Metachromatic Granule Staining

Metachromatic Granule Staining

Aim: To stain the metachromatic granules by Albert’s staining method

Approach:

        Metachromatic granules were first discovered in Spirillum volutans, so the name volutin granules. They are also called Babes-Ernst granules, after their inventors and metachromatic granules, because they show metachromatic effect. (Gr. Meta = change; chroma = color) appear red when stained with blue dyes such as Methylene blue or Toludine blue. Chemically, volutin granules are polyphosphates and strongly basophilic demonstrated by using special staining techniques such as, Albert's method. By electron microscopy they appear as electron dense bodies.

       Starvation of the cells for any nutrient leads to volutin formation. Sulphate starvation is particularly effective and leads to a rapid and massive accumulation of polyphosphate.
Volutin granules have been observed in a wide variety of bacteria and blue green algae. The bacteria include species of Lactobacillus, Corynebacterium, Spirillum, Pseudomonas, and Desulfovibrio.

        The formation of polyphosphate occurs by the sequential addition of phosphate residues to pyrophosphate, ATP serves as phosphate donor.

       Metachromatic granules have a strong affinity towards basic dye, Toludine blue, Methylene blue. When basic dye is used to stain the organism that contain the metachromatic granules are stained much more intensely (dark) than the other part of the cell. The cell typically looks like a string of beads because of more deeply stained granules. In some cells granules occur at the end of the cell resulting in bipolar staining appearance.

Principle:
Albert staining is a type of differential stain used for staining the volutin granules also known as Metachromatic granules or food granules found in Corynebacterium diphtheriae.
Albert stain is basically made up of two stains that is Toluidine blue ‘O’ or Methylene Blue and Malachite green both of which are basic dyes with high affinity for acidic tissue components like cytoplasm. The pH of Albert stain is adjusted to 2.8 by using acetic acid which becomes basic for volutin granules as pH of volutin Granule is highly acidic.
Therefore, on applying Albert’s stain to the smear, Toluidine blue ‘O’ or Methylene Blue, stains Volutin Granules i. e the most acidic part of cell and Malachite green stains the cytoplasm blue-green. On adding Albert’s iodine due to effect of iodine, the metachromatic property is not observed and granules appear blue in colour.

Requirements:

1.      Bacterial Culture or curd sample

2.      Albert’s stain

Solution A

    Methylene blue / Toludine blue        -       0.15 gm

    Malachite green                            -       0.2 gm

    95 % ethanol                                -       2 ml

Solution B

    Glacial acetic acid                         -       1 ml

    Distilled water                            -       100 ml

Mix solution A & B

 

3.      Albert’s iodine

        Iodine                                        -       2 gm

        Potassium iodide                           -       3 gm

        Distilled water                             -       300 ml

Procedure:                                 

1. Prepare the smear of the given bacterial suspension or curd sample, air dry and heat fix it.

2. Treat the smear with xylene fir 30 second to 1 minute, if curd sample is used.

3. Discard the xylene & cover the smear with Albert’s stain for 5 - 7 minutes.

4. Drain of the excess stain do not water wash the slide with water.

5. Cover the smear with Albert’s iodine for 1 to 2 minutes.

6. Wash smear with tap water, air dry it & observe under the oil immersion objective.

Observation table:

Result:

        By Albert’s method, if Corynebacterium diphtheria is present in the sample it appears green colored rod-shaped bacteria arranged at angle to each other, resembling English letter ‘L’, ‘V’ or Chinese letter pattern along with bluish black metachromatic granules at the poles.

Conclusion:

        Metachromatic granules are reserved food materials. They are highly acidic than the cytoplasm. Due to this character, the metachromatic granules get stained with Toludine blue or M. B. present in the Albert’s stain which is basic in nature. The cytoplasm is less acidic so it gets stained by malachite green. The Albert’s iodine acts as mordant. The acetic acid acts as fixative.



Antibiotic Sensitivity

 

Disc Diffusion Method

Aim:  To test the pathogens for antibiotic sensitivity by disc diffusion method.

Theory:

Once the causative organism of a specific disease in a patient has been isolated, it is up to the attending physician to administer a chemotherapeutic agent that will inhibit or kill the pathogen without causing serious harm to the individual. The method must be relatively simple to use, be very reliable, and yield results in as short a time as possible. The Kirby – Bauer method of sensitivity testing is such a method.

