Microbiology: Principles and Explorations Jacquelyn G. Black Microbiology: Principles and Explorations Sixth Edition Chapter 3: Microscopy and Staining Copyright © 2005 by John Wiley & Sons, Inc.

Principles of Microscopy Microscopy is the technology of making very small things visible to the human eye Table 3.1 Milli = one thousandth 10-3 m Micro = one millionth 10-6 m Nano = one billionth 10-9 m

Relative Sizes of Objects

In order for the microscopy technique to be good there must be a staining process for the microorganisms to be visualized under the microscope Resolution power (RP) : the ability to see 2 objects beside each other clearly ( more resolution power more clear ) RP unaided human eye < RP compound microscope < RP scanning electron microscope (SEM) < RP transmission electron microscope (TEM) Resolving Power (RP) of a lens in a numerical measure of the resolution that can be obtained with that lens The smaller the distance between objects that can be distinguished, the greater the resolving power of the lens

Resolution

Light Microscopy Refers to the use of any kind of microscope that uses visible light to make specimens observable The compound light microscope has more than one lens Objective lens Ocular lens Monocular vs. Binocular

The Compound Light Microscope

Different Types of Microscopy Bright-Field Microscopy Dark-Field Microscopy Phase-Contrast Microscopy Nomarski (Differential Interference Contrast) Microscopy Fluorescence Microscopy Confocal Microscopy Digital Microscopy Electron Microscopy

Bright Field vs. Dark Field Illumination

A comparison of the illumination in bright field and dark-field microscopy The condenser of the bright-field microscope concentrates and transmits light directly through the specimen. The dark-field microscope condenser deflects light rays so that they reflect off the specimen at an angle before they are collected and focused into an image.

dark-field microscope is also important in the case of Light sensitive organisms, to examine specimens that would lack contrast with their background in a bright field under other illumination when dark field illumination is used. A microscope adapted for dark-field illumination has a condenser that prevents light from being transmitted through the specimen but instead causes the light to reflect off the specimen at an angle .When these rays are gathered and focused into an image, a light object is seen on a dark background An example is Treponema pallidum ( it can’t be stained )

Phase contrast microscopy is used for alive non stained microorganisms Nomarski microscopy is a higher resolution phase contast In Fluorescence Microscopy ultraviolet light is used to excite molecules Some organisms, such as Pseudomonas, fluoresce naturally when irradiated with ultraviolet light ( it contains fluorescein ). Other organisms, such as Mycobacterium tuberculosis and Treponema pallidum (the cause of syphilis), must be treated with a fluorescent dye called a fluorochrome Acridine orange is a fluorochrome that is heated and to bind to nucleic acids of the microorganism , when it’s exposed to UV light, it colors bright green, orange green, or yellow,.

Fluorescent antibody staining is now widely used in diagnostic procedures to determine whether an antigen (a foreign substance such as a microbe) is present. Antibodies ( molecules produced by the body as an immune response to an invading antigen) are found in many clinical specimens such as blood and serum. If a patient’s specimen contains a particular antigen, that antigen and the antibodies specifically made against it will clump together. However, this reaction is ordinarily not visible. Therefore, fluorescent dye molecules are attached to the antibody molecules. If the dye molecules are retained by the specimen, the antigen is presumed to be present

Confocal microscopy use beams of ultraviolet laser light to excite fluorescent chemical dye molecules into emitting (returning) light helpful in studying communities of microbes without disturbing them, as in examining living biofilms ( microorganisms culture has an architecture shape that we don’t want to disturb , so we examine the biofilm after staining it with a fluorescent dye )

Electron Microscopy Uses a beam of electrons instead of a beam of light electromagnets rather than glass lenses to focus the beam Produce electron micrographs with great detail Two most common types of electron microscopy: Transmission electron microscopy (TEM) Scanning electron microscopy (SEM) Atomic force microscope is a type of scanning tunneling microscope let us see actual molecules and even individual atoms ( top graphic ) which gives us the stage of growth

Electron Microscope Cross-section

Freeze-Fracturing and Freeze-Etching

Scanning Tunneling Microscopy thin wire probe made of platinum and viridium used to trace surface of substance Run probe in straight line reveals highs and lows of individual molecules or atoms in a surface Individual atoms of an element can be clearly distinguished

Techniques of Light Microscopy Wet mounts: A drop of medium containing organisms is placed on slide and used to view living microorganisms ( dark field microscopy ) Smears: Microorganisms are spread onto the surface of a glass slide and used to view destroyed organisms Heat fixation: destroys the organisms, causes organism to adhere to slide, and alters organism to accept stains (dyes)

The Hanging Drop Technique This technique is used to see the motility of the microorganism without staining ( wet mount ) using the dark field or phase contrast microscope ( living microorganism )

