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Togs Introduction
What is TOGS?
STEPPI Technology And Background
What is STEPPI Technology?
Intra-Thermic Incubation (ITC)
In-Process Incubator (IPI)




TOGS - Introduction

The development of chromogenic/fluorogenic reagents to conduct microbial testing has opened the door to better and faster testing protocols. In addition, these products provide the additional opportunity to use technology such as optical spectroscopy to conduct microbiological analysis.

Spectrophotometric analyses are very sensitive and hence can detect the presence of a very low concentration of color producing components of interest in liquid samples (in parts per million). Visually, the human eye can only detect the color when these components are present in very high concentration, thus the need for incubation periods ranging from 18 to 72 hours. The time required to identify or estimate the presence of coliforms in water samples can be drastically reduced when combining incubation with photometric analysis.

While the use and advantages of spectrophotometry for microbial analysis of liquid samples has been cited in numerous literatures [1-8], with all other instrumentation the test has been done outside the incubation chamber by drawing an aliquot of sample from the incubation vessel to photometric tubes at various intervals and measuring using standard spectrometers. This is not only time consuming but requires separate incubators, spectrometers and technical personnel to conduct the test and in some cases robotic sampling systems. An additional risk of cross contamination and human error exists, if proper care is not applied in conducting the analysis.

Tests conducted with the Aquasure PRO 3000 are based on visual detection of the change in the color/fluorescence produced in the test solution. The end point of the test is evaluated by human eye. In order for us to visually observe and distinguish a color in a solution (between 400-800 nm in the visible region of the electromagnetic spectrum), or fluorescence emission under long UV light, requires a very high concentration of the specific chromogenic/fluorogenic components (chromophore/fluorophore). \Therefore, for low level detection of microbial indicators in water samples (e.g., 1 CFU/100 mls-drinking water) the incubation time required at 35 °C to produce that concentration of the chromophore/fluorophore in the test medium can be anywhere from 18-24 hours.

Studies have shown that the incubation time required to detect visually the fluorescence emission intensities with a long wavelength UV lamp should be longer than 18 hours. In order to shorten the time, sensitive instruments such as a spectrofluorometers [8] are required. This approach has shown to reduce the incubation and detection time by a factor of 2 [4,6,8].

A rapid method should be able to produce the result in the shortest possible time and for microbial testing it should be within the same working day. In addition, it should have sensitivity and specificity equal or greater than standard methods [2]. According to Dr Fung of Kansas State University an instrument, which can automatically and continuously monitor color change and turbidity of a liquid sample in the presence of a microbial growth can be considered as a rapid detection system for the presence of microorganisms [1]. TOGS is such a system.

By virtue of its design STEPPI Technology facilitated greatly the ability to take the Aquasure PRO 3000 to this next level, transforming it into our TIME OF GROWTH SPECTROPHOTOMETER - TOGS System. TOGS is not only automated but the results can be deduced much faster than that which is achievable by visual detection or any other currently approved method.


WHAT IS TOGS? TIME OF GROWTH SPECTROMETER

Traditionally spectrometers are used to measure the light energy (intensity). When light is passed through a medium (sample) it may absorb some of the energy and the difference between the intensity before and after passing the sample is a measure of the light-absorbing component in the sample. Thus detection and quantification is related to the intensity of the light absorbed or emitted. According to the Beer-Lambert law there exists a linear correlation between the intensity of the light absorbed and the concentration of the component, which absorbs the light.

In microbiology, the microbes are cultured to provide useful information. Culturing is the process whereby the microbes are allowed to reproduce (multiply) in a controlled environment – namely, constant temperature (incubation) and nutrient (food). Microbes multiply exponentially and as a rule of thumb they double every 20 minutes. Thus a single microbe produces millions of microbes after an elapsed time of incubation. Multiplying microbes forms clusters called "colonies". With time the colonies grows bigger and bigger and can be detected by naked eyes or by microscopes; if cultured on a solid medium such as a filter paper. A 24-hour incubation (standard practice for membrane filtration method) thus produces colonies, which can be detected and counted.

In our Time of Growth Spectrophotometer- TOGS we combine the spectrometer and the microbial culturing in a liquid medium (rather than in a solid medium - Membrane Filtrate method). We use the spectrometer to detect and measure the initial microbial quantity and use incubation to multiply the microbes. In the presence of a selective reagent, the microbial growth is accompanied by the production of chromophores and fluorophores. Chromophore is a chemical which absorbs visible light intensity of specific wavelength (color) while fluorophore is a chemical which when absorbs light intensity of specific wavelength will emit light of different wavelength (fluorescence). Though we monitor and measure the intensity of the light absorbed or emitted, the quantification of the initial microbes (called population), unlike conventional spectrometric analysis, is achieved based on a time at which a known intensity is observed.

Since microbes multiply exponentially, a typical growth curve (instantaneous population- time plot) is exponential. A typical growth curve is shown below. As expected, the growth curve shows a lag, an exponential and a stationery phase. Under a controlled and constant incubation, the microbial growth is proportion to the increasing concentration of chromophore and or fluorophore (the spectrometer detection units) in the sample.



