Microscopy and Staining

Important words and concepts from Chapter 3, Black, 2002 (3/28/2003):

by Stephen T. Abedon (abedon.1@osu.edu) for Micro 509 at the Ohio State University

Course-external links are in brackets

Click [index] to access site index

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(1) Chapter Title: Microscopy and Staining

(a) "Microscopy is the technology of making very small things visible to the human eye."

(b) See Figure 3.2, Relative sizes of objects, for a look at the relative sizes, on a log scale, of microorganisms and other objects of biological relevance

(d) Note that a compound light microscope is only capable of viewing down to the smallest common bacteria (i.e., Chlamydia in the figure) whereas the human eye can see down to only the largest of individual cells

(e) See additionally: [microscopy (Google Search)] [history of the light microscope (Thomas E. Jones)][microscope glossary (Microbiology 12 - City College of San Fransico)] [Standard microscopy terminology (Center for Interfacial Engineering - University of Minnesota)] [Microscopy links (Light Microscopy Forum - Ron Neumeyer)] [index]

INTRODUCTION TO COURSE, NOTES, AND MICROORGANISMS (SUPPLEMENT)

(2) Studying tips:

(a) Throughout this course (i.e., these notes) I will be doing my best to supply you with links to supplemental material found on the World Wide Web

(i) If you have additional interest in presented material or need additional exposure to concepts, consider following these links (on line, of course)

(ii) I will regularly include Google searches that may be followed for abundant additional information on material—however, always keep in mind that your first, best reference will usually be your text book

(iii) There also exists an index to this site called MicroPort that is found at http://www.phage.org/microbiology.htm or by pressing [index] throughout these notes

(b) Read over assigned material in your text before coming to lectures

(i) Read your text well so that, minimally, you have made an attempt at understanding the presented concepts

(i) Read lecture notes well so that you have made an attempt at understanding the presented concepts

(ii) Make an effort to memorize the supplied vocabulary

(d) Come to class prepared to ask questions

(e) After class, organize the material, integrating the notes that you take during class

(f) REMEMBER, CLASS ONLY MEETS TWICE A WEEK SO BLOWING OFF A LECTURE OR NOT STUDYING PRIOR TO AND AFTER A LECTURE IS EQUIVALENT TO BLOWING OFF HALF OF A WEEK OF STUDYING

(g) Study for the first exam in this course harder than you have ever studied for an exam before

(i) Triage the material you will be studying such that you don’t waste your time studying the material you already know/understand

(ii) Make sure that you have extensively been through the material and have organized it before you begin to study

(iii) Don’t put off your studying to the last minute

(iv) Don’t count organizing and learning your material as exam study time—studying for an exam involves making sure that you have memorized and can lucidly regurgitate the material, not simply becoming familiar with it

(v) Simply reading over notes again and again is not necessarily equivalent to doing the hard work of learning

(h) Don’t forget that labs are worth a good chunk of your grade

(i) Don’t blow off labs

(ii) Read labs before you come to laboratories and as you are doing them

(iii) Also read your lab schedule for tips on how to do labs

(iv) Labs are much (much, much) easier to do when you are familiar with them; I will be able to tell when you are unfamiliar with labs; I will reserve the right to quiz on lab preparation if I get the impression that students are coming to laboratories unprepared

(v) Answer questions and make notes while the material is still fresh in your mind

(i) For tips on how to study for microbiology, see: [microbiology and "study tips" (Google Search)] [microbiology study tips (need to skip down a bit to find) (Gary E. Kaiser)]

(j) Links to other on-line microbiology courses (and course-like sites): [microbiology and course (Google Search)] [Microbiology Webbed Out (Kenneth Todar)]

(k) Microbiology link collections can be found at: [microbiology links (Google Search)]

(l) For an overview of chemistry, click here.

(3) What is Microbiology?

(a) Microbiology is "the study of microbes, organisms so small that a microscope is needed to study them."

