Antibacterial Activities and Phytochemical Screening of Crude Extracts of Calotropis gigantea (Giant Milk Weed)

The root and leaf of were screened for its antimicrobial and phytochemical activities. The solvents used for the roots and leaves extraction were ethanol and water. The extracts were tested against infectious disease causing bacterial such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus, Klebsiellaaerogenesand α-haemolysin using the well diffusion method. The aqueous extracts of root of Calotropis gigantea against all the test bacteria ranged from 6.0 mm to 20.0 mm diameter zones of inhibition. The ethanolic extract of root of Calotropis gigantea inhibition against some the test microbe ranges from 6 mm to 14.0 mm diameter inhibitory zone. The ethanolic leaf extract of C. gigantea also showed an inhibition of 8mm to 20.0 mm. In present study, bacterial extract showed a varying zone of inhibition of the growth of tested organism than ethanol. Phytochemical properties of root and leaves of Calotropis gigantea obtain from ethanol and aqueous extracts were investigated. The results confirmed that presence of antibacterial activity and phytochemical in the shade dried extract of Calotropis gigantea against the human pathogenic organisms.

Procedural Revision to the AOAC Germicidal Spray Products as Disinfectants Test Method: Establishment of Minimum and Maximum Log Density Values for Test Microbes on Inoculated Carriers

The AOAC Germicidal Spray Products as Disinfectants test method (AOAC Official Method 961.02) is used to measure the efficacy of spray products on hard inanimate surfaces; however, the method does not provide procedures to determine the population of the test microbe on inoculated glass slide carriers (e.g., carrier counts reported as CFU/carrier). Without a method to measure and monitor carrier counts, the associated efficacy data may not be reliable and repeatable. This report provides a standardized procedure to address this issue and, based on carrier count data collected by four laboratories from 2000 to 2010, proposes a specific range for the mean log density per carrier as a requirement. Laboratory-based carrier count data were collected concurrently with 116 Method 961.02 efficacy tests conducted on spray products bearing claims against Pseudomonas aeruginosa and Staphylococcus aureus. For many of the tests a soil load (SL) was added to the inoculum (as specified on the product label claim). Six carriers were assayed per test for a total of 696 carriers. All but two of the 116 mean log densities were at least 5.0 (a geometric mean of 1.0 × 105 CFU/carrier). Across the four combinations of microbes and SL treatments, the mean TestLD (mean log density across all enumerated carriers in a test) ranged from approximately 6.0 (a geometric mean of 0.9 × 106 CFU/carrier) to 6.3 (a geometric mean of 2.0 × 106 CFU/carrier). Across all microbes and SL treatments, the mean log density (±SEM) was 6.2 (±0.07) per carrier (a geometric mean of 1.5 × 106 CFU/carrier). The mean log density for six carriers per test showed good repeatability (0.32) and reproducibility (0.34). The proposed requirement for S. aureus tests and P. aeruginosa tests is a mean log density (across six carriers) between 5.0 and 6.5. A separate 2009 study at three laboratories was conducted to evaluate the persistence of P. aeruginosa, S. aureus, and Salmonella enterica on glass carriers. Based on the persistence data, a 2 h use period is proposed for using the inoculated carriers post drying. The persistence data set was also used to assess the carrier counts for S. enterica. The carrier counts were approximately one log lower for S. enterica compared to S. aureus and P. aeruginosa; a range of 4.0 to 5.5 logs is proposed as a requirement for S. enterica tests.

Procedural Revision to the Use-Dilution Methods: Establishment of Maximum Log Density Value for Test Microbes on Inoculated Carriers

