V. Post-Lab Questions- Acid-Fast Stain 1. Complete this table for the acid-fast
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Question
V. Post-Lab Questions- Acid-Fast Stain 1. Complete this table for the acid-fast stain Step A-F bacteria Non A-F after this step bacteria after Chemical Purpose this step Primary Stain red rad cels Mordant red red Decolorizer Counterstain red blue N0 2. Clinical Connection: A patient comes to the emergeney room in obvious respiratory distress complaining of painful breathing and deep coughs which produce bloody sputum. The patient's medical history indicates that they have tested positive on a past TB (tuberculosis) skin test. A chest X-ray and acid-fast stain of the sputum are ordered. The chest X-ray shows fluid in the lungs. The acid-fast stain shows the presence of blue-stained cocei in pairs and chains. The cocci were later determined to be Gram-positive and produced a capsule. A diagnosis of typical bacterial pneumonia was made. Information about TB and opical bacterial pneumonia can be found in your textbook. (a) How do the results of the stain rule out active (b) Assuming the cocci are the cause, what is the most likely genus/species causing this pneumonia?
Explanation / Answer
1. Acid fast stain can be done in Zeihl- Neelsen method as well as modified method which is Kinyoun method.
Let me discuss the differences in the procedure and according to that you can choose which one to use and write :
Primary stain : basic aniline dye carbol fuchsin. The stain has affinity towards mycolic acid present in the tubercle bacilli and stains red. The AF bacteria will be red and non AF bacteria too will be stained red.
Mordant : ZN stain uses physical mordant which is heat and kinyoun method needs chemical stain which is the high concentration of phenol present in the carbol fuchsin. Mordant is dye fixative which fixes the stain into the cell membrane forming coordination complex with dye. Both AF and non AF bacteria are red after the step.
Decolourizer : acid-alcohol. For ZN 20% sulphuric acid and 95% ethanol. Kinyoun method uses counterstain Gabott's methylene blue which contains acid alcohol in it and that is why for kinyoun method there are no separate decolourization step. This step decolourizes the stain but acid fast organisms resist decolourization and remain red whereas non AF organisms loose their colour and become colourless.
Counterstain : loeffler's methylene blue is used in ZN stain and Gabott's methylene blue is used in kinyuon method. Counterstain gives contrasting colour with primary stain so that stained structure can be visible clearly , moreover in differential staining like AFB it is used to differentiate between stained and non stained. The AF organism, due to the decolourization resistance retained the primary stain and be red after the step . Non AF organisms and the background materials like pus cells take up the colour of the stain and become blue.
2.a. In active pulmonary TB , often chest xray shows cavities in the upper lung which was not reported in this case. The causative agent of tuberculosis is Mycobacterium tuberculosis, which should be interpreted as red rod after the sputum staining but clearly it was stated that the staining showed blue stained cocci in pairs and chains which is the characteristics of non acid fast organisms. Later it was determined the resultant organism was gram positive which possesses capsule. Most likely the organism is Streptococcus pneumoniae which is capsular , gram positive and also the pneumoniae caused by the bacteria shows lung consolidations in chest x-ray.
b. As already discussed above the gram positive capsular cocci with lung consolidations are of genus Streptococcus and species pneumoniae, so the organism is Streptococcus pneumoniae, most causative agent of bacterial pneumoniae which was already diagnosed in the case. The cocci can be seen as diplococci in pairs or in short chains.
c. A positive tuberculin test indicates hypersensitivity to tuberculoprotein, denoting infection with the tubercle bacillus or BCG immunisation, recent or past, with or without clinical disease. Patient had past history of TB and probably patient got immunized.
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