The Kirby-Bauer method is not restricted to antibiotics. It may also be used to measure the sensitivity of any microorganism to a variety of antimicrobial agents such as sulfonamides and synthetic chemotherapeutics. Antibiotics are chemotherapeutic agents of low molecular weight produced by microorganisms that inhibit or kill other microorganisms. Drugs, on the other hand, are antimicrobic agents that are man – made.

Principles:

        One method that is used to determine antibiotic susceptibility is the sensitivity disk method of Kirby – Bauer (named after W. Kirby and A. W. Bauer in 1966). The Kirby-Bauer assay is a standardized assay used to determine the susceptibility of bacteria to various antibiotics. The assay uses a filter paper disk impregnated with an antibiotic that is placed on agar. The antibiotic diffuses from the disk into the agar. The concentration of the antibiotic decreases as it diffuses away from the disk. The solubility of the antibiotic and its molecular size will determine the size of the area of infiltration around the disk. If an organism is placed on the agar it will not grow in the area around the disk if it is susceptible to the antibiotic. This area of no growth around the disk is known as a “zone of inhibition”. At the point around disk where bacterial growth begins the antibiotic as reached its “minimum inhibitory concentration”. Further away from the disk the antibiotic concentration is too low to inhibit the grow of the bacteria.

        Antibiotic susceptibility patterns are called antibiograms. Antibiograms can be determined by comparing the zone diameter obtained with the known zone diameter size for susceptibility. For example, a zone of a certain size indicates susceptibility, zones of a smaller diameter or no zone at all show that the bacterium is resistant to the antibiotic. Frequently one will see colonies within the zone of inhibition when the strain is antibiotic resistant.

        This method is also useful for determining the minimal inhibitory concentration (MIC) which is determined by measuring the diameter of growth inhibition (clear) zone surrounding the antibiotic disc.

Many factors are involved in sensitivity disk testing and must be carefully controlled. These include size of the inoculum, distribution of the inoculum, incubation period, depth of the agar, diffusion rate of the antibiotic, concentration of antibiotic in the disk, and growth rate of the bacterium. If these factors are carefully controlled, this type of testing is highly satisfactory for determining the degree of susceptibility of a bacterium to a certain antibiotic.

Requirements:

1.      Nutrient Agar

a.      Peptone                             1.0 g

b.      Meat or Beef Extract              0.5 g

c.      Nacl                                 0.3 g

d.      Agar                                  2.5 g

e.      Distilled water                     1,00.0 ml

2.      Nutrient broth culture of Bacteria

3.      Sterile filter paper disc

4.      Antibiotic solution or Antibiotic disc

5.      Spreader

6.      Alcohol

Procedure:

1.      On nutrient agar plates, add 0.1 ml of bacterial culture in aseptic condition.

2.      With the help of disinfected spreader, spread the bacterial culture on the nutrient agar.

3.      Pick up a sterile filter paper disc by the outer edge using flamed sterile forceps and dip the opposite edge of the disc in the antibiotic dilutions.

4.      Place the disc near the edge of the agar surface of the inoculated plate.

5.      Press gently with sterile forceps to ensure firm contact with agar surface.

6.      Keep these plate in refrigerator for 5 minutes to diffuse the antibiotic solution.

7.      Incubate all plates at 37 °C for 24 to 48 hours in an inverted position.

 

Observation:

1.      Examine all the plates for the zone for inhibition surrounding the discs.

2.      Measure diameter of zone of inhibition in millimeters.

Bacterial Culture	Diameter of Zone Inhibition in millimeter
	Penicillin	Chloramphenicol
Staphylococcus aureus
Bacillus subtilis

Results:

 

 

 

 

 

 

Microscope

Demonstration of Equipment’s

Compound Microscope: -

Aim: - To study various parts of Compound Microscope and their uses.

Theory: -

Part ‘A’: Different parts of microscope and their USES: -

Definition: - “Microscope defined as an optical instrument consisting of optical lenses for making enlarged image of small objects, cannot be seen by naked eyes”.

        Compound microscope has been designed to observe small objects which are too small to be seen with naked eyes. Microscope is a Greek work, here micro means too small and scope means to view.

