ESTIMATION of MICROBIAL NUMBERS FROM PURE BACTERIAL CULTURES and FROM MINCED BEEF SAMPLES BY REFLECTANCE COLORIMETRY WITH OMNISPEC^TM 4000¹

Abstract

Tristimulus reflectance colorimetry (RC) is a rapid method with which microbial activities can be monitored by the mediation of dye pigmentation changes. Dyes that produce color changes because of shifts of pH and oxidation/reduction (O/R) potential or free amino groups can be used. To estimate total microbial numbers from pure coliform and Listeria cultures and from minced beef samples, the appropriate color endpoint values were selected for each sample type (b*=+2 for coliforms, L*=+ 11 for Listeria, and a*=+ 4 and a*=+ 6 for minced beef samples) and used to estimate color detection times (CDTs). the regression lines of data between aerobic plate count (APC; conventional and also Spiral System with minced beef samples) and RC were calculated. Reliability of the calibration lines was good; r = 0.90–0.99 for bacterial cultures and r = 0.82 (0.80) for minced beef (a*=+ 4) and r = 0.80 (0.78) for minced beef (a*=+6) when RC was compared with APC (Spiral). The calibration line for minced beef samples (1/10 dilution) could be used to classify samples above and below a certain specified microbial level (e.g., log 6.0 CFU/g) and to marginal samples. Using these scheme with a*=+4 as the color endpoint value, 70% of the tested 40 minced beef samples were correctly classified. the calibration line could also be used to approximate plate count determinations (e.g., CDT of 12 h will result in an estimated count of log 5.9 CFU/g). Results were obtained in shorter time (ranging from 0–15 h for Enterobacter aerogenes and E. coli, from 2–17 h for H. alvei, from 1.5–25 h for Listeria monocytogenes ATCC 35152 and Scott A, from 6–39 h for slowly growing L. monocytogenes ATCC 19919, and from 2–15 h for minced beef, depending on the initial cell concentration) than required by conventional plate counting (24 h for coliforms, 24–48 h for Listeria, and 48 h for total counts). Time for further confirmation varies according to bacterial species. the RC technique was sensitive for low levels (as low as 1 CFU/mL) of microbes. Colorimetric microtest plate technology provided in the OmnispecTM Bioactivity Monitor System simplifies the analysis, saves labor and materials, and provides high capacity.

RAPID DETECTION OF LISTERIA MONOCYTOGENES USING A REFLECTANCE COLORBMETRIC METHOD WITH MEMBRANE FRACTIONS FROM OXIDATIVE BACTERIA

“…OmniSpec Bioactivity System Figure 1 shows the times required for serial dilutions of L. monocytogenes Scott A to decrease the L* color values from 18 to 4. The color values changed from light yellow to black according to the hydrolysis of esculin in Fraser broth, and L* = + 11 was the suitable endpoint value for all Listeria strains (Manninen and Fung 1992). Each dilution curve was separated clearly and perpendicular to the x-axis at L* = 11.…”

“…To enumerate the bacteria before each test. 0.1 mi of the pure culture suspension was spread in duplicate onto BHI and modified Oxford (MOX) agar plates (McClain and Lee 1989) and incubated at 37C with 30 min reading interval for 48 h. The color value for the determination of a CDT was at L* = I 1 (Manninen and Fung 1992) with color change from L* = 20 to 3 (light yellow to black). Each membrane fraction was used in amounts equivalent to the activity of Oxyrase at 0.1.…”

Rapid Methods and Automation in Microbiology

“…The change of color or hue (a*, b*, L*) can be monitored similar to impedance and conductance curve. Manninen and Fung (1992b) evaluated this system in a study of pure cultures of Listeria monocytogenes and food samples and found high correlation coefficients (r) of 0.90 to 0.99 for pure cultures and 0.82 for minced beef between the colony counts predicated by the colorimetric technique and the results of the traditional plate count method. They also showed that detection times for bacterial cultures were substantially (2 to 24 h) shorter using the instrument and concluded that the Omnispec system simplifies the analyses, saves labor and materials and provides a high sample capacity.…”