(b) Microbiology, as a science, may be differentiated along organism lines ("the variety of kinds of microbes") and in terms of techniques and goals ("the kind of work microbiologists do")

(c) [microbiology (Google Search)] [index]

(4) The variety of kinds of microbes (microbes, microorganisms)

(a) Microbes are typically (but certainly not always) either unicellular organism (e.g., bacteria) or acellular "organisms" (e.g., viruses)

(b) Typically a microbiologist will differentiate microbes into the following categories:

(i) bacteria

(ii) algae

(iii) fungi

(iv) viruses

(v) protozoa

(vi) helminths

Supplemental Material – Types of Microorganisms & their General Properties
organism:	types:	description:	Nutrition type (-trophs):	durable state:	some diseases:
algae:	brown, red, green, diatoms, dinoflagellates, euglenoids	photosynthetic aquatic eucaryotes, cell walls, unicellular and multicellular	photoauto-	---	---
bacteria:	eubacteria, archaeabacteria, Gram-negative, Gram-positive, acid fast, cyanobacteria	procaryotes, absorbers, wet conditions, animal decomposers, cell walls, unicellular	chemohetero- photohetero- chemoauto- photoauto-	endospores (some)	tetanus, botulism, gonorrhea, chlamydia, tuberculosis, etc., etc., etc.
cyano-bacteria:	blue-green algae	photosynthetic aquatic procaryotes, green lake scum, cell walls	photoauto-	---	---
fungi:	yeasts (unicellular fungi), molds (filamentous fungi)	eucaryotes, absorbers, dry conditions, plant decomposers, cell walls, ~100 human pathogens	chemohetero-	spores	mycoses: candida, ringworm, athlete's foot, jock itch, etc.
helminths:	Flatworms (platyhelminths), roundworms (nematodes)	metazoan (multicellular animal) parasites, engulfers and absorbers	chemohetero-	---	tape worm, trichinosis, hook worm, etc.
protozoa:	Unicellular and slime molds, flagellates, ciliates	eucaryotes, parasites, engulfers and absorbers, wet conditions, no cell wall, ~30 human pathogens	chemohetero-	cysts (some)	malaria, giardiasis, amoebic dysentery, etc.
viruses:	Enveloped, non-enveloped	acellular, obligate intracellular parasites	not applicable	virion particles, encased in durable state of host	common cold, flu, HIV, herpes, chicken pox, etc.

(5) Bacteria (chapters 4, 6, 7, 8, 9)

(a) The bacteria have the following characteristics:

(i) Relatively small

(ii) Single-celled

(iii) No nucleus or other membrane-bound organelles

(iv) Simple morphologies

(v) Primarily synthesizers or absorbers (i.e., not engulfers)

(b) Most bacteria do not cause human diseases, but most infectious diseases are caused by bacteria (and viruses)

(d) [bacteria (Google Search)] [index]

MICROSCOPY – SOME THEORY

(2) Resolution

(a) The object of microscopy is not just to increase magnification, but to do so while retaining sufficient resolution

(b) Resolution is the ability to see two items as two separate things, i.e., two dots as two separate dots

(c) The resolution a microscope is capable of achieving is the smallest distance between two dots such that the two dots may be observed (resolved) as separate entities

(d) In less technical terms, lower resolution means an increased degree of fuzziness, i.e., less focusable [sic?] specimens

(e) See Figure 3.5, Resolution

(f) [microscope resolution (Google Search)] [resolution (Standard Microscopy Terminology)] [index]

(3) Wavelength [l]

(a) Viewing things through a microscope usually means passing something (e.g., light) through a specimen (an object)

(b) The shorter the wavelength, the higher the resolution theoretically one can achieve with a microscope

(d) Blue light, for example, has a shorter wavelength than red light (blue light is also more energetic than red light)

(e) See Figure 3.4, The electromagnetic spectrum

(f) Thus, a light microscope that was limited to employing blue light can theoretically achieve a higher resolution than an otherwise similar light microscope that employs only red light (or all wavelengths of visible light)

(g) [wavelength and light (Google Search)] [wave length (Standard Microscopy Terminology)] [index]