The lag phase, which represents the metabolic adjustment time of the bacteria, will vary with initial bacterial count. It usually shows a flat profile indicating very less activity (no growth). Once pass the lag phase, the signal starts to increase (or decrease) rapidly in an exponential manner (growth). The time at which the detector output signal deflection is significant (detection limit) is the Time of Significant Deviation - (TSD).

The detection time (tdet), which is defined as the time taken to reach a pre-defined detectable population size, has been used to estimate bacterial growth parameters [9]. The detection time has been shown to be inversely proportional to the logarithm of the inoculum (initial population of the microbe) level [2, 9].

where X0 = initial bacterial population.

This value provides important information:

  • Presence of bacteria under investigation.
  • Initial population level.

Since the increase in population size is directly proportional to the change in optical density, the optical density at tdet can be used to measure the initial population [2, 9].

Since the population size at detection limit (TSD) is predefined, the time required to reach TSD should corresponds to tdet as in equation {1}. TSD also depends on the initial concentration of the bacteria in the sample and higher the initial bacterial count shorter the TSD and vice versa.



Therefore, in addition to detection, TSD can provide quantitative information of the bacterial population.

The slope of the lag (rate of hydrolysis) has been shown to increases linearly with increasing initial concentration and the rate of hydrolysis can provide semi-quantitative information of the target bacteria [2]. A plot of the rate of hydrolysis as a function of log CFU gave a correlation with r2 =0.65 [2]. This provides semi-quantitative information.

Thus Time-of-Growth represents the monitoring of the microbial growth with time under a given isothermal condition. In our system we achieve this real-time growth monitoring using multiple spectrometers. We use a spectrometer to generate a real-time growth curve and use the growth curve to calculate the initial population of the microbe under investigation. While a conventional spectrophotometer method of analysis is used to measure the instantaneous concentration of the analyte, this approach uses a spectrometer method to measure time. Moreover, we use multiple spectrometers to measure different microbial activities simultaneously.

STEPPI Technology And Background

STEPPI Technology (Single Test Precision Portable Incubation) STEPPI Technology delivers proportional heating and continuous temperature monitoring of a given sample. The unique design provides STEPPI with a level of quality control that is unsurpassed.

The Aquasure Pro 3000 is a patented microbial testing system which eliminates transportation and holding time errors and delays, provides Rapid Test Results and which provides a sterile environment for testing, even in rural and remote areas. Aquasure Pro 3000 is utilized by governments, educators, water treatment and service personnel in Canada, the U.S., Mexico, Asia, Africa and Australia.

What is STEPPI Technology?

STEPPI Technology or Single Test Precision Portable Incubation is the cornerstone of the Aquasure PRO 3000 incubator as well as our New, Full Featured TOGS Systems (TOGS 9000).


With traditional batch sampling, the sample is heated from the outside, whereas with STEPPI Technology the water is heated and monitored from inside the sample itself for temperature accuracy. STEPPI Technology takes advantage of the scientific fact that water is a good insulator and a poor conductor. With the STEPPI approach to incubation, heat loss is minimized thus providing for a more precise temperature control of the sample. Calibration verification can be conducted easily on a blank sample or at the end of the incubation period, with a thermometer in the sample itself. STEPPI Technology utilizes a software driven microprocessor to provide precision temperature control, which minimizes the effect of temperature fluctuation of the surrounding environment on maintaining a constant temperature of the water at the set range (e.g., 35 C plus or minus 0.5 C). The temperature profile, which is reproducible test after test, is independent of the outside environment such that it can be operated in an environment of between 5 C - 35 C.

The STEPPI approach to incubation requires low power consumption meaning that the equipment can be operated using batteries, making it an ideal instrument for use in rural and remote applications where power supplies may not be available or are unreliable for continuous operation.

Although simple to operate, this innovative technology represents a major breakthrough in precision testing of microbials making it possible for both total coliform and e. coli testing to be conducted simultaneously, by on-site even non -technical personnel. Results are obtainable within 24 hours of sample collection, at a fraction of the cost of a traditional analysis.

Intra-Thermic Incubation (ITC)

STEPPI Technology uses the principle called intra-thermic incubation to provide precision temperature control of the microbial culture medium. The STEPPI technology design allows the thermal energy to be provided directly to the culture medium when placed inside the culture bottle and allows the temperature of the medium to be monitored and controlled. See figure above.



In-Process Incubator (IPI)

The unique patented design of the STEPPI Technology is such that each sample bottle becomes an incubator when placed within the holding device, providing controlled thermal energy to the culture medium for the growth of the microbe under investigation.

The sample bottle when place inside the holder meets the criteria of an incubator.

  • The heater and the temperature probe reside inside the bottle.
  • The bottle is thermally insulated by the isothermal chamber surrounding the bottle.
  • The thermal energy is provided, monitored and controlled inside the bottle.

An in process incubator (IPI) is an apparatus which possesses all the qualities of an incubator during a process (analysis) only. Each unique patented bottle (PRO 3000 and TOGS 9000) is an in-process incubator.

In addition, each bottle has the following attributes. It is a:

  • Sample collection vessel
  • Culturing vessel
  • Photometric cuvette (in TOGS 9000).

Thus each bottle provides a disposable but sterile sampler and incubator in one unit.


For refrences or more information please visit our documentation page here


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