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Microorganisms in the histology lab

Bacteria Tiny, single celled organisms
Bacteria ~0.2-10 micrometers
Spherical or ovoid bacteria Cocci
Cause of TSS (Toxic Shock Syndrome) Staph Aureus
Cocci Staph aureus, Streptococcus pneumoniae, Nisseria gonorrhoeae, and Neisseria meningitdis
Rod shaped bacteria Bacilli
Bacilli Clostridium tetani, Clostridium botulinum, and bacillus anthracis.
Coccobacilli Haemophilus influenzae, and Chlamydia trachomatis
Spiral shaped bacteria Spirochetes
Spirochetes Syphillus:Treponema pallidum, and Borrelia burgdorferi: Lyme disease
What stains are primarily used to identify Spirochetes? Silver stains
Stain for presence of bacteria Giemsa and Methylene Blue
Acid fast techniques are of value in the detection of what? Mycobacteria, rod shaped organisms that sometimes exhibit filamentous (fungus like) growth
Fungi Unicellular or multicellular primitive plants that have a distinct membrane bound nucleus containing genetic material.
Filamentous fungi are also called molds
Yeasts Single round or oval cells that reproduce by "budding"
Classified as a yeast Crytococcus neoformans
Yeastlike fungi that does not detach from parent cell "pseudohyphae" Candida albicans
Virus Composed of either DNA or RNA protein
Virus Protein coated genes that need living cells to provide energy and the machinery for duplication.
Virus ~20-30 nm
Most viruses can only be seen with a _ microscope. Electron
Protozoans Single celled microorganisms that are functionally complex structures
Protozoans Entamoeba histolytica, Giardia lamblia, and Toxoplasma gondii.
Kinyoun Acid Fast Detect presence of Acid-fast bacteria
Kinyoun Acid Fast Lipoid capsule or organism takes up carbol fuchsin and deists decolorization with dilute mineral acid.Acid alcohol used to decolorize to get an even decolorization.
Kinyoun Acid Fast 10% NBF (Carnoy can't be used)
Kinyoun Acid Fast 4-5 micrometers
Kinyoun Acid Fast control Tissue containing acid fast organisms
Kinyoun Acid Fast 1. Carbol fuchsin 2. Water 3. 1% acid alcohol 4. water 5. Methylene Blue 6. rinse 7. Dehydrate and clear
Kinyoun Acid Fast results Acid-fast bacteria: Bright red Background: Blue
Ziel-Neelsen Detection of acid fast bacteria
Ziel-Neelsen Afixed tissue with exception of Carnoy
Ziel-Neelsen 4-5 micrometers
Ziel-Neelsen control Tissue containing acid fast organisms
Ziel-Neelsen results Acid-fast bacteria: Bright red Background: Blue
Ziel-Neelsen microwave results Acid fast organisms: Red Erythrocytes: Pink Mast cells: Blue Other tissue elements: Pale blue
Fite Detection of mycobacterium leprae
Fite Lipoid capsule of organism takes up carbol fuchsin and resists decolorization with dilute mineral acid.
Fite 10% NBF and others with exception of Carnoy
Fite 4-5 micrometers
Fite control Tissue containing leprosy organisms
Fite 1. Xylene peanut oil 2. Blot 3. Ziel Nelson carbol fuchsin 4. wash 5. 1% acid alcohol 6. Wash 7. Methylene blue 8. Rinse 9. Blot 10. Mount with synthetic resin
Fite results M leprae and other acid fast bacteria: Bright red Background light blue
Auramine Rhodamine Detection of M tuberculosis or other acid fast organisms
Auramine Rhodamine 10% NBF
Auramine Rhodamine 4-5 micrometers
Auramine Rhodamine control Tissue containing Acid fast mycobacteria
Auramine Rhodamine 1. Auramine rhodamine 2. rinse 3. Acid alcohol 4. rinse 5. 0.3% eriochrome black T 6. Rinse 7. Dry 8. Dip in xylene and mount
Auramine Rhodamine results Acid fast organisms: Reddish-yellow fluorescence Background: Black
Gram stain (Brown-Hopps) Demonstration of gram positive and gram negative organisms
Gram stain (Brown-Hopps) 10% NBF
Gram stain (Brown-Hopps) 4-5 micrometers
Gram stain (Brown-Hopps) control Sections containing bothe Gram positive and gram negative organisms should be used
Gram stain (Brown-Hopps) 1. Crystal violet 2. rinse 3. iodine 4. rinse 5. decolorize 6. rinse 7. basic fuchsin 8. rinse 9. Gallego solution 10. rinse 11. Acetone 12. Picric acid-acetone 13. Acetone 14. Acetone-xylene 15 Mount
Gram stain (Brown-Hopps) results Gram +: blue-black Gram -: Red Background tissue: Yellow Nuclei: light red
Giemsa can be used to demonstrate T gondii, rickettsias, and bacteria
Diff Quick Identification of H pylori
Diff Quick Romanowsky stains "neutral" dyes combing basic and acid dyes creates a wide range of color.
