Practical Microbiology
a) Microscope – handling & care of parts, uses, handling
microscopes Identify and care of microscopes
common microbes b) Observation of staining procedure, Specimens Slides
under the microscope preparation and examination of
slides and smears
c) Identification of common
microbes under the microscope for
morphology of different microbes

INTRODUCTION TO LABORATORY TECHNIQUES

MICRSCOPE is a tool for studying Microbe’s.

In which magnification is possible due to the formation of specific precursors, since these powerful lenses are arranged, the shape, type, movement and group of micro-organism can be known.

Micro = Small,

Scope = View

Invented by Antony van Leeuwen Hock in 1975, it has since been improved to become the modern microscope. In a simple microscope, the object can be magnified 4 to 40 times.

1,00,000 times magnification is possible in Electron Microscope.

100 to 430 times magnification can be done in Compound Microscope which is widely used microscope (used in solution).

Part of Microscope

There are three main parts,

(1) stand

(2) body and

(3) optical system.

(1) Stand :- It is a part made of Solid Iron like horseshoe. The supporting part is designed in such a way. So that the alignment of optical system gets support.

(2) Body :- “C” shaped part is called body. Through which the microscope can be lifted. in its composition

(a) Stage :- Provides a platform to the Specimen. It has a central operator to mount the specimen. It makes it possible to pass light rays through the slide. A clip is kept on the stage to keep the slide in position. Screws are provided for the movement of the horizontal and vertical planes of the slide.

(b) Sub stage :- It is below the stage. And the screw works to raise and lower,

(c) Mirror :- The light reflected on the specimen can be turned in any direction. It has two surfaces.

(i) Plain – Used for Sunlight,

(ii) Concave is used for artificial light.

3) Optical System

:- Eye piece, object glass makes a condenser Hull optical system

(a) Body tube :– Wide tube connects objective lens ocular piece. It does up-down through two different arrangements. Attached to the upper part of the “C” shape of the body

(b) Coarse adjustment :- To focus the specimen by sliding the body tube downwards.

(c) Fine adjustment :- For delicate focussing movement. Picture helps to slide the draw tube for precision and clarity.

(d) Nose piece :- Located at the lower end of the draw tube. Object lenses of different magnifying powers can be fitted in its sockets.

(e) Objective lenses :- Immediately above the object. It gives a magnified image. Its number is 3. Which are of increasing less magnifying power. (10× 45×, 100×)

(f) Eye pieces :- At the upper end of the draw tube, through which the examiner can see the picture (magnified) of the slide.

(g) Limb or Arm :– Illuminating apparetus, stage and observation tube are there.

(h) Draw tube :- Located inside the body tube, which can be slid up and down.

Mechanical stage :– “c” shape is attached to the lower end of the body, on which the slide can be arranged. The slide can be moved left-right by the slide adjustment screw.

Glass slide:-

Arranged on mechanical stage.

Condenser :-

It is formed below the mechanical stage. Which helps to magnify microbes with the help of lenses.

Diaphragm :- Located at the bottom of the condenser. Through which light rays pass from the slide.

Mirror :-Reflects the light and throws it on the object through the condenser.

Care of Micro Scope-

As a valuable tool, maintain and use it.

It should be lifted with the help of two hands only.

Support under the base with the palm of the left hand. Hold the “C” shaped part of the body with the help of the right hand.

Both are less hair-changing.

It should be arranged in a place where there is enough light.

While sliding the draw tube down it should be seen that it does not come into contact with the mechanical stage.

A flannel duster should be kept to keep the Micro Scope and its stage always dry.

To clean the lenses with lance paper.

When the lenses are damaged by immersion oil, first clean them with dry lens paper, then clean them with lens paper soaked in xylol and dry them again with dry lens paper.

After use the least powerful lens should be moved a little higher on the stage, and the condenser should be moved a little lower.

Mirror should always be kept clean and dry.

