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HT/HTL: Chap10

Microorganisms

Special StainPurposePrincipleFixative/TechniqueQ.C.Reagents/ProcedureResultsNotes
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;
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
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
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;
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
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
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
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
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
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
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
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;
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?
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
Created by: Miellee