Staining is a valuable technique used in microscopy to enhance contrast in the microscopic image. Stains are used to highlight structures in clinical specimens, often when viewed with the aid of different microscopes. Stains have different affinities for different organisms and are used to differentiate types of organisms or to view specific parts of organisms. Staining involves the sample preparation onto slides, fixation (which aims to preserve the shape of the cell), the staining with dyes, and the observation under the microscope. Common Errors During Staining Procedure The duration of each step may vary depending on the concentration and formulation of staining solutions and other reagents. Follow the manufacturer’s instructions where possible. Rinsing stepThe use of tap water is not recommended when making the smears or whenperforming rinse steps in some staining protocols, for example, in the Ziehl-Neelsen protocol, Mycobacterium gordonae has been found in tap water and may interfere with the accurate assessment of the specimen to be stained. Deionized or distilled water is recommended.Excess rinsing between steps could also cause error in a staining procedure. Decolourising stepMany laboratories do not adhere to a fixed decolorizing time for staining protocols and so results may vary. In some laboratories, laboratory staff is taught to add the decolorizing reagent drop by drop until it runs clear. Difficulties in interpreting stain resultsThe staining technique is one factor that affects results. This may be due to differences in applying the steps in the protocols which might warrant analysis if problems in interpretation persist. Standardization of the protocols will minimize variation in results. Other issues that may affect results are:• when cultures have not been sufficiently mixed to break up clumps of cells, the resulting smear can be difficult to read because individual cells are not discernible • partially acid-fast bacteria may also contribute to confusion during a smear evaluation• the type and quality of specimen/smear. Smears that are too thick will not be readable and those that are too thin may result in false negatives or result in the need to repeat the procedure• expired reagents• preparation of reagents – this includes confirming the expiration dates ofreagents and confirming protocols to ensure proper reagent concentrations.Difficulty in reading stains can occur when reagents are not prepared to their right concentrations• improper operation of the microscope Bacteria Staining Procedures 1 Auramine-phenol stain – 1 (acid fast bacilli) This staining technique is used to demonstrate the presence of acid-fast bacilli (Mycobacterium species). These organisms have waxy envelopes that make them difficult to stain and decolorize. A fluorescent stain is used in this method. Auramine stain show higher sensitivity and specificity than Ziehl-Neelsen’s method. It is a better method for screening samples from suspected cases of tuberculosis especially pulmonary and extrapulmonary cases where bacilli count is usually low. Method • prepare smear and heat to fix• flood the slide with Auramine-phenol (1:10v/v) and leave for 10min• gently rinse with water (ensure water is either deionized or distilled)• decolorize with 1% acid alcohol for 3-5min• gently rinse with water as above• repeat acid alcohol step until no further stain seeps from the film • counterstain with 0.1% potassium permanganate or thiazine red for 15sec (this ensures a dark background for the fluorescing alcohol and acid-fast bacilli (AAFB) which are easier to see). KMNO4 stains all epithelial cells making it more difficult to see AAFB• gently rinse with water as above and air dry. Do not blot dry• examine slides using ultraviolet epi-fluorescence microscopy at 25 x or 40 x magnification (the use of a 40 x magnification non-cover-glass (NCG) objective lens will avoid the need to apply a cover glass) InterpretationPositive resultAcid fast bacilli vary from 0.5-10µm in length and stain bright yellow-green against a dark background.Negative resultNo fluorescence observed. Non-acid-fast cells appear dark. Quality control organismsPositive controlMycobacterium species.Negative controlA proven negative smear may be used as a negative control. 2 Gram stain The Gram stain is complex and differential staining technique that remains a useful test performed in microbiology laboratories. The staining procedure differentiates organisms of the domain bacteria according to the cell wall structure. Organisms are classified according to their Gram staining reaction – Gram-positive and Gram-negative. The name “Gram” comes from its inventor, Hans Christian Gram. Gram-positive bacteria have thicker and denser peptidoglycan layers in their cell walls. Iodine penetrates the cell wall in these bacteria and alters the blue dye to inhibit its diffusion through the cell wall during decolorization. Gram-positive bacteria must have an intact cell wall to produce a positive reaction. Gram-negative cells which do not retain the methyl/crystal violet are stained by a counterstain. Neutral red, safranin, or carbol-fuchsin may be used as the counterstain. This technique has also been used for staining of certain fungi such as Candida and Cryptococcus which are observed as Gram-positive yeasts. MethodHucker’s modification of Gram staining technique for the examination of smears• prepare a smear and heat gently to fix• flood the slide with 0.5% crystal violet and leave for 30sec• tilt the slide, and rinse slide gently with water• flood on sufficient (1%) Lugol’s iodine (also known as Gram’s iodine) to rinse off excess water, cover with fresh iodine and allow to remain for 30sec• tilt the slide and wash off the iodine with water• decolorize with 95 – 100% ethanol or acetone until color ceases to run out of the smear• rinse with water• flood the slide 0.1% counterstain safranin and leave to act for about 30sec to 1min Note: It can be counterstained for longer if using other dyes, for example,neutral red for about 2min• wash briefly with water and blot dry• examine the slide using an oil immersion objective to observe cell morphology and Gram reaction InterpretationPositive resultGram-positive organisms stain deep blue/purple.Negative resultGram-negative organisms stain pink/red.Note: Other counterstains (such as carbol fuchsin) used may give more intense colours.Quality control organismsA culture containing Gram-positive and Gram-negative organisms may be used for quality control. Common errors in the Gram staining procedureThese are the errors that arise depending on the method and techniques