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=100 For high power: mag power= 10 x 40= 400 For 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