Lecture 6: Microscopy and Staining PDF

Lecture 6 Microscopy and Staining Copyright © 2015 John Wiley & Sons, Inc. All rights reserved. Historical microscopy Anton Van Leuwenhoek (Holland) in 1600 was probably the first to see microorganisms (bacteria and protozoa). 100 to 300 times bigger using powerful magnify...

Lecture 6 Microscopy and Staining Copyright © 2015 John Wiley & Sons, Inc. All rights reserved. Historical microscopy Anton Van Leuwenhoek (Holland) in 1600 was probably the first to see microorganisms (bacteria and protozoa). 100 to 300 times bigger using powerful magnifying glass Send letter to inform Royal society of London to inform of his findings which he called “animalcules” Microscopy- technology of making very small things visible to human eye. Copyright © 2015 John Wiley & Sons, Inc. All rights reserved.  Anthony van Leeuweenhoek (1632-1723) Dutch cloth merchant First microscope 3 Copyright © 2015 John Wiley & Sons, Inc. All rights reserved. Letter to Royal Society of London in Dutch 4 Copyright © 2015 John Wiley & Sons, Inc. All rights reserved.  5 Copyright © 2015 John Wiley & Sons, Inc. All rights reserved.  419 microscopes with samples. 100 send to Royal Society. Following his death in 1723, microbiology field slowed down. However, through his letters, the existence of microbes was revealed to the scientific community 6 Copyright © 2015 John Wiley & Sons, Inc. All rights reserved. Relative sizes of objects Copyright © 2015 John Wiley & Sons, Inc. All rights reserved.  Light Properties (λ) Wavelength (represent by lambda ; λ) The sun produces continuous spectrum of electromagnetic radiation. White light-combination of visible lights Copyright © 2015 John Wiley & Sons, Inc. All rights reserved. Light Properties (RP) Resolving Power – a) two dots resolved – b) two dots not resolved Resolution - ability to see two items as separate and discrete units. Key to resolution-light of shorter wavelength to fit between object we want to see separately. Copyright © 2015 John Wiley & Sons, Inc. All rights reserved.  Shorter wavelength to improve resolution. Visible light (500nm) cannot resolve object separated by less than 220nm. Ultraviolet light (100-400nm) can resolve separations as small as 110nm. Thus cellular structure can be determined by UV 10 Copyright © 2015 John Wiley & Sons, Inc. All rights reserved.  Properties of Light : Light and object Reflection – Light bounces back when strike an object. Transmission – Is the passage of light through an object Absorption – Light absorbed by the object Copyright © 2015 John Wiley & Sons, Inc. All rights reserved. Light Travel Refraction – Bending of light when it passes from one medium to another of different density Diffraction – is the bending of light waves as they pass through small opening Copyright © 2015 John Wiley & Sons, Inc. All rights reserved.  13 Copyright © 2015 John Wiley & Sons, Inc. All rights reserved. Light Microscopy Types Light microscopy-any kind of microscope that uses visible light to make specimens observable. These days there are many complex designs of them which have been developed with the aim of improving resolution and sample contrast. Copyright © 2015 John Wiley & Sons, Inc. All rights reserved. a) Bright Field Compound light microscope Condenser allows light to be transmitted directly through specimen. b)Dark Field Condenser prevents light from being transmitted through the specimen Copyright © 2015 John Wiley & Sons, Inc. All rights reserved.  Phase Contrast – Has a special condenser and obj lenses that can emphasize small differences of various structures – No staining – The changes in the speed of light are seen as different brightness. Phase contrast image- Nomarski Amoeba, a protozoan (160x) – Uses differences in the refractive index to visualize unstained cells and structures. – Much higher resolution – Has short depth of field- thickness of specimen to focus at one time Nomarski Image- The protozoan Paracineta is attached – 3 dimensional image By a long stalk to green algae Spongomorpha (400x) Copyright © 2015 John Wiley & Sons, Inc. All rights reserved.  Fluorescence microscopy: – Ultraviolet light is used to excite molecules so that they release light of longer wavelength – Some microorganisms produce fluorescence/some must be treated with dye – Fluorescent antibody staining – Antigen and antibody (stained) bind together. – Can confirm presence of Flouorescent antibody staining antigen by visualizing the fluorescent dye. Copyright © 2015 John Wiley & Sons, Inc. All rights reserved.  