LIM BROTH

Cat. no. L57 LIM Broth, 16x100mm Polycarbonate Tube, 5ml 20 tubes/box

INTENDED USE

Hardy Diagnostics LIM Broth is recommended for the selective enrichment of group B streptococci ( Streptococcus agalactiae ) from anovaginal specimens from pregnant women.

SUMMARY

Approximately 10-35% of women are asymptomatic carriers of group B streptococci (GBS) in the genital and gastrointestinal tracts. (1) GBS remains a leading cause of serious illness and death in newborn populations, and therefore, the detection of GBS in the vaginal-anorectal area is critical to the prevention of neonatal GBS disease. Several surveys have been conducted that show the incidence of neonatal sepsis and meningitis due to GBS is currently 0.5-3 cases per 1,000 live births, although there are substantial geographical and racial differences. (2) The case-fatality ratios are now declining due to prompt recognition and proper treatment. (3) Because group B streptococci produce asymptomatic disease in adult women, it is important to identify the carrier state so that effective therapeutic intervention can be implemented.

The Center of Disease Control (CDC) recommends the screening of all pregnant women for vaginal and rectal GBS colonization between 35 and 37 weeks of gestation using an enrichment broth followed by subculture to a Blood Agar.

Szilagy, Lim, and Jones, et al., in 1978, 1982, and 1983-84, respectively, conducted studies using Todd Hewitt Broth with various supplements for use in the rapid identification of maternal carriers and infants colonized with group B streptococci. (8-11) Szilagy, et al., supplemented the broth with 5% sheep blood, gentamicin and nalidixic acid. (8) Lim, et al., added colistin and nalidixic acid while Jones, et al., utilized Todd Hewitt Broth with yeast extract. (9-11)

Hardy Diagnostics LIM Broth is a further modification of Lim's formula. The medium is made from a Todd Hewitt Base made up of beef heart infusion, peptone and yeast extract. The basal medium is formulated to provide the necessary nutrients for the development of streptococci. Carbon and energy are supplied by glucose. Disodium phosphate and sodium carbonate serve as buffers which ultimately protect the hemolysin from the acids produced during the fermentation of the carbohydrate. Colistin and nalidixic acid are added as inhibitory agents toward gram-negative microorganisms.

FORMULA

Ingredients per liter of deionized water:*

Todd Hewitt Broth 30.0gm
Yeast Extract 10.0gm
Nalidixic Acid 15.0mg
Colistin Sulfate 10.0mg

Final pH 7.6 +/- 0.2 at 25ºC.

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

STORAGE AND SHELF LIFE

PRECAUTIONS

PROCEDURE

Specimen Collection: If performing a Strep B screening of pregnant women, obtain one or two swabs of the vaginal/rectal areas. Cervical cultures are not acceptable. Vaginal cultures should be collected with a dacron, rayon or calcium alginate swab between 35 and 37 weeks gestation. The swab(s) should be inoculated into an appropriate transport medium and submitted directly to the laboratory for immediate processing. If specimen is to be delayed more than 24 hours, it must be refrigerated at 2-6ºC. (13)

Method of Use:

1. Inoculate swab(s) directly to LIM Broth.

2. Incubate aerobically for 18-24 hours at 35-37ºC.

3. Following incubation, subculture the broth to a Blood Agar plate (Cat. no. A10).

4. Incubate the subculture for 18-24 hours at 35-37ºC. in an aerobic atmosphere.

5. Observe for growth of beta-hemolytic or non-hemolytic, gram-positive, catalase-negative colonies.*

6. Using isolated colonies obtained from the Blood Agar plate, perform a particle agglutination test or tests recommended for the detection of group B streptococci antigen (e.g. genetic probe or fluorescent antibody) adhering to the manufacturers procedure.

* If group B streptococci is not identified after 18-24 hours incubation of the Blood Agar subculture, reincubate and inspect at 48 hours.

INTERPRETATION OF RESULTS

Consult the manufacturers package insert for interpretation of the test method employed for the detection of group B streptococci antigen.

LIMITATIONS

This media is a selective enrichment broth. Additional tests including subculture to an appropriate agar media as well as biochemical and/or serological tests using pure cultures should be performed for complete identification. For more information, consult appropriate references.

MATERIALS REQUIRED BUT NOT PROVIDED

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

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Streptococcus agalactiae
ATCC ® 12386
A 24hr 35°C Aerobic Growth; beta-hemolytic colonies upon subculture to Blood Agar
Proteus mirabilis
ATCC ® 12453
B 24hr 35°C Aerobic Partial to complete inhibition
Escherichia coli
ATCC ® 25922
B 24hr 35°C Aerobic Partial to complete inhibition
Pseudomonas aeruginosa
ATCC ® 27853
B 24hr 35°C Aerobic Partial to complete inhibition

User Quality Control

PHYSICAL APPEARANCE

LIM Broth should appear clear, and light amber in color.

S. agalactiae and P. mirabilis incubated in LIM broth

LEFT:
Streptococcus agalactiae (ATCC ® 12386) growing in LIM Broth (Cat. no. L57). Incubated aerobically for 24 hours at 35ºC.

RIGHT:
Proteus mirabilis (ATCC ® 12453) growth inhibited in LIM Broth (Cat. no. L57). Incubated aerobically for 24 hours at 35ºC.

REFERENCES

1. Regan, J.A., Klebanoff, M.A., Nugent, R.P. 1991. The epidemiology of group B streptococcal colonization in pregnancy. Vaginal Infections and Pregnancy Study Group. Obstet. Gynecol.; 77:604-10.

2. Schrag, S.J., E.R. Zell, R. Lynfield, et al. 2002. A population-based comparison of strategies to prevent early-onset group B streptococcal disease in neonates. N. Engl. J. Med. 25;347(4):233-9.

3. Schuchat, A. 2001. Group B streptococcal disease: from trials and tribulations to triumph and trepidation. Clin. Infect. Dis. 5;33(6):751-6.

4. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

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

6. 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.

7. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

8. Szilagyi, G., et al. 1978. Journal of Clinical Microbiology; 8:410-412.

9. Lim, D.V., et al. 1982. Current Microbiol.; 7:99-101.

10. Jones, E.E., et al. 1983. Journal of Clinical Microbiology; 18:558-560.

11. Jones, et al. 1984. Journal of Clinical Microbiology; 20:438-440.

12. Physician Office Lab News-5, The quality touch: CDC recommends procedures for group B strep. screens.

13. Rosa-Fraile, et al. 2005. Specimen Storage in Transport Medium and Detection of Group B Streptococci by Culture. J. Clin. Microbiol.; 43:928-930.


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