Microorganisms Notes EXAM 4

Bacteria ex (1)

• Staph aureus

• Strep pneumonia

• Enterococcus

• Neisseria

• Meningitides

• Neisseria gonorrhoeae

1

Bacteria (2)

• Clostridium perifgens

• tetani

• botulinum

• salmonella, shingella

• pseudomonas

• H. pylori

2

Bacteria (3)

• Influenzae

• chlamydia

• pneumophilia

• borrelia burgdoferi (lyme dx)

• pallidum (syphilis)

• Leptospira interrogans

(3)

Spirochetes characteristic

Argyrophilic: demonstrated w/silver techniques

Bacteria examples (4)

• rickettsiae rickettsii

• typhi

• chlamydiae trachomatis

• actinomyces israelii

(4)

Mycobacteria are…

resistant to decolorization.

• Mycobacterium tuberculosis, leprae, avium intracellulare

Fungi EX

• Aspergillus fumigatus/niger, stachybotrys

• cryptococcus neoformans

• candida albicans

• Pneumocystis jiroveci

• blastomyces dermatitidis

• histoplasma capsulatum

• coccidiodies immitis

Fungi info

• Unicellular (yeasts), multicellular (molds)

• Argentaffin - rx w/metal and CAN reduce to metallic form

• Wall contains chitin (neutral polysaccharide) and glycoproteins

Hyphae

multicellular filiments

Mycelium

mass of interwoven filaments

Sporangia/conidia

spore-forming fruiting bodies that only form if fungus reaches an open cavity, body surface, or luminal surface (bronchi)

Yeasts

• Budding fungi

• Unicellular

Pseudohyphae

elongated buds that form filamentous structures

Dimorphic fungi

Exists as mold or yeast development based on temp

• Room = mold (multicellular)

• Body = yeast (unicellular)

Viruses

DNA or RNA and protein, protein coated genes

• Negri bodies (rabies), hepatitis (ground glass), Herpes (HSV) & cytomegalovirus = owl eyes and intranuclear inclusions

Negri bodies are associated with …

rabies

Ground glass is associated with …

hepatitis

Owl’s eyes and intranuclear inclusions are associated with…

Herpes (HSV) and cytomegalovirus

Protozoans/Parasites EX:

• Entamoeba histolytics, malarial…, giardia, toxoplasma gondii

• leishmania tropica, trapanosoma gambiense

• Pneumocystis jirovecii

Protozoans/Parasites info

Single celled that are motile and feed on organic compounds

Locomotion is classification

IDed with H&E or giemsa diff quik

General acid fast procedure

1. Carbol-fuchsin dye = all red

   • Phenol and alcohol are enhancers

2. Acid alcohol = decolorization/differentiation.

   • Removes dye from non-A.F.

3. Methylene blue = blue stains non-A.F.

General acid fast procedure fixation and other notes

• No alcohol fixatives, NBF is good

• Lipofuscin will stain

General acid fast procedure results

• Mycobacteria - bright red

• background - blue

Kinyoun (cold) and Ziehl-Neelsen (hot/microwave) purpose

Acid fast mycobacteria

• NO dead organisms

• NO leprae

Fite acid fast stain purpose

All mycobacterium

• Yes leprae bc of fragile capsule

Fite acid fast stain procedure

Same as other but…

• Deparaffinize w/xylene-peanut oil → protects fragile waxy capsule

• Sections are AIR DRIED bc alcohol will dissolve capsule

Auramine-Phodamine fluorescence purpose

Acid fast mycobacteria

ALSO dying/non-viable mycobacteria

• Also most sensitive and specific mycobacteria

Auramine-Phodamine fluorescence results

• Mycobacteria - fluoresce yellow

• background - black

Gram + bacteria

Teichoic acid

• compound lipids, glycolipids, peptidoglycans

Gram - bacteria

Lipoplysaccharides, structure is complex

• compound lipids, glycolipids, peptidoglycans

Gram Stain purpose

• Stains + and - that contribute to infections

• also stains fibrin

Gram Stain procedure

1. Crystal violet (basic) → all blue

2. Iodine → mordant that traps dye molecule inside gram + bacteria

3. Acetone or other organic solvent→ dissolve liposaccharide layer of gram - bacteria // also removes dye from dead bacteria

   • Decolors - or dead bacteria

4. Red dye (basic fuchsin, safranin O, neutral red) → stains gram negative and fibrin

5. Yellow dye → counterstain

Gram Stain results

• + = blue/purple

• - and fibrin = red

• Background = yellow-orange

Giemsa method/modified diff quik stain purpose

• Demonstrates H. pylori

• plus gram +/-, rickettsia, amoebae, A.F. bacilli, malarial parasites, fungi, plasmodium