Principle of Compound Microscope: -

        “The objective lens system forms an enlarge, real, inverted, primary image of the object which is further magnified by the eye piece system to form the enlarge virtual image”.

Construction of Compound Microscope: -

        The parts of a compound microscope are of two categories as given below:

(i) Mechanical Parts:

These are the parts, which support the optical parts and help in their adjustment for focusing the object.

1.      Base or Metal Stand:

The whole microscope rests on this base. Mirror, if present, is fitted to it. It is U or horseshoe-shaped metallic structure that supports the whole microscope.

    2.      Pillars:

        It is a short upright part that connects to base as well as arm.

3.      Curved Arm:

        It is a curved metallic handle held by the pillars. It holds the stage, body tube, fine adjustment and coarse adjustment.

4.      Inclination joint:

        It is a movable joint, through which the body of the microscope is held to the base by the pillars. The body can be bent at this joint into any inclined position, as desired by the observer, for easier observation.

5.      Body Tube:

        It is usually a vertical tube holding the eyepiece at the top and the revolving nosepiece with the objectives at the bottom. The length of the draw tube is called ‘mechanical tube length’ and is usually 140-180 mm (mostly 160 mm). The body tube has an internal pathway for the passage of light rays which form the enlarged image or microscopic objects.

6.      Draw Tube:

        It is the upper part of the body tube, slightly narrower, into which the eyepiece or ocular lens is slipped during observation.

7.      Coarse Adjustment:

        It is a knob with rack and pinion mechanism to move the body tube up and down for focusing the object in the visible field. As rotation of the knob through a small angle moves the body tube through a long distance relative to the object, it can perform coarse adjustment. In modern microscopes, it moves the stage up and down and the body tube is fixed to the arm.

8.      Fine Adjustment:

        It is a relatively smaller knob. Its rotation through a large angle can move the body tube only through a small vertical distance. It is used for fine adjustment to get the final clear image. In modern microscopes, fine adjustment is done by moving the stage up and down by the fine adjustment.

9.      Stage:

        It is a horizontal platform projecting from the curved arm. It has a hole at the center, upon which the object to be viewed is placed on a slide. Light from the light source below the stage passes through the object into the objective.

10.  Mechanical Stage (Slide Mover):

        Mechanical stage consists of two knobs with rack and pinion mechanism. The slide containing the object is clipped to it and moved on the stage in two dimensions by rotating the knobs, so as to focus the required portion of the object.

11.  Automatic Stop:

        It is a small screw fitted at lower end or rack and pinion. It is meant for stopping the downward sliding of the body tube so as to prevent the damage of objective lens and the slide.

12.  Revolving Nosepiece:

        It is a rotatable disc at the bottom of the body tube with three or four objectives screwed to it. The objectives have different magnifying powers. Based on the required magnification, the nosepiece is rotated, so that only the objective specified for the required magnification remains in line with the light path.

(ii) Optical Parts:

        These parts are involved in passing the light through the object and magnifying its size. The components of optical parts include the following:

1.      Light Source (Mirror):

        Old models, a mirror is used as the light source. It is fixed to the base by a binnacle, through which it can be rotated, so as to converge light on the object. The mirror is plane on one side and concave on the other. Plane side is used in strong light and concave side in weak light. But Modern microscopes have in-built electric light source in the base. The source is connected to the mains through a regulator, which controls the brightness of the field.

2.      Diaphragm:

        If light coming from the light source is brilliant and all the light can pass to the object through the condenser, the object gets brilliantly illuminated and cannot be visualized properly. Therefore, an iris diaphragm is fixed below the condenser to control the amount of light entering the condenser. Diaphragm is of two types, disc and iris

3.      Condenser:

        The condenser or sub-stage condenser is located between the light source and the stage. It has a series of lenses to converge on the object, light rays coming from the light source. After passing through the object, the light rays enter into the objective lens.

        The ‘light condensing’, ‘light converging’ or ‘light gathering’ capacity of a condenser is called ‘numerical aperture of the condenser’. Similarly, the ‘light gathering’ capacity of an objective is called ‘numerical aperture of the objective’. If the condenser converges light in a wide angle, its numerical aperture is greater and vice versa.

If the condenser has such numerical aperture that it sends light through the object with an angle sufficiently large to fill the aperture back lens of the objective, the objective shows its highest numerical aperture (Figure). Most common condensers have numerical aperture 1.25.

