

Distilled water 1 liter

Boil to dissolve peptone, phosphate, and agar in 1 liter of water. Add water to make original volume. Dispense in 100 or 200 ml portions and autoclave 15 min at not over 121°C. Final pH, 7.1 ± 0.2.

Before use, melt, and to each 100 ml portion add:

a.       5 ml sterile 20% lactose solution;

b.      2 ml aqueous 2% eosin Y solution; and

c.       4.3 ml 0.15% aqueous methylene blue solution.

When using complete dehydrated product, boil to dissolve all ingredients in 1 liter water. Dispense in 100 or 200 ml portions and autoclave 15 min at 121°C. Final pH, 7.1 ± 0.2.

 

MacConkey Agar

 

http://www.cfsan.fda.gov/~ebam/m91.html

 

 

Proteose peptone
or polypeptone

3 g

Peptone or gelysate

17 g

Lactose

10 g

Bile salts No. 3
or bile salts mixture

1.5 g

NaCl

5 g

Neutral red

0.03 g

Crystal violet

0.001 g

Agar

13.5 g

Distilled water

1 liter

 

 

 

 

 

 

 

 

 

 Suspend ingredients and heat with agitation to dissolve. Boil 1-2 min. Autoclave 15 min at 121°C, cool to 45-50°C, and pour 20 ml portions into sterile 15 x 100 mm petri dishes. Dry at room temperature with lids closed.

DO NOT USE WET PLATES. Final pH, 7.1 ± 0.2

Mannitol Salt Agar

 

http://www.cfsan.fda.gov/~ebam/m97.html

 

Beef extract

1 g

Polypeptone

10 g

NaCl

75 g

Mannitol

10 g

Phenol red

0.025 g

Agar

15 g

Distilled water

1 liter

 

Heat with agitation to dissolve agar and boil 1 min. Dispense 20 ml portions into 15 x 100 mm petri dishes. Autoclave 15 min at 121°C. Final pH, 7.4 ± 0.2.

 

MR-VP Broth

 

http://www.cfsan.fda.gov/~ebam/m104.html

 

Medium 1

Buffered peptone-water powder
(Difco or BBL)

7 g

Glucose

5 g

K2HP04

5 g

Distilled water

1 liter

 

Medium 2

Pancreatic digest of casein

3.5 g

Peptic digest of animal tissue

3.5 g

Dextrose

5.0 g

Potassium phosphate

5.0 g

Distilled water

1 liter


Dissolve ingredients in water with gentle heat if necessary. Dispense 10 ml into 16 x 150 mm test tubes and autoclave 15 min at 118-121°C. Final pH, 6.9 ± 0.2.

Medium 3

Peptone

5.0 g

Glucose

5.0 g

Phosphate buffer

5.0 g

Distilled water

1 liter

 

Dissolve ingredients in water. Dispense 10 ml into 16 x 150 mm test tubes and autoclave 15 min at 121°C. Final pH, 7.5 ± 0.2.

For Salmonella: Dispense 10 ml into 16 x 150 mm test tubes and autoclave 12-15 min at 121°C.

For use with halophilic Vibrio spp., add NaCl to a final concentration of 2-3%

Nitrate Broth

 

http://www.cfsan.fda.gov/~ebam/m108.html

 

 

Beef extract

3 g

Peptone

5 g

KNO3 (nitrite-free)

1 g

Distilled water

1 liter

 

Dissolve ingredients. Dispense 5 ml portions into 16 x 125 mm tubes. Autoclave 15 min at 121°C. Final pH, 7.0 ± 0.2.

 

 

Nitrate Reduction Medium and Reagents

 

http://www.cfsan.fda.gov/~ebam/m110.html

Culture medium

Prepare nitrate broth (M108) from nutrient broth (M114) containing 1.0 g/liter potassium nitrate.

Reagent A. Dissolve 0.5 g alpha-naphthylamine (a carcinogen) in 100 ml 5 N acetic acid by gently heating. Prepare 5 N acetic acid by adding distilled water to 28.7 ml glacial acetic acid (17.4 N) to give final volume of 100 ml.

Reagent B. Dissolve 0.8 g sulfanilic acid in 100 ml 5 N acetic acid by gently heating.

