CulGenex™

LB AGAR MEDIA

Cat. no. G81 LB Agar with 100ug/ml Ampicillin, 15x100mm Plate, 26ml 10 plates/bag
Cat. no. G186 LB Agar with 50ug/ml Ampicillin, 15x100mm Plate, 26ml 10 plates/bag
Cat. no. G194 LB Agar with 50ug/ml Kanamycin, 15x100mm Plate, 26ml 10 plates/bag
Cat. no. G401 LB Agar, 15x100mm Plate, 26ml 10 plates/bag
Cat. no. G406 LB Agar with 100ug/ml Ampicillin, 60µg/ml X-GAL and 0.5mM IPTG, 15x100mm Plate, 26ml 10 plates/bag
Cat. no. G407 LB Agar with 100ug/ml Carbenicillin, 15x100mm Plate, 26ml 10 plates/bag
Cat. no. G408 LB Agar with 25ug/ml Tetracycline, 15x100mm Plate, 26ml 10 plates/bag

INTENDED USE

Hardy Diagnostics CulGenex™ LB Agar Media formulations are recommended for the cultivation of bacteria used in molecular biology studies.

This product is not intended to be used for the diagnosis of human disease.

SUMMARY

LB, or "lysogeny broth", media formulations have been widely used for the cultivation of Escherichia coli since the 1950s, and have become an industry standard in molecular microbiology applications for the preparation of plasmid DNA and the growth of recombinant strains. (3,5-8) LB medium was originally formulated by Giuseppe Bertani and published in 1951 and has since been modified by Miller, Lennox and Luria: the formulations differ in the concentration of sodium chloride, which provides for greater selectivity. (3) LB Agar, Miller medium contains 10gm of sodium chloride; LB Agar, Lennox contains 5gm of sodium chloride; and Luria Agar, Miller contains 0.5gm of sodium chloride. (3,5-8) Low salt formulations, such as those by Lennox and Luria, are ideal for salt-sensitive applications.

Adapted by J.H. Miller, LB Agar is a nutritionally rich medium designed for the growth and culture of pure recombinant strains used in genomic testing. (8) Hardy Diagnostics LB Agar media formulations are based on the original recipe by Miller and contain casein peptone and yeast extract for amino acids, vitamins and essential minerals. Sodium chloride provides sodium ions for transport and helps maintain osmotic balance. Agar is the solidifying agent.

Additional agents such as ampicillin, carbenicillin, tetracycline, or kanamycin are ideal for selective applications; sucrose or glucose are added to some formulas to provide an additional level of selection. The chromogen X-GAL (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) used with IPTG (isopropyl beta-D-thiogalactopyranoside) can be utilized to distinguish lacZ transformed cells, making it easy to differentiate between lac+ (blue) and lac- (white) colonies.

FORMULA

Ingredients per liter of deionized water:*

LB Agar (Cat. no. G401):
Casein Peptone 10.0gm
Sodium Chloride 10.0gm
Yeast Extract 5.0gm
Agar 15.0gm

Final pH 7.0 +/- 0.2 at 25ºC

LB Agar with 100ug/ml Ampicillin (Cat. no. G81) also contains:

Ampicillin 0.1gm

Final pH 7.0 +/- 0.3 at 25ºC.

LB Agar with 50ug/ml Ampicillin (Cat. no. G186) also contains:

Ampicillin 0.05gm

Final pH 7.0 +/- 0.3 at 25ºC.

LB Agar with 50ug/ml Kanamycin (Cat. no. G194) also contains:

Kanamycin 0.05gm

Final pH 7.0 +/- 0.3 at 25ºC.

LB Agar with 100ug/ml Ampicillin, 60ug/ml X-GAL and 0.5mM IPTG (Cat. no. G406) also contains:

Ampicillin 0.1gm
X-GAL 0.06gm
IPTG 0.12gm

Final pH 7.0 +/- 0.2 at 25ºC.

LB Agar with 100ug/ml Carbenicillin (Cat. no. G407) also contains:

Carbenicillin 0.1gm

Final pH 7.0 +/- 0.2 at 25ºC.

