Staining

Simple Staining

• uses only 1 dye

• the color of the dye is the resulting color

• differentiation is impossible

Indirect/Relief/Negative

• the background and not the organism is stained

• bacteria will appear colorless

Indirect/Relief/Negative Example

• India ink

• Nigrosin

Special Staining

demonstrate special features of the cell

Capsular stains

• Hiss

• Anthony’s

Spore stains

• Dorner’s

• Schaeffer and Fulton

• Wirtz Conklin

Schaeffer and Fulton

• spores will appear green

• primary dye:

  • malachite

• counterstain:

  • safranin

    • stains other structures to emphasize presence of spores

Flagellar stains

• Gray’s

• Fisher and Conn

• Leifson

Metachromatic granules

• Albert’s

• Neisser

• Ljubinsky

• Ponder

• Methylene Blue

Differential staining

differentiate one organism from another

Differential staining Example

• Gram stain

• Acid-fast stain

Smear Preparation

organism in media is called culture

1. liquid

2. solid

Smear in liquid media

1. Put the bacteria from the tube on a slide

2. allow to air dry

3. fixate by heat

Smear in solid media

1. get a slide and apply NSS

2. pick up

3. transfer colonies onto a slide and mix

4. allow to dry

5. fixate by heat

Gram Stain

1. Crystal Violet

2. Gram’s Iodine

3. Alcohol

4. Safranin

Primary Stain: Gram Stain

Crystal Violet

• wash w/ tap water after 1 min

Mordant/Fixative: Gram Stain

Gram’s Iodine

• wash w/ tap water after 1min

Decolorizer: Gram Stain

Alcohol, acetone, or alcohol acetone

• wash w/ tap water after 30seconds

Counterstain/Secondary stain: Gram Stain

Safranin

• 3-5 mins

Gram (+)

Violet/purple

Gram (-)

Red/pink

All cocci are gram (+), except:

1. Neisseria

2. Veillonella

3. Branhamella

All bacilli are gram (-), except:

1. Mycobacterium

2. Clostridium

3. Corynebacterium

4. Bacillus

5. Erysipelothrix

6. Listeria

7. Lactobacillus

Higher forms of organisms are gram (?)

Gram (+)

• Actinomyces

• Streptomyces

• Yeast

• Mold

not Gram Stained

• Rickettsia

• Chlamydia (intracellular)

• Mycoplasma

• Ureaplasma (wall less)

• Spirochetes (can’t resolve by bright field)

Acid Fast Staining

1. Carbolfuchsin

2. Heat/Steam

3. Alcohol or 3% HCl

4. Methylene Blue

Hucker’s Modification

gram staining technique for fungi

Ways to improve gram staining of GRAM (-) organisms

• 0.1% or 0.2% Carbol Fuchsin instead of Safranin

• Prolong contact of stain to the organism

Acid fast Organisms (AFO)

• Red against blue bg

• will take the color of the primary dye

  • red against blue bg

Non Acid Fast Organisms (NAFO)

• blue/green

• will take the color of counterstain

  • blue/green

Mycolic acid or hydroxymethoxy acid

makes AFO

• difficult to stain, and

• difficult to decolorize once stained

All bacteria are NAFO, except:

Genus Mycobacterium

Partially AFO

Nocardia

Facilitate AFS

• Steaming process

• Increasing conc. of phenol & basic fuchsin

• Prolonging contact of stain w/ the material

• Addition of wetting agent (Tergitol) prior to the stain solution

Steaming Process

removes mycolic acid temporarily

Use of wetting agent

e.g tergitol

removes mycolic acid temporarily

2 Most Common Methods of AFS

1. Ziehl Neelsen Method

2. Kinyoun’s Method

Ziehl Neelsen

• Hot Method

• Best for Direct Sputum Smear Microscopy (DSSM)

Kinyoun’s

• Cold Method

• Best to detect AF Bacilli in tissue specimens

Primary Dye: Ziehl Neelsen & Kinyoun’s

Carbol Fuchsin

Mordant: Ziehl Neelsen

heat/steaming

Mordant: Kinyoun’s

wetting agent:

• tergitol

Decolorizer: Ziehl Neelsen & Kinyoun’s

3% acid alcohol (HCl + ethanol)

Counterstain: Ziehl Neelsen & Kinyoun’s

Methylene Blue or Malachite Green

Other Methods of AFS

1. Pappenheim’s

2. Baumgartens

3. Fite Faraco’s

   1. Fluorochrome Staining

Pappenheim’s

Differentiates Mycobacterium smegmatis (blue) from M. tuberculosis (red)

Baumgartens

Differentiates Mycobacterium leprae (blue) from M. tuberculosis (red)

Fite Faraco’s

Mycobacterium leprae

Counterstain: Fite Faraco

hematoxylin

Fluorochrome Staining

• Auramine-Rhodamine

• Most sensitive

Primary dye: Fluorochrome

Auramine-Rhodamine dye

Mordant: Fluorochrome

None

Decolorizer: Fluorochrome

1.5% Acid alcohol (HCl+alcohol)

Counterstain: Fluorochrome

Quenching agent

0.5% Potassium Permanganate

AFO Result: Flurochrome

Yellow against black bg