Microorganisms
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Giemsa (Diff-Quik) | permit i.d. of HELICOBATER PYLORI | (regular Giemsa: bacteria, rickettsias and Toxoplasma gondii)Diff-Quik is a Romanowsky stain, "neutral" combine basic dye methyline bue and acid dye eosin; | 10%NBF; paraffin at 4-5u | Sections w/ H.pylori | DiffQuik kit solution I & II and Acetic acid; hydrate to distilled H20; dip in Sol I; dip in Sol II; rinse; diff in acetic H2O; dehydrate continue to xylene and synthetic resin | H.PYLORI= dark blue; OTHER BACTERIA= blue; nuclei= dark blue; cytoplasm = pink | Solution I = buffered eosin Y; solution II= cationic dye mixture of azure A and methylene blue; careful of decolorization;
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Hotchkiss-McManus PAS Rxn | demonstrate FUNGI | similar to PAS for polysaccharides | 10%NBF or Bouin or Zenker; paraffin 4-5u | section w/ fungi | Periodic Acid; wash; Schiff; wash in sulfurous rinse; running tap water; couter w/ fast green; rinse; run through | FUNGI= rose; background = green | green counterstain is better contrast for fungi, as does light green; can use diastase; make sure solutions are not too old
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Gridley | demonstrate FUNGI | modified Bauer, chromic acid oxidizes adjacent glycol groups to aldehydes; chromic acid leaves fewere reactive aldehyddes w/ chiff reation; less intense, less background; aldehyde fuchsin acts as an aldehyde reinforcing depth of the stain | 10%NBF; paraffin at 4-5u | sections w/ fungi | oxidize in 4% chromic acid; wash; stain in schiff; wash; rinse in 70%ROH; adehyde fuchsin; rinse stain in 95%; rinse in h2o; counter w/ metanil yellow solution; rinse; run through | MYCELIA = deep purple; CONIDIA= deep rose to purple; background = yellow; elastic fibers and mucin = deep purple | very old fungi not stained well; remember that chromic acid is a VERY strong oxidizer
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Grocott Methenamine-Silver Nitrate | demonsrate FUNGAL ORGANISMS | Chitin is oxidized to aldehydes by chromic acid; aldehydes reduces methenamine-silver to visible metallic silver; methenamine gives solution alkaline properties necessary for proper reaction; sodium borate buffer; gold chloride, sodium thio | 10%NBF(NO GLUTARALDEHYDe); 4-5u paraffin; 6u frozen | need section w/ fungi, use pneumocystis control if staining for pneumocystis; | oxidize in chromic acid; wash; rinse in sodium bisulfite; wash; incubate in methenamine silver; check microscopically; rinse; tone in gold chloride; rinse; remove unreduced silver in sodium thio; wash, counter w/ lt grn; dehydrate to xylene | FUNGI= sharply delineated in black; mucin = taupe to dark gray; background = green | do not overheat silver; also demonstrates actinomyces, nocardia asteroides and other certain encapsulated bacteria;
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Warthin-Starry (Cat Scratch) | demonstrate SPIROCHETES | ARGYROPHIL method; can bind Ag ions from solution reduced to silver by hydroquinone | 10%NBF; paraffin 4-5u | tissue w/ spirochetes | incubate sections to acidulated water then 1%silver nitrate; developer (silver nitrate, gelatin, hydroquinone; wash, run trhough | spirochetes = black; other bacteria = black; background = pale yellow to light brown | if overdeveloped trate w/ iodine and sodium thiosulfate; anybackteria ar nonselectively blacked by silver impregnation methose demosntrating small, weakly gram(-); very sensitive
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Dieterle | demonstrate SPIROCHETES or causative organism of LEGIONELLOSIS | ARGOPHILIC method adsorb the Ag; hydroquinone is the "developer" | 10%NBF; paraffin 4-5u | tissue w/ spirochetes of legionella organisms; chemically clean gassware | place i preheated 5% alcoholic uranyl nitrate; dip in distilled h20; dip 95%ROH; place in distilled then dry; place in silver nitrate; rinse; develop; dip; formic acid; runt through | SPIROCHETES, BACTERA = brown to black; background = pale yellow or tan | note others may also stain
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Microwave Steiner and Steiner | demostrate SPIROCHETES, H. PYLORI, or causative organism of LEGIONELLOSIS | argyrophillic!; hydroquinone is the reducer/developer | 10%NBF best; avoid mucruial and chromium fixatives; cut paraffin 4-5u | tissue w/ spirochetes, hpylori or legionella pneumophila | sensitize sections by placing in rmtp 1% aqueous uranyl nitrate; heat; rinse in DI;tm temp silve nitrate and heat; rinse; rinse in 95 then 100; gum mastic; air dry; rinse; reduce in hydroquinone; rinse; run through to xylene | SPIROCHETES, H. PYLORI, LEGIONELLA PNEUMOPHILA AND OTHER NONFILAMENTOUS BACTERIA = dark brown to black; background = light yellow | uses microwave; careful of xylene substitutes
