Microscopy & Microscopy & Staining Staining Dr Chinmay
May 25, 2015
Microscopy & StainingMicroscopy & Staining
Dr Chinmay
MICROSCOMICROSCOPYPY
Why do we use a microscope?
For study of structural details that are too small to be observed with unaided eye
Antony Van Leeuwenhoek (1632-1723)
1st Microscope
Resolving power of lens :
It is the smallest distance by which 2 points can be separated & still be observed as distinct points.
Unaided human eye has resolution limit of 0.1mm.
Magnification:
Magnification is the process of enlarging something only in appearance, not in physical size.
MA= Mo ˟ Me
where Mo is the magnification of the objective and Me the magnification of the eyepiece
The two types of microscopes based on the principle of magnification-
Light or Optical MicroscopeMicroscopy in which magnification is obtained by a system of optical lenses
-Bright field Microscope-Dark Field Microscope-Fluorescence Microscope-Phase contrast Microscope
Electron MicroscopeMicroscope in which magnification is obtained by a beam of electrons
Optical or Light Microscope
• Commonly used.
• 2 lens system.
• Initial magnification by objective lens
followed by ocular lens or eye piece.
Bright field Microscope
• Modified form of compound light microscope.
• Transmitted light is used to observe stained/unstained preparations.
• Depends upon differential absorption of light by different parts of the specimen.
Uses :-
• To observe stained smears of specimen e.g. gram smear, acid fast smear etc.
• Hanging drop / Wet mount to observe the shape, arrangement, motility and approximate size of the cells.
Dark Field Microscopy
• Reflected light is used instead of the transmitted light
• A specialized condenser is used. (Having a central circular stop) which illuminates the object with a cone of light, without letting any ray of light fall directly on the objective lens
• Light rays falling on the object are reflected or scattered on to the objective lens
• And the object appears self luminous against a dark background
OBJECTIVE
STAGE
CONDENSOR
LIGHT
SPECIMEN
Uses- To demonstrate extremely slender organisms Treponema spp. Leptospira spp
Fluorescence Microscope
• Uses fluorochromes that absorb light of a high intensity source which excites the flourescent agent, which in turn emits a low energy light of a longer wavelength that produces the image.
• Examples of fluorochromes are :
Acridine Orange,
Fluoroscein Isothiocyanate
MycobacteriumMalaria parasite
Use : oTo visualize Mycobacterium tuberculosis, Gonococci, Meningococci, Malaria parasite (Plasmodium spp) and other microorganisms.
oDetection of antigen and antibody in serum.
Phase Contrast Microscope
In this, the phase difference i.e. difference in thickness or refractive index of the structures within a cell is converted into differences in intensity of light. Thus , producing a light & dark contrast in the image.
Uses-
• It is a method to observe unstained living organisms with good contrast and high resolution.e.g. Vibrio cholerae in stool, Trichomonas vaginalis in the specimen etc
• Useful for the structures and structural changes in larger microorganisms and tissue cells but not for small or slender objects like spirochaetes
Same image with Phase Contrast Objectives. White dots inside each cell are the nuclei.Regular Bright-field Objective
Air bubbles
Two specialized types of microscopes are
Interference Microscope
Which is not only reveals the cell organelles but also enables quantitative measurement of the chemical constituents of cells such as lipids, proteins and nucleic acid
Polarization Microscope
which enables the study of intracellular structures using difference in birefringence.
Electron Microscope
Principle:• Instead of ordinary light, a beam of electrons is used
•Circular electromagnets-Analogous to lenses
•Object held in path of the beam scatters the electrons and produce an image
•Image is focused on a fluorescent viewing screen
• Wavelength of electrons used is approx 0.005nm
• Resolving power of the electron microscope-0.1nm
• Techniques used for the microscopy-
• Shadow casting
• Negative staining ( with phosphotungstic acid)
• Freeze-etching
• Two types:
• Transmission Electron Microscope(TEM)
• Scanning Electron Microscopes (SEM)
• Advantage:
To visualize very small organisms such as viruses (Rotavirus) or structures of microbial cells which are smaller than o.2µ.
The nucleoid of bacteria consists of a tangled circular DNA molecule without a nuclear membrane.Transmission electron microscopy (TEM) image of staphylococci
The straight rodbacteria with rounded endsshown here are coli bacteria
(SEM).
STAINING
Staining
• Artificial colouration of a substance.
• In microbiology used for: Demonstrating microorganisms & human cells. Elucidating structures such as spores or flagella. Differentiating organisms on basis of property
such as cell wall chemistry.
Types of stains used are :
• Simple stains (Monochrome stain)-
Watery solution of a simple basic dye is used. They provide the colour contrast but impart same colour to all microbes in the smear.
Example- Methylene blue, Carbol fuschin.
Methylene Blue Stain
• Differential Stain:
The staining which differentiate two types of organisms, are known as differential staining. They impart different colour to different bacteria or bacterial structures, e.g.
Gram Stain
Acid Fast Stain
Gram Positive Cocci Gram Negative Bacillus
Pus Cells
Acid Fast bacilli
• Special staining methods :
» For capsule
» For flagella
» For granules
» For spores
India ink method: Negative Staining for capsule
In this, the background is stained and the organism is appear as colourless objects against a dark background.
Use- Demonstration of capsules
Albert Stain for Metachromatic Granules
Corynebacterium diphtheriae
Modified ZN Stain for Spores
Silver Impregnation Stain
Organisms which are very thin to be seen under light microscope, are rendered visible by increasing their thickness by impregnating silver on their surface.
Use- To demonstrate organisms like spirochaetes and bacterial flagella
---Must know--- ---Must know---
Principles and uses of different
Principles and uses of different microscopesmicroscopes Principles of various staining
Principles of various staining techniquestechniques
QUESTIONSQUESTIONS
• Short NotesShort Notes
» Dark Field MicroscopeDark Field Microscope
» Fluorescent MicroscopeFluorescent Microscope
» Electron MicroscopeElectron Microscope
» Differential StainingDifferential Staining
» Negative StainingNegative Staining
» Silver Impregnation StainingSilver Impregnation Staining
Reference
• Textbook of Microbiology. Ananthanarayan and Paniker: 9th Edition
• Textbook of Microbiology. Prof C. P. Baveja: 4th Edition