Microscopy & Staining

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