Uses of Twincubator in Line Probe Assay (LPA):

• Different steps of the hybridization (shown in figure below) and detection procedure with DNA strips in reverse blot hybridization assays.
• In the amplification/ post-amplification area.

Figure: Twincubator used in Hybridization

Features of Twincubator: ​

In the Line Probe Assay (LPA) lab, the water bath is used for warming reagents used in the Hybridization Room.

It is electrically heated and electronically controlled.

Components:

• Recessing push/ turn control for simple operation of the bath
• Visual temperature indication​
• Push button to set desired temperature​
• Electrically heated and electronically controlled​
• Water trough​

Installation of Water Bath (Figure):

Thermocycler is also known as Polymerase Chain Reaction (PCR) Machine.

Use of Thermocycler

Thermal cycler is used for amplification of nucleic acid by PCR​.

The thermocycler should be placed in the amplification/ post-amplification area. 

The features of a thermocycler are listed below.

Polymerase Chain Reaction (PCR) hood is a workstation enclosed by three sides.

Use of Polymerase Chain Reaction (PCR) Hood:

• Used in the Master Mix Reagent Preparation and DNA Amplification Room
• Ultraviolet (UV) light (Figure) in PCR hood denatures nucleic acids​
• Used to decontaminate reagents and equipment prior to PCR reaction​
• Provides protection against exogenous DNA, contaminants or inhibitors from entering the reaction tubes

Use of Micro Centrifuge

• Designed for the separation of materials through sedimentation using centripetal force​
• Used in DNA Extraction Room​ to concentrate specimens during processing

Components and Use (Figure)

Use of Hot Air Oven 

• For heating non-explosive substances and objects ​ 
• For dry heat sterilization  
• During DNA extraction step​; kills Mycobacterium tuberculosis and partially lyses the cells at 95^oC

​Installation of the Hot Air Oven (Figure 1)

Use of Micropipette  

• Micropipette (Figure) dispenses and aspirates liquid (reagent, sample). ​ 
• IT is used in the DNA Extraction Room, Master Mix Room, Amplification Room and Hybridization Room to dispense reagents for Line Probe Assay (LPA). ​ 
• A micropipette should be dedicated to each room and labelled accordingly. 

Figure: Micropipette

Cleaning:  

Procedural biosafety measures include: 

• Biosafety measures taken during the Line Probe Assay (LPA) procedures ​ 
• Aerosol generation and preventive measures​ 

Manipulations that produce aerosols in DNA Extraction Room are: 

• Pipetting​ 
• Centrifugation​ 
• Vortexing​ 
• Discarding micropipette tips after use​ 

Measures to Prevent Aerosol Generation ​ 

Line Probe Assay (LPA) lab personnel must comply with Good Laboratory Practices for their biosafety.  

These include technical competency in: 

• Performing tests 
• Using Personal Protective Equipment (PPE) 
• Using equipment 
• Handling infectious materials 
• Keeping work areas clean 
• Using disinfectants 
• Waste management practices 
• Understanding responsibility for protecting themselves and others 

Do’s and Don’t of Good Laboratory Practices

The refrigerated centrifuge is used in the mycobacteriology laboratory for concentrating bacterial cells, following the processing of sputum or other specimens. It is also used for spinning down reagents from walls of containers. The temperature maintained within the refrigerator centrifuge is 4°C to reduce the percentage of mycobacteria killed due to heat generated during centrifugation. The required G force is 3000 x g (not RPM).

Essential Features of the Refrigerated Centrifuge