Antony van Leeuwenhoek (1632-1723)• Inventor of the first microscope
• Leeuwenhoek earned his living as a draper, but spent much ofhis spare time constructing simple microscopes composed ofdouble convex glass lenses held between two silver plates (figurebelow)• his microscope could magnify around 50 to 300 times,
• A microscope is an instrument used to see objects that are toosmall for the naked eye. The science of investigating small objectsusing such an instrument is called microscopyThere are many types of microscopes, the most common twomain type,1. light microscope2. electron microscope .
Light Microscopy • Bright-field Microscopes • Dark-field Microscopes • Phase -contrast Microscopes • Fluorescence MicroscopesMicroscopy Electron Microscopy • SEM • TEM
Light MicroscopeIn the light microscopy there is a use of visible light and glasslenses for the magnification. Microbiologist currently employ avariedly light microscopes in their work. The most commonly usedbasic types of microscopes include;a. Bright-field microscopes,b. Dark-field microscope,c. Phase-contrast microscopesd. Fluorescence microscope
• Modern microscopes are all compound microscopes, that is the magnifiedimage formed by the objective lens is further enlarged by one or moreadditional lenses like eyepiece (ocular) . A compound microscope with a singleeyepiece is said to be monocular; one with two eyepiece is said to be binocular•The part of a modern compound microscope and the part light takes through itare shown in the above figure. light enters the microscope from a light sourcein the base and often passes through a blue filter, which filter out the base andoften passes light, leaving the shorter wavelengths and improving resolution. Itthen goes through a condenser, which gathering the light beams so that theypass through a specimen. The iris diaphragm controls the amount of light thatpasses though the specimen and into the objective lens. The higher themagnification, the greater the amount of light needed to see the specimenclearly. The objective lens magnifies the image before it passes though thebody tube to the ocular lens in the eyepiece. The ocular lens (eyepiece) furthermagnifies the image. A mechanical stage allows precise control of moving theslide , which is especially useful in the study of microbes. The focusingmechanism consist of a coarse adjustment knob, which changes the distancevery slowly. The coarse adjustment knob is used to locate the specimen. Thefine adjustment knob is used to bring it into sharp focus.
ResolutionThe resolving power(RP) of a lens is a numerical measurement ofthe that can be obtained with that lens. We can calculate the RPof a lens if we know its numerical aperture(NA) and thewavelength(λ) of the electromagnetic radiation used. The formulafor calculating resolving power is; RP = λ/2NA
Numerical AperturesThe numerical aperture is more difficult to understand. That isdefined as half aperture angle, the angle of the cone of lightentering an objective, θ θ Thus, NA = ½ n sinθBut, the angle of the cone of light that can enter a lens depends on the refractiveindex(n) of the medium in which the lens work.The refractive index for air is 1.00 in case of dry objective.
a. Bright-field microscopesSpecial features:- Use visible light Simple to use Least expensiveAppearance:-Coloured or clear specimen on light back groundUses:-Observation of dead stained organisms or live ones with sufficient naturalcolour contrast.
b. Dark-field microscopeSpecial features:-Uses visible light with special condenser that causes light rays to reflect offspecimen at an angleAppearance:-Bright specimen on dark backgroundUses:-Observation of unstained living or difficult to stain organisms. Allows one to seemotions
c. Phase-contrast microscopesSpecial features:-Use visible plus phase shifting plate in objective with a special condenser thatcauses some light rays to strike specimen out of phase with each otherAppearance:-specimen has different degrees of brightness and darknessUses:-Detail observation of internal structure of living unstained organisms
d. Fluorescence microscopeSpecial features:-Uses ultraviolet light to excite molecules to emit light of different wavelengths, often brilliant colour, because UV can burn eyes, special lens materialsare usedAppearance:-Bright fluorescent colour specimen on dark back groundUses:-Diagnostic tool for detection of organism or antibiotic s in clinical specimens orfor immunological studies
Special features:- Uses electron beams and electromagneticlenses, inexpensive, preparation requires considerable time andpractice.Appearance: -Three dimensional view of surfaces.Uses:-Observation of exterior of cells or of internal surfaces• To visualise the internal structure - TEM• Electrons instead of light• Greater resolving power• Disadvantages: Expensive A high vacuum is required Very thin sections (<100 nm thick)
Figure:- Bacterial cells viewed with light microscope
Figure:- Bacterial cell viewed with electron microscope
STAINING OF BACTERIAWhy to stain bacteria?Because bacteria are semitransparent and difficult to see in the unstained state.It is necessary to make bacteria visible to find out their shape, arrangementsand other morphological characters. Staining will make the cells more visibleand help in revealing their internal structures.Types of stains and dyesA large number of colored organic compounds are available for stainingmicroorganisms. Based on the chemical behavior of the dye, these can beclassified into three., acidic, basic or neutral. An acidic dye is one in which thechange on the dye ion is negative. An basic dye is one in which the change onthe dye ion is positive. A neutral dye is a complex salt of a dye acid with a dyebase. Acid dyes generally stain basic cell components, and basic dyesgenerally stain acidic cell components.
Process of stainingthe process of staining may be involve ion-exchange reactions between the stain andactive sites at the surface of or within the cell. The colored ions of dye may replace otherions on cellular components. For example the ionic exchange which takes place duringstaining can be represented by the following equation, in which the (MB+) actionreplaced the (NA+ action in the cell. )(Bacterial cell ˉ) (NA ) + (MB+) (Clˉ) ------- (Bacterial cell ˉ) (MB ) + (NA ) (Clˉ) + + +In this MB is methylene blue dye, which is actually methylene blue chloride.Staining methods can be divided into two groups.1. simple staining and 2. differential staining.Under differential staining comes.• gram’s staining• acid-fast staining• endospore staining• capsule staining• flagella staining• cytoplasmic inclusion staining• giemsa staining