(4) Electrons

(a) Light is not the only thing that has a wavelength

(b) All objects have an associated wavelength, and the larger the object, the shorter the wavelength

(d) Electrons thus have much smaller wavelengths than light (especially visible light) so consequently a microscope that employs electrons rather than light has a much higher theoretically (and actually) achievable resolution

(e) [electrons and waves, electron and waves (Google Search)] [index]

(5) Numerical aperture [NA]

(a) Numerical aperture is a partial measure of the resolving power a lens is capable of; basically a measure of the quality of the lens

(b) The greater the numerical aperture, the greater the resolving power of a lens

(d) [numerical aperture (Google Search)] [resolution page—scroll down for information on numerical aperture] [numerical aperture (Standard Microscopy Terminology)] [index]

(6) Refraction

(a) Another thing that can influence resolution (in addition to wavelength and numerical aperture) is refraction

(b) The occurrence of refraction causes objects to appear fuzzy (i.e., lowers resolution)

(d) To do this, one employs immersion oil between objects and lenses

(e) [refraction (Google Search)] [refraction (Standard Microscopy Terminology)] [index]

(7) Immersion oil (index of refraction)

(a) Immersion oil possesses an index of refraction (that is, the speed with which light passes through a substance) that is identical to that of glass, with both different from that of air

(b) When light passes from a glass slide into air, the light bends; this bending causes a scattering of the light exiting a specimen so consequently there is a reduction in resolution (i.e., there is an increase in fuzziness)

(c) Because immersion oil possesses the same index of refraction as glass, light passing out of a specimen and slide will pass through the oil without bending and then will go directly into the immersed lens

(d) See Figure 3.9, Immersion oil

(e) Reminder: A teaching microscope typically has only a single lens that can be immersed in immersion oil; all other lens can be damaged by immersion oil; always double check that you are using the correct lens when employing immersion oil and always clean the microscope of immersion oil when you are done

(f) [immersion oil (Google Search)] [immersion media (nice image) (Molecular Expressions)] [immersion of a lens—immersion medium—immersion liquid (Standard Microscopy Terminology)] [index]

(g) [index of refraction (Google Search)] [index]

LIGHT MICROSCOPY

(8) Light microscopy

(a) Light microscopy is the use of light as the substance that passes through the specimen (i.e., contrast electron microscopy)

(b) Optical or light microscopes are the tools of light microscopy

(9) Compound light microscope

(a) A compound light microscope employs at least two lenses through which light passes going from the specimen to the eye

(b) Compound light microscopes can be monocular or binocular (one eye piece or two, respectively; we will use binocular scopes exclusively)

(i) Light source

(ii) Illuminator (light source)

(iii) Condenser

(iv) Iris diaphragm

(v) Specimen (on stage)

(vi) Objective lens

(vii) Body tube

(viii) Ocular lens (eye piece)

(d) See Figure 3.12, The compound light microscope

(e) [compound light microscope (Google Search)] [cells and the light microscope] [light microscopy techniques and light microscopy instrumentation] [light microscopy page] [microscope parts page] [light microscopy] [light microscopy forum] [index]

(10) Light source

(a) One controls the level of illumination of an object at the light source

(b) The level of contrast, on the other hand, is controlled using the iris diaphragm (which also impacts the level of object illumination)

(c) Not having the light source sufficiently illuminating is a common source of error; make sure you at least are aware that the light source is not sufficiently illuminating if you choose to avoid using the microscope in this way

(d) [microscope and "light source" (Google Search)] [index]

(11) Condenser

(a) The condenser emits light as parallel beams to reduce scattering

(b) For what we will be doing in the laboratory it will make sense to raise the condenser as far as it will go and then to leave it there; though those of you who are into fine tuning, you may want to lower the condenser slightly under oil immersion

(c) Make sure that you are at least aware that you are lowering the condenser if you choose to use the microscope in this way

(d) [microscope and condenser and introduction (Google Search)] [condenser (Standard Microscopy Terminology)] [index]