Giemsa method/modified diff quik stain reagents

Polychromatic and neutral dyes → wide color range

Acid water (aggressive) or alcohol = differentiator

Giemsa method/modified diff quik stain results

• H. pylori and nuclei = dark blue

• background = pink-blue

Alcian Yellow-Toluidine Blue Method purpose

H. pylori + demonstration of glycoproteins

Alcian Yellow-Toluidine Blue Method reagents

• Periodic acid = oxidized neutral mucosubstances so they will strain w/cationic alcian yellow dye

• Alcian yellow (basic monoazo) → rx with glycoproteins

• Toluidine blue → Metachromatic basic dye that stains H. pylori and nuclei (also mast cells)

Alcian Yellow-Toluidine Blue Method results

• H. pylori = dark blue

• Mucin = yellow

• Background = pale blue

Hotchkiss-McManus PAS (PAS-Fungus) purpose

Pathological fungi, glycogen, polysaccharides

Hotchkiss-McManus PAS (PAS-Fungus) reagents

• PA oxidizes polysaccharides in fungal walls to aldehydes

• Schiffs → rx with aldehydes = leucofuchsin

• Water rinse → restore chromophore

• Counterstain with fast/light green

Hotchkiss-McManus PAS (PAS-Fungus) results

• Fungi, glycogen, polysaccharides = bright red

• Background = green

Hotchkiss-McManus PAS (PAS-Fungus) other notes

• NO degenerated or non-viable fungi

• Yes = glycogen, polysaccharides, amyloid, glycolipids, colloid, zymogen granules

Chromic acid-Schiff stain will…

overoxidize acidic glycoproteins and GaGs so they will NOT be demonstrated

→ cleaner background

Grocott (Grocott-Gomori) Methenamine Silver Nitrate Method (GMS) purpose

Fungi, degenerated and non-viable fungi

** More specific than PAS-F

GMS reagents

1. Chromic acid (stronger and will X polysaccharides) or Periodic acid = oxidizer

   • Chromic acid = no polysaccharides

   • PA will show them

2. Sodium bisulfate = removes excess chromic acid

3. Methanamine silver solution for impregnation

   • Borax for alkalkine pH

   • Heating not necessary bc fungi is argentaffin

4. Gold chloride = toner

5. Sodium thiosulfate = unreduced silver

6. Light green counterstain

GMS and Warthin Starry Steiner & Diertle Technique fixatives

10% NBF ok

NO metal

GMS results

• Fungi (alive, dead), bacteria, acidic polysaccharides = black

• Background = green

Pneumocystis jirovecii organisms will…

require longer impregnation time.

Insufficient removal alcohol after deparaffinization and hydration will result in…

reduction of chromic acid → shift in solution color from orange to brown.

Warthin Starry Steiner and Diertle Technique purpose

Spirochetes and other bacteria

Warthin Starry Steiner and Diertle Technique reagents

Bc agryophilic, reducer is needed.

1. Uranyl nitrate = sensitizer

2. Silver nitrate = impreg

3. Hydroquinone = reducer

4. Gum mastic = stabilizer

Warthin Starry Steiner and Diertle Technique Results

• Spirochetes and bacteria = black

• Background = yellow-brown bc no toner

How to increase specificity of Warthin Starry Steiner and Diertle Technique?

Shortening impregnation and decreasing the pH

Genta triple stain

• Warthin/Starry/Steiner/Diertle

• Alcian blue

• Hematoxylin and Eosin

Other stains that show bacteria and fungi:

• Mayer Mucicarmine

• PAS

• Colloidal iron

• Alcian Blue

• Cresyl violet

Other stains that show Hep B surface antigen:

• Orcein

• Aldehyde fuchsin

Other stains that show Viral inclusion bodies:

• Phloxine-tartrazine

• Giemsa

• H&E

Other stains that show parasites

• Iron hematoxylin

• PAS

• Giemsa

• Trichrome

• Carbol fuchsin

• GMS

• H&E

Artifactual pigments/artifacts:

• Mercury - B5, Zenker, Helly

• Chrome - acid alcohol

• Formalin - acid hematin

• Malarial pigment

How to remove Mercury

Iodine and sodium thiosulfate/hypo

How to remove Chrome

1% HCl in 70% alcohol

How to remove Formalin and malarial pigment

Alcoholic picric acid or alkaline alcohol

Hemosiderin are…

“storage iron granules”

• Byproduct of hemoglobin degradation

• Hemorrhage hemolytic anemia

• Distinguished with Prussian Blue

Biliverdin and Bilirubin

• Biliverdin = green

• Bilirubin = yellow

• -verdin is reduced to -rubin

• Argentaffin

• Hemolytic diseases, cholesetasis and jaundice

Hematoidin is…

an ironless pigment similar to bilirubin

Melanin

Argentaffin and + w/Schmorl rxn

• Bleaching may be necessary

  • Ox reagents: hydrogen peroxide (1-2 days)

