COMMON STAINING PROCEDURES
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-touse 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 aspirates 10% 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 preparations 1. Albert stain I Toluidine blue 0.15 gm Malachite green 0.20 gm Glacial acetic acid 1.0 ml Alcohol(95%) 2.0 ml Distilled 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 staining Bacteria 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 techniques Cells 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 stains Fluorescence 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. coli25177 25922 Pink red bacilli Blue bacilligram E. coli S. aureus25922 25923 Gram -ve bacilli Gram +ve cocciiodine solution Formalin treated stool specimen with cysts Visible cyst nuclei