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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR CHARACTERIZATION AND GASTROINTESTINAL TRACT ECOLOGY OF COMMENSAL HUMAN FOOD-BORNE BACTERIAL PATHOGENS IN THE CHICKEN

Location: Poultry Microbiological Safety Research

Title: Colorimetric assay for bioWlms in wet processing conditions

Author
item Arnold, Judy

Submitted to: Journal of Industrial Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 30, 2008
Publication Date: September 1, 2008
Citation: Arnold, J.W. 2008. Colorimetric assay for bioWlms in wet processing conditions. Journal of Industrial Microbiology and Biotechnology. 35(11):1475-1480.

Interpretive Summary: The bacterial pathogen, Listeria monocytogenes, with a 20 percent death rate among its victims, can cause diarrhea, fever, and chills. Most of the food product recalls in the last two years occurred because of contamination with L. monocytogenes. Our goal was to develop methods to grow and assess the pathogen during culture that mimics food production and processing environments. Our model included stainless steel surfaces incubated in liquid suspensions containing minimal nutrients and exposed to bacteria under temperature and humidity conditions similar to food production facilities. To determine cell counts and mass of the bacteria on the stainless steel coupons, culture material collected was measured separately for each coupon by aerobic plate counts of viable bacterial cells and for biofilm production by the crystal violet assay. L. monocytogenes formed biofilms on all of the surfaces tested. The log of the mean of the cell counts was 5.90 based on 24 coupons for the experiment. During incubation from 24 to 144 h, the numbers of viable cells was greater for each succeeding 24-h, but did not increase significantly. The biofilm assay, a modified version of the crystal violet assay, resulted in different statistical groupings than the cell counts. There was not a significant increase in bacterial mass from 24 to 48 h, from 48 to 72 h, or from 72 to 144 h. However, there was a significant difference between 24 h and 72 h, because the 144-h sample was less than 72 h. The variability for the assay was somewhat high. The overall average was 0.50. The sequence of biofilm development, followed on glass coupons from 24 to 144 hrs, followed a change from dispersed single cells to increased numbers of clumps to an all-over pattern of clumps with few to no dispersed single cells. Bacterial counts taken from free-swimming bacterial cultures at 24, 48, 72, and 144 h confirmed that L. monocytogenes remained viable throughout the time course of the experiment. The logs of the viable cell counts were 8.01, 8.03, 7.69, and 6.66, respectively. Because of the urgent need for control of this pathogen, the collected data will be used to grow stable biofilms of Listeria spp. for further study.

Technical Abstract: Aims: The bacterial pathogen, Listeria monocytogenes, causes a high death rate among its victims and many food product recalls. Our goal was to develop methods to quantitatively assess the pathogen under conditions that mimic food environments. Methods and Results: Stainless steel and glass coupons were incubated in aqueous media containing minimal nutrients and exposed to bacteria under static temperature and humidity conditions. Samples were measured separately for each coupon by aerobic plate counts, a modified crystal violet assay (CV), and spectrophotometry. The mean of the log10 density (cfu/cm2) was 5.90, and the std dev ranged from 0.127 to 0.438 on 24 coupons. The CV assay resulted in different t groupings than the cell density. The mean was 0.50, and the error % was 0.595. The typical sequence of biofilm development, followed on glass coupons, exhibited a change from dispersed single cells to an all-over pattern of clumps with few dispersed. Bacterial counts from planktonic cultures at 24, 48, 72, and 144 h confirmed that L. monocytogenes remained viable throughout the experiment. The cell density log10/ml was 8.01, 8.03, 7.69, and 6.66, respectively. Conclusions: L. monocytogenes formed biofilms on all of the substrata tested. The variability for the CV assay was somewhat high, with std dev ranging from 0.156 to 0.394. The data will be used to grow stable biofilms of Listeria spp. for further study. Significance and Impact of the Study: The study will provide data for risk assessment of L. monocyotogenes. This is the first use of the crystal violet assay for measurement of bacterial biofilms on stainless steel. The methods tested are applicable to other bacteria and substrata.

Last Modified: 7/24/2014
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