(12) Iris diaphragm

(a) The iris diaphragm controls how much light enters the specimen

(b) Note that the more light that enters the specimen, the less contrast you will observe, i.e., the less detail you will be able to observe [contrast page] [contrast (Standard Microscopy Terminology)] [index]

(c) Typically you will want to close the iris diaphragm as far as possible to give maximum contrast while still allowing sufficient light through to see the object; note that if your light source is not sufficiently powerful (or is turned down too far) then you may fail to achieve adequate illumination simultaneously with achieving adequate contrast

(d) Very typically students will attempt to observe a specimen using their iris diaphragm too-far opened, allowing too much light can wash out detail to an amazing degree; make sure that you are at least aware that you are not sufficiently closing the iris diaphragm if you choose to use the microscope in this way

(e) [The iris diaphragm is the most important single control on the microscope. There is a misconception that it is used to regulate the amount of light. The light intensity control is the sole means to adjust the brightness. The iris diaphragm is the resolution verses contrast control. It does this by varying the size of the numerical aperture of the objective lens. Usually, lenses such as those found on cameras have the iris diaphragm built in the objective lens. In a microscope objective the iris diaphragm would have to be very small, which would be difficult to manufacture. So the optical engineers put the iris diaphragm at the optical equivalent of being in the objective lens, in the condenser assembly. This is one of the reasons why the condenser lens has to be set at the correct distance to the objective. In addition the iris diaphragm controls the depth of field.] [index]

(f) [microscope and "iris diaphram" (Google Search)] [index]

(13) Mechanical stage

(a) Our microscopes posses a mechanical stage that allows us to adjust the position of the specimen without touching the slide

(b) Make sure that you properly place the slide in the clip; a common mistake is to incorrectly attach the slide to the stage mechanism

(14) Focusing

(a) Our microscopes are focussed by moving the objective lenses up and down rather than moving the stage up and down

(b) There are two adjustments, the coarse adjustment and the fine adjustment

(d) Note that the microscopes we own are approximately ten-year-old Nikons whose focusing mechanisms both tend to fail and are not repairable; if you are having extreme difficulty focusing your microscope, by all means call over your instructor for assistance—it is at least plausible that the problem may be found in the mechanism of your microscope

(e) Note also that it is possible to focus onto things other than your specimen; this can fool you into thinking that you have your specimen in focus when this otherwise is not the case; remember that your specimen will move as you move your mechanical stage about and if what you have focussed in on does not move with your stage, then it is not your specimen

(f) [focus (Standard Microscopy Terminology)] [index]

(15) Objective lenses

(a) Our microscopes have four objective lenses ranging in power from 4x (i.e., four-fold increase in magnification) to 100x

(b) These lenses are called “objectives” because they are closest to the "object"

(d) The 40x lens represents the "high and dry" lens, i.e., high power without the oil

(e) [objective lenses ()] [objective (Standard Microscopy Terminology)] [index]

(16) Parfocal

(a) Viewing an object typically requires the use of more than one objective lens

(b) First one centers and focuses on the specimen using a low power lens (e.g., 3x or 10x depending on the size of the specimen)

(c) Then one changes objectives, by rotating the nosepiece of the microscope containing the objective lenses, to bring the specimen into higher magnification

(d) Because the microscope is parfocal, the specimen will stay in approximate focus even as you change objectives

(e) For much of our work in microbiology we will ultimately be employing the oil immersion lens, but we will employ some or all of our lower-power objectives to find and initially bring the specimen into focus

(f) [parfocal microscope (Google Search)] [parfocal objectives (Standard Microscopy Terminology)] [index]

(17) Bright-field microscopy

(a) We will be employing almost exclusively bright-field illumination in our microscopy laboratories

(b) In bright-field microscopy the background is bright and the specimen illuminated from below and therefore appears darker than the background

(c) [bright-field microscopy, bright-field microscope (Google Search)] [bright field setup] [bright field illumination (Standard Microscopy Terminology)] [index]

(18) Dark-field microscopy

(a) Dark-field microscopy using a different technique of illuminating the specimen so that it appears light on a dark background (i.e., lighter than the background, which appears black)

(b) See Figure 3.14, A comparison of bright-field and dark-field images

(c) [dark-field microscopy, dark-field microscope (Google Search)] [dark field illumination (Standard Microscopy Terminology)] [dark field] [dark field] [index]

(19) Phase-contrast microscopy

(a) Phase-contrast microscopy is employed to view living, unstained microorganisms

(b) We will have a demonstration of phase-contrast microscopy in our laboratory

(c) See Figure 3.18, Images of the same organism (Paramecium, 600X) produced by four different techniques

(d) [phase-contrast microscopy, phase-contrast microscope (Google Search)] [phase-amplitude contrast (Standard Microscopy Terminology)] [phase contrast] [phase contrast] [botany online: phase contrast microscopy] [index]

ELECTRON MICROSCOPY

(20) Electron microscopy (EM)

(a) Electron microscopes use electrons instead of photons (light) to illuminate a specimen

(b) This allows a dramatic increase in magnification and resolution over what is possible using light microscopes

(d) Electron microscopy comes in two types

(i) Transmission electron microscopy (TEM)

(ii) Scanning electron microscopy (SEM)

(e) See Figure 3.20, Light and electron microscopy images compared

(f) See Figure 3.21, Shadow casting

(g) See Figure 3.23, A freeze-etch preparation

(h) [electron microscopy, electron microscope (Google Search)] [index]

(21) Transmission electron microscopy (TEM)

(a) This form of EM transmits the electrons through the specimen thus revealing internal structures, e.g., such as the structures within cells

(b) Specimens must be cut very thinly and treated with substances (metals) that scatter electrons

(d) [transmission electron microscopy, transmission electron microscope, TEM (Google Search)] [index]

(22) Scanning Electron Microscopy (SEM)

(a) This form of EM supplies less magnification but is good for observing the surface of relatively large objects (i.e., thin sectioning is not necessary)

(b) A heavy metal must be deposited on the surface of the specimen and it is actually this metal whose image is formed

(c) See Figure 3.25, Colorized SEM photos of representative microbes

(d) [scanning electron microscopy , scanning electron microscope, SEM (Google Search)] [scanning electron microscope (Museum of Science)] [Dennis Kunkel’s microscopy] [index]

STAINING TECHNIQUES

(23) Techniques of light microscopy

(a) Successful light microscopy requires specimen preparation (wet mount, smearing) and staining (various); we consider these various techniques as follows

(b) "Microscopes are of little use unless the specimens for viewing are prepared properly. Here we explain some important techniques used in light microscopy. ¶ Although resolution and magnification are important in microscopy, the degree of contrast between structures to be observed and their backgrounds is equally important. Nothing can be seen without contrast, so special techniques have been developed to enhance contrast."

(c) [techniques of light microscopy (Google Search)] [contrast page] [contrast (Standard Microscopy Terminology)] [index]

(24) Wet mount

(a) A wet mount is a not-dried specimen, typically a drop of specimen-containing medium

(b) Wet mounts do not provide good contrast (i.e., it is difficult to see the microorganism) when using bright-field microscopy

(25) Smears

(a) A smear is small volume (a loopful) of specimen-containing medium that is spread (smeared) onto a microscope slide

(b) "If you make smears too thick, you will have trouble seeing individual cells; if you make them too thin, you may find no organisms. If you stir the drop of medium too much as you spread it on the slide, you will disrupt cell arrangements."

(26) Heat fixing

(a) To kill the smeared organism as well as to adhere the smear to the slide, some form of fixing must be employed, and heat fixing is the most common form used

(b) "Heat fixation accomplishes three things: (1) it kills the organisms; (2) it causes the organisms to adhere to the slide; and (3) it alters the organisms so that they more readily accept stains (dyes). If the slide is not completely dry when you pass it through the flame, the organism will be boiled and destroyed. If you heat-fix too little, the organism may not stick and will wash off the slide in subsequent steps. Any cells remaining alive will stain poorly. If you heat-fix too much, the organisms may be incinerated, and you will see distorted cells and cellular remains. Certain structures, such as capsules found on some microbes, are destroyed by heat-fixing so this step is omitted and these microbes are affixed to the slide just by air-drying."

(27) Stains

(a) A stain is a substance that adheres to a cell, giving the cell color

(b) The presence of color gives the cells significant contrast so are much more visible as a consequence

(d) They thus may be used to differentiate different types of organisms or to view specific parts of organisms

(e) [bacteria stains (Google Search)] [index]

(28) Simple stain

(a) A stain using only a single dye that does not differentiate between different types of organisms

(b) That is, there typically is only a single staining step and everything that stains is stained the same color

(29) Differential stain

(a) A differential stain uses more than one dye and stains different kinds of organisms different colors

(b) Typical differential stains include

(i) The Gram stain

(ii) The Ziehl-Neelsen acid fast stain

(30) Gram stain

(a) The Gram stain distinguishes between organisms with Gram-positive and Gram-negative cell envelope types

(b) We will consider the details of Gram staining in the laboratory

(c) Note that the reason that Gram staining works is because the Gram-positive bacteria retain the stain (a combination of crystal violet stain and iodine mordant) while the Gram-negative cannot retain this stain

(d) See Figure 3.28, The Gram stain

(e) [The Gram staining method, named after the Danish bacteriologist who originally devised it in 1844, Hans Christian Gram, is one of the most important staining techniques in microbiology. It is almost always the first test performed for the identification of bacteria. The primary stain of the Gram's method is crystal violet. Crystal violet is sometimes substituted with methylene blue, which is equally effective. The microorganisms that retain the crystal violet-iodine complex appear purple brown under microscopic examination. These microorganisms that are stained by the Gram's method are commonly classified as Gram-positive or Gram non-negative. Others that are not stained by crystal violet are referred to as Gram negative, and appear red. ¶ Gram staining is based on the ability of bacteria cell wall to retaining the crystal violet dye during solvent treatment. The cell walls for Gram-positive microorganisms have a higher peptidoglycan and lower lipid content than gram-negative bacteria. Bacteria cell walls are stained by the crystal violet. Iodine is subsequently added as a mordant to form the crystal violet-iodine complex so that the dye cannot be removed easily. This step is commonly referred to as fixing the dye. However, subsequent treatment with a decolorizer, which is a mixed solvent of ethanol and acetone, dissolves the lipid layer from the gram-negative cells. The removal of the lipid layer enhances the leaching of the primary stain from the cells into the surrounding solvent. In contrast, the solvent dehydrates the thicker Gram-positive cell walls, closing the pores as the cell wall shrinks during dehydration. As a result, the diffusion of the violet-iodine complex is blocked, and the bacteria remain stained. The length of the decolorization is critical in differentiating the gram-positive bacteria from the gram-negative bacteria. A prolonged exposure to the decolorizing agent will remove all the stain from both types of bacteria. Some Gram-positive bacteria may lose the stain easily and therefore appear as a mixture of Gram-positive and Gram-negative bacteria (Gram-variable). ¶ Finally, a counterstain of basic fuchsin is applied to the smear to give decolorized gram-negative bacteria a pink color. Some laboratories use safranin as a counterstain instead. Basic fuchsin stains many Gram-negative bacteria more intensely than does safranin, making them easier to see. Some bacteria which are poorly stained by safranin, such as Haemophilus spp., Legionella spp., and some anaerobic bacteria, are readily stained by basic fuchsin, but not safranin. The polychromatic nature of the gram stain enables determination of the size and shape of both Gram-negative and Gram-positive bacteria. If desired, the slides can be permanently mounted and preserved for record keeping.]

(f) [Gram stain (Google Search)] [Gram stain (University of Pennsylvania Health System)] [Gram’s stain and meningitis] [cell differentiation by Gram stain] [Gram stain] [index]