  • Potassium permanganate followed by oxalic acid/potassium metabisulfate within hours will clear it

Lipofuscin

Bleaching may be needed (oxidizer potassium permanganate w/oxalic acid)

• PAS, Sudan black B, carbol fuchsin, Schmorl’s

Melanosis coli

Lipofuscin-laden machrophages

Ceroid

Lipofuscin-like pigment

Urate depostis/Gout

disorder of uric acid metabolism, particularly purines

• NO aqueous solutions

• Birefringence

• Argentaffin

Calcium

Stain basophilic in H&E (Undecalcified)

Inflammatory conditions

Copper

Normally small amounts present and are undetectable

Accumulation = Wilson’s disease

Adrenal chromaffin cells

Adrenaline producing cells: adrenal medulla

• Orth best

• Excessive is pheochromocytomas

Argentaffin cells in GI epithelium and Islets of Langerhan (NO alcohol solutions)

All demonstrated with argyrophil/argentaffin techniques

Prussian Blue purpose

Ferric ions

Prussian Blue principle

1. HCl denatures ferric ions

2. Ferric ions rx with acid potassium ferrocyanide → Prussian blue pigment (ferric ferrocyanide)

3. NFR counterstain

Prussian Blue and Turnbull Blue fixatives

NBF or alcohol

NO acids or chromates

• EDTA is rec for decal

Prussian Blue and Turnbull Blue results

• Ions = blue

• Background = red

Turnbull Blue purpose

Ferrous ions

Turnbull Blue principle

1. HCl

2. Ferrous rx with acidic potassium ferricyanide → insoluble Turnbull Blue pigment (ferrous ferricyanide)

Melanin and ferric ions

Melanin will form complexes with ferrous ions.

Schmorl Technique and Fonatana-Masson purpose

Argentaffin substances (bile, melanin, argentaffin granules, chromaffin)

→ melanoma and neuroendocrine tumors

Schmorl Technique principle

1. Argentaffin substance reduce ferric ions → ferrous ion

2. Ferrous ion combine with potassium ferricyanide.

3. Forms Ferrous Ferricyanide (turnbull blue pigment)

4. Mucicarmine optional for acidic glycoproteins

5. Metanil yellow for counterstain or NFR if no mucicarmine used

Schmorl Technique /// Fonatana-Masson /// Grimelius and Churukian-Schenk Method fixation

NBF

• NO ALCOHOL

Schmorl Technique results

• Argentaffin substances (bile, melanin, chromaffin), lipofuscin = blue

• Mucin = pink/red

• background = yellow

Fonatana-Masson principle

1. Ammonical silver nitrate = impregnation (no ox or sensitizer)

2. Gold chloride = toner

3. Hypo - removes excess silver and gold

4. NFR counterstain

Fonatana-Masson results

• Argentaffin and lipofuscin = black

• Background = pink

Grimelius and Churukian-Schenk Method purpose

Argyrophil AND argentaffin substances

Differentiates neuroendocrine tumore, carcinoid tumor

Grimelius and Churukian-Schenk Method principle

silver method, chem reducer needed

1. Silver nitrate = impregnation

2. Hydroquinone = reducer

3. NFR counterstain

To differentiate btwn argentaffin and argyrophil → perform Fontana-Masson

Grimelius and Churukian-Schenk results

• Argentaffin and argyrophil substance = black

• Nuceli = pink

• Background = yellow-tan

GMS for urate purpose

Urate crystals (argentaffin)

GMS for urate principle

1. NO Oxidizer

2. Methenamine silver = impregnation, no heat bc no reducer needed

3. Sodium thio = remove unred silver

4. Light green counterstain

GMS for urate results

• Urate crystals = black

• background = green

Calcium may be demonstrated if reduced by light

Hall’s Bile stain Fouchet’s technique purpose

Distinguish bilirubin/hematoidin (yellow) from lipofuscin

For liver transplant patients

Hall’s Bile stain Fouchet’s technique principle

Bilirubin (yellow) is oxidized to biliverdin (green) in an acidic solution

1. Fouchet reagent = acidic ferric chloride → oxidizes bilirubin to biliverdin

2. van Gieson counterstain

Hall’s Bile stain Fouchet’s technique Results

• Bile/hematoidin = green

• Muscle (picric acid) = yellow

• Collagen = red

Van Kossa Stain purpose

Calcium phosphate salts

Van Kossa Stain principle

1. Silver iron rx with phosphate and carbonate anions (not specific to calcium and only the anions associated with calcium)

2. Silver phosphate is reduced to metallic silver with light.

3. Sodium thio removes unreduced silver (removed silver carbonate)

4. NRF counterstain

Van Kossa Stain fixatives

NBF good

Alcohol is preferred

NO acidic fixatives and decal solutions

Van Kossa Stain results

• Calcium phosphate and argentaffin substances = brown black

• Background = pink