ENDOSPORE STAINING

ENDOSPORE STAINING (Schaeffer-Fulton method)

Aim:  

To determine the spore forming (Endospore) bacteria by Schaeffer-Fulton method

Principle :

Endospore staining is used to visualize specialized cell structures. The endospore stain is used to determine the highly resistant spores of certain microorganisms within their vegetative cells. The multiple thick coats of the spore made the endospore resistant to stain with most dyes. In Schaeffer-Fulton method, the primary stain, Malachite Green, is added over the heat fixed bacterial smear and heated over a steam bath for few minutes. This will soften the hard outer coverings of the spore and the primary stain gets stick to the spore. When taken from the steam bath followed by further cooling hardens the outer layer of the spore. During this stage both the spore and vegetative cells appear as green in color. But later the thick outer layer makes the spore resistant to the action of decolorizing agent (water), but however, water can easily decolorize the vegetative cells.  When counterstained with Safranin, vegetative cells are easily stained with Safranin, and the cells appear in red or pink color.

Materials required

Clean glass slide in box

Inoculation loop

Test organism

Absorbent paper

Boiling water in water bath

Water in wash bottle

Microscope

Primary Stain – Malachite green

Counter stain - Safranin

Procedure

1. Aseptically transfer the bacterium with an inoculating loop to a clean glass slide and prepare a thin smear of the bacterium.

2. Air dry and heat-fix the bacterial smear.

3. Cover the bacterial smear with a piece of absorbent paper cut to fit the smear and slide.

Place the slide over a container of boiling water.

4. Saturate the absorbent paper with malachite green stain solution and steam for 5 minutes.  Keep the paper moist by adding more stain as required.

5. Remove the absorbent paper using forceps, allow the slide to cool, and rinse the slide with water for 30 seconds.

6. Wash the slide with water.

7. Counter stain with safranin for 30 seconds.

8. Wash the slide with water and blot dry the slide.

9. Examine the slide under the oil immersion lens for the presence of endospores.

Result: 

 On microscopic observation endospores appear in green color and the vegetatives cells as pink.

Micrometry

           Determination of Microbial Cell Size by Micrometry

Aim:

            To measure the dimensions of microorganisms with the help of a microscope.

 Principle: 

Microorganisms are microscopic objects that are visible only with the help of a microscope.  Sometimes it is necessary to measure its dimensions (length breadth and diameter) for its identification process. But, determination of the size of a microorganism is not an easy process. Micrometry refers to the measurement of dimensions of the desired microorganisms under a microscope which uses two micro-scales known as ‘micrometers’. At first, the diameter of the microscopic field must be established with the help these micrometers namely ocular micrometer and stage micrometer. Ocular micrometer with microscopic graduations etched on their surfaces is a circular glass disc that fits into the circular shelf inside the eyepiece of the microscope. It has 100 equally spaced divisions marked as 0 to 10. Depending on the objective being used, the distance between these graduations will vary that determines the size of the field. The stage micrometer is clipped to the stage of the microscope.  In the centre of the stage micrometer a known 1mm distance is etched into100 equally spaced divisions making each division equals 0.01 mm or 10 µm. 

Calibration:

            The calibration procedure for the ocular micrometer to requires that the graduations on both micrometers to be superimposed on each other. The number of ocular divisions that corresponds to the known distance in the stage micrometer is determined. Finally the calibration calculated as follows.

                                                              Number of divisions on stage micrometer

One division of ocular micrometer = ____________________________________ x10

                                                               Number of divisions on ocular micrometer

After calibration, the ocular micrometer measures the size of various microbes including its length, breadth, and diameter. First count the number of spaces occupied by the organism on ocular micrometer graduations. Then multiply this number by calibration factor.

Materials Required:

Stage micrometer

Ocular micrometer

Microscope

Microscopic slide

Microbial culture

Procedure

1. The eyepiece is removed from the microscope, and its top lid is unscrewed. The lid is removed. Carefully, the eye lens is removed. The ocular micrometer (a circular etched glass piece, which slips into the eye piece) is placed carefully into the eyepiece. The eye lens is placed back and the top lid is screwed to its original condition. The eyepiece is placed back in the microscope.
2. The stage micrometer is clipped to the stage and the etchings centered by moving the mechanical stage.
3. Adjust ocular and the objectives (10x, 45 x, 100x) which is to be calibrated.
4.  Superimpose the graduation of the slide with that of the ocular such that the line on the slide at one end exactly coincides with first line on the ocular. This done by rotating ocular and moving the stage micrometer.
5. Count the number ocular meter divisions coinciding between two lines of both ocular micrometer and stage micrometer.
6. The size of the bacterial cell is determined by counting the number of divisions in ocular micrometer occupies by single cell.

Result:

The size of the given microbial cell determined as ___________ (length, breadth) in 10x/40x/100x.