Rapid Microbial Testing - Pharmaceutical Technology

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PharmTech Europe

Rapid Microbial Testing
In this technical forum, experts describe different methods of rapid microbial testing and their applications.

Pharmaceutical Technology
Volume 36, Issue 5, pp. s14-s19

Growth-Based System for Rapid Microbial Testing

Ruth Eden, president, BioLumix

Currently available growth-based rapid microbiological methods provide either a quantitative cell count, estimation of viable cell concentration, information regarding the presence of a specific microorganism, or a microbial identification. However, the ability of these rapid microbiological methods technologies are limited in scope in that they cannot be used to perform all the required assays using a single technology platform. A new automated growth-based system simultaneously detects microbial growth, provides an estimation of viable cell counts, and identifies the presence of specified micro-organisms.

Technology. The system is based upon detection of optical variations due to microbial metabolism in liquid medium within a two-zone ready-to-use test vial. An optical sensor monitors optical changes within the vial's reading zone, which is physically separated from the incubation zone. This two-zone approach prevents masking of the optical pathway by product or microbial turbidity and, therefore, eliminates product interference. Separate test vials are used to automatically detect the presence of viable microorganisms and to estimate the concentration of viable counts by monitoring changes in CO2 production during cellular growth. The CO2 sensor is composted of a matrix of a polymeric material that is transparent to light and contains an indicator agent sensitive to CO2 gas generated by micororganisms, changing the indicator color from blue-green to yellow. Alternatively, the hydolysis of fluorogenic synthetic substrates by bacterial enzymes can cause an increase in fluorescence. Also, pH changes can be monitored calorimetrically. Each of these applications can be simultaneously performed using the same instrumentation and at the same time.

The sensitivity of the system is a single viable cell per sample vial; when a single cell replicates to a specific detection threshold level, a positive response is recorded. The threshold level is ~100,000 cells/mL for bacteria and ~10,000 cells/mL for yeast and molds. Additionally, the system yields significantly faster results than the plate count method; one bacterial cell is usually detected within 8–18 hours, a single yeast cell is detected in 20–30 hours, and mold cells require 35–48 hours.

Figure 1
The system creates dynamic patterns as the microorganisms grow in the medium. As shown in Figure 1, the green curve shows a pattern where no growth had occurred. The curve is flat without any significant increase in the signal and no detection time (DT) is observed. The blue curve shows the pattern generated when the microorganism grows in the vial (a DT of 11 hours is observed).

In highly contaminated samples, bacteria are typically detected in 8–12 hours, yeast in 16–24 hours, and mold in 24–35 hours, providing timely warning of contamination.

The system . Each BioLumix instrument has a capacity of 32 sample locations with a single incubating temperature. Multiple instruments (up to 32 instruments) can be attached to a single computer with Windows-based program that controls the operation of the instrument(s) and is barcode capable. The software is validated to meet 21 CFR Part 11 requirements, provides an audit trail, operator identification (log in and log out), trend analysis, and provides various data reports. Detection events are automatically displayed.

A critical element of the technology is the two-zone detection vial with an upper incubation zone where the sample is added and a lower reading zone that remains optically clear and free of turbidity from microorganisms and sample components. This two-zone vial design eliminates interference of the optical pathway during color and fluorescence monitoring by the sample and microbial growth.

Table I
Comparative testing of products: The data generated for the comparison of the growth-based rapid method to USP <61> Microbial Examination of Nonsterile Products: Microbial Enumeration Tests, is summarized in Table I. The table shows the specification ranges tested, the number of samples, and the percent agreement between the new method and the USP plate count method using soybean casein digest agar.

Total aerobic count: (i) Naturally contaminated samples: Two hundred and one nonsterile over-the-counter medicine samples such as vitamins, antacids, suppositories, laxatives, ibuprofin, and aspirin were tested. All noninoculated samples were below the detection level by both methods. There was 100% agreement between the two methods in classifying samples as above or below the specification levels. This indicates the ability of the BioLumix system to yield equivalent results to the plate count method when detecting growth over the range of 10 cfu/g to 1000 cfu/g.

(ii) Inoculated products: Fifty-nine products were inoculated with all the organisms cited in USP <61>. Ten inoculated samples had counts below the specification level and all were correctly classified as the challenge organism by the BioLumix system. There was 100% agreement between the two methods in determining whether samples were above or below the specification level.

As shown in Table I, similar data were obtained for yeast and mold as well as Gram negative bile-tolerant bacteria. The data indicate the ability of the system to yield equivalent results to the plate-count method over the range of 10–1000 cfu/g for both of these assays.

The method has good specificity in detecting target organisms and excluding nontarget flora, and the detection limit for the system equals or is slightly better that the limit for the plate count method. High precision or repeatability was obtained for all three assays tested. It can be used to detect the presence or absence of organisms, total aerobic count, the presence of yeast and mold, enterobacterial count, and absence of objectionable organisms in 10 grams of product. By encompassing both USP types of testing, the system offers a complete screening solution, making the existing microbial testing simpler, faster, and automated.


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