Fraser Broth Base, Modified (Half Fraser Broth)

Intended use

Recommended as a primary enrichment medium for the isolation and enumeration of Listeria monocytogenes from food and animal feeds. The composition and performance criteria of this media is as per the specification laid down in ISO 11290-1:2017, ISO 11290-2:2017 and 11133:2014 (E) /Amd. :2020.

Composition**

Final pH (at 25°C): 7.2±0.2

**Formula adjusted, standardized to suit performance parameters # - Equivalent to Enzymatic digest of animal tissues $ - Equivalent to Enzymatic digest of casein ## - Equivalent to Meat extract

Directions

Suspend 54.97 gram (the equivalent weight of dehydrated medium per litre) in 1000 ml purified/distilled water. Heat if necessary to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to 45-50°C and aseptically add rehydrated contents of 2 vials of Fraser Supplement (FD141). Mix well and dispense in tubes or flasks as desired.

Principle And Interpretation

Listeria species are widely distributed in the environment. They have been isolated from soil, decaying vegetable matter, silage, sewage, water, animal feed, fresh and frozen poultry, meats, raw milk, cheese and asymptomatic human and animal carriers (1). L. monocytogenes primarily causes meningitis, encephalitis or septicemia in humans (2,3). In pregnant women, L. monocytogenes often causes influenza like bacteremic illness that, if untreated, may leaded to amnionitis and infection of the fetus, resulting in abortion, still birth or premature birth. Contaminated foods are the primary vehicles of transmission (4). Fraser Broth Base is based on the formulation of Fraser and Sperber (1) is used for the detection of Listeria species in food products (5). Fraser Broth Base, Modified is formulated so as to provide optimum conditions for the growth of Listeria. This medium is recommended by ISO for primary enrichment of Listeria species (6,7,8).

Peptone, Tryptone, yeast extract, and HM extract make the media highly nutritive by providing essential nutrients including carbonaceous and nitrogenous substances. Phosphates maintain the buffering capacity of the medium. All Listeria species exhibit beta-glucosidase activity which is evident by the blackening of the media. Listeria species hydrolyze esculin (substituted glucoside) to glucose and esculetin. The latter combines with ferric ions of ferric ammonium citrate (FD141), resulting in the formation of 6-7 dihydroxycoumarin, a black brown complex. Ferric ammonium citrate also enhances the growth of L. monocytogenes (8). The high salt tolerance (of sodium chloride) of Listeria is used as means to inhibit the growth of Enterococci. Lithium chloride is also used to inhibit Enterococci, which also possess the ability to hydrolyze esculin. Growth of accompanying bacteria is largely inhibited by the addition of Nalidixic acid and Acriflavin hydrochloride.

Type of specimen

Food samples

Specimen Collection and Handling

1. Initial suspension This broth is used as an dilution fluid for the preparation of initial suspension 25 grams/25 ml of sample to 225 ml of the medium (M1764I + 2 vials of FD141).
2. Primary enrichment The dilution prepared in Half Fraser broth is incubated at 30°C ± 1°C for 24-26 hours. The preenriched sample after incubation can be stored at 5°C for a maximum of 72 hours before transfer to Fraser Broth (secondary enrichment), A black colouration can develop during incubation.

The sample from primary enrichment and secondary enrichment is then subcultured on HiCrome™ Listeria Ottaviani-Agosti Agar Base (M1540I) and on Listeria Oxford Medium Base (M1145) or Listeria Identification Agar Base (PALCAM) (M1064I). Incubate at 37 ± 1 °C for 24 ± 2 hours. Additional incubation at 37 ± 1 °C for 24 ± 2 hours is recommended for Listeria spp. other than L. monocytogenes for recovery of more species (6,7,8).

Warning and Precautions

Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/ face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling specimens. Safety guidelines may be referred in individual safety data sheets.

Limitations

1. Presence of L. monocytogenes is often masked by other Listeria species like L. inocua and L. ivanovii.
2. Further subculture of organisms on selective media is required.

Performance and Evaluation

Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.

Quality Control

Appearance Cream to yellow homogeneous free flowing powder

Colour and Clarity of prepared medium Fluorescent yellow coloured clear solution.

Reaction Reaction of 5.5% w/v aqueous solution at 25°C. pH : 7.2±0.2

pH 7.00-7.40

Cultural response

Cultural characteristics observed after an incubation at 30 ± 1°C for 25 ± 1 hour, with added Fraser Supplement (FD141). Further subculture is carried out on M1540I at 37 ± 1°C for 48± 4 hours.

Storage and Shelf Life

Store the dehydrated powder and prepared medium on receipt between 2-8°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition Seal the container tightly after use. Product performance is best if used within stated expiry period.

Disposal

User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with sample must be decontaminated and disposed of in accordance with current laboratory techniques (4,9).

Reference

1. Fraser and Sperber, 1988, J. Food Prot., 51:762-76
2. Nieman R. E., and Lorber B., 1980, Rev. Infect. Dis. 2 : 207-2
3. Schuchat A. B., Swaminathan and C. V. Broome, Clin. Microbiol., Rev. 4 : 169-1
4. Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.
5. Salfinger Y., and Tortorello M.L., 2015, Compendium of Methods for the Microbiological Examination of Foods, 5th Ed., American Public Health Association, Washington, D.C.
6. Microbiology of the food chain — Horizontal method for the detection and enumeration of Listeria monocytogenes and of Listeria spp. - Part 1 , Detection method ; ISO 11290-1:2017.
7. Microbiology of the food chain — Horizontal method for the detection and enumeration of Listeria monocytogenes and of Listeria spp. - Part 2 , Detection method ; ISO 11290-2:2017
8. Microbiology of food, animal feeding stuffs and water- Preparation, production, storage and performance culture media, EN ISO 11133:2014 (E) /Amd. :2020.
9. Cowart R. E. and Foster B. G., 1985, J. Infect. Dis.; 151:17
10. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.