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Microorganism

Staining

QuestionAnswer
Cocci bacteria arrangements: diplococci - paired, staphylococci - grape-clusters, streptococci - chains
Some pathogenic cocci include: Staphylococcus aureus (TSS), streptococcus pneumonia, Neisseria gonorrhea, Neisseria meningitidis, methicillin-resistant staph aureus
Bacilli bacteria Rod-shaped bacteria; pathogenic: Clostridium tetani, C. botulinum, C. difficile, C. perfringens (food poisoning), bacillus anthracis
Coccobacilli bacteria Rod-shaped, short & wide bacteria; pathogenic: Hemophilus influenza, Chlamydia trachomatis
Gram-negative bacteria: Don't stain; Salmonella, Shigella dysenteriae, E. coli, Pseudomonas aeruginosa (pneumonia, UTI), N. meningitidis, N. gonorroeae, S. aureus
Gram-positive bacteria: Stain blue due to cell wall of teichoic acid, include - Clostridium botulinum, C. tetani, Corynebacterium diptheriae, Actinomyces
Spirochetes are: Spiral/corkscrew bacteria; pathogenic: Leptospira (Weil's disease), Triponema pallidium (syphilis), Borrelia burgdorferi (Lyme disease), argyrophil stains
Some bacteria types that don't have typical bacterial attributes include: Rickettsiae, mycoplasmas
Which bacteria can only reproduce w/i living host cell? Rickettsiae & chlamydiae
Rickettsiae causes: Rocky mountain spotted fever, typhus, tick-borne diseases
Mycoplasmas: Don't form cell walls, can cause pneumonia, still birth, abortion
Which stains are commonly used to screen for the presence of bacteria? Methylene blue, Giemsa
Mycosis is: disease caused by fungi
Mycology is: study of fungi
Molds are: Filamentous fungi, easy to see septate hyphae in Aspergillus fumigatus
Yeasts are: Single round/oval "budding" cells, Cryptococcus neoformans (meningitis)
Yeast-like fungi are: Filamentous, reproduce by budding to form pseudohyphae, Candida albicans (yeast infection, thrush)
Dimorphic fungi: Have yeast-like morphology at 37 °C, filamentous morphology on artificial media @ 25°C, Blastomyces dermatitidis, Coccidioides immitis (Valley fever), Histoplasma capsulatum
Viruses are: Protein-coated RNA or DNA, seen w/ EM, may form cellular inclusions visible w/ light microscope, e.g. Negri bodies of rabies & cytomegalovirus
Protozoans are: Single-celled microorganisms, psuedopods, flagella, or cilia, Entamoeba histolytica, Giardia lamblia, Toxoplasma gondii
Pneumocystis carinii: aka Pneumocystis jirovecii, used to be considered protozoan, more closely related to yeast
Acid-fast bacteria are: Mycobacteria, rod-shaped, may exhibit filamentous growth, cell walls have lots of lipids so once stained resists acid decolorization, Mycobacterium tuberculosis, mycobacterium leprae
Kinyoun & Ziehl-Neelsen stains facts Detect presence of acid-fast mycobacteria, 10% NBF, no Carnoy, QC: tissue w/ acid-fast organisms
Kinyoun stain solutions Kinyoun Carbol-Fuchsin + acid alcohol + methylene blue
Kinyoun & Ziehl-Neelsen stain results acid-fast bacteria: bright red - background: light blue
When cutting slides to test for acid-fast bacteria must use: Millipore filtered water in bath, no tap or dH2O, use negative control from same day/bath/microtome
Kinyoun Carbol-Fuchsin solution basic fuchsin + phenol crystals (enhance stain) + 95% + filtered water filter before use
Methylene blue solution methylene blue + 95% + H2O
Kinyoun vs. Ziehl-Neelsen Carbol-Fuchsin solutions Kinyoun has more basic fuchsin & phenol than Ziehl-Neelsen
Ziehl-Neelsen method solutions Ziehl-Neelsen Carbol-Fuchsin + acid alcohol + methylene blue
Ziehl-Neelsen Carbol-Fuchsin solution basic fuchsin + phenol crystals + 100% + filtered H2O filter before use
