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Tb-color modified - Staining kit for the detection of mycobacteria (AFB) by hot staining method

Merck Staining kit for the detection of mycobacteria

Tb-color modified staining set uses the classical Ziehl-Neelsen hot staining procedure with  methylene blue counter-straining.
Mycobacteria are difficult to stain because of the high proportion of lipid and wax in their cell walls. Up to now, in order to carry out the classical Ziehl-Neelsen staining, the test material to be heated with carbol fuchsin solution to produce the mycolic acid fuchsin compound.
Once stained, acid fast mycobacteria keep their colouring even after treatment with strong Hydrochloric acid in ethanol as HCl-ethanol. They remain red after counterstaining with methylene blue, whereas the micro-organism susceptible to acid take on the blue.
The method can be used for a wide range of sample material as heat-fixed smears of sputum, FNAB, lavages, imprints, body fluids, exudates, pus, liquid, solid cultures and histological sections. It is really important that the sample material is fixed in the following way:
Fixation is carried out over the flame of a Bunsen burner (2-3 times, avoiding excessive heating).
It is also possible to fix the smears in an oven at 100-110 °C for 20 min. Impairment of staining must be expected if a higher temperature or longer heating is employed.
For mucus-rich sputum is the following pre-treatment helpful:
Sputum should be pre-treated with Sputofluol® in order to free the mycobacteria from surrounding mucus. The active ingredient in Sputofluol® is hypochlorite, which oxidatively dissolves the organic material while sparing the mycobacteria for the greater part.
In a centrifuge tube mix 1 part of the sample (at least 2 ml) with 3 parts of a 15% Sputofluol® solution prepared with distilled water, and leave to react for 10 min shaking vigorously from time to time. Centrifuge at 3000-4800 U/ min for 20 min, decant the supernatant, smear out the sediment allow to dry.
Punction and lavage material, sediments are used in the lowing way-after carrying out the appropriate enrichment measures smear out samples on the microscopic slides and allow to air dry. For histological sections - deparaffinize the sections in the typical manner and rehydrate in a descending alcohol series.

Procedure on a staining rack:

1. Cover specimens completely with Tb-color modified carbol-fuchsin solution. Carefully heat 3 times from below with a Bunsen burner to steaming and keep hot for 5 min. Do not allow the stain to boil.
2. Wash with tap water until no further colour is given off.
3. Cover completely with Tb-color modified hydrochloric acid in ethanol and, depending on the thickness of the specimen, allow to stand for 15 – 30 sec
4. Wash immediately with tap water
5. Counter-stain for 30 sec in Tb-color modified methylene blue solution or 1 min in diluted Tb-color modified solution(dilution:1+9 with dist. water)
6. Wash well with tap water
7. Dry

Dehydrate histological specimens (ascending alcohol series), clear in xylene or Neo-Clear®) and mount with Entellan® new or Neo-Mount®.


Acid fast bacteria  red
Background   light blue

For further information about the wide range of products available from Merck KGaA for Mycobacteria (AFB) detection, please request here : Request for more information

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Description Presentation Cat. No. Promo Price Qty.
Tb-color modified staining set Solution 1 Tb-color modified carbol-fuchsin solution 500 ml
Solution 2 Tb-color modified hydrochloric acid in ethanol
Solution 3 Tb-color modified methylene blue solution
Ziehl-Neelsen Carbol fuchsin solution 100 ml 1.09215.0100
Ziehl-Neelsen Carbol fuchsin solution 500 ml 1.09215.0500
Ziehl-Neelsen Carbol fuchsin solution 2.5 l 1.09215.2500
Löffler's methylene blue solution 500 ml 1.01287.0500
Hydrochloric acid in ethanol 1 l 1.00327.1000
Hydrochloric acid in ethanol 5 l 1.00327.5000
Sputofluol® 1 l 1.08000.1000