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Preliminary: In vitro analysis of antimicrobial activity of wheat grass components

Aim: This study will be conducted to determine if different components of wheat grass-containing solutions have antimicrobial activity against Campylobacter rectusActinobacillus actinomycetemcomitans (reclassified as Aggregatibacter actinomycetemcomitans, Aa), Candida albicans, Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum and Streptococcus mutans in possibly two types of assays, agar diffusion and biofilm formation.

Experimental Design: For the agar diffusion assay, blood agar (for the 3 anaerobic bacteria, 3 facultative bacteria and 1 aerobic yeast; Fisher Scientific) plates will be individually swabbed with fresh 24 h broth cultures of C. rectus, Aa, C. albicans, P. gingivalis, P. intermedia, F. nucleatum andS. mutansto provide a confluent lawn of microbial growth. Five minutes after swabbing the plates (to allow the inoculum to absorb into the agar), the test and control specimens (see below) will be aseptically pipetted (5 ul) onto designated places on the plates and allowed to adsorb (or adhere, in the case of the undiluted sera) into the agar. Assays are conducted in duplicate and undiluted and 1:5 and 1:10 dilutions of the solutions and sterile saline (negative control) and chlorhexidine (CHX) used. The plates will be incubated agar-side up at 37oC in a GasPak anaerobic jar or in 5% CO2 for 72 h. Plates will be examined at 72 h for zones of inhibition of growth of the microbes. The diameter of each zone will be measured in mm. The zones of inhibition of each dilution sample will be compared to the zones of control saline without antimicrobial components. If the zones of the solutions are the same size (or smaller) than the inhibition zones from the control saline than there will be judged no inhibition of antimicrobial activity by the antimicrobial solutions. Conversely, if the zones of the solutions are larger than the inhibition zones from the control saline than there will be judged significant antimicrobial activity by the solutions. It is anticipated that there will be significant inhibition of microbial growth by at least some of the antimicrobial wheat grass components.

In addition, a biofilm model will be conducted in this study where 1:5 and 1:10 dilutions of each component will be made in BHI-YE. 190 ul of each dilution in triplicate will be pipetted into wells of a sterile 96 well microtiter plate. 10 ul of a 24 h BHI-YE culture of each bacterial species will be inoculated into each dilution and the plates incubated in a GasPak jar or a 5% CO2 incubator at 37oC for 48 h. To correct for any possible absorbance of the components the identical wells will be prepared but without the wheat grass component and the absorbance values subtracted. Total absorbance (planktonic and biofilm), planktonic and biofilm measurements will be obtained providing minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC) for each component.

Test Specimens: Undiluted wheat grass components and 1:5 and 1:10 dilutions in sterile saline.

Control Specimens: Sterile saline (negative control) and CHX.

Richard L. Gregory, Ph.D.
Mucosal Biology Laboratory
Indiana University School of Dentistry
415 Lansing Street, Room OH120
Indianapolis, Indiana 46202