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Microorganisms

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Special Stain
Purpose
Principle
Fixative/Technique
Q.C.
Reagents/Procedure
Results
Notes
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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