The staining of microorganism requires preparation and proper fixation of smear on glass slide.
Smears should be spread evenly covering an area of about 15-20 mm diameter on a slide.
- From purulent specimens (Pus, turbid urine etc.): Take a loopful of specimen with the sterile wire & spread thinly on the slide.
- From non purulent specimens: Centrifuge the specimen & make a smear from the drop of a well mixed deposit.
- From sputum: Use a piece of clean stick to transfer & spread purulent & caseous material on a slide.
- From culture plate: Put a drop of saline on a clean slide. With the help of help of sterile wire, individual colony is picked up & mixed with saline drop to prepare a thin emulsion.
Labeling: Every slide should be labeled with number or letters by diamond or grease pencil.
Fixation: Smear is fixed to preserve microorganisms & to prevent being washed from slides during staining.
Method of smear fixation:
- Heat fixation:
- Allow the smear to air-dry.
- Rapidly pass the smear 3 times above the flame of spirit lamp or Bunsen burner.
- Allow the smear to cool before staining.
- It can damage the organism & alter the staining reaction especially when the excessive heat is used.
- It damages leucocytes. So it is unsuitable for the smear which contain intracellular organism like N. gonorrhoeae & N. meningitidis
- Alcohol fixation:
- Allow the smear to air-dry.
- Add 1-2 drops of absolute methanol or ethanol.
- Leave the alcohol on the smear for 2 minutes or until it evaporate.
- Less damage to microorganisms than heat.
- Pus cells are well persevered so recommended for observation of intracellular organisms.
- It is more bactericidal than heat, so it is preferable to fix the smear prepared from sputum. (M. tuberculosis).
- Other chemical fixatives:
- Potassium permanganate for Anthrax bacilli
- Formaldehyde vapour for Mycobacterium species.
The microscopic examination of clinical materials is important because of the following reasons;
- The number and percentage of neutrophils that are present usually indicate the magnitude and type of inflammatory response.
- The quality of the specimen can be validated and the observation of bacteria, mycelial elements, yeast forms, parasitic structures, or viral inclusions may provide sufficient information to tender an immediate presumptive diagnosis, leading to specific therapy.
- Direct microscopic examination may also give immediate presumptive evidence that species of anaerobic bacteria are present.
A number of techniques may be used in the direct microscopic examination of clinical specimens, either to demonstrate the presence of microorganisms or to observe certain biochemical, physiological, or serologic characteristics.
- Saline mounts:
- It is used to determine biologic activity of microorganisms, including motility or reactions to certain chemicals, or serologic reactivity in specific antisera.
- Hanging-drop procedure:
- The hanging-drop mount serves the same purpose as the saline mount, except there is less distortion from the weight of the cover slip and a focus into the drop can be achieved. This technique is generally used for studying the motility of bacteria.
- Iodine mounts:
- Iodine mount is usually used in parallel with saline mount when examining feces or other materials for intestinal protozoa or Helminthes ova. The iodine stains the nuclei and intracytoplasmic organelles so that they are more easily seen.
- KOH mounts:
- The KOH mount is used to aid in detecting fungus elements in thick Mucoid material or in specimen containing keratinous material, such as skin scales, nails, or hair. The KOH dissolves the background keratin, unmasking the fungus elements to make them more apparent.
- India ink preparation:
- India ink or nigrosin preparation is used for the direct microscopic examination of the capsules of many microorganisms. The fine granules of the India ink or nigrosin give a semiopaque background against which the clear capsules can easily be seen. This technique is particularly useful in visualizing the large capsules of Cryptococcus neoformans in CSF, sputum, and other secretions.
- Dark field examination:
- Dark field examinations are used to visualize certain delicate microorganisms that are invisible by bright field optics and stain only with great difficulty. This method is particularly useful in demonstrating spirochetes from suspicious syphilitic chancre for T. pallidum.
Stains in microbiology:
Supravital stain: Stain which is toxic and kill the cell on staining.
Vital stain: Stain which is non-toxic and the cell remains viable during staining.
Types of stain:
- Simple stain:
- Negative stain:
- Silver impregnation stain:
- Differential stain:
- Special stain
Biologic stains are generally required to visualize bacteria adequately and demonstrate the finer detail of internal structures. Stain consists of aqueous or organic preparation of dyes or groups of dyes that impart a variety of colors to microorganisms.
The following is a brief description of the stains most commonly used.
- Loeffler’s methylene blue:
- This is a simple direct stain used to stain a variety of microorganisms, specifically used to detect bacteria in CSF smears in suspected case of bacterial meningitis.
- Gram stain:
- This is a differential stain used to demonstrate the staining properties of bacteria of all types.
- Gram-positive bacteria retain the crystal violet dye after decolorization and appear deep blue. Gram-negative bacteria are not capable of retaining the crystal violet dye after decolorization and are counterstained red by saffranine dye. Gram-staining characteristics may be atypical in very young, old, dead, or degenerating cultures.
- Ziehl-Neelsen acid-fast stain:
- Acid-fast bacilli are so called because they are surrounded by a waxy envelope that is resistant to staining. Either heat or detergent is required to allow the stain to penetrate the capsule. Once stained, acid-fast bacteria resist decolorization with sulphuric acid and appear red due to crystal violet stain where other bacteria are destained with the acid alcohol and appear blue due to counterstain methylene blue.
- This fluorochrome dye stains mycobacteria selectively by binding to the mycolic acid in the cell wall. This stain demonstrates mycobacteria better than conventional acid-fast stains and permits screening of smears at lower magnification because organisms are more easily seen.
- Acridine orange is a stain particularly well adapted for the demonstration of bacteria in blood culture broth, CSF, urethral smears, or other smears where they may be present in relatively small numbers or when they are obscured by a heavy background of leukocytes or other debris. At pH below 4.0, bacteria and yeast cells stain brilliant orange against black, light green or yellow background.
- Wright’s-Giemsa is commonly used for staining the cellular elements of the peripheral blood smear. It is useful in microbiology for the demonstration of intracellular organisms such as H. capsulatum, Leishmania, and Malaria species. The stain is also useful in demonstrating intracellular inclusions in direct smears of skin or mucous membranes, such as corneal scrapings for trachoma.
- Lactophenol cotton blue:
- Because of sulphonic groups, the dye is strongly acidic and has been used as a counterstain for unfixed tissues, bacteria, and protozoa, in combination with other dyes. Currently it is most commonly used for the direct staining of fungal mycelium and fruiting structures, which take on a delicate light blue color.