Confocal Microscopy (UV) – Use beams of UV laser light to excite fluorescent chemical dye molecules into emitting light – Less out of focus light Standard fluorescent microscopy Confocal microscopy Digital Microscopy – Built in digital camera and software Copyright © 2015 John Wiley & Sons, Inc. All rights reserved. Electron Microscopy (EM) To overcome problem by light microscope which could not resolve objects separated by 0.2um It uses electron instead of light- higher resolving power Transmission (TEM) – Internal structure of microbes – Resolve 1nm Scanning (SEM) – Surface of specimen – Exterior of cell Copyright © 2015 John Wiley & Sons, Inc. All rights reserved. EM Images TEM SEM Copyright © 2015 John Wiley & Sons, Inc. All rights reserved. EM Images Colourized SEM : a) fingus:Aspergillus, b) Actinomyces, c) radiolorian from Indian ocean d) diatom: Cyclotella meneghiniana There are density related technique to do the colouring of the images Copyright © 2015 John Wiley & Sons, Inc. All rights reserved. Light Microscope Specimen Preparation Wet Mounts – Drop medium on slide to view living microorganisms. Smears – Placement of cells (smear) – Air drying (allow to air dry completely) – Heat fixation (use open flame and passed three to four times) Kills organisms Microbe adhere to slide Alters the microorganims so that they are more readily to accept stain (dyes). Copyright © 2015 John Wiley & Sons, Inc. All rights reserved.  Staining Principles Staining techniques make the microorganisms stand out against their background. Dyes are cationic (positively charged) or basic (Methylene blue, crystal violet, safranin). Dyes are attracted to the negatively charged cell components- cell membranes Simple Stains – Single dye – Reveals basic cell shapes and cell arrangements – Methylene blue, safranin, crystal violet Differential Stains – 2 or more dyes – To distinguish between two kind of organisms or between two different parts of organism Special stains – To identify specialized structure Copyright © 2015 John Wiley & Sons, Inc. All rights reserved. Gram staining https://www.youtube.com/watch?v=9Bnak_ITqck 24 Copyright © 2015 John Wiley & Sons, Inc. All rights reserved. Differential staining-Gram Stain Common technique – Gram positive ( cell wall retain crystal violet) – Gram negative (cell wall do not retain crystal violet) Significance – Cell wall anatomy – Diagnosis Copyright © 2015 John Wiley & Sons, Inc. All rights reserved.  Differential staining: Ziehl-Neelsen acid-fast stain Acid Fast Bacteria – for acid fast bacteria are organisms with wax-like nearly impermeable cell walls with mycolic acid and large amount of fatty acids, waxes and complex lipids. Eg: Mycobacterium (cause TB) – Acid fastness is a physical property that gives a bacterium the ability to resist decolorization by acids during staining procedures. – Acid-fast bacteria retain the bright red colour. This stain produces vivid red colour in acid-fast organisms such as Mycobacterium leprae that cause leprosy Copyright © 2015 John Wiley & Sons, Inc. All rights reserved. Differential stain: Negative staining Negative stains are used when a specimen or part of it resist taking up stains such as capsule – Background around the organisms is filled with a stain such as India ink or nigrosin. – The unstained object will stand out against the dark background Negative staining for capsules reveal clear area-capsule Streptococcus pneumoniae Copyright © 2015 John Wiley & Sons, Inc. All rights reserved. Special stain-Flagellar staining Flagella for locomotion Too thin to be seen easily with the light microscope. Hence must be coated for viewing. Flagella appear as dark lines with silver or red with carbolfuchsin or metal staining such as silver Copyright © 2015 John Wiley & Sons, Inc. All rights reserved. Endospore staining Heat-resistant endospores – Malachite green stain – Vegetative cells are red stained with safranin Schaeffer-Fulton stain Medical significance – Cause tetanus, gas gangrene, botulism The Schaeffer-Fulton spore stain- Endospores of Bacillus megaterium are visible as green Copyright © 2015 John Wiley & Sons, Inc. All rights reserved. Thank you Copyright © 2015 John Wiley & Sons, Inc. All rights reserved.