LT-Microscopy

Sputum microscopy diagnostic services under the National TB Elimination Programme (NTEP) are provided by the Designated Microscopy Centres (DMCs) established at the Peripheral Health Institution (PHI) level, where a functional binocular microscope and a trained Laboratory Technician (LT) is available. Light Emission Diode Fluorescent microscopes are provided to high-load PHI-DMCs such as that of the medical colleges. 

The National TB Elimination Programme (NTEP) utilizes different coloured bags for the segregation of waste generated in TB laboratory settings as shown in the table below.

The National TB Elimination Program (NTEP) recommends freshly preparing reagents from commercial procured products, at the District TB units to be used for microscopy. Chemicals need to be carefully monitored for potency and the calculation needs to be accurate while preparing reagents for smear microscopy.

For Preparing 1% Carbol Fuchsin (500ml): The required chemicals and materials for preparing 500 ml Carbol Fuchsin are:

1. Basic Fuchsin

Standard Operating Procedure (SOP) for Packaging Specimen during the transportation of biological specimens for TB diagnosis, also known as Triple Layer Packaging, is as follows:

The Lab Technicians (LTs) at the Designated Microscopy Centres (DMCs) should be trained to carefully pack the sputum samples in the cold box to avoid spillage of the samples. Before packing, personal protection measures such as wearing hand gloves (double gloves preferred), goggles and masks are to be followed by the LTs to prevent contracting the infection.

The Healthcare Worker (HCW) needs to carefully explain how to collect a good quality sputum specimen. He/she needs to demonstrate how to bring up sputum from the chest, what a good sputum specimen looks like, and the quantity of sputum required.  

Characteristics of a Good Sputum Sample 

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Resources

Disinfectants used in lab settings include:

1% Sodium Hypochlorite

• Broad spectrum antimicrobial action
• Used to disinfect surfaces
• Used to disinfect infectious material and disposal of used Truenat consumables (reagent bottles, cartridges, tips, chips) 
• Hazardous and corrosive, to be used with care
• Is highly alkaline so can corrode metal
• Waste soaked in Sodium Hypochlorite should not be discarded by autoclaving. 

70% Alcohol

Reagent preparation is an important activity and it’s essential to use certified chemicals and reagents. One should always check the potency of the chemicals used and calculate the amount to be weighed accordingly.

The following are the steps for reagent (primary stain and counter stain) preparation for Florescence Microscopy:

0.1% Auramine–O (1 L), the primary stain

It is important to store slides, after reading as these will be required for Quality assurance - internal rechecking by the supervisor, and for External Quality Assurance (EQA) as per National TB Elimination Programme (NTEP) guidelines.

• Prior to storage slides need to be dried by gently placing the slide gently face down on the tissue paper or by wrapping it in the tissue paper and leaving it overnight to remove excess oil (Figure 1).

The following are the measures to be kept in mind when adding immersion oil to the slide:

• Before adding immersion oil to the slide, ensure that the smear is facing upwards (Figure 1).

Figure 1: Slide should Face Upwards when Mounted on the Microscope Stage