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Microwave Methenamine-Silver Nitrate | rapid method on cytospin preparations or frozen sections for PNEUMOCYSTIS CARINII, for fungi as well, | refer to GMS | cytospin in 95%, frzen in 40% formaldehyde, paraffin sections same as GMS | Frozens at 6u; paraffin at 4-5; sections tend to wash off in this procedure! | basically it's the same as GMS only refer to the boock | Fungi and P carinii = black; background = green | careful of leaving too long in the methenamine-silver soluion
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Kinyoun Acid-Fast | Detect acid-fast MYCOBACTERIA | Lipoid (waxy) capsule of acid-fast takes up CARBOL-FUSCHIN, resists decolorization; stain enhanced by phenol and alcohol; carbol-fuschin provide a specific way of identifying mycobateria | 10%NBF preferred; NO CARNOY; 4-5 u | tissue w/ acid-fast bacteria; Millipore filtered h2o for water bath; negative control from same days workload; NO TAP OR REGULAR H2O | Rinse in Millipore-H2O; Stain in Kinyoun Carbol-Fuschin incubate warm; Wash; differentiate in w changes acid alcohol until pale pink; wash; counter w/ methylene blue; rinse; run through | ACID-FAST BACTERIA: bright red; background = light blue | Section from block of uterus good negative control; careful of overcounterstaining masks organisms (take back to acid-alcohol to destain and re-counter); do not let dry after carbol-fuschin, compound is insoluble and won't remove; not good for leprosy
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Ziehl-Neelsen | detect acid-fast MYCOBATERIA | See Kinyoun | well-fixed, NO CARNOY; 4-5 u | Acid-fast organisms; Millipore H2O, negative control from same day's workload (uterus); NO TAP H2O | Millipore; fresh/filtered carbol-fuschin; wash; decolorize w/ 1% acid ROH until pale pink ; rwash; counter w/ methylene blue; was; run through | ACID-FAST bacteria = bright red; background = light blue | preferred by many laboratorians
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Microwave Ziehl-Neelsen | detect acid-fast MYCOBATERIA | See Kinyoun | 10%NBF prefered, NO CARNOY; 4-5 u | ACID-FAST positive tissue; Millipore; uterus as negative control work day's workload; no tap | Carbol-Fuschin w/ parasoaniline!! (1) Milippore; carbol- fuschin microwave; wash; decolor w/ acid alcohol; wash; counter w/ methylene blue; wash; run through | Acid fast incuding mycobaterior avium intracellulare = Red; Erythrocytes = pink; Mast cells = blue; other tissue elements = pale blue | can do at room temp
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Fite | detect MYCOBATERIUM LEPRAE | Lipoid capsule of organism takes up carbol fuschin and resists decoloriaztion w/ dilute mineral acid; Leprosy organisms have lipoid capsules sensitive to alcohols and xylene, need to protect capsule | 10% NBF preferred; NO CARNOY; 4-5 u paraffin | tissue w/ Leprosy organisms; millipore h2o; negative control; no tap water | Xylene-peanut oil mixture; drain blot to opacity; stain in fresh Ziehl-Neelsen carbol-fuschin; wash; diff w/ 1% acid ROH until faint pink; wash; counter w/ methylene blue; rinse, blot dry competely NO ALCOHOL OR XYLENE | M.LEPRAE and other ACID-FAST bacteria = bright red; background = light blue | For NOCARDIA; use sulfuric acid to decolorise; avoid alcohol for both leprae and nocardia;
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Microwave Auramine-Rhodamine | detect MYCOBACTERIUM TUBERCULOSIS or other acid-fast organisms | Fluoresence technique; use of 2 basic dyes that fluoresce at whort wavelengths | 10%NBF prefered; 4-5u | tissue w/ acid-fast mycobateria; millipore h2o; negative control from same day's workload; no tap water! | Millipore h2o; place in auramine o-rhodamine b solution and microwave; rinse; diff in 2 changes of acid ROH; rinse; stain in 0.3% criochrom black T; rinse; air dry; xylene | ACID-FAST ORGANISMS = reddish-yellow fluoresence; background = black | extremely sensitive--freq. false-positives; can restain w/ carbol-fuschin; CAN STAIN DEAD AND DYING ORGANISMS; can't really use w/ zinc salts?
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Brown-Hopps Modification of Gram Stain | demonstrate Gram-negative AND Gram-positive bacteria | Crystal violet + iodine = lake; both gram-neg and pos stained; pos bacteria have thicker cell wall and isn't decolorized retaining crystal violet-iodine complex; counterstain is applied to color the gram-neg organisms | 10%NBF; paraffin at 4-5u | sections w/ both Gram-positive and Gram-negative | Stain w/ crystal violet; rinse; stain w/ Gram's Iodine; rinse; blot and decolor QUICKLY in acetone; rinse; stain w/ basic fuchsin; diff w/ Gallego (form, acetic acid); rinse; acetone; picric-acetone; acetone; acetone-xylene; synthetic resin | GRAM-POSITIVE= blue; GRAM-NEGATIVE= red; Background tissue generally = yellow; nuclei = light red | SEE BOOK
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Created by:
Miellee
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