Microwave Ziehl-Neelsen method solutions Carbol-Fuchsin soln. + acid alcohol + methylene blue w/ acetic acid
Microwave Ziehl-Neelsen method results acid-fast bacteria (incl. mycobacterium avium intracellulare): red - rbc: pink - mast cells: blue - other: pale blue
Carbol-Fuchsin solution pararosaniline (basic fuchsin) + phenol crystals + isopropanol + filtered H2O filter before use
Fite stain facts To detect presence of Mycobacterium leprae, 10% NBF, no Carnoy, QC: section w/ leprosy organisms
Fite stain solutions xylene-peanut oil + acid alcohol + Ziehl-Neelsen Carbol-Fuchsin soln. + methylene blue w/ acetic acid
Fite stain results M. leprae, acid-fast bacteria: bright red - background: light blue
Xylene-peanut oil is used to: Protect capsule of leprae organism from dissolving during deparaffinization
Microwave auramine-rhodamine fluorescence technique facts To detect presence of Mycobacterium tuberculosis & acid-fast bacteria, 10% NBF QC: section w/ acid-fast mycobacteria
Microwave auramine-rhodamine fluorescence technique solutions auramine O-rhodamine B soln. + acid alcohol (decolorize) + Erichrome black T 0.3% (quench autofluorescence)
Microwave auramine-rhodamine fluorescence technique results acid-fast bacteria: reddish-yellow fluorescence - background:black
Auramine O-rhodamine B solution auramine O (fluorescent dye) + rhodamine B (fluorescent dye) + glycerol + phenol + filtered H2O
Alcian yellow-T blue facts To ID H. pylori, no glyoxal QC: tissue w/ H. pylori
Alcian yellow-T blue solutions periodic acid (ox. cell wall) + alcian yellow (stain mucin) + T blue (stain wall) + sodium metabisulfite
Alcian yellow-T blue results H. pylori: deep blue - mucin: yellow
Gram stains purpose ID Gram+ & - bacteria, QC: section w/ Gram +& - bacteria
Gram stains solutions crystal violet (stain all bacteria) + basic fuchsin (stain Gram -) + Gram iodine (mordant) + 100% or acetone (diff.) + tartrazine or picric acid-acetone soln. (counterstain) if overdecolorize, Gram + no stain
Gram stains results Gram + : purple - Gram - : red
Brown-Hopps modification of Gram stain solutions crystal violet in dH2O + basic fuchsin + Gram iodine + Gallego solution + picric acid-acetone
Brown-Hopps modification of Gram stain results Gram + : blue - Gram - : red - background: yellow - nuclei: light red
Gallego solution formalin + glacial acetic acid + dH2O
Giemsa stains can demo: Bacteria, rickettsias, Toxoplasma gondii
Modified Diff-Quik Giemsa Stain purpose ID H. pylori, QC: section w/ H. pylori, Romanowsky-type
Modified Diff-Quik Giemsa Stain solutions Diff-Quik soln. I + Diff-Quik soln. II + acetic acid water
Modified Diff-Quik Giemsa Stain results H. pylori, nuclei: dark blue - other bacteria: blue - cytoplasm: pink
Hotchkiss-McManus PAS reaction, Gridley, & Grocott-methenamine-silver nitrate stains purpose Demo fungi, QC: section w/ fungi
Hotchkiss-McManus PAS reaction solutions periodic acid + Schiff reagent + sulfurous rinse (remove excess leucofuchsin) + fast green w/ acetic acid (or light green)
Hotchkiss-McManus PAS reaction results fungi: rose - background: green
Gridley stain solutions chromic acid + Schiff + aldehyde fuchsin (reacts w/ Schiff like an aldehyde) + metanil yellow
Gridley stain results Mycelia, elastic fibers, mucin: deep purple - conidia: deep rose to purple - background: yellow
Grocott-methenamine-silver nitrate stain facts 4-5 μm paraffin, 6 μm frozen, QC: for Pneumocystis use Pneumocystis control, use chemically clean glassware & nonmetallic forceps
Grocott-methenamine-silver nitrate stain solutions chromic or periodic acid (ox. walls to aldehyes) + methenamine-silver nitrate working + gold chloride + sodium bisulfite (remove acid) + sodium thiosulfate + light green w/ acetic acid