Reagent C. Dissolve 1 g alpha-naphthol in 200 ml acetic acid.

Zinc powder.

Cadmium reagent. Place zinc rods in 20% solution of cadmium sulfate for several hours. Draw off precipitated cadmium and add to 1 N HCl.


Nutrient Broth

 

http://vm.cfsan.fda.gov/~ebam/m114.html

 

 

 

Beef extract

3 g

Peptone

5 g

Distilled water

1 liter

 

Heat to dissolve. Dispense 10 m1 portions into tubes or 225 ml portions into 500 ml Erlenmeyer flasks. Autoclave 15 min at 121°C. Final pH, 6.8 ± 0.2.

 

OF Glucose Medium, Semisolid

 

http://www.cfsan.fda.gov/~ebam/m116.html

 

 

Tryptone (trypticase)

2 g

NaCl

5 g

Dipotassium phosphate

0.3 g

Bromthymol blue dye

0.03 g

Agar

2 g

Glucose

10 g

Distilled water

1 liter

 

Boil ingredients to dissolve.

 

For halophilic Vibrio spp., add 15 g NaCl (2% NaCl, final concentration) to medium.

 

Dispense 5 ml into 13 x 100 mm tubes and autoclave 15 min at 121°C. Final pH, 6.8 ± 0.2.


*To use OF medium with sugars other than glucose, prepare medium without glucose in 900 ml water, sterilize as above, and cool to 45-50°C. Add 100 ml of 10% solution of filter-sterilized sugar to basal medium. Aseptically dispense 5 ml into sterile 13 x 100 mm tubes.

For Campylobacter, prepare half the tubes with glucose and half without.

For use with halophilic Vibrio spp., add NaCl to a final concentration of 2-3%

 

Oxidative-Fermentative (OF) Test Médium

 http://www.cfsan.fda.gov/~ebam/m117.html

  

Base

Peptone

2 g

NaCl

5 g

K2HPO4

0.3 g

Bromthymol blue

0.03 g

Agar

2.5 g

Distilled water

1 liter

 

 

Heat with agitation to dissolve agar. Dispense 3 ml portions into 13 x 100 mm tubes. Autoclave 15 min at 121°C. Cool to 50°C; pH, 7.1.


Carbohydrate stock solution

Dissolve 10 g carbohydrate in 90 ml distilled water. Sterilize by filtration through 0.22 µm membrane. Add 0.3 ml stock solution to 2.7 ml base in tube. Mix gently and cool at room temperature.

Inoculate tubes in duplicate. Layer one tube with sterile mineral oil. Incubate 48 h at 35°C.

For use with halophilic Vibrio spp., add NaCl to a final concentration of 2-3%.

Sabouraud's Dextrose Broth and Agar

 

http://www.cfsan.fda.gov/~ebam/m133.html

 

 

Polypeptone or neopeptone

10 g

Dextrose

40 g

Distilled water

1 liter

 

Dissolve completely and dispense 40 ml portions into screw-cap bottles. Final pH, 5.8. Autoclave 15 min at 118-121°C. Do not exceed 121°C.

For Sabouraud's dextrose agar, prepare broth as above and add 15-20 g agar, depending on gel strength desired. Final pH, 5.6 ± 0.2. Dispense into tubes for slants and bottles or flasks for pouring plates. Autoclave 15 min at 118-121 °C.

 

Selenite Broth Cystine

 

http://www.cfsan.fda.gov/~ebam/m134.html

 

Medium 1 (modification of Leifson's formulation for selenite broth)

 

Tryptone or polypeptone

5 g

Lactose

4 g

Sodium acid selenite (NaHSeO3)

4 g

Na2HPO4

10 g

L-Cystine

0.01 g

Distilled water

1 liter

 Heat to boiling to dissolve. Dispense 10 ml portions into sterile 16 x 150 mm test tubes. Heat 10 min in flowing steam. DO NOT AUTOCLAVE. Final pH, 7.0 ± 0.2. The medium is not sterile. Use same day as prepared.


