The Osmosis Unit   

www.Osmosis-Unit.co.uk

26 Cotlands Park, Longniddry East Lothian Scotland EH32 0QX

44 (0)1875 852761                 

 

 

The Bug Scanner

The Problem

 

One developing problem within the Health Service is the increase in Methicillin Resistant Staphylococcus aureus (MRSA) in hospitals and clinics. This is an organism that has developed resistance to  most of the commonly used antibiotics. Any outbreak results in closure of the affected area for 5-7 days while the areas is decontaminated. The greatest part of this delay is the time taken for microbiological incubation and identification. The ability to speed up this process would greatly reduce the shut down time in these clinical areas.    

Understanding Bacteria

 

William Stewart, the Surgeon General in the US declared in the early 1960’s that the time had come to close the book on infectious diseases (Economist 1995; May 20th : p15). Since the discovery of the first antibiotics by Erlich in Germany and the discovery of Penicillin by Flemming in Britain, there was a growing belief that we had cracked the treatment of bacteriological infection. People had underestimated the adaptability of bacteria.

 

Bacteria are amazing. The healthy skin of one individual is home to about one trillion bacteria. There are 100 trillion more in every individuals gut. Most people have ten times as many bacteria as they have cells. These organisms are generally not only harmless but co exist symbiotically with their host. They multiply roughly every 20 minutes and normally produce perfect copies of themselves. However, about every million divisions they produce a mutant. This rarely provides benefit for the species but occasionally an individual is produced with an advantage. This advantage may include antibiotic resistance. More worryingly bacteria have a method of sharing the genetic code that provides this resistance through a process known as plasmid transfer. Any bacteria can take little pieces of genetic information and share it with all other bacteria. Much of antibiotic resistance is believed to be spread in this way. Antibiotic development is therefore a race between the pharmaceutical companies and microbiological mutation. At present the microbes are winning. As we cannot kill this agent with antibiotics we have to remove it from contaminated areas by disinfection. Infected areas are washed from floor to ceiling with disinfectant. After leaving time for the disinfectant to work the area is swabbed and the swabs incubated to ensure that the infection has been removed. The aim of the process is not to cure the infected patient but to prevent the spread of the infection to other patients.   

 

 

Bacteriological Incubation

 

Microbiological testing at present involves sample collection, spreading this onto settle plates, transferring any areas of growth to secondary culture tubes, further incubation and colourimetric identification. The methods used have changed little since Robert Koch first grew anthrax bacteria in 1862. Koch, a country GP in Germany, worked out how to grow the organisms first on slices of cooked potatoe and then, after being shown how to make jelly by one of his wife’s friends, on agar jelly.  

Agar jelly is still the standard growth medium used in microbiology.  

 

Present microbiological techniques

 

The infected area is first swabbed with a cotton wool swab. This is then wipes over the surface of a sterile agar plate. This is then incubated at about 37 deg C for 2-3 days. Any visible areas of growth are then cut out of the agar and transferred to a broth. This is incubated for a further two days. The broth is then plated out into an OPI well disk which contains a range of different colour chemicals. The organism is identified depending on the resultant colour pattern produced. Various other methods of early bacterial identification are presently being tested. These are listed in table 1

 

 

 

Table 1 Methods of early identification of bacteria presently being tested

 

Method

Comments

HPLC

Identifying fatty acid methyl ester groups

Requires initial culture on settle plates to acquire enough material

Micro raman spectroscopy

Expensive and difficult to perform

Requires initial culture on settle plates to acquire enough material

DNA analysis

BLAST
CLUSTAL
ORSAY

Required DNA amplification

Then involves DNA sequencing and matching to a table of norms

At least £300 per test

Multispectral

Using fluorescent signals from 16S ribosomal DNA

False positives extremely high

Expensive

Difficult to perform

CDS/ISIS

Computer program to identify bacteria according t o shape, O2 requirement, motility, gram stain, resting stage, sulphur utilisation

ELISA

Antibody antigen reaction

ROSCO system

Colourimetric enzyme identification system

Similar to OPA system

Requires initial plating out and secondary broth culture before colourimetric testing

Voltametric

Using change in conductivity across the plate

Chemometric

 

Liquid chromatography mass spectrometry

Ridiculously expensive

Surface IR spectroscopy

Expensive

Scanning electron microscopy

Ridiculously expensive

Lectin based biosensor array

Expensive

 

The equipment used in many of these tests (HPLC, mass spectroscopy, scanning electron microscopy etc) costs several hundreds of thousands of pounds, requires specially trained staff and requires expert interpretation. These tests are not viable for the routine quality control lab. The other tests are focusing on DNA analysis. At present these cost in the region of about £300 per test. Again this is too expensive to consider for routine quality control. A simpler, cheaper test is required.    
How the bug tester works

 

The bug tester utilizes the fact that bacteria are identified visually. For a growth of organisms to be detected against the colour of the agar plate they need to visibly different ie a different colour. Their growth either produces an identifiable colour difference in the culture or in the growth media. Either way, their recognition depends on them being visible. The Bug tester records a digitized image of the plate at standard time intervals over a 24-48 hour period. The colour of every pixel is compared to a standard colour chart on the scanner and any differences in colur developing over time are automatically detected. Changes in colour over time are characteristic of particular organisms.  

 

 

Operation of the Initial Prototype

 

The initial prototype was designed to fit onto the platen of an A4 scanner (see fig 1). The incubator box was designed so that six settle plates could be scanned during each run. There was a fixed clearance between the settle plates and the scanner glass. Six settle plates were placed agar side up on the platen of the scanner. A watch and a thermometer were also added to enable the times and temperatures of each scan to be recorded. The bug tester was then placed over the plates and the heating circuit turned on. The scanner was connected to the computer and the plates scanned at hourly time intervals.    

 

 

Initial results

 

Good initial results were obtained from buccal samples grown on blood agar plates. Growth could be observed as gray white coloration against a red background. Growth was detectable at eight hours at room temperature. Measurement of the change in colour values of the growth areas was performed in Adobe photoshop.  A second run was performed using clear agar plates which had been spread with either Staph aureus of pseudomonas aeruginosa. The system therefore has the potential for identification of organsism at an early state during their incubation.

 Comments on the operation of the first pilot bug scanner

 

The Bug Tester has the potential to revolutionise microbiological testing and remove the necessity for manual plate counting. By monitoring the changes in RGB rather than undertaking a plot count bacteriological identification should be more rapid and automated.  Furthermore it provides a permanent record of culture growth and development which is essential for most quality control records.

 

The principle of colony counting for the analysis of bacteriological growth is well known and well established. Sophisticated automatic machines also exist, capable of managing and monitoring this growth. This proposed technology is an extension to this technique.

 

 Benefits of the bug tester over conventional techniques

bulletIdentification of growth sooner than conventional “colony counters”
bulletEnables identification of bacterial strain which should enable better targeting of antibiotic therapy where appropriate.
bulletIn the pharmaceutical industry it should enable faster analysis of antibiotic effectiveness during the antibiotic development process
bulletIt requires no significant changes to the methods of preparation, or the subsequent handling of samples for analysis
bulletUses existing techniques of preparation- little additional user training
bulletBuilds on existing principles- rapid, cost effective Route to Market

 

Back to previous page                                Home

 

Send mail to with questions or comments about this web site.
Last modified: 07/05/06