The AOAC Use-Dilution Methods, 955.15 (Staphylococcus aureus) and 964.02 (Pseudomonas aeruginosa), were revised in 2009 to include a standardized procedure to measure the log density of the test microbe and to establish a minimum mean log density value of 6.0 (geometric mean of 1.0 × 106 CFU/carrier) to qualify the test results. This report proposes setting a maximum mean log density value of 7.0 (geometric mean of 1.0 × 107 CFU/carrier) to further standardize the procedure. The minimum value was based on carrier count data collected by four laboratories over an 8-year period (1999–2006). The data have been updated to include an additional 4 years' worth of data (2006–2010) collected by the same laboratories. A total of 512 tests were conducted on products bearing claims against P. aeruginosa and S. aureus with and without an organic soil load (OSL) added to the inoculum (as specified on the product label claim). Six carriers were assayed in each test, for a total of 3072 carriers. Mean log densities for each of the 512 tests were at least 6.0. With the exception of two tests, one for P. aeruginosa without OSL and one for S. aureus with OSL, the mean log densities did not exceed 7.5 (geometric mean of 3.2 × 107 CFU/carrier). Across microbes and OSL treatments, the mean log density (±SEM) was 6.80 (±0.07) per carrier (a geometric mean of 6.32 × 106 CFU/carrier) and acceptable repeatability (0.28) and reproducibility (0.31) SDs were exhibited. A maximum mean log density per carrier of 7.0 is being proposed here as a validity requirement for S. aureus and P. aeruginosa. A modification to the method to allow for dilution of the final test cultures to achieve carrier counts within 6.0–7.0 logs is also being proposed. Establishing a range of 6.0–7.0 logs will help improve the reliability of the method and should allow for more consistent results within and among laboratories.

Improving the AOAC Use-Dilution Method by Establishing a Minimum Log Density Value for Test Microbes on Inoculated Carriers

The AOAC Use-Dilution methods, 955.14 (Salmonella enterica), 955.15 (Staphylococcus aureus), and 964.02 (Pseudomonas aeruginosa), are used to measure the efficacy of disinfectants on hard inanimate surfaces. The methods do not provide procedures to assess log density of the test microbe on inoculated penicylinders (carrier counts). Without a method to measure and monitor carrier counts, the associated efficacy data may not be reliable and repeatable. This report provides a standardized procedure to address this method deficiency. Based on carrier count data collected by four laboratories over an 8 year period, a minimum log density value is proposed to qualify the test results. Carrier count data were collected concurrently with 242 Use-Dilution tests. The tests were conducted on products bearing claims against P. aeruginosa and S. aureus with and without an organic soil load (OSL) added to the inoculum (as specified on the product label claim). Six carriers were assayed per test for a total of 1452 carriers. All 242 mean log densities were at least 6.0 (geometric mean of 1.0 106 CFU/carrier). The mean log densities did not exceed 7.5 (geometric mean of 3.2 107 CFU/carrier). For all microbes and OSL treatments, the mean log density (SEM) was 6.7 (0.07) per carrier (a geometric mean of 5.39 106 CFU/carrier). The mean log density for six carriers per test showed good repeatability (0.29) and reproducibility (0.32). A minimum mean log density of 6.0 is proposed as a validity requirement for S. aureus and P. aeruginosa. The minimum level provides for the potential inherent variability that may be experienced by a wide range of laboratories and the slight effect due to the addition of an OSL. A follow-up report is planned to present data to support the carrier count procedure and carrier counts for S. enterica.

Enumeration Procedure for Monitoring Test Microbe Populations on Inoculated Carriers in AOAC Use-Dilution Methods

The AOAC Use-Dilution methods do not provide procedures to enumerate the test microbe on stainless steel carriers (penicylinders) or guidance on the expected target populations of the test microbe (i.e., a performance standard). This report describes the procedures used by the U.S. Environmental Protection Agency to enumerate the test microbe (carrier counts) associated with conducting the Use-Dilution method with Staphylococcus aureus (Method 955.15) and Pseudomonas aeruginosa (Method 964.02) and the examination of historical data. The carrier count procedure involves the random selection of carriers, shearing bacterial cells from the carrier surface through sonication, and plating of serially diluted inoculum on trypticase soy agar. For each Use-Dilution test conducted, the official AOAC method was strictly followed for carrier preparation, culture initiation, test culture preparation, and carrier inoculation steps. Carrier count data from 78 Use-Dilution tests conducted over a 6-year period were compiled and analyzed. A mean carrier count of 6.6 logs (approximately 4.0 × 106colony-forming units/carrier) was calculated for both S. aureus and P. aeruginosa. Of the mean values, 95% fell within ±2 repeatability standard deviations. The enumeration procedure and target carrier counts are desirable for standardizing the Use-Dilution methods, increasing their reproducibility, and ensuring the quality of the data.