Diff Quick 10% NBF
Diff Quick 4-5 micrometers
Diff Quick control Tissue with H pyori
Diff Quick 1. Diff Quick solution I 2. Diff Quick solution II 3. Rinse 4. Acetic water 5. Rinse 6. Dehydrate, clear, and mount
Diff Quick results H pylori: Dark blue Other bacteria: Blue Nuclei: Dark Blue Cytoplasm: Pink
Solution I in Diff Quick Buffered solution of Eosin Y (Anionic Dye, stains cytoplasmic elements pink)
Solution II in DIff Quick Cationic dye mixture of azure A and methylene blue, stains nuclei and bacteria blue
Alcian yellow-toulidine blue Detection of H pylori
Alcian yellow-toulidine blue Alcian yellow is a monoazo dye that reacts similar to alcian blue, staining mucin yellow. Toulidine blue is a basic dye and metachromatic stain that stains H pylori and nuclei blue
Alcian yellow-toulidine blue 10% NBF
Alcian yellow-toulidine blue 4-5 micrometers
Alcian yellow-toulidine blue control Tissue containing H pylori
Alcian yellow-toulidine blue 1. Periodic acid 2. wash 3. Sodium metabilsulfite 4. rinse 5, Alcian yellow 6. wash 7. Toluidine blue 8. wash 9. blot 10 dehydrate, clear, mount
Alcian yellow-toulidine blue results H pylori: Blue Mucin: Yellow Background: Pale blue
Hotchkiss-McManus periodic acid Schiff Demonstration of fungi
Hotchkiss-McManus periodic acid Schiff Polysaccharides present in the fungal cell walls are oxidized by the periodic acid to aldehydes. Aldehydes react with Schiff reagent to yield rose colored fungi.
Hotchkiss-McManus periodic acid Schiff 10% NBF, Bouin or Zenker
Hotchkiss-McManus periodic acid Schiff 4-5 micrometers
Hotchkiss-McManus periodic acid Schiff control Section containing fungi
Hotchkiss-McManus periodic acid Schiff 1. periodic acid 2. rinse 3. Schiff 4. rinse with sulfurous acid 5. wash 6. counterstain light green 7. rinse 8. Dehydrate, clear and mount
Chromic acid Schiff Demonstration of fungi
Chromic acid Schiff 10% NBF
Chromic acid Schiff 4-5 micrometers
Chromic acid Schiff control Section containing fungi
Chromic acid Schiff 1., Chromic acid 2. rinse 3. Schiff 4. rinse in sulfurous acid 5. rinse 6. Counterstain with harris heme or light green 7. wash 8. Dehydrate, clear and mount
Chromic acid Schiff results Fungi: Deep rose to purple Nuclei if heme is used: Blue Background in light green is used: Green
Gridley Demonstration of fungi
Gridley 10% NBF
Gridley 4-5 micrometers
Gridley control Section containing fungi
Gridley 1. Chromic Acid 2. rinse 3. schiff 4. rinse 5. 70% alcohol 6. Aldehyde fucchsin 7. rinse in 95% alcohol 8. rinse 9. counterstain with mental yellow 10. rinse 11. Dehyrate, clear, mount
Gridley results Mycelia: Deep purple Conidia: Deep rose to purple Background: Yellow Elastic fibers: Deep purple
Grocott Methenamine Silver Nitrate Demonstration of fungi
Grocott Methenamine Silver Nitrate 10% NBF
Grocott Methenamine Silver Nitrate 4-5 micrometers
Grocott Methenamine Silver Nitrate control Section containing fungi
Grocott Methenamine Silver Nitrate 1. Chromic Acid 2. rinse 3. Sodium bisulfite 4. rinse 5. Methnamine silver 6. rinse 7. gold chloride 8. rinse 9. sodium thiosulfate 10 rinse 11. counterstain 12. dehydrate clear mount
Grocott Methenamine Silver Nitrate results Fungi: Crisp black Mucin: Taupe to dark grey Background green
Mayer mucicarmine & Alcian Blue stains for C neoformans, Blastomyces dematidis, and Rhinosporidium
Warthin-Starry Demonstration of spirochetes
Warthin-Starry Argyrophil method Hydroquinone is used to reduce
Argyophil Have the ability to bind silver ions from a solution but they do not have the ability to reduce the silver to a visible form.
Argentaffin Have the ability to bind and reduce silver ions from a solution
Warthin-Starry 10% NBF
Warthin-Starry 4-5 micrometers
Warthin-Starry control Tissue that contains spirochetes
Warthin-Starry 1. Silver Nitrate 2. Developer 3. wash 4. Dehydrate clear mount
Warthin-Starry results Spirochetes: Black Other bacteria: black Background: Pale yellow to light brown
If sections are over developed they can be treated with what and then restrained? Iodine or sodium thiosulfate for color removal
Dieterle Demonstration of Spirochetes and Legionella organisms
Dieterle Argyrophillic
Dieterle 10% NBF
Dieterle 4-5 micrometers
Dieterle control Tissue containing spirochetes or legionella
Dieterle 1. Alcohol uranyl nitrate 2. rinse 3. 95% 4. Distilled Water 5. Silver nitrate 6. rinse 7. Developer 8. rinse 9. 10% formic acid 10. rinse 11. dehydrate, clear, mount
Dieterle results Spirochetes, bacteria: Brown to black Background: pale yellow or tan
Steiner & Steiner Demonstation of spirochetes, h pylori, or legionella
Steiner & Steiner Argyrophillic
Steiner & Steiner 10% NBF (Chromate fixatives should be avoided)
Steiner & Steiner 4-5 micrometers
Steiner & Steiner control Tissue with spirochetes, H pylori, or L pnuemonphila must be used
Steiner & Steiner 1. Uranyl nitrate 2, rinse 3. silver nitrate 4. rinse 5. 95% 6. 100% 7. Gum mastic 8. Air dry 9. rinse 10. reduce 11. rinse 12. dehydrate clear mount
Steiner & Steiner results Spirochetes, H pylori, L pnuemophila and other non filamentous bacteria: Dark brown to black Background: Light yellow
Created by: Ziek98



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