LETHEEN MEDIA

Cat. no. G42 Letheen Agar, 15x100mm Plate, 18ml 10 plates/bag
Cat. no. Q18 Letheen Agar, 20x125mm Tube, 18ml Deep 20 tubes/box
Cat. no. K106 Letheen Broth, 16x125mm Tube, 10ml 20 tubes/box
Cat. no. U39 Letheen Broth, 8oz Wide Mouth Jar, 90ml 12 jars/box
Cat. no. U40 Letheen Broth, 180ml Wide Mouth Jar, 99ml 12 jars/box
Cat. no. U371 Letheen Broth, 1L polypropylene bottle, 1000ml 10 bottles/box

INTENDED USE

Hardy Diagnostics Letheen Agar is used for evaluating the bactericidal activity of quaternary ammonium compounds. Letheen Broth is used for determining the phenol coefficient of cationic surface-active materials.

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

SUMMARY

Quaternary ammonium compounds are molecules which contain a nitrogen atom with four other atoms bonded to it. Most quaternary ammonium compounds are organic compounds and have biological activity. These compounds often work well as disinfectants, offering bactericidal and bacteriostatic effects.

Letheen Media is formulated according to the guidelines set by the AOAC for the determination of phenol coefficient of quaternary ammonium compounds. (1) The medium contains beef extract and peptone to provide a nutrient rich medium supporting the growth of a variety of microorganisms. Quaternary ammonia compounds are neutralized by lecithin while phenolic disinfectants and hexachlorophene are neutralized by Tween ® 80. Together, lecithin and Tween ® 80 neutralize ethanol.

Letheen Broth is used in the AOAC for use with disinfectants containing cationic surface active materials. (1) Letheen Agar and Letheen Broth are specified for use by the American Society for Testing Materials (ASTM) in the Standard Test Method for Preservatives in Water Containing Cosmetics. (4)

Letheen Broth is available in a 99ml fill volume for preparation of 1:100 test dilutions.

FORMULA

Ingredients per liter of deionized water:*

Letheen Agar
Tween ® 80 7.0gm
Pancreatic Digest of Casein 5.0gm
Beef Extract 3.0gm
Dextrose 1.0gm
Lecithin 1.0gm
Agar 15.0gm

Final pH 7.0 +/- 0.2 at 25ºC.

Letheen Broth
Peptic Digest of Animal Tissue 10.0gm
Tween ® 80 5.0gm
Sodium Chloride 5.0gm
Beef Extract 5.0gm
Lecithin 0.7gm

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 Letheen Media 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

Allow media to warm to room temperature before inoculation.

Please consult AOAC for methods using Letheen Media. (1)

The Letheen Agar, Deep (Cat. no. Q18) can be used for plate counts using either the spread plate method or pour plate method. See listed references for more information. (2,3)

Spread Plate Method:

1. Warm media to room temperature.

3. Prepare decimal dilutions in sterile diluent to obtain 30-300 CFU per plate.

4. Aseptically inoculate agar surface with 0.1ml of well mixed diluted sample.

5. Spread the dilution evenly over the surface of the medium.

6. Using a sterile spreader device, distribute the inoculum evenly over the agar surface.

7. Incubate plates aerobically for 48 +/- 2.0 hours at 35ºC.

Pour Plate Method:

1. Melt agar by placing in a boiling waterbath until liquified.

2. Cool media to 45-50ºC. Maintain in a 45-50º waterbath until ready to pour.

3. Prepare decimal dilutions in sterile diluent to obtain 30-300 CFU per plate.

4. Place a 1ml inoculation into a sterile petri plate.

5. Aseptically pour approximately 18ml of the cooled media (45-50ºC.) over the inoculum. Carefully swirl the plate to mix the inoculum evenly.

6. Allow to solidify.

7. Incubate plates aerobically for 48 +/- 2.0 hours at 35ºC.

INTERPRETATION OF RESULTS

See listed references for the interpretation of growth in Letheen Media. (1-4)

Plate Counts:

Following incubation, examine the plates for growth. Count the number of colonies and express in number of colony forming units (CFU) per gram or milliliter of sample (take into account the dilution factor). If duplicate plates were set-up, express the average for the two plates in terms of the number of microorganisms per gram or milliliter of sample. Consult listed references for additional information on interpretation and enumeration of microbial growth on this medium. (1-4)

LIMITATIONS

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
Letheen Agar
Staphylococcus aureus
ATCC ® 25923
A 24-48hr 35°C Aerobic Growth
Escherichia coli
ATCC ® 25922
A 24-48hr 35°C Aerobic Growth
Salmonella typhimurium
ATCC ® 14028
A 24-48hr 35°C Aerobic Growth
Letheen Broth
Staphylococcus aureus
ATCC ® 25923
A 18-48hr 35°C Aerobic Growth; turbidity
Escherichia coli
ATCC ® 25922
A 18-48hr 35°C Aerobic Growth; turbidity
Salmonella typhimurium
ATCC ® 14028
A 18-48hr 35°C Aerobic Growth; turbidity

User Quality Control

Physical Appearance

S. aureus growing on Letheen Agar

Staphylococcus aureus (ATCC ® 25923) colonies growing on Letheen Agar (Cat. no. G42). Incubated aerobically for 24 hours at 35ºC.

E. coli growing on Letheen Agar

Escherichia coli (ATCC ® 25922) colonies growing on Letheen Agar (Cat. no. G42). Incubated aerobically for 24 hours at 35ºC.

Letheen Agar

Uninoculated plate of Letheen Agar (Cat. no. G42).

REFERENCES

1. Association of Official Analytical Chemists. Official Methods of Analysis , AOAC, Washington, D.C.

2. U.S. Food and Drug Administration. Bacteriological Analytical Manual. Arlington, VA
http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm2006949.htm

3. The Official Compendia of Standards. USP-NF . United States Pharmacopeial Convention, Rockville, MD.

4. American Society for Testing Materials . Standard test method for preservatives in water-containing cosmetics. 1991. E: 640-78. Annual Book of ASTM Standards, Philadelphia, PA.


ATCC is a registered trademark of the American Type Culture Collection.
Tween is a registered trademark of ICI Americas, Inc.

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