STAINING of MICROORGANISMS

BACTERIA

These are infectious microorganisms that lack nuclei but have a cell wall composed of two phospholipid bilayer membranes separated by a peptidoglycan layer

• staphylococci

• streptococci

• listeria

• clostridium

GRAM-POSITIVE BACTERIA:

• escherichia

• citrobacter

• salmonella

• shigella

• klebsiella

• enterobacter

• proteus

• bacteroides

• neisseria

• pseudomonas

• brucella

GRAM-NEGATIVE BACTERIA:

Negative Staining

In this staining technique, an acidic, anionic dye is used to change the color of the background, not the cells, causing the cells to stand out

Simple Staining

Are single dyes used to stain the organism and it has limited clinical application.

The dye is negative and the bacterium is positively charged and they will get stained due to its interaction with a negatively charged dye.

Differential Staining

procedure that takes advantage of differences in the physical and chemical properties of different groups of bacteria.

1. Gram Stain

2. Gram-positive bacteria

3. Gram-negative bacteria

1. The basis for the differential stain reaction is due to a difference in the permeability structure and chemical components of the bacterial cell wall

2. bacteria have a thicker cross-linked wall of peptidoglycan and are not lipid-rich

3. bacteria have very little peptidoglycan but do have an outer layer of lipopolysaccharide and they have a greater lipid content in their cell walls

Gram-Twort Stain

Stain for bacteria

This stain can be used effectively in place of neutral red as a counterstain in the basic Gram method

The green counterstain facilitates the detection of red-staining Gram-negative organisms.

Mycobacteria

These bacteria are difficult to demonstrate by Gram's technique because they have a fatty acid capsule that influences the penetration and resistance to removal of the stain by acid and alcohol

1. Mycobacterium tuberculosis

2. Mycobacterium avium intracellulare

MYCOBACTERIA:

1. associated with caseating granulomas and the presence of 1-2 µm, blunt-ended, acid and alcohol fast bacilli. an opportunistic infection associated with AIDS.

2. a group of intracellular opportunistic bacteria that cause an often lethal infection in the immunosuppressed patients, particularly in association with AIDS

ACID-FAST STAIN

is a differential stain that distinguishes organisms with waxy cell walls that can resist decolorization with acid alcohol.

BROWN-BRENN STAIN

For gram-pos and gram-neg bacteria in paraffin sections; Nocardia and Actinomyces​

ZIEHL-NEELSEN METHOD

For acid fast bacilli

Auramine-rhodamine stain

MYCOBACTERIA:

The most sensitive stain for mycobacteria which requires a fluorescence microscope for viewing.

WADE-FITE / FITE STAINS

Modification of Ziehl-Neelsen method

Is a specific way to identify mycobacteria, which are not readily demonstrated by other methods including Gram stain

Stain for Leprosy bacilli and nocardia

Helicobacter pylori

The the organism that is strongly implicated as a cause of chronic gastritis. It is seen in endoscopic biopsies as small, weakly hematoxylin-staining bacilli in the lumen of gastric glands, often adherent to the luminal surface of epithelial cells.

1. Toluidine Blue Stain

2. Cresyl Violet Acetate Method

STAIN/S FOR Helicobacter pylori

(2)

Legionella pneumophilia

a small Gram-positive coccobacillus that was first identified in 1977 as the cause of a sporadic highly lethal type of pneumonia

It is generally spread in aerosols from stagnant water reservoirs, usually in air-conditioning units.

1. Treponema pallidum

2. Leptospira interrogans

SPIROCHETES

1. This is the spirochetes that causes syphilis.

2. This causes leptospirosis or Weil’s disease

Dieterle Method

Modified Steiner Silver Stains

Stains that demonstrate Spirochetes and Legionella

(2)

Warthin-Starry Method

For demonstrations of spirochetes are colored black while golden yellow background

Modified Steiner and Steiner

Stains for Spirochetes, Donovan Bodies, Fungi, and Bacteria

with Black color as its result

Grocott Methenamine Silver

Microsporum, Trichophyton, Aspergillus and Candida albicans are well demonstrated in this stain

Polysaccharides in the fungal cell wall are oxidized to aldehydes by chromic acid, a strong oxidant that further oxidizes many of the newly released aldehyde groups to breakdown products that will not react

Stains these fungi black

Lendrum’s Phloxine-Tartrazine Method

For Viral Inclusions

All tissue is stained red with Phloxine which is then differentiated for displacement with counterstain, tartrazine.

Orcein Method

For Hepatitis B Surface Antigen

This method is based on permanganate oxidizing the sulfur-containing proteins to sulfonate residues that can then react with orcein

Giemsa Stain

For Parasites

Diluted version of this stain is recommended for blood and bone marrow parasites inclusion conjunctivitis, Toxoplasma, spirochetes and other bacteria.