Keep it in its cabinet with plastic cover.

Use of Micro Scope

→ Arrange the microscope towards the edge of the table in such a way that the “C” shape of its body comes towards our chest. And the Mirror should come in the opposite direction. The sunlight or lamp light should come from the opposite side in such a way that it falls directly on the lens.

.→ Arrange the Condenser in such a way that the rays of light entering it enter the mechanical stage and illuminate the required part of the slide.

→ Stained slides require more powerful lenses to examine

.→ First of all focus on less powerful lenses slide, while doing so bring the body tube down with the help of coarse adjustment. Be careful not to touch the slide. After that adjust the lenses for the necessary magnification. Look in the slide through the eye pieces and adjust the tube up and down as needed.

→ Then make fine adjustment.

Staining of micro-organisms

Procedure (staining of slide) for biological study of microbes is done by air drying, chemical fixation and staining with aniline dye.

Bacteria are distinguished from surrounds. So that the examiner can easily see and the slide can be kept for study for a long time.

Smear

Micro-organisms are spread on the slide like a thin film from the growth culture media through an inoculating loop.

A loop of nichrom wire with insulated handle is used. The loop is sterilized by heating or flaming before use.

It is then used when the loop cools down. After the smear is taken, the loop is sterilized again.

Bacterial suspension is spread on a clean slide and after making a smear it is kept for air/dry.

Dry smear is then passed through Bunsen burner flare several times called heat fix. Heat fixing changes the properties of enzymes of bacteria. And cell destruction stops. Heat causes bacteria to adhere to the slide.

Simple stain

Most of the stains used are synthetic dyes (aniline) made of a coaltar derivative derived from benzene. Dyes are usually salts. Sometimes they contain ions of acids or bases.

The stain dye containing positive ion is called basic stain and the dye containing negative ion is called acidic stain.

Most of the bacteria are stained in basic stain. The staining procedure in which only one stain is used is called simple stain.

The simple stain that stains bacteria is called direct stain.

When the simple stain stains the back ground and not the bacteria, it is called negative stain.

. Simple stain is used to study the morphology and arrangement of bacteria.

Gram stain technique

Given by Hans Christian gram in 1884, this is a very useful stain for identifying and classifying bacteria. From this stain, bacteria can be separated into a gram positive gram negative group.

Staining technique

Apply primary stain (crystal violet) all bacteria will stain purple.

Apply mordent (grains iodine) This will intensify the bonding of bacteria and primary.

Apply decolorizing (ethyl alcohol, or ethyl alcohol acetone) it will wash out the primary stain of bacteria. while some will be unaffected.

Apply secondary stain or counter stain (sofranin) – this will make bacteria red color. After this procedure bacteria are easily decolorized by examining the slide. It is gram negative and bacteria that retain primary stain. They are considered positive

Acid fast staining

The Ziehl – ​​Ncolson procedure is a commonly used technique everywhere. Smear is done in this.

Flooded with Carbolfuchsin – which binds to the chemical body of bacteria.

Ziehl introduced carbolfuchisi containing 5% phenol as dry instead of aniline.

The slide is heated along with the smear for penetration of dry into the bacteria. After washing the smear with acid-alcohol mixture, most of the bacteria stain loses its color. But acid fast organisms do not lose that color.

Hanging drop method

→ Take the hedging drop slide. If there is no hanging drop slide, take a plain slide and make a depression-like part on the slide with the help of plasticin. Take a small amount of petroleum jelly about the size of a 50 paise coin in your palm, spread it on the slide as a smear of the same size.

->Hold the cover slip by its edge and scrub the petroleum jelly on the cover slip. Scrub the remaining three slides with petroleum jelly as well. Be careful to apply the petroleum jelly to the same side of the cover slip each time.

→ Place the cover slip on a paper towel with the petroleum jelly side up.

Place one drop of Slide Organic infusion or suspension on the slip