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COMMON STAINING COMMON STAINING PROCEDURESPROCEDURES

COMMON STAINING COMMON STAINING PROCEDURESPROCEDURES

Staining of the clinical material or the bacteria from colonies on laboratory media provide a direct visualization of the morphology of the organisms as well as their reactions to the chemicals present in stains. This is an invaluable and easy-to-use tool for establishing the identity of various microorganisms.

Staining techniques• To enhance contrast• To identify bacteria• To identify bacterial structures• Rapid and cost effective

Microscopy • Light microscopy (10X, 40X, 100X)

• Magnification, resolution and contrast

• Phase contrast microscopy• Dark field microscopy• Fluorescent microscopy• Electron microscopy

Light Microscope

Phase contrast microscopy

Dark field microscopy

Wet mounts• Determine the cellular composition,

morphology of the organisms, their structure and motility

e.g. wet mount for stool, urine and other aspirates10% KOH for fungal hyphae in skin

scrapings, hairs and nails

Chemical basis of staining

• Cellular material and organisms are themselves transparent

• Best distinguished by dyes (chemicals or biological)

• Chromophoric groups in the dyes are responsible for the color

• Auxophores in the dyes give it the affinity to bind to the cellular structures

Making films/ smears• Cover slip or a clean dry glass slide; should

be grease free• Fluid material or solid material• Take a loopful and spread thinly• Let dry• Fix the smear• Stain • Impression smears from biopsy material

Types of staining techniques

• Simple stains• Differential stains• Negative staining• Impregnation techniques

Simple stains• Show presence of organisms and

nature of cellular exudate• Loeffler’s Methylene Blue• Polychrome Methylene Blue• Dilute Carbol Fuchsin• Borax Methylene Blue

Differential Stains• Impart different colors to different

bacteria or bacterial structures• Gram’s Stain• Zeihl Neelsen Stain• Albert’s Stain• Spore stain• PAS stain• Romanowsky stains

Gram Staining

ZN Staining• Acid fast organisms• Mycolic acids in the cell wall• Resist decolorisation by dilute acid

Ingredients and preparations

Carbol fuchsin 1%Sulphuric acid 20%Methylene blue 0.1%

• Make a smear from yellow purulent portion of the sputum using a bamboo stick. A good smear is spread evenly, 2 cms x 3 cms in size and is neither too thick nor too thin. The optimum thickness of the smear can be assessed by placing the smear on a printed matter, the print should be readable through the smear.

• Let the smear air-dry for 15-30 minutes. • Fix the smear by passing the slide over the flame

3-5 times for 3-4 seconds each time. • Place the fixed slide on the staining rack with the

smeared side facing upwards. • Pour filtered 1% carbol fuchsin over the slide so

as to cover the entire slide.

• Heat the slide underneath until vapours start rising. Do not let carbol fuchsin to boil or the slide to dry. Continue the process up to five minutes.

• Allow the slide to cool for 5-7 minutes. • Gently rinse the slide with tap water to remove the

excess carbol fuchsin stain. At this point, the smear on the slide looks red in colour.

• Decolor the stained slide by pouring 20% sulphuric acid on the slide and leaving the acid for 2-4 minutes.

• Lightly wash away the free stain. Tip the slide to drain off the water.

• If the slide is still red, reapply sulphuric acid for 1-3 minutes and rinse gently with tap water.

• Counter stain the slide by pouring 0.1% methylene blue solution onto the slide and let it stand for one minute.

• Gently rinse the slide with tap water and tip the slide to drain off the water.

• Place the slide in the slide tray and allow it to dry.

• Examine the slide under a microscope using 40 x lens to select the suitable area of the slide and examine under 100 x lens using a drop of immersion oil.

Albert staining

Ingredients and preparations1. Albert stain I • Toluidine blue 0.15 gm

Malachite green 0.20 gmGlacial acetic acid 1.0 mlAlcohol(95%) 2.0 mlDistilled water 100 ml

2. Albert stain II • Iodine 2.0 gm

Potassium iodide 3.0 gm Distilled water 300 ml

Staining procedure

• Cover the heat-fixed smear with Albert stain I. Let it stand for two minutes.

• Wash with water. • Cover the smear with Albert stain II. Let it stand

for two minutes. • Wash with water, blot dry and examine. Uses• To demonstrate metachromatic granules in C.

diphtheriae. These granules appear bluish black whereas the body of bacilli appear green or bluish green.

Alberts Staining

Romanowsky stains• It is a polychromatic stain that contains a mixture

of methylene blue, azure B (from the oxidation of methylene blue), and eosin Y dissolved in methanol. The eosin ions are negatively charged and stain the basic components of the cells orange to pink, while the other dyes stain the acidic cell structures various shades of blue to purple.

• It is primarily used for the differentiation of intracellular and extracellular circulating blood parasites (e.g., Plasmodium, Babesia, and Leishmania spp.), fungi (e.g., Histoplasma spp., yeast cells, Pneumocystis spp.), rickettsiae, chlamydiae, and viral inclusions.

Spore stains

PAS stain

Negative stainingBacteria are mixed with stains that

impart a uniform colored background against which the unstained bacteria or organisms stand out. Used for capsules and cryptococcus

• India Ink• Nigrosin Stain

Impregnation techniquesCells and structures too thin to be

seen under ordinary microscopes may be rendered visible if they are thickened by impregnation of silver on the surface. Used especially for flagella and spirochaetes

Fluorescent stainsFluorescence is dependant on the ability of

fluorophores or fluorochromes to absorb the energy of UV light and emit energy in the form of longer visible wavelengths which appear as fluorescence in a microscope

• Acridine orange• Auramine rhodamine • Calcofluor white • FITC

Fluorescent microscope

• Quality control of stains• Test all stains at appropriate intervals for their ability to

distinguish positive and negative organisms and document the results. The performance standards for Ziehl-Neelsen and Gram staining are as given below: Stain Control organism/ material ATCC No* Expected result Ziehl-Neelsen Mycobacterium spp.E. coli2517725922 Pink red bacilliBlue bacilliGram E. coliS. aureus2592225923 Gram -ve bacilliGram +ve cocciIodine solution Formalin treated stool specimen with cysts Visible cyst nuclei