Flagella staining is a technique which examines the presence and arrangement of bacterial flagella under the microscope. It is a specialized staining method, which requires a combination of special reagents to stain the bacterial flagella. The flagella appear as a narrow appendage, which cannot be visualized by employing common stains.
Therefore, the flagella staining makes the use of mordant to thicken the bacterial flagella that are too thin to be stained. Then, the flagella are stained by special flagella stains and observed under the light microscope. The flagella staining can be done by two methods, wet-mount staining or Ryu method and Dried-smear method or Leifson’s staining technique.
Content: Flagella Staining
- Bacterial Flagella
- Result Interpretation
Flagella staining can define as one of the specialized staining techniques that make the use of mordant and special stains to visualize the flagella. It is quite a complicated method, which requires specific considerations, proper handling and operation, due to the fragility of the bacterial flagella.
The purpose of flagella staining is to check whether a bacteria is motile or non-motile. Secondly, through this special staining technique, the shape, size, location and arrangement of the bacterial flagella help in genus and species differentiation.
Bacterial flagella are much thinner and simpler in structure. It possesses a thread-like, twisted appendage that attaches through the cell membrane of the bacterial cell and protrudes out to facilitate motility. The localization of the bacterial flagella can be polar or lateral, depending upon different species. Generally, the bacterial flagella comprise three components, namely a basal body, hook and the filament.
The basal body connects the flagella to the cell membrane and cell wall. A hook is generally short, and the filament appears as a whip-like structure that is several times longer than the bacterial cell. The filament consists of a protein called flagellin. The length of bacterial flagella varies from 20 µ to 200 µ. Flagella are mainly found in rod shaped bacteria and very few in cocci shape bacteria.
Methods of Flagella Staining
It involves two standard techniques, namely wet mount and Leifson’s staining method.
Wet Mount Method
It is a more successful and easy method to practice for routine use. It also refers as “Ryu method” that makes the use of Ryu flagella stain when the arrangement and number of flagella are critical to identifying the species of motile bacteria. For visualization, a mordant is used in staining procedures so that the stain sticks in layers to the flagella.
Preparation of Ryu stain
Ryu stain involves the preparation of two solutions (Solution-I and II).
Solution-I includes the following components in a defined amount:
- 5% aqueous solution of phenol: 10 ml
- Tannic acid: 2 g
- An aqueous solution of aluminium potassium sulphate-12 hydrate: 10 ml
Solution-II contains a saturated ethanoic solution of crystal violet, where 12 g of crystal violet is mixed in 100 ml of 95% ethanol.
The final stain is prepared by mixing solution-I and II in a ratio of 10:1. Then the coarse precipitated particles are removed from the stain via filtration through the filter paper, and then stored at room temperature.
- The organism needs to grow at room temperature on a blood agar medium for 16 to 24 hours.
- Put a drop of saline onto the microscope slide.
- Now take a sterilized inoculating loop and remove little inoculum from the culture plate by touching the margin.
- Then add the inoculum into the drop of water placed on the glass slide.
- After that, the glass slide is left undisturbed for about 15-20 minutes.
- Then place the coverslip to the faintly turbid drop of water, and then immediately view it under the power of 40-50X of the objective lens.
- If the motile cells are visible, then, the process is followed by staining the bacterial culture. A drop of Ryu flagella stain is added towards the one edge of coverslip, which will penetrate the bacterial suspension via capillary action.
- Then after 10 minutes, observe the glass slide under the light microscope upto the power of 100X.
- Finally, note down the results by examining the presence, number and arrangement of the flagella.
As flagella are too thin hair-like structure, so it’s not possible to visualize under the microscope. Therefore, this technique makes the use of Leifson dye by mixing a mordant (tannic acid) and (stain) basic fuschin formed in an alcohol base.
When the Leifson’s stain is treated with cell, the tannic acid available gers attached to flagella and after that available alcohol got evaporated and after evaporation of alcohol, flagella thickness got increased because of deposition of tannic acid. After the above treatment, cells are required to be treated by Methylene blue stain. This will stain the cell.
Preparation of Leifson’s Stain
To prepare leifson stain, we require the following contents in the given amount:
Ammonium or potassium alum, saturated water solution: 20 ml
Tannic acid in 20% water solution: 10 ml
Distilled water: 10 ml
95% of ethyl alcohol: 15 ml
Saturated ethyl alcohol solution of basic fuschin: 3 ml
- Firstly take flagellated cell culture slant and put this for 2 hours and then by using a sterile pipette, put two to three droplets of distilled water into the culture slant slowly without disturbing cell growth.
- Now incubate the slant for 20 minutes after adding water into it.
- Now take one drop from above-prepared suspension and put it on a clean slide and then place the slide in an inclined position.
- Then drop needs to flow from one end to another end of the slide to restrict the flagella folding on cell.
- Now the smear is allowed to dry in the air only.
- After complete drying of slide in the air, it needs to be flooded in the Leifson’s stain until we get a shiny thin film.
- Then wash the slide gently with water.
- Now the treatment of slide is required for 1 minute with 1 % methylene blue.
- Finally, observe the slide by putting a drop of oil immersion, after washing slide with water and air-drying.
Therefore, the flagella staining can be used to check the motility in the bacteria and to classify them depending upon its presence, absence, number and arrangement on the bacterial cell, under the light microscope.