Method of bacteria examination in microbiology

Microscopic Examination of Bacteria

Microscopic examination of stained or I unstained preparation of bacteria is usually one of the essential steps in the long process of isolating and identifying bacteria. Microscopy of stained smears helps to differentiate cellular constituents and render them more visible. Staining also helps to classify organisms by placing them in their separate groups, based on the staining reaction of the organism.

Method of bacteria examination  in microbiology

WIRE LOOPS

Wire loop used in microbiology

The microbiological wire loops are used to make smears. They are made of platinum or nichrome wires. 

• The platinum wire used is actually a mixture of platinum (90%) and iridium (10 %). Platinum on its own is too soft to be of any use. 
• The nichrome wire is cheaper and more flexible. Disposable plastic loops are also available. 

• Wire loops are made in various sizes of between 1.5 mm and 3 mm in diameter with the handle or holder about 6-8 cm long (Fig. 3.1a).

The wire loops may be purchased commercially though making a wire loop is not very difficult

1. Wind the wire once round a rod of appropriate diameter. 

2. With a pair of scissors, cut one arm of the wire wound around the rod, forming a loop. 

3. Bend back the loop with a pair of forceps to centre it. 

4. Insert into a metal wire loop holder.

Making a wire loop for bacterial examination

Making a Wet Preparation for Microscopy

1. On a clean microscope slide, place a drop of the material to be examined, e.g., urine, or mix a small portion of the specimen such as vaginal secretion in a drop of saline. 

2. Carefully place a cover-slip over the preparation, making sure that there are no air bubbles trapped under the cover-slip. 

3. Examine immediately. If there is going to be a delay in examining the preparation, it is recommended to seal the edges of the coverslip with nail varnish or petroleum jelly.

Hanging Drop Preparation

The hanging drop is a microscopic technique used to determine the motility of bacteria when suspended in a fluid. 

• True motility is when bacteria actively move from one position to another in a haphazard manner. 

• It should not be confused with Brownian movement which is the vibration caused by molecular bombardment, or the one-directional movement caused by convectional currents. 

1. Make a ring of plasticine or vaseline about 2 cm in diameter on a clean slide. 

2. Place a loopful of culture in the centre of a clean 22 mm square cover-slip. 

3. Carefully press the ring of plasticine on to the cover-slip with the drop of culture in the centre of the ring and not touching the slide. 

4. With a quick movement invert the slide so that the cover-slip is uppermost (Fig. 3.2).

Hanging drop preparation for bacteria examination

5. Examine under the microscope, first using the low power objective (16 mm) to focus on the edge of the 'drop', then observe motility with high-power (4 mm) objective. 

6. At the end of the examination, discard the whole preparation into a jar of disinfectant and allow to soak to kill the microorganisms.

Note

1. A well-slide' with a depression in the centre can be used in place of the slide with a ring of plasticine. Remember to seal the cover-slip with vaseline or nail varnish. 

2. With experience, one can use the ordinary wet preparation to determine motility microscopically.

PREPARATION OF SMEARS

Stained preparations are needed to examine microorganisms microscopically in order to study their morphology and observe their cellular constituents. 

• Smears (or tissue sections) are made and stained by anyone of the recognized staining methods.
• Smears can be made from liquid or solid cultures or from clinical specimens.

Smears from Solid Media

1. Sterilize the wire loop in bunsen flame 

2. Place one drop of sterile saline on a clean slide with the sterilized loop. 

3. Re-sterilise the loop. 

4. With the wire loop, pick a small portion of bacterial growth and emulsify it in the drop of saline and spread to give a thin homogeneous film or smear on the slide (Fig. 3.3).Sterilize the loop. 

5. Allow the smear to dry in air, fix and stain.

Bacterial smear

Smears from Liquid Media

1. Sterilise wire loop in the bunsen flame.

2. Using aseptic technique, remove a loopful of the culture.

3. Place the culture on a clean slide and spread it with the loop to give a fairly thick film of culture. Sterilise the loop.

 4. Allow the film to dry, fix and stain.

Note

1. Always allow the film to dry on its own in air without heating before fixing it. This is to prevent the smear from being washed off during staining.

2. From solid media the smear should be thin and from liquid media, it should be thick.

Smears From Clinical Specimens

Smears are made from original clinical specimens directly on the slide if it is a swab or with a loop if it is fluid.

Fixation of Bacterial Smears

The smear having been made and allowed to dry, is fixed by quickly passing it two or three times over a bunsen flame to a temperature of about 55 to 60 °C. 

• This is to coagulate the bacterial proteins which makes them adhere to the slide. 

• Fixation should be such that the bacterial morphology remains intact, preventing the smear from being washed off during staining. Overheating will lead to distortion of morphology.

STAINING OF BACTERIA

Dyes from which stains are made, are either natural or synthetic products. Most of the dyes used for bacterial smears are synthetic. The natural dyes are mostly used in histopathology. The synthetic dyes are all derivatives of benzene and are usually referred to as aniline dyes.

Stains are mostly salts, comprising a base and an acid. They are classified into acid, basic and neutral stains. 

Basic stains are those in which the colouring substances are contained in the basic radical with the acid radical being colourless, while those stains in which the colouring substances are in the acidic radical and base components being colourless, are Acid stains. An aqueous mixture of some acid and basic dyes results in Neutral stains, with the colouring substances contained in both acid and base components.

• The theory of staining reaction is not fully understood but it is generally believed to be a combination of chemical and physical reactions. 
• The bacterial cell which is rich in nucleic acid, has affinity for basic stains and so is stained by the basic stain. 
• The acid stain is useful for basic components and as a background stain to give a good contrast.

Mordant used in microbiology

Some organisms (or tissues) do not take up the stain except in the presence of a mordant--a substance that forms a link between the organism and the stain to bring about a staining reaction. 

• The mordant forms a 'lake with the dye which is capable of attaching itself firmly to the organism. 
• A popular mordant in bacteriology is the Lugol's iodine.
•  The mordant should not be confused with an accentuator which is a substance that greatly increases the staining power and selectivity of a stain. 
• It does not combine or form a lake with the dye. 

Examination of stained smears All stained smears are examined with oil immersion objective (x 100).