Automated methods in the Microbiology Lab

FARIS KMSc MLT MicrobiologyGovt: Medical collegeTrivandrum

Isenberg et.al.- pioneered the development of automated systems in 1960s.

Automation is the replacement of human efforts in the performance of a given process by mechanical and instrumental devices that are regulated by feedback of information so that an apparatus is self monitoring or self adjusting.

Basic principles in automation Turbidity as growth indicator. Colorimetric determination of growth.

Radioactive method of detection. Bioluminescence assays. Reflectance measurement. Electrical impedance. Measurement of ions in system. MALDI TOF

Titles Section 1 - ABCS

Section 2 – Rapid identification system1 – api 20 E

Section 3 - Rapid identification system with AST

1– Vitek 22 - BD Phoenix3 - Microscan Walkaway (Dade Behring)

Section 4 - MALDI TOF – vitek MS

Section 1CBCMS/ABCS

Blood culture automation ABCS are blood culture system that

uses instrumental devices to incubate, agitate, and/or monitor blood culture bottles for microbial growth.

Use advanced culture media and better detection time.

Suppliers: BacT/Alert (BioMerieux), BACTEC FX (BD), Versa TREK

Media

Various readymade media bottles available.

High enriched media Aerobic, anaerobic, mycobacteria,

paediatric, fastidious, Brucella- Specialized media

PES (0.25 %) Additives (charcoal, resins, ionic

exchangers, etc.) Barcode and colour code

BACTEC

BacT/Alert

Principle BacT/Alert and BACTEC System utilizes a

colorimetric sensor and reflected light to monitor the presence and production of carbon dioxide (CO2) dissolved in the culture medium. If microorganisms are present in the test sample, carbon dioxide is produced as the organisms metabolize the substrates in the culture medium. When growth of the microorganisms produces CO2, the color of the gas-permeable sensor installed in the bottom of each culture bottle changes from blue-green to yellow.

Detection The lighter color results in

an increase of reflectance units monitored by the system. Bottle reflectance is monitored and recorded by the instrument every 10 minutes.

Alarm Then do a sub culture

from bottle and identify organism

VersaTREK principle VersaTREK use a special media – redox

medium (REDOX 1® and REDOX 2® bottles)  Only two formulations, VersaTREK REDOX 1

aerobic and VersaTREK REDOX 2 anaerobic bottles, are needed to support the growth of a wide range of organisms.

If organism grow gas either produced or utilized.

The istrument monitor gas production or gas consumption in the medium by a special electrode.

Section 2 Rapid identification system

API 20 E A standardized identification system for

Enterobacteriaceae and other non-fastidious, Gram negative rods which uses 21 miniaturized biochemical tests and a database.

The complete list of those organisms

that it is possible to identify with this system is given in the Identification Table provided with package insert.

Principle API 20 E strip consists of 20 micro tubes containing

dehydrated substrates.

These tubes are inoculated with a bacterial suspension that reconstitutes the media.

During incubation, metabolism produces color changes that are either spontaneous or revealed by the addition of reagents.

The reactions are read according to the Reading Table and the identification is obtained by referring to the Analytical Profile Index or using the identification software.

MIKRO-LA-TEST® ERBA

Section 3 – Rapid identification system with AST

Rapid identification system with AST

Suppliers1. Microscan Walkaway (Dade Behring)2. Vitek2 (BioMerieux) 3. BD Phoenix (BD)

Principle Detection, enumeration, and identification of

bacteria and yeasts in clinical specimens. The biological basis for the system resides in

lyophilized, highly selective and specific media enclosed in wells of a disposable plastic curette, or a cartridge.

Introduction of a suitable specimen rehydrates and inoculates the media in the wells.

An automated optical system monitors, and the computer interprets, changes in the media, with enumeration and identification results automatically.

AST= Serial dilutions of antibiotics to determine MIC

Section 4MALDI TOF

MALDI-TOF- Matrix assisted LASER Desorption Ionisation-Time of Flight Mass spectrometry

Protein based spectral identification of bacteria

Identifications available in literally minutes – not hours

Tiny amount of bacterial growth needed – not affected by media or incubation conditions

Minimal cost per test (in cents not even dollars), virtually no consumables

Suppliers : Bruker, BioMerieux ( Vitek MS)

Principle Once the sample slide has been prepared, it is

introduced into a high vacuum environment. The sample is ionized with a precise laser burst, releasing a “cloud” of proteins. These proteins are accelerated using an electric charge, and the time of flight is recorded; lighter proteins travel faster, heavier proteins travel slower.  At the end of their travel, the proteins are detected with a sensor, which creates a spectrum representing the protein makeup of each sample. By comparing the spectrum from a particular sample against a large database of spectra from precisely characterized bacteria and fungi, identifications can be made at the species, genus and family level. The entire sample acquisition process typically takes less than a minute

Steps In MALDI TOF MS (Vitek MS) The target slide is prepared and

introduced to a high-vacuum environment.

A precise laser burst ionizes the sample.

A “cloud” of proteins is released and accelerated by an electric charge.

Contin.. After passing through the ring electrode, the proteins’

Time of Flight is recorded using a formula from the time recorded.

Proteins are detected with a sensor to create a spectrum that represents the protein makeup of each sample.

A spectrum from a particular sample is compared against a large database of spectra from bacteria and fungi that have been precisely characterized, such that identifications can be made at the family, genus, and species levels.

MALDI TOF MS

Issues with MALDI TOF

Need pure cultureIn mixed cultures, will pick up the

dominant organismAntimicrobial susceptibility not

possibleViruses not identifiedIdentifies only organisms in database

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Thank you for listening

The list is not complete

There are many other areas in microbiology automationE.g. Viable counting

Urine bacteriologySlide preparation and StainingCulture media preparationAutoclaveinoculation methods.Full microbiology lab automation, etc.Biochip.Molecular Techniques.

A detailed discussion about all of these are beyond the limits.