Special Stains (A-G) Flashcards
(22 cards)
Acid Fast Bacilli Principle
Mycobacterial cell walls contain a waxy substance composed of mycolic acids. These are ß-hydroxy carboxylic acids with chain lengths of up to 90 carbon atoms. The property of acid fastness is related to the carbon chain length of the mycolic acid found in any particular species (Lyon H 1991).
Basic fuchsin binds to negatively charged groups in bacteria. The mycolic acid (and other cell wall lipids) present a barrier to dye entry as well as elusion (washing out with solvent) and this is partly overcome by adding a lipophilic agent to a concentrated aqueous solution of basic fuchsin and partly by heating.
Acid Fast Bacilli Method
Place the working solution in a coplin jar and pre-heat in 58 -60 ºC waterbath for 10 minuntes.
Deparaffinise sections, bring to water.
Stain in the pre-heated working solution in the water bath for 15 minutes.
Place the COPLIN JAR containing the slides into running cold tap water for 2 minutes.
Remove the slides from the coplin jar and wash in running water for 1 minute.
Differentiate in acid alcohol until no more colour runs from the slide.
Wash briefly in water to remove the acid alcohol
Counterstain with methylene blue for 15 to 30 seconds.
Wash in water, dehydrate, clear and mount in DPX.
Results
Acid Fast Bacilli Results
Acid fast bacilli …………………………….. Red
Nuclei ………………………………………. Blue
Other tissue constituents ………………….. Blue
Alcian Blue Principle
Alcian blue stains acid mucosubstances and acetic mucins. Excessive amounts of non-sulfated acidic mucosubstances are seen in mesotheliomas, certain amounts occur normally in blood vessel walls but increase in early lesions of atherosclerosis. Strongly acidic mucosubstances will be stained blue, nuclei will be stained pink to red, and cytoplasm will be stained pale pink.
Alcian Blue Method
- Deparaffinize slides and hydrate to distilled water.
- Stain in alcian blue solution for 30 minutes.
- Wash in running tap water for 2 minutes.
- Rinse in distilled water.
- Counterstain in nuclear fast red solution for 5 minutes.
- Wash in running tap water for 1 minute.
- Dehydrate and through 95% alcohol, 2 changes of absolute alcohol, 3 minutes each.
- Clear in xylene or xylene substitute.
- Mount with resinous mounting medium.
Alcian Blue Results
Strongly acidic sulfated mucosubstances ————– blue
Nuclei ------------------------------------------------------ pink to red Cytoplasm ------------------------------------------------- pale pink
Alcian Blue and PAS Staining Principle
This is a combined method utilising the properties of both the PAS (see PAS) and Alcian blue (see Alcian blue) methods to demonstrate the full complement of tissue proteoglycans.
The rationale of the technique is that by first staining all the acidic mucins with Alcian blue, those remaining acidic mucins which are also PAS positive will be chemically blocked and will not react further during the technique. Those neutral mucins which are solely PAS positive will subsequently be demonstrated in a contrasting manner. Where mixtures occur, the resultant colour will depend upon the dominant moiety.
The combined result from the port-manteau method demonstrates both acidic- neutral- and mixtures of acidic and neutral mucins.
Alcian Blue and PAS Method
Bring sections to distilled water.
Stain with Alcian blue 15 mins
Wash well in running tap water 2 mins
Rinse in distilled water
Treat with periodic acid 5 mins
Wash well in distilled water
Stain with Schiff’s reagent 10 mins
Wash well in running tap water 5 mins
Stain nuclei with haematoxylin 1 min
Wash in running tap water 2 mins
Differentiate with acid alcohol
Wash and blue nuclei in Scott’s tap water
Wash in water
Dehydrate, clear and mount
Alcian Blue and PAS Results
acidic mucins …………………………. blue
neutral mucins ……………………….. magenta
mixtures of above ……………………. blue/purple
nuclei ………………………………… deep blue
Bennhold’s Congo Red Principle
This Bennhold’s Congo Red stain is used for the detection of amyloid on formalin-fixed, paraffin-embedded tissue sections with amyloidosis, and may be used for frozen sections as well. The amyloid deposits will be stained red and the nuclei will be stained blue. The thickness of sections is usually 5 um. But in case of inadequate amyloid deposits, 10um thick sections will be more satisfactory. Congo red stains amyloid in tissue sections.
Bennhold’s Congo Red Method
Deparaffinize and hydrate sections to distilled water.
- Stain in Congo red solution for 30-60 minutes.
- Rinse in distilled water.
- Differentiate rapidly (5-10 dips) in alkaline alcohol solution.
- Rinse in running tap water for 5 minutes.
- Counterstain in Gill’s hematoxylin for 30 seconds.
