The simple staining technique is a widely employed method for detection of bacterial cell shape, size and arrangement. Most of the bacteria contain a unique shape, that falls into three important morphological categories such as Spherical shape (coccus), rod shape (bacillus), and spiral or corkscrew shape (spirillium).
In simple staining we use different types of dyes, based on the type of strains. Some example of dyes are Crystal violet for detection of gram-positive bacteria, safranin for detection of gram-negative bacteria, carbol fuchsin for acid fast bacteria, and methylene blue for non-acid fast bacteria, Malachite green for endospore, etc.
Objective of Simple Staining
The main purpose of simple staining is to determine the cell shape, size, and arrangement of bacterial cells.
Principle of Simple Staining
In simple staining, the bacterial cells are first fixed on a clean, oil-free slide and then flooded with stain (safranin, methylene blue, carbol fuchsin, Crystal violet, etc ). This stain will produce a distinctive contrast between the organism and its background so that we can easily distinguish them.
These stains will readily give up a hydroxide ion or accept a hydrogen ion, as result they become positively charged. Hence, the bacterial cell wall components and cytoplasm are negatively charged, which strongly attracts with the and binds to the cationic chromogen.
- 24 hours old bacterial culture (Bacillus subtilis, Escherichia coli, and Staphylococcus aureus.)
- Methylene blue or crystal violet or safranin
- Staining tray
- Glass slide
- Inoculating loop
- Bunsen burner/spirit lamp
- Blotting paper
Simple staining procedure
- Take a clean slide and make it grease-free or oil-free by washing it with soap and water.
- Air-dry the slide and label it with the organism names.
- Sterilize the inoculating needle by holding the loop tip on the Bunsen burner flame, until the loop tip becomes hot red. Allow the tip to cool.
- Use the other hand to hold the bacterial culture tube and remove the cap. Now, hold the tube tip on flame for 20 sec, it will kill the air-born microorganism. It will prevent contamination.
- Now dip the loop tip into the culture tube and transfer a loop full of bacterial culture on the slide. Spread the bacterial culture on the slide and prepare the smear. After the transfer of culture media, again reflame the tub tip, replace the cap and store the culture media in incubator.
- Air-dry the smear.
- Heat fix the smear by passing it through the blue flame of Bunsen burner for 3 times.
- Now take placed the slide on a staining tray and flood the slide with any of these stains: carbol fuchsin,(15 to 30 seconds), crystal violet (20 to 60 seconds), methylene blue(1 to 2 minutes).
- Gently wash the slide with slowly running tap water, it will remove the excess stain from the slide.
- Blot dry the slide by using blotting paper. Do not wipe the slide
- Repeat these steps for other organisms.
- Now slide is ready to observe under microscope.
Observe the slide under microscope by using the oil-immersion technique and make a drawing of each organism. On the basis of observation write a description of each organisms; indicating color, shape and arrangement of cell.
Simple staining Result Interpretation
- Bacilli and diplobacilli will appear in rod-shape and in purple color (crystal violet).
- Spirilla will appear in spiral-shaped and in purple color (crystal violet).
- Cocci will appear in spherical-shaped and in purple color (crystal violet).
The color of organism can be changed based on the type of stain you used during the staining.