Automation in Microbiology

 

Preface:

 

Automation in microbiology is much difficult as compare to those applied in biochemistry and haematology. Automated methods in microbiology are mainly used for rapid results and to reduce the manpower.

However, the technology can never be an alternative of manual work done by human resources in microbiology laboratory; because,

           - The machine can neither perform skillful techniques nor make critical decision.

           - Agar plates are still the main modalities for observation of bacterial growth;

           - Microscopic methods provide the best tool for observation of bacterial morphology & for its identification.

 

The scope of automation in microbiology

 

Following are the fields where automation is widely used and accepted in clinical microbiology laboratory

 

1. Continuous bacterial and fungal growth monitoring system. (For detection of bacterial / fungal growth in clinical samples)

2. Identification and antimicrobial susceptibility testing for bacteria and fungi.

3. Immunological techniques.

4. Nucleic acid analysis.

 

 

Following are the fields where automation is still under development phase  OR  mainly used at research level and less widely used in diagnostic microbiology laboratory.

 

1. Media preparation - plate pouring machines

2. Speciemen inoculation: Plate streakers

2. Urine analyzer for bacteriology

3. Protein based spectral identification of bacteria

 

 

1.    Continuous growth monitoring system

 

i. automated blood culture system

 

          Specimens like blood, CSF, Pleural fluid, Abdominal fluid, Synovial fluid etc. are always otherwise sterile in normal person. In any infectious condition, when we suspect presence of infecting bacteria or fungi in such specimen, we need to inoculate them in a liquid enriched medium for isolation of bacteria. For detection of whether the viable bacteria are present or not in these inoculated specimen, we can utilize automated methods which can detect growth of these viable bacteria. These technologies allows fully automated testing using continuous monitoring instrument. This instrument agitates and incubates blood culture bottles, resulting in earlier detection of bacterial growth. The principle of detection of these systems is based on the release of CO2 in the presence of microorganism metabolism.

 

Following systems are commercially available:

 

a.     BACTEC FX / 9000 ( BD Microbiology, Cockeysville, MD ) : This system works on the unique fluorescent sensor technology. Blood or fluid sample is inoculated in specified bottle containing liquid culture medium containing fluorescent agent in it; The inoculated bottle is incubated in the BACTEC instrument. The bottles have a sensor at the bottom. Any bacterial or fungal growth will release CO2 and make the sensor at the bottom to emit fluorescent light which is detected by light sensitive diode in the machine. As the fluorescent emission increases above threshold value, the machine signals positive result.

The BD blood culture bottles contain resin particles in the medium, which has neutralizing effect on bacterial growth inhibitors (i.e. antibiotics). This favors growth of bacteria and increase the isolation rate.

 

b.    BacT Alert 3D (biomerieux, Durham, N.C.): The bottles used in this system are having a chemical sensor in the bottom. As the CO2 increase in the bottle due to growth of organism, the colour of sensor will change from green to yellow. This will be measured by a photosensitive detector.

    The BacT/ALERT blood culture bottles contain charcoal / polymeric resin particles in the medium, which has neutralizing effect on bacterial growth inhibitors (i.e. antibiotics). This favors growth of bacteria and increase the isolation rate.

 

c.     Versa TREK (Trek diagnostic systems, Cleveland, Ohio): This system is based on pressure change in the bottle headspace due to bacterial metabolism and gas production.

 

 

ii. Automated tuberculous culture system

 

          Mycobacterium tuberculosis is very slow growing bacteria. These bacteria are isolated on LJ medium which needs a period of 4 – 12 weeks for incubation and for appearance of growth. However, in automated methods its growth in liquid medium like Middle brooks medium can be detected earlier by one of the following growth detection system

 

a.     BACTEC MGIT ( Mycobacterium growth indicator tube ): The MGIT is a tube containing Middle brook 7H9 broth together with florescence quenching based oxygen sensor. If the specimen inoculated in this tube is having viable Mycobacteria, its growth will produce fluorescence. The fluorescence above threshold level will be detected by the machine and indicate the positive signal.

 

b.    BacT Alert 3D (biomerieux, Durham, N.C.): This system is based on detection of CO2 as an indicator of mycobacterial growth. This works on the principle of colorimetric detection system.

 

Both of the above methods can also be used for anti tuberculosis drug sensitivity. For drug sensitivity, the mycobacterial growth is incubated along with specified concentration of anti-tuberculous drug. If inoculated bacteria can grow further in presence of drug, indicates bacterial resistance.

 

2.    Automated system for identification &antimicrobial sensitivity

These systems can be applied for pure growth of

          i. Bacteria

          ii. Yeast or yeast like fungi i.e. candida sp.

 

The automated methods provides

          i. Identification up to species level - by detection of biochemical properties of bacteria or yeast

          ii. Antimicrobial drug susceptibility; It can also detect minimum inhibitory concentration of many antimicrobial agents against the bacteria and yeast.

 

Automated system available:

a.  VITEK 2 (biomerieux)

b. Microscan (Siemens Healthcare)

c.  BD Phoenix ^TM (Becton Dickinson)

 

 

3.    Immunological assay:

Various automated systems are available which works on the principle of antigen antibody reactions

a.     Automated ELISA system

b.    Automated RIA system

c.     Automated Chemiluminescence system

 

 

4. Nucleic acid Techniques:

-Real time PCR is an example of automated system. The system recognizes the presence or absence of target nucleic acid as well as it also measures the initial concentration of target sequence in the specimen.

- Automation system for extraction of gene (RNA / DNA)  is also available.

-Fully automated systems including extraction of gene, amplification & identification over one platform are also available