Medium 2 (North-Bartram modification)

 

Polypeptone

5 g

Lactose

4 g

Sodium acid selenite (NaHSeO3)

4 g

Na2HPO4

5.5 g

KH2PO4

4.5 g

L-Cystine

0.01 g

Distilled water

1 liter

 Heat with agitation to dissolve. Dispense 10 ml portions into sterile 16 x 150 mm test tubes. Heat 10 min in flowing steam. DO NOT AUTOCLAVE. Use same day as prepared

 

 

 

 

 

 

 

 

 

Simmons Citrate Agar

http://www.cfsan.fda.gov/~ebam/m138.html

 

Sodium citrate*

2 g

NaCl

5 g

K2HPO4

1 g

NH4H2PO4

1 g

MgSO4

0.2 g

Bromthymol blue

0.08 g

Agar

15 g

Distilled water

1 liter

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Heat gently with occasional agitation. Boil 1-2 min until agar dissolves. Fill 13 x 100 or 16 x 150 mm screw-cap tubes 1/3 full. Autoclave 15 min at 121°C. Before medium solidifies, incline tubes to obtain 4-5 cm slants and 2-3 cm butts. Final pH, 6.8 ± 0.2.

 *Difco does not specify waters of hydration.

Triple Sugar Agar (TSI)

 

http://www.cfsan.fda.gov/~ebam/m149.html

 

Medium 1

Medium 2

Polypeptone

20 g

Beef extract

3 g

NaCl

5 g

Yeast extract

3 g

Lactose

10 g

Peptone

15 g

Sucrose

10 g

Proteose peptone

5 g

Glucose

1 g

Glucose

1 g

Fe(NH4)2(SO4)2•6H2O

0.2 g

Lactose

10 g

Na2S2O3

0.2 g

Sucrose

10 g

Phenol red

0.025 g

FeSO4

0.2 g

Agar

13 g

NaCl

5 g

Distilled water

1 liter

Na2S2O3

0.3 g

 

Phenol red

0.024 g

Agar

12 g

Distilled water

1 liter

These two media are interchangeable for general use.

Suspend ingredients of Medium 1 in distilled water, mix thoroughly, and heat with occasional agitation. Boil about 1 min to dissolve ingredients. Fill 16 x 150 mm tubes 1/3 full and cap or plug to maintain aerobic conditions. Autoclave Medium 1 for 15 min at 118°C.

 

Prepare Medium 2 in the same manner as Medium 1, except autoclave 15 min at 121°C. Before the media solidify, incline tubes to obtain 4-5 cm slant and 2-3 cm butt. Final pH, 7.3 ± 0.2 for Medium 1 and 7.4 ± 0.2 for Medium 2.


For use with halophilic Vibrio spp., add NaCl to a final concentration of 2-3%.

 

Trypticase Tryptic (Soy Agar)

 

http://www.cfsan.fda.gov/~ebam/m152.html

 

 

Trypticase peptone

15 g

Phytone peptone

5 g

NaCl

5 g

Agar

15 g

Distilled water

1 liter

Heat with agitation to dissolve agar. Boil 1 min. Dispense into suitable tubes or flasks. Autoclave 15 min at 121°C. Final pH, 7.3 ± 0.2.

For use with halophilic Vibrio spp., add NaCl to a final concentration of 2-3%.

 

Violet Red Bile Agar (VRBA)

 

http://www.cfsan.fda.gov/~ebam/m174.html

 

 

Yeast extract

3.0 g

Peptone or gelysate

7.0 g

NaCl

5.0 g

Bile salts or bile salts No. 3

1.5 g

Lactose

10.0 g

Neutral red

0.03 g

Crystal violet

0.002 g

Agar

15.0 g

Distilled water

1.0 liter

Suspend ingredients in distilled water and let stand for a few min. Mix thoroughly and adjust to pH 7.4 ± 0.2. Heat with agitation and boil for 2 min. Do not sterilize. Before use, cool to 45°C and use as a plating medium. After solidification, add a cover layer above the agar of approximately 3.0 to 4.0 ml to prevent surface growth and spreading of colonies.

Voges-Proskauer Medium (Modified)

 

http://www.cfsan.fda.gov/~ebam/m177.html

 

 

Proteose peptone

7 g

NaCl

5 g

Dextrose

5 g

Distilled water

1 liter

Dissolve ingredients in water and adjust pH if necessary. Dispense 5 ml portions into 20 x 150 mm tubes. Autoclave 10 min at 121°C. Final pH, 6.5 ± 0.2.