- Rinse in tap water for 1 minute.
- Dip in ammonia water (add a few drops of ammonium hydroxide to tap water and mix well) for 30 seconds or until sections turn blue.
- Rinse in tap water for 5 minutes.
- Dehydrate through 95% alcohol, 100% alcohol
- Clear in xylene and mount with resinous mounting medium.
Bennhold’s Congo Red Results
Amyloid, elastic fibers, eosinophil granules ————- red
Nuclei -------------------------------------------------------- blue
Fontana Masson Principle
Fontana-Masson stains argentaffin granules and melanin. Melanin is a nonlipid, nonhematogenous pigment. It is a brown-black pigment present normally in the hair, skin, retina, iris, and certain parts of CNS. Argentaffin granules are found in carcinoid tumors.
Fontana Masson Method
- Deparaffinize and hydrate slides to distilled water.
- Place slides in Fontana silver nitrate working solution and leave in a 56 C oven for 2 hours. Slides may be checked after 1 hour.
- Rinse in 3 changes of distilled water.
- Tone in gold chloride working solution for 1 minutes.
- Rinse in distilled water.
- Place in 5% sodium thiosulfate solution for 1 minutes.
- Rinse in distilled water.
- Counterstain with nuclear fast red solution for 2-5 minutes
- Rinse thoroughly in distilled water twice.
- Dehydrate, clear and coverslip, using a synthetic mounting medium
Fontana Masson Results
Argentaffin cell granules or melanin ——— black
Nuclei -------------------------------------------- pink-red Cytoplasm --------------------------------------- pale pink
Giemsa’s Protocol
- Bring sections to distilled water
- Stain with diluted Giemsa’s stain made up fresh (see technical point 1)
- Rinse in distilled water
- Differentiate with 0.5% aqueous acetic acid (see technical point 2)
- Dehydrate rapidly
- Clear and mount
Giemsa’s Results
bile pigments…………………………………………………green
collagen, muscle, bone..............................................pale pink
micro-organisms, fungi, parasites.................................purplish-blue
starch granules, cellulose............................................sky blue
pigments (native colour is yellow/brown, or if fixed in dichromate containing fixative)...green
nuclei. .................................................................dark blue to violet
erythrocytes. .........................................................salmon pink
cytoplasm…………………………………………………………..varying light blue shades
Gordon & Sweet’s Method
Deparaffinise sections with xylene then take through alcohols to water.
2 Oxidise in acidified potassium permanganate for 3 minutes
3 Rinse in distilled water.
4 Decolourise with 2% oxalic acid for 1 min
5 Rinse in distilled water.
6 Mordant in 4% iron alum for 10 minutes
7 Rinse in distilled water.
8 Impregnate in ammoniacal silver solution for 11 seconds
9 Rinse quickly in distilled water.
10 Immediately reduce with 10% aqueous formalin for 2 minutes
11 Wash in running tap water for 2 minutes
12 Tone in 0.2% gold chloride (Not liver cores-see technical point 4) for 2 minutes
13 Rinse in distilled water.
14 Fix with 2% aqueous sodium thiosulphate (hypo) for 2 minutes
15 Wash in water for 2 minutes
16 Counterstain with neutral red (optional-see technical note 5) for 2 minutes
17 Dehydrate, clear and mount.
Gordon & Sweet’s Results
Reticulin fibres ……………………….Black
Nuclei …………………………………Red
Grocott’s Methenamine Silver Principle
Chromic acid oxidation forms aldehydes from fungal cell wall polysaccharide components, which are subsequently demonstrated by reduction of an alkaline hexamine-silver complex
Grocott’s Methenamine Silver Method
Bring sections to distilled water.
- Oxidise with 4% aq chromic acid at room temperatur 1 hr
- Wash in water for a few seconds.
- Treat sections with 1% sodium metabisulphite 1 min
- Wash in running tap water 3 mins
- Rinse thoroughly in distilled water.
- Place in pre-heated working silver solution in a water bath at 60 °C for 15 to 20 mins until section turns yellowish-brown (Check microscopically after washing in distilled water – fungi should be dark brown).
- Rinse well in distilled water
- Tone sections with 0.2% gold chloride 2 mins
- Rinse in distilled water
- Treat sections with 2% sodium thiosulphate 2 mins
- Wash with running tap water 5 mins
- Counterstain in working light green 15 sec
- Rinse excess light green off slide with alcohol
- Dehydrate, clear and mount.
Grocott’s Methenamine Silver Results
fungi, Pneumocystis carnii, histoplasma spp ——– black
inner parts of mycelia and hyphae ——————– old rose
leishmania spp, toxoplasma spp ———————- negative
mucin —————————————————- dark grey
background ———————————————- pale green