Grocott-methenamine-silver nitrate stain results fungi: sharply delineated in black - mucin: taupe to dark gray (if black, methenamine too hot) - background: green
Microwave methenamine-silver nitrate procedure purpose Best to demo on cytospin preps or frozen diagnosis Pneumocystis carinii
Microwave methenamine-silver nitrate procedure facts cytospin prep fixed in 95%, frozen fixed in 40% formaldehyde, 6 μm, paraffin routine QC: pneumatocyst for P. carinii, fungi for fungi
Microwave methenamine-silver nitrate procedure solutions chromic acid + methenamine-silver nitrate working + gold chloride + sodium thiosulfate + sodium bisulfate (clear) + light green w/ acetic acid
Microwave methenamine-silver nitrate procedure results fungi & P. carinii: black - background: green
Methenamine-silver working solution methenamine + AgNO3 + Borax + dH2O
What stains can demo Crytococcus neoformans? Mayer mucicarmine & alcian blue; may demo Blastomyces dermatitidis & Rhinosporidium seeberi
Warthin-Starry, microwave Warthin-Starry, & Dieterle purpose Demo spirochetes, legionella, QC: section w/ spirochetes or legionella
Warthin-Starry Technique solutions AgNO3 2% w/ acidulated water + AgNO3 w/ acidulated water, gelatin soln. & hydroquinone
Warthin-Starry Technique results spirochetes, other bacteria: black - background: pale yellow to light brown
Acidulated water triple-distilled H2O + citric acid pH4.0
Microwave Warthin-Starry Method solutions glycine-acetic acid pH 4.2 + silver nitrate-gelatin-hydroquinone developer w/ glycine-acetic acid
Microwave Warthin-Starry Method results alipia felis (cat-scratch bacillus), legionella pneumophila, nocardia asteroides, H. pylori: black - nuclei, rbc: brown
Dieterle Method solutions alcoholic uranyl nitrate + alcoholic gum mastic (protect organism) + AgNo3 + developer + formic acid
Dieterle Method results spirochetes, bacteria: brown to black - background: pale yellow or tan Melanin, chromatin, formalin pigment, foreign materials may also stain brown to black
Dieterle developer solution alcoholic gum mastic + acetone + hydroquinone + pyridine + sodium sulfite + formaldehyde + dH2O
Microwave Steiner & Steiner procedure purpose Demo spirochetes, H. pylori, legionella QC: section w/ what staining for
Microwave Steiner & Steiner procedure solutions uranyl nitrate (sens.) + AgNo3 + gum mastic + reducing soln.
Microwave Steiner & Steiner procedure results Spirochetes, H. pylori, Legionella, other nonfilamentous bacteria - dark brown to black background - light yellow
Microwave Steiner & Steiner reducing solution gum mastic + hydroquinone (red.) + abs. alcohol
When staining for spirochetes avoid what? Mercurial & chromate fixatives, 10% NBF preferred
Sulfurous rinse solution HCl + potassium metabisulfite + dH2O
Argentaffin stains Gomori methenamine-silver, Grocott methenamine-silver, Fontana Masson, von Kossa
Argyrophil stains Reticulin, Holmes, Bielschowsky, Warthin-Starry, Dieterle, Steiner & Steiner, Grimelius, Churukian-Schenk
Sayeed's Stain purpose & reagents Detect H. Pylori, periodic acid + Schiff + Mayer's hemalum
Plasmodium parasites Cause malaria which deposits pigment closely related to formalin pigment
Cryptosporidium Protozoa, causes severe outbreaks of diarrhea among AIDS patients
Listeria monocytogenes Cause rare form of meningitis, stain variably with Gram stain, appear as intracellular rods in "Chinese letter" formation
Ziehl-Neelson may also demonstrate Lipofuscins if technique is modified to include heat and prolonged staining
Created by: CCF
 

 



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