Tryptic Soy Agar with Lecithin

Intended Use

These media are recommended for the detection and enumeration of microorganisms present on surfaces of sanitary importance. Prepared plates are provided for environmental monitoring. Sterile Pack and Isolator Pack RODAC™ prepared plates are particularly useful for monitoring surfaces in clean rooms, Isolator Systems and other environmentally-controlled areas and are also recommended for use in air sampling equipment such as the Surface Air System. Finger Dab™ Sterile Pack and Isolator Pack plates are intended for sampling gloved hands.

Summary and Explanation

These media may be employed to establish and monitor cleaning techniques and schedules.^1-4 Collection of “samples” from identical areas before and after treatment with disinfectant yields data useful in evaluating cleaning procedures in environmental sanitation. Tryptic (Trypticase) Soy Agar with Lecithin and Polysorbate 80 is recommended for the Aerobic Plate Count (Microbial Limit Test) for water-miscible cosmetic products containing preservatives.⁵

RODAC (Replicate Organism Detection and Counting) and contact plates are used in a wide variety of surface sampling programs and may be employed to establish and monitor cleaning techniques and schedules.^1-4,6 The presence and number of microorganisms on a flat impervious surface is determined by the appearance of colonies on the surface of the medium following application to the test surface and incubation.^7,8 The RODAC plate has a marked grid to facilitate counting organisms. The RODAC SL (Secure Lid) has three lugs on the base, providing a tight fit between lid and base to reduce accidental contamination.

The 100 × 15 mm and the 150 × 15 mm style plates can be used for active and passive air sampling. These plates are also designed for personnel monitoring of finger tips (Finger Dab).

Principles of the Procedure

Casein and soy peptones are a source of nutrients required for the replication of microorganisms. Sodium chloride maintains osmotic equilibrium. Lecithin and polysorbate 80, two commonly used neutralizers, are reported to inactivate residual disinfectants when the sample is being collected.⁷ Lecithin is incorporated to neutralize quaternary ammonium compounds and polysorbate 80 is used to neutralize substituted phenolic disinfectants.^9-12 Agar is the solidifying agent.
Trypticase Soy Agar with Penicillinase and Trypticase Soy Agar with Lecithin, Polysorbate 80 and Penicillinase contain 50 mL/L of penicillinase, which inactivates antibiotics such as penicillins and cephalosporins.

With the Sterile Pack and Isolator Pack plates, the entire double-wrapped (Sterile Pack) or triple-wrapped (Isolator Pack) product is subjected to a sterilizing dose of gamma radiation, so that the contents inside the outer package(s) are sterile.¹³ This allows the inner package to be aseptically removed without introducing contaminants. Since the agar medium has been sterilized after packaging, the presence of microbial growth after sampling and incubation can be relied upon to represent true recovery and not pre-existing medium contaminants. A third rolled sterile bag is included as a transport device. Isolator Pack plates have been validated to protect the medium from vaporized hydrogen peroxide when used in an Isolator System.

User Quality Control

Identity Specifications
Tryptic Soy Agar with Lecithin and Polysorbate 80 (Microbial Content Test Agar)
Dehydrated Appearance: Beige, free-flowing, homogeneous, may appear moist.
Solution:                        4.57% solution, soluble in purified water upon
                                     boiling with frequent gentle swirling. When hot,
                                     solution is medium amber, slightly opalescent
                                     with a resuspendable precipitate.
Prepared Appearance:    Light to medium amber, slightly opalescent,
                                    may have a precipitate.
Reaction of 4.57%
Solution at 25°C:           pH 7.3 ± 0.2
Cultural Response

Tryptic Soy Agar with Lecithin and Polysorbate 80 (Microbial Content Test Agar)
Prepare the medium per label directions. Test the medium in parallel with Plate Count Agar, using the pour plate method. Apply disks impregnated with varying dilutions of a quaternary ammonium compound to the medium surface. Incubate plates at 35 ± 2°C for 40-48 hours and inspect for zones of inhibition.

*Interpretation: The smaller zones of inhibition indicate neutralization of the quaternary
ammonium compound by the medium.

Formula

Tryptic Soy Agar with Lecithin and Polysorbate 80 (Microbial Content Test Agar)
Approximate Formula* Per Liter
Pancreatic Digest of Casein.......................................... 15.0 g
Soy Peptone................................................................. 5.0 g
Sodium Chloride........................................................... 5.0 g
Lecithin........................................................................ 0.7 g
Polysorbate 80............................................................. 5.0 g
Agar.......................................................................... 15.0 g
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

1. Suspend 45.7 g of the powder in 1 L of purified water. Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
3. Autoclave at 121°C for 15 minutes. Cool to approximately 45°C.
4. In RODAC plates, use 16.5-17.5 mL per plate.
5. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

100 × 15 mm and 150 × 15 mm-Style Plates
1. If specimen is being cultured from a swab, roll the swab directly on the medium surface.
2. Incubate all plates at 35-37°C for 48 hours, and 25°C for 7 days or as required.
3. When incubation has been completed, count the colonies.
RODAC™/Contact Plates
Selected surfaces are sampled by firmly pressing the agar medium against the test area. Hold the plate with thumb and second finger and use index finger to press plate bottom firmly against surface. Pressure should be the same for every sample. Do not move plate laterally; this spreads contaminants over the agar surface making resolution of colonies difficult. Slightly curved surfaces may be sampled with a rolling motion.
Areas (walls, floors, etc.) to be assayed may be divided into sections or grids and samples taken from specific points within the grid.
Grid method:
1. Subdivide surface (floor or wall) into 36 equal squares per 100 square feet of area by striking five equidistant dividing lines from each of the two adjacent sides.
2. These dividing lines intersect at twenty-five points.
3. Number these intersections consecutively in a serpentine configuration.
4. Use red numerals for odd numbers, black numerals for even numbers.
5. Omit number 13 which falls in the center of the total area.
6. Sample odd points at one sampling period, even points at the next sampling period.
7. For areas greater than 100 square feet, extend grid to include entire area.
8. For areas smaller than 25 square feet, divide the areas into twenty-five equal squares (sixteen intersections). Sample eight even-numbered or odd-numbered intersections at each sampling period.
9. For areas between 25 and 100 square feet, divide into 36 equal squares as in #1.
10. Mark plates with intersection numbers. Incubate exposed plates at 35-37°C for 48 hours, and 25°C for 7 days or as required.

Expected Results

Because interpretations are relative, each laboratory should establish its own values for what constitutes a clean area. Count all developing colonies. Spreading colonies should be counted as one but care should be taken to observe other distinct colonies intermingled in the growth around the plate periphery or along a hair line. These should also be counted as one colony, as should bi-colored colonies and halo type spreaders.
It is generally agreed that 200 colonies is the approximate maximum that can be counted on contact plates.

Colony counts may be recorded by:
1. Simply keeping individual counts.
2. Number of viable particles per square foot (agar area is 3.97 square inches).
3. Means and standard deviations.
Subculture colonies of interest so that positive identification can be made by means of biochemical and/or serological testing.

Limitations of the Procedure

The effectiveness of preservative neutralization with this medium depends on both the type and concentration of the preservative(s).

*Store at 2-8° C.

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