LB Agar with 25ug/ml Tetracycline (Cat. no. G408) also contains:

Tetracycline 0.025gm

Final pH 7.0 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.


PRECAUTIONS


PROCEDURE

Consult listed references for recommended test procedures. (2,5,8-10)

INTERPRETATION OF RESULTS

Growth is evident by the formation of isolated colonies or a confluent lawn of growth.

Colonies of lacZ gene transformed cells grown on media containing X-GAL and IPTG should appear blue in color.

LIMITATIONS


MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

The following organisms are routinely used for testing at Hardy Diagnostics:

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Cat. no. G401:
Escherichia coli
ATCC ® 25922
A 24hr 35°C Aerobic Growth

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Cat. no. G81, G186, G194, and G406**:
Pseudomonas aeruginosa
ATCC ® 27853
A 18-24hr 35°C Aerobic Growth; colorless**
Escherichia coli
ATCC ® 25922
A 18-24hr 35°C Aerobic Partial to complete inhibition

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Cat. no. G407:
Klebsiella pneumoniae
ATCC ® 13883
A 18-24hr 35°C Aerobic Growth
Escherichia coli
ATCC ® 25922
B 18-24hr 35°C Aerobic Partial to complete inhibition
Pseudomonas aeruginosa
ATCC ® 27853
B 18-24hr 35°C Aerobic Partial to complete inhibition

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Cat. no. G408:
Candida albicans
ATCC ® 10231
A 1-3 days 35°C Aerobic Growth
Escherichia coli
ATCC ® 25922
B 24hr 35°C Aerobic Inhibited

USER QUALITY CONTROL


Physical Appearance

LB Agar (Cat. no. G401), LB Agar with 100ug/ml Ampicillin (Cat. no. G81), LB Agar with 50ug/ml Ampicillin (Cat. no. G186), LB Agar with 100ug/ml Ampicillin, 60ug/ml X-GAL and 0.5mM IPTG (Cat. no. G406), LB Agar with 100ug/ml Carbenicillin (Cat. no. G407) and LB Agar with 25ug/ml Tetracycline (Cat. no. G408) should appear translucent, and light amber in color.

LB Agar with 20ug/ml Chloramphenicol (Cat. no. G183), LB Agar with 10ug/ml Chloramphenicol (Cat. no. G285), LB Agar with 50ug/ml Kanamycin (Cat. no. G194) and LB Agar with 10ug/ml Chloramphenicol (Cat. no. H29) should appear clear and slightly opalescent, and light to medium amber in color.

LB Agar with 15ug/ml Kanamycin (Cat. no. G184) should appear clear, with a slight precipitate, and light to medium amber in color.

REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl. 1994. Current Protocols in Molecular Biology . Vol. 1. Current Protocols, New York, N.Y.

3. Bertani, G. 1951. Studies on Lysogenesis: The Mode of Phage Liberation by Lysogenic Escherichia coli . J. Bacteriol. ; 62:293-300.

4. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

5. Lennox, E.S. 1955. Transduction of Linked Genetic Characteristics of the Host by Bacteriophage P1. Virology ; 1:190.

6. Luria, S.E., and J.W. Burrous. 1957. Hybridization Between Escherichia coli and Shigella . J. Bacteriol. ; 74:461-476.

7. Luria, S.E., J.N. Adams, and R.C. Ting. 1960. Transduction of Lactose-Utilizing Ability Among Strain of E. coli and S. dysenteriae and the Properties of the Transducing Phage Particles. Virology ; 12:348-390.

8. Miller, J.H. 1972. Experiments in Molecular Genetics . Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

9. Sambrook, J., E.F. Fritsch and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual , 2nd ed. Cold Spring Harbor Laboratory. Cold Spring Harbor, N.Y.

10. Sambrook, J. and D.W. Russell. 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory. Cold Spring Harbor, N.Y.


ATCC is a registered trademark of the American Type Culture Collection.

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