 

 

If the numerical aperture of the condenser is smaller than that of the objective, the peripheral portion of the back lens of the objective is not illuminated and the image has poor visibility. On the other hand, if the numerical aperture of condenser is greater than that of the objective, the back lens may receive too much light resulting in a decrease in contrast.

There are three types of condensers as follows:

(a) Abbe condenser (Numerical aperture=1.25): It is extensively used.

(b) Variable focus condenser (Numerical aperture =1.25)

(c) Achromatic condenser (Numerical aperture =1.40): It has been corrected for both spherical and chromatic aberration and is used in research microscopes and photomicrographs.

The total magnification of the microscope is the product of objective and eye piece lenses. Magnification of microscope depends on following factors:

i)                  Optical tube length,

ii)                 The focal length of objective lenses and

iii)              Magnifying power of eye piece.  

4.      Objective lens:

        It is the most important lens in a microscope. Usually three objectives with different magnifying powers are screwed to the revolving nosepiece.

        There are three types of objective lenses:

a)      Low power objective lens (10 X): It produces ten times magnification of the object.

b)     High power objective lens (45 X): It gives a magnification of forty-five times.

c)      Oil immersion objective lens (100 X): It gives a magnification of hundred times, when immersion oil fills the space between the object and the objective.

        In microbiology, oil immersion objective lens is generally used for observations of micro-organisms.

Principle of Oil Immersion Objective: -

        Because of it’s high magnification it is frequently used in microbiology. The numerical aperture of the instrument is depending on the angle of the light that admits from the object. Hence any factor that reduces the entry of rays in objective. In case of oil immersion objective, such a factor is the air between the object from dense medium, they are refracted in such great angle that large amount of light is lost which affects the brightness and clarity of image. Therefore, the space between objective lens and the glass slide or object is filled with cedar wood oil as the refractive index of oil and glass are nearly same (i.e., 2.5). So, the maximum light rays will pass through the objective lens and makes the image bright and clear.

 5.      Eye piece / Ocular lens: -

        The eyepiece is a drum, which fits loosely into the draw tube. It magnifies the magnified real image formed by the objective to a still greatly magnified virtual image to be seen by the eye. The most commonly used ocular is10 X. They are the Huygenian, the hyper plane and the compensating. Among them, the Huygenian is very widely used and efficient for low magnification. In this eyepiece, two simple Plano-convex lenses are fixed, one above and the other below the image plane of the real image formed by the objective. The convex surfaces of both the lenses face downward.

Function: -

        The main function of ocular system is magnification of primary image and formation of enlarge virtual image. It can also carries scale, marker and pointer.

Part ‘B’: Care of compound microscope:

Handling of compound microscope:

        Precautions to be measured are special in maintaining compound microscope as follows: -

1.      Always clean microscope and lens system before and after use with muslin cloth soaked in xylene.

2.      Never leave a slide on microscope’s stage, when it is not in use. Remove oil from oil immersion objective after it’s use. I not remove, oil will harden on it and if too much xylene used to clear the same, it will dissolve the cements holding the lens.

3.      Always keep the stage clean and dry. Dry the stage with cloth. Do not tilt microscope assembly by working with oil immersion objective, it may fall down from microscopic stage. Then, it is very difficult to remove oil dried on sub-stage.

4.      When the microscope is not in use, keep it covered in the microscope box to avoid breaking of microscopic lenses.

5.      Before and after using a microscope, never force the microscope. All the adjustments should work easily.

6.      Never allow the objective lens touch the glass slide.

7.      Never exchange ocular and objective of different microscope under any circumstances.

8.      Keeping the microscope in the box, low power objective should be in focus and be sure that, the mechanical stage doesn’t extend beyond the end of the stage.

Working of microscope:

        Before observing any object under the microscope, first light of microscope must be adjusted by using 10 X objective lens, without placing slide on stage so as to observe bright illumination in eye piece lens by adjusting the mirror by which maximum light passes through the objective lens while looking through the eye piece. Then place a slide on stage and center the object which is to be examined. Slowly lower objective lens with coarse adjustment until the object is in focus, then bring the object in sharp focus with iris diaphragm and condenser. Always watch the objective lens from the slide while lowering it and then focus by raising the body tube slowly. Focusing object using oil immersion requires more concentration.