Microscopy• Compound light microscope is

composed of: 1- stand 2- stage 3- substage ( condenser, diaphragm) 4- body tube (carrying lens system) 5- light source ( electric lamp)

Lens system• Ocular ( eye piece)• Objective carried on revolving nose piece (low

power, high power, oil immersion lens) Magnification power= magnification power of

ocular lens (10X) x magnification power of objective lens

For low power: mag power= 10 x 10=100For high power: mag power= 10 x 40= 400For oil immersion lens : mag power= 10 x 100=1000 As magnification power increase, focal

length decrease. oil immersion lens is brought very close to the specimen (immersion)

Objective lenses

In addition to magnification, microscopes must distinct between 2 adjacent points after magnification. This is known as resolving (resolution) power.

-Resolving power of a microscope depends on: wave length of light and refractive index.

-When light passes from a material of one refractive index to material of another, as from glass to air or from air to glass, it bends (refract), so that as objects are magnified the images become less and less distinct.

-With "dry" objective lenses this loss of resolution prevents using magnifications of above 400x.

-We place a drop of oil with the same refractive index as glass( cedar wood oil or paraffin oil) on the specimen

to eliminate the two different refractive

surfaces, so that magnifications of 1000x or greater can be achieved while still preserving good

resolution.

NB: Oil must be cleaned from the oil immersion

lens using organic solvent: xylene, after terminating sample

examination

Light microscope can be classified into

• Bright field microscope• Dark field microscope• Phase contrast microscope• Fluorescent microscope

Preparing heat fixed bacterial smear

• A heat fixed smear is a thin layer of the bacterial specimen dried and fixed onto the slide

Procedure: 1- A circle should be marked on the under side of a slide

with a glass marker. Several circles can be located on the same slide.

2- To prepare a smear from a suspended culture, aseptically transfer 3- 4 loopfuls of the culture (after shaking), place directly on the slide and spread. To prepare a smear from a dry culture, a very small drop of distilled water should be placed over the circled area. After aseptically removing material from a culture it is them mixed with the drop of water

3- Air dry or dry over the flame 4- heat fixation : by passing the slide three quick passes

through the flame.

Heat fixation• Heat fixation accomplishes three things: (1) it kills

the organisms; (2) it causes the organisms to adhere to the slide; and (3) it alters the organisms so that they more readily accept stains (dyes). If the slide is not completely dry when you pass it through the flame, the organism will be boiled and destroyed.

• If you heat-fix too little, the organism may not stick and will wash off the slide in subsequent steps.

• If you heat-fix too much, the organisms may be incinerated, and you will see distorted cells and cellular remains

Staining• Even with the microscope, bacteria

are difficult to see unless they are treated in a way that increases contrast between the organisms and their background. The most common method to increase contrast is to stain part or all of the microbe

Staining• There are several staining methods that are used

routinely with bacteria. These methods may be classified as:

Simple stain: use only one dye, it will react with different types of bacteria in an identical fashion

Differential stain: use 2 or more dyes, that react differentially with different types bacteria giving varying results depending on the organism being treated

Structural stain: Special stain to examine an internal or external structure of bacterial cell: capsular stain, flagellar stain

Types of dyes (stain)• Stains are chemicals containing chromophores, (groups

that impart color). Their specificity is determined by their chemical structure and charge they carry

• Accordingly there are 3 types of dyes: basic dye (cationic dye) is a stain that has positively

charged chromophore. Examples of basic dyes are crystal violet, safranin, basic fuchsin and methylene blue.

Acid dye (anionic dye) has a negatively charged chromophores.Examples of acid dyes are Nigrosine and sodium eosinate.

Neutral dye:Has both a negatively and positively charged chromophores (net charge is neutral) Example: eosin methylene blue

Mechanism of staining

• The surface of bacteria is somewhat negatively charged • When we use cationic dye (crystal violet) it dissociates

in aqueous solutions into CV+ and chloride (Cl – ) ions. These ions penetrate through the cell wall and cell membrane of bacterial cell The CV+ ion interacts with negatively charged components of bacterial cells and stains the cells purple, while the background is unstained. This is called Direct simple stain

• When we use an anionic dye (Nigrosine) it is repelled by the bacterial surface. It stain the background and leave the bacteria transparent. This is called Negative stain

Direct simple stain procedure

• Prepare a heat fixed bacterial smear• Leave to cool• Using a dropper, cover the film with crystal

violet (methylene blue or safranin)• Leave for 30 sec in case of using crystal

violet,2- 3 min for safranin or methylene blue• Wash gently, dry between 2 filter papers• Add oil and examine using oil immersion lens

Negative stain procedure

• Prepare an air dried bacterial smear• Do not heat fix !!!• Add one drop of nigrosine on the side of the

slide• Holding a second slide at a 45degree angle,

allow the drop to spread along the angled slide. • Allow the dye to thoroughly air dry. • Do not wash•  Apply immersion oil to the smear and observe

under light microscope. •  

Microscopical examination

Staphylococcus aureus

Simple stain with crystal violet

Escherichia coli

Simple stain with Safranin

Staphylococcus aureus

Negative stain

Scheme for discription of M.O

• Name of M.O : Staphylococcus aureus• Name of stain: direct simple stain, or

negative stain• Shape : cocci• Size : small• Arrangement : bunches or clusters• Color: according to the stain used

• Name of M.O : Escherichia coli• Name of stain: direct simple stain,

or negative stain• Shape : Bacilli or rods• Size : small• Arrangement : single• Color: according to the stain used