TRYPTIC SOY AGAR (TSA) WITH LECITHIN AND TWEEN® 80

Cat. no. Q13 TSA with Lecithin and Tween® 80, 20x125mm Tube, 18ml Deep 20 tubes/box
Cat. no. U174 TSA with Lecithin and Tween® 80, 500ml Polycarbonate Bottle, 400ml 10 bottles/box
Cat. no. U412 TSA with Lecithin and Tween® 80, 16oz. Glass Bottle, 400ml 12 bottles/box
Cat. no. W41 TSA with Lecithin and Tween® 80, 15x100mm Plate, 27ml 10 plates/bag

INTENDED USE

Hardy Diagnostics Tryptic Soy Agar (TSA) with Lecithin and Tween® 80 is recommended for the isolation of microorganisms from environmental surfaces and is used primarily to monitor microbial contamination, enumerate the number of microbial colonies growing on a variety of surfaces sanitized with quaternary ammonium compounds, and to assist in determining surface sanitation.

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

SUMMARY

Hardy Diagnostics Tryptic Soy Agar (TSA) with Lecithin and Tween® 80 contains digests of soybean meal and casein that provide amino acids and other nitrogenous compounds, making it a nutritious medium for many microorganisms. Sodium chloride is added to maintain cellular osmotic equilibrium. Lecithin and Tween® 80 are added to the formulation to neutralize germicidal or disinfectant residues. Neutralization of these residues reduces their inhibitory effect which ultimately results in lowering of microbial count. Quaternary ammonia compounds are neutralized by lecithin, while phenolic disinfectants and hexachlorophene are neutralized by Tween® 80. Together, lecithin and Tween® 80 neutralize ethanol.(1,2)

When the contact surface is flat and the expected number of recoverable microbes is between 30-300 colonies, the contact plate method is most useful. The contact plate method was one of several methods developed in the 1930's to monitor surfaces for contamination. In this method, a petri dish with a specified grid molded into the bottom of the plate and a diameter of 60mm is slightly overfilled with nutritive media. Surface contact is made easy by raising the meniscus above the rim.

FORMULA

Ingredients per liter of deionized water:*

Pancreatic Digest of Casein 15.0gm
Peptic Digest of Soybean Meal 5.0gm
Sodium Chloride 5.0gm
Tween® 80 5.0gm
Lecithin 0.7gm
Agar 15.0gm

Final pH 7.3 +/- 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

Specimen Collection: TSA with Lecithin and Tween® 80 is not recommended for primary isolation. Consult listed references for information on specimen collection.(1,2)

Method of Use: Allow medium to warm to room temperature prior to inoculation. Consult listed references for information concerning testing procedures.(1,2)

For melting bottled and tubed media: Autoclave at 121ºC. for 1-3 minutes or until melted. Alternatively, a covered, boiling waterbath (100ºC.) can be used. There should be enough water in the waterbath to reach the media line. A covered waterbath will help to reach and maintain the temperature. Heat in waterbath until melted. Cool media to 45-50ºC. and dispense as desired.

Contact Plate Method:

1. Hold the contact plate with thumb and second finger and use index finger to press plate bottom firmly against the selected test surface. The same amount of pressure should be applied for every sample. Do not move plate laterally. Lateral movement spreads contaminants over the agar surface, thus making resolution of colonies difficult. A rolling motion may be used for slightly curved surfaces.(1)

2. Incubate the plates aerobically at 35ºC. for 48 hours. Using adequate light and magnification, count the number of colonies within the squares of the grid area. Take care not to count a square more than once.

Spread Plate Method:

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

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

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

4. Incubate plates at 35ºC. for 48 hours.

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 inoculum 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.

Note: Do not heat media longer than 3 hours at 45-50ºC. Sterile solidified medium can only be remelted once.

6. Allow plate(s) from step 5 to solidify.

7. Incubate plates aerobically at 35ºC. for 48 hours.

Data should be collected and recorded according to a designed monitoring system that statistically provides for the accurate acquisition of data.

INTERPRETATION OF RESULTS

Similar appearing colonies growing in close proximity, but not touching, should be counted as individual colonies. Colonies with different morphology or color should be counted as individual colonies. When spreading colonies are present, a representative portion of colonies in a spread-free area should be counted. This is only done if the area covered by the spreaders does not exceed one-half of the plate area.

Consult listed references for more detailed information concerning plate count methods.(1,2)

LIMITATIONS

Accurate counting can be made difficult by molds or spreading colonies.

Results can be uninterpretable or misleading unless a statistical method for monitoring is designed.

Sampling challenges may occur as a result of irregular, porous, rough or textured media surface.

Microbial contamination on a surface cannot be completely characterized by a single assay.

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

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Staphylococcus aureus
ATCC® 25923
A 24hrs 35°C Aerobic Growth
Aspergillus brasiliensis
ATCC® 16404
G 1-5 days 20-25°C Aerobic Growth
Escherichia coli
ATCC® 25922
A 24hrs 35°C Aerobic Growth

USER QUALITY CONTROL

Physical Appearance

TSA with Lecithin and Tween® 80 should appear clear to slightly hazy, and light amber in color.

REFERENCES

1. American Public Health Association. Standard Methods for the Examination of Water and Wastewater, APHA, Washington, D.C.

2. APHA Technical Committee on Microbiological Methods for Foods. Compendium of Methods for the Microbiological Examination of Foods, APHA, Washington, D.C.

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

041816gr