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Microbiology Applications

Spiral Plating
The Autoplate® system was designed to help laboratorians utilize their time and materials more effectively.  The ideal customer plates samples regularly (>25 a week), uses a sample with a microbial concentration greater than 1,000 cfu/ml, and has sample available in volumes greater than 200µl.  Any application where counts are high and standardization and reproducibility are key is an opportunity for spiral plating1.

The Autoplate is perfect for testing for pathogens and spoilage bacteria in food and dairy products.  In the Pharmaceutical industry it can be used for Preservative Challenge testing (PET), Kill Curves, and susceptibility testing with SGE.  The Agriculture and Environmental industries utilize spiral platers for bio-pesticide challenges, frost prevention and leaf studies.  Mutagenicity testing in the Medical field, oral microbiology in dental, and fecal contamination in food and pediatric applications all utilize Autoplate systems.

Colony Counting
Colony counting and spiral plating go hand-in-hand.  All the applications mentioned above can also be applied to colony counting.  Using QCount®, microbiologists get sharp, error-free plate counts in as little as one second. So how does that compare to what most microbiologists do now? Ask any microbiologist to name the most boring, mind-numbing task of their job and nine out of 10 laboratorians will say “counting plates”.

Remembering that most samples are run in duplicate or triplicate; imagine the discomfort resulting from manually reading plates for 8 hours a day - every day.

Your eyes hurt and your neck aches from the tedious work of counting a few hundred tiny little colonies on each plate. Many colonies overlap one another, so you begin to ask yourself… “Is it one colony, two colonies, or three?”  Or worse, you lose track of which colonies you’ve already counted. The only way to make manual counting easier is to reduce the number of colonies that must be counted on each plate. Now, you have to make a number of time-consuming dilutions before plating. Still there will probably be 50-500 colonies per plate. After dilutions have been made, your count must be multiplied by a conversion factor to estimate the actual number of CFUs (colony forming units) in the original sample.

Both research and quality assurance personnel find that using QCount enables them to process large samples rapidly with consistently reliable results.  Benefits realized can include a 150% increase in productivity, a 70% reduction in preparatory activity, and a 33% improvement in sample information turnaround time2.

Anaerobic Culturing
Anaerobic bacteria are common causes of infection and will be completely missed in clinical diagnosis unless special precautions are taken for their isolation and culture.  With anaerobic culture, microbiologists are not only challenged with obtaining a good specimen, but also with ensuring that the specimen does not come in contact with air3.

Anaerobic and micro-aerophilic bacteria can also be beneficial to humans.  These bacteria are used as “waste digesters” by industry to clean oil spills, methane gas production in waste management, and can actually be a culprit in the spoilage of beer.  

The cultivation of microorganisms in these oxygen-depleted atmospheres can take place in a CO2 incubator or an anaerobic glove cabinet.  These conventional methods are marred by many disadvantages, like late discovery of leaks, slow anaerobiasis or chemical waste.  Massive space-occupying chambers with fixed environments consume huge quantities of gas.  Servicing is costly and inconvenient.  Additionally, they are often incapable of cultivating micro-aerophilic and anaerobic organisms at the same time.

Alternatively for anaerobic incubation, agar plates can be placed in a sealed jar made anaerobic by evacuating and replacing the atmosphere with an oxygen-free gas mixture.  The Adcanced Instruments  Anoxomat™ is a microprocessor controlled evacuation-replacement system for removing oxygenated environments from jars.  The Anoxomat will replace the oxygenated environment with an anaerobic, micro-aerophilic, or a programmed gas mixture. 

Antibiotic Sensitivity Testing
In normal medical practice and research, bacteria are isolated to aid in diagnosis, decide therapy, or to determine their susceptibility to anti-microbial agents.  Tests in the clinical lab can be performed on automated, high-throughput instrumentation available from Trek Diagnostics, bioMerieux Diagnostics and BD Diagnostic Systems.  But in research, their sensitivity to a particular agent is most commonly determined by the agar diffusion method.  Other methods include broth dilution, disk diffusion assays (Kirby-Bauer) and the Spiral Gradient Endpoint™ (SGE™) Test from Advanced Instruments.

Minimum Inhibitory Concentration (MIC) tests are used to determine the minimum concentration of antimicrobial agent required to inhibit the growth of a microorganism. So why do MIC testing?  The MIC test may be performed in liquid (macrotube dilution broth method and microtube dilution broth method) or on an agar medium. The endpoint (MIC) is taken as the lowest concentration of drug at which the microorganism tested does not show visible growth.

The MIC is defined as the lowest concentration of an antibiotic that will inhibit the in vitro growth of an infectious organism. The results of microdilution susceptibility tests are reported in micrograms per mL. The interpretation of in vitro data is based on the achievable serum concentrations, which may vary depending on dose, route of administration, degree of protein binding, site of infection, age and weight of the patient, state of health of the patient, and other factors.

Reporting of MICs can provide the physician or researcher with precise information regarding the infectious organism's degree of susceptibility. When this information is coupled with the physician's knowledge of the site and severity of the infection, as well as the pharmacology of antibiotics, a rational choice of the most appropriate antibiotic can be made to suit the individual patient.  The same theory can be applied to research to discover the most efficient concentration of agent for the destruction of the bacteria.
 
The Spiral Gradient Endpoint (SGE) assay provides highly sensitive and repeatable MIC determinations through a unique agar dilution method.  Using an automated spiral plater, a concentration gradient of antimicrobial is produced across a 150 mm plate.  The resultant gradient spans a four Log range and eliminates the need for incremental dilutions.  Up to 15 test strains can then be directly analyzed radially across this antimicrobial gradient.

Laboratory evaluations show equivalence of SGE MIC's with MIC's obtained by the standard agar dilution method, affording superior repeatability and greater sensitivity by surveying a continuous gradient. The SGE software includes analysis programs for quality control purposes and for evaluating the correlation between MIC's obtained by SGETM and other methods.  Strain and antimicrobial profiles are further compiled to produce comparative and rank ordered analyses, and subsequent data is easily exported for additional examination and display.

Measuring Minimum Inhibitory Concentrations (MICs) by standard two-fold agar dilution techniques is laborious and imprecise.  The Spiral Gradient Endpoint (SGE) test is more sensitive than standard methods and reduces time and materials needed to obtain MICs. 

1 Scher, Fran. “Spiral Plating and Automated Colony Counting.” A2C2. January 2000.
2 Scher, Fran.  “Spiral Plating Adds New Spin to an Old Standard.” Food Testing and Analysis.  December 1999.
3 Brock, Thomas D and Madigan, Michael T, Biology of Microorganisms (Englewood Cliffs, NJ: Prentice-Hall, Inc), p. 479.