USP 51 Antimicrobial Effectiveness Test

"USP <51>" Refers to chapter 51 of the United States Pharmacopeia (USP), which is a detailed description of the USP method of preservative efficacy testing, which is also sometimes called "challenge testing. If you would like to learn more about the USP <51> preservative challenge test, you are in the right place! Below,you will find a summary of the USP 51 method, along with some of its strengths and weaknesses. If you're here to learn, also be sure to read the page called "Getting the Most from Preservative Efficacy Testing." If you are a product formulator and would like Antimicrobial Test Laboratories to run a preservative challenge test for you or your company, simply call the lab or click here to get a same day price quote.

Summary of the USP <51> Antimicrobial Effectiveness Test:

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USP <51> challenges (inoculates) a formula with 5 different microorganisms, separately. Three bacteria and two fungal strains are used for each USP <51> test. Test microorganisms are grown in liquid or on solid medium, depending on the microorganism. Microorganisms used for a USP 51 test follow: Candida albicans (a yeast...yeasts are a form of fungus) Aspergillus brasiliensis (a filamentous mold...also a fungus) Escherichia coli (a bacterium...better known as "E. coli") Pseudomonas aeruginosa (a bacterium....very problematic industrially) Staphylococcus aureus (a bacterium...better known as "Staph") The test microorganisms are either harvested by centrifugation from broth culture or by washing surface growth from a solid medium into a sterile vessel. The concentrations of test microorganisms are standardized by resuspending harvested microorganisms in sterile saline to yield ~1 X 10^8 CFU/ml. A recovery analysis is performed to verify that microorganisms present in a sample can be adequately recovered and enumerated using the chosen dilution and plating scheme. A sufficient volume of test product (typically 10ml) is distributed into each of 5 separate containers, and each container is inoculated with a separate test microorganism (mentioned above). The initial concentration of viable microorganisms in the test product is determined by standard dilution and plate count methods.

Inoculated test products are incubated at 22.5 2.5C and sampled to determine microorganism concentration at 7, 14 and/or 28 day intervals depending on the product category into which the formulation falls. The microorganism concentration at each interval is compared to the initial concentration, and then preservative effectiveness is determined based USP guidelines.

Strengths of the USP <51> Preservative Challenge Method:

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The USP 51 Antimicrobial Effectiveness Test specifies the initial target inoculum concentration, which allows for a fairly reproducible comparison of products that fall in the same product category. The USP 51 method challenges preserved products with a variety of microorganisms representing a broad spectrum of manufacturing, nosocomial and household contaminants, including gram-negative and gram-positive bacteria, yeast and mold. The initial inoculum concentration is relatively high, providing an indication of how the product will fare in "real life" should it be inadvertently contaminated with microorganisms during manufacturing or after sale.

Weaknesses of the USP <51> Preservative Challenge Method:

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The USP 51 test method covers a broad range of microbial physiologies and metabolic capabilities, but microorganisms may be encountered during manufacturing or normal consumer use that can defeat the preservative. For this reason, some customers choose to supplement USP 51 studies with separate inoculations of organisms that have proven problematic for them in the past. Antimicrobial effectiveness criteria with respect to C. albicans and A. niger is liberally defined across the 4 categories; a sample taken from 14 and 28 day intervals can have the same microorganism concentration as the initial inoculum, and still meet the "pass" criteria for these two microorganisms.

Antimicrobial Test Laboratories has a great deal of expertise in preservative efficacy testing, and in particular the USP <51> challenge test. For more information about the <USP 51>Preservative Challenge Method, Contact the Lab Today!

How do you prepare nutrient agar plates? ?

I need the materials needed and the basic, concise procedures on how to do it.
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Agar is pronounced awger (sounds like fogger without the f). Agar is a gelling agent extracted from red seaweed. Nutrient agar is a commonly used food medium for microbial cultures. Nutrient agar contains: o beef extract (provides carbohydrates, nitrogen, vitamins, salts) o peptone (helps control pH) o agar (a carbohydrate used as a solidifying agent) o distilled water (an agent for distributing food materials to growing colonies of micro-organisms) Materials: Agar Powder distilled water flask or beaker glass stir rod lab thermometer sterile Petri dishes (plastic) flame or boiling mixture heat resistant hand protection NOTE: Keep sterile Petri dishes closed until ready to pour agar into them. Air-borne contaminants can easily invade an open Petri dish. Procedure: 1. Measure agar and distilled water into clean flask or beaker. Recipe: Agar + Distilled Water = Yield Agar + Distilled Water = Yield 23 g 1000 ml 50 plates 11.5 g 500 ml 25 plates 9.2 g 400 ml 20 plates 4.6 g 200 ml 10 plates 2. Flame sterilize a clean glass stir rod to stir the medium as it melts. 3. While wearing heat resistant hand protection, hold the flask or beaker over the flame. Swish or stir the mixture constantly while heating. 4. Boil the mixture for 1 minute. Remove from heat. 5. Place a sterile lab thermometer. in the mixture and monitor the temperature until it falls to approximately 45 - 50 C or if a lab thermometer is not available, cover and let stand a few minutes. 6. Pour enough melted agar into each sterile plastic petri dish to cover the bottom - about 1/8" to 1/4" deep. Replace the lid immediately. 7. Place agar plates on a counter top to cool and set. Agar medium will set like stiff gelatin at room temperature. 8. The agar medium is now ready for storage or use. Storage: Stack agar plates upside down in the refrigerator. Do Not Freeze! The purpose of placing the plates upside down is to prevent condensation from dripping down onto the agar surface which could then facilitate movement of organisms between colonies. Preparing the Plates 1. If plates have been refrigerated, set them out and allow them to warm to room temperature. 2. Sterilize the loop

o To sterilize the loop, hold the handle with a pot holder and place the tiny looped wire in a flame until it turns bright red o Allow the loop to cool for 3 - 5 seconds before touching the collection area. o Resterilize the loop after each inoculation. o Do not allow the loop to touch any surface other than the collection area and the agar. 3. Uncover each agar plate just long enough to inoculate the medium. hold the petri dish lid directly over the petri dish (or tilt the lid just enough to allow the loop inside) while inoculating the medium to help prevent contamination from air-borne particles. Do not allow the loop to touch the petri dish. 4. Do not dip the loop in the agar; let it glide over the surface. 5. Make a pattern of inoculation lines (parallel lines, tic-tac-toe, zig-zag, initials, etc.) to help determine that what is growing is what you put there and not an air-borne contaminant. 6. Place the cover back on the plate immediately. Incubation: Turn the plates upside down and put them in a warm place. The ideal temperature for incubation is 32 C or 90 F. Bacterial growth should start to become visible in about 2 -3 days.