Simple staining technique
Introduction
It is very difficult to observe microorganisms by our naked eyes because they are very minute and transparent as well as colorless when they are suspended in aqueous medium. The refreactive index of microorganism is not very different from the medium in which they grow, due to which they cannot be observed in unstained preparation. Staining helps to observe the organism clearly.
Chemically a stain (dye) can be defined as an organic compound containing an aromatic compound ie, benzene ring, chromphore and auxochrome group. According to the nature of stain, they are classified into three groups.
- Acidic dye: those dyes which ionizes to give anionic chromogen portion and has a strong affinity towards positively charged constituents of the cell wall are called acidic dyes. Eg. eosin, picric acid, india ink etc
- Basic dye: those dyes which ionizes to give cationic chromogen portion and therefore has strong affinity for negatively charged constituents of the cell wall are called basic dyes. Eg. Crystal violet, methylene blue
- Neutral dye: they are formed by the suitable mixing of two types of dyes, so they contain both cationic and anionic chromogens. They produce precipitate with the cellular components. They are used to stain nucleic acids and cytoplasm. Eg. Sudan IV, eosinate of methylene blue.
Principle
Simple staining technique uses a single stain to visualize the bacteria, which produces a distinctive contrast between the organism and its background. Basic stains (such as methylene blue, crystal violet, and carbol fuchsin) with a positively charged chromogen are preferred in simple staining because bacterial nucleic acids and certain cell wall components carry a negative charge that strongly attracts and binds to the cationic chromogen. They stains the microbial cell, not the background.
The purpose of simple staining is for visualization of morphological shape, size, and arrangement of microbial cells.
Requirements:
- Fresh culture sample: 24-hour agar culture of Staphylococcus epidermidis/ 24-hour agar culture of Bacillus subtilis/ 24-hour agar culture of Escherichia coli
- Stains (Methylene blue or Safranin or crystal violet)
- Bunsen burner
- Inoculating loop
- Microscope
- Distilled water
- Soft cotton or tissue paper
- Toothpicks
- Microscopic Slides
- China-marking pencil or permanent marking pen
Procedure
- Take a clean, grease free glass slide. In order to make the slide grease free, wash it, rinse with alcohol and then clean with filter paper to make it dry.
- Prepare separate bacterial smears of the given sample organism.
- Smears must be heat fixed prior to staining.
- Place a slide on the staining tray and flood the smear with one of the indicated stains, using the appropriate exposure time for each: carbol fuchsin, 15 to 30 seconds; crystal violet, 20 to 60 seconds; methylene blue 1 to 2 minutes.
- Gently wash the smear with distilled water to remove excess stain. During this step, hold the slide parallel to the stream of water; in this way you can reduce the loss of organisms from the preparation.
- Using bibulous paper, blot dry, but do not wipe the slide. Or let it for air dry sometime
- Examine the stained slides under 10X, 40X and then under oil immersion respectively.
- Observe the morphology of the organisms with reference to their shapes (bacilli, cocci, spirilla) and arrangements (chains, clusters, pairs).
- Record your observation in record file.
References
- Cappuccino, J.G and Welsh C. Microbiology: a laboratory manual (2018). Pearson education limited, England. 11 edition.
- Manandhar S, Sharma S (2006): practical approach to microbiology, Graphic plus printers, Kathmandu
- Shah P.K, Dahal P.R, Amatya J (2009): Practical microbiology, Delta offset press, Thapathali, Kathmandu.