Principles of Staining Stain or dye: A molecule that can bind to a cellular structure and give it color (contrast) 1. Cationic or Basic Dyes +ve charge Anionic or Acidic Dyes –ve charge Cationic or basic dyes such as methylene blue, crystal violet, safranin, and malachite green anionic or acidic dyes such as eosin and picric acid The cell membranes of most bacteria have negatively charged surfaces and thus attract the positively charged basic dyes , so anionic dye is used to stain the background

Simple stain: makes use of a single dye and reveals basic cell shapes , sizes and arrangements Differential stain: makes use of two or more dyes and distinguishes between organisms based on structural differences such as gram stain

The Gram Stain : (Hans Christian Gram ) The Gram stain used as a differential stain Four groups of organisms can be distinguished with the Gram stain: (1) Gram-positive organisms, whose cell walls retain crystal violet stain ( violet ) (2) Gram-negative organisms, whose cell walls retain safranin stain ( red ) (3) Gram-nonreactive organisms, which do not stain or which stain poorly (4) Gram-variable organisms, which stain unevenly

In Gram staining and after smearing, bacterial cells take up crystal violet. Iodine is then added; it acts as a mordant, a chemical that helps retain the complex stain ( Iodine + crystal violet) in certain cells without being washed out. Those structures that cannot retain crystal violet are decolorized with 95% ethanol or an ethanolacetone solution, rinsed, and subsequently stained (counterstained) with safranin ( colored with red ) The structures that were stained in purple  gram +ve The structure that were stained in red  gram –ve The differentiation between Gram-positive and Gram-negative organisms reveals a fundamental difference in the nature of thecell walls of bacteria

Gram-variable organisms have somehow lost their ability to react distinctively to the Gram stain. Organisms from cultures over 48 hours old (and sometimes only 24 hours old) are often Gram-variable, probably because of changes in the cell wall with aging. Therefore, to determine the reaction of an organism to the Gram stain, you should use organisms from cultures 18–24 hours old. It also could be a contaminated culture that cause the gram variable

The Gram Stain

The Ziehl-Neelsen Acid-Fast Stain A differential stain depend on the cell wall Distinguishes members of the genera Mycobacterium and Nocardia from other bacteria Acid-fast bacteria retain carbolfuchsin and appear red. Non–acidfast bacteria accept the methylene blue counterstain and appear blue Slides of organisms are covered with carbolfuchsin and are heated, rinsed, and decolorized with 3% hydrochloric acid (HCl) in 95% ethanol, rinsed again, and then stained with Loeffler’s methylene blue. Most genera of bacteria will lose the red carbolfuchsin stain when decolorized. However, those that are ‘‘acid-fast’’ retain the bright red color

The cell wall of these microorganism is very hydrophobic that resist staining , so we use heating to force staining , once it’s stained it resist de staining even with the strongest decolorizing agents ( such as HCL with ethanol ) Bacteria that are not acid fast lose the red color and can therefore be stained blue with the Loeffler’s methylene blue counterstain.

Negative Staining. specimen or a part of it, such as the capsule resists taking up a stain. The capsule is a layer of polysaccharide material that surrounds many bacterial cells and can act as a barrier to host defense mechanisms. ( prevent the phagocytosis) It also repels stains. In negative staining, the background around the organisms is filled with a stain, such as India ink, or an acidic dye, such as nigrosin. Or eosin

This process leaves the organisms themselves as clear, unstained objects that stand out against the dark background. A second simple or differential stain can be used to demonstrate the presence of the cell inside the capsule. Thus, a typical slide will show a dark background and clear, unstained areas of capsular material, inside of which are purple cells stained with crystal violet or blue cells stained with methylene blue.

Flagellar Staining Flagella, appendages that some cells have and use for locomotion, are too thin to be seen easily with the light microscope. When we need to determine their presence or arrangement, flagellar stains are prepared to coat the surfaces of the flagella with dye or a metal such as silver. These techniques are very difficult and time consuming Flagella appear as dark lines with silver, or red with carbolfuchsin Indicates presence of flagella by building up layers of stain on their surface

Endospore Staining A few types of bacteria (gram +ve under certain conditions) produce resistant cells called endospores. Endospore walls are very resistant to stains. When a simple stain is used, the spores will be seen as clear, glassy, easily recognizable areas within the bacterial cell. Thus, it is not absolutely necessary to perform an endospore stain to see the spores. However, the differential Schaeffer-Fulton spore stain makes spores easier to visualize. Heat fixed smears are covered with malachite green and then gently heated until they steam.

The slide is then rinsed with water for 30 seconds to remove the green dye from all parts of the cell except for the endospores, which retain it. Then a counterstain of safranin is placed on the slide to stain the non–sporeforming, or vegetative, areas of the cells. Cells of cultures without endospores appear red; those with endospores have green spores and red vegetative cells. Bacteria Form endospores ( protection ) while fungi form exospores ( reproduction ) Some types of bacteria that form endospores are clustridium and baccilus