chapter 3: observing microorganisms through a telescope

1 kilometer (km)

1000m

1 decimeter (dm)

1/10m = 0.1m = 10^-1m

1 centimeter (cm)

1/100m = 0.01m

1 millimeter (mm)

1/1000m = 0.001m

1 micrometer (um)

1/1,000,000m = 0.000001m = 10^-6m

1 nanometer (nm)

1/1,000,000,000m = 0.0000000001m = 10^-9m

lenses

focus light rays at a specific place called the focal point

• distance b/w center of lens and focal point is the focal length

• strength of lens is related to focal length

  -shorter the focal length = more magnification

refraction

bending or change in the angle of the light rays as it passed through a medium such as a lens

lenses & the bending of light

light is refracted (bent) when passing from one medium to another

refractive index: a measure of how greatly a substance shows the velocity of light OR a measure of the light-bending ability of a medium

• direction & magnitude of bending is determined by the refractive indexes of the two media forming the interface

• to achieve high magnification, objective lens must be small, but a small lens will lose much of a bending light. so immersion oil is used that has the same refractive index as glass so light will not bend any further

refraction in the compound microscope using an oil immersion objective lens

• the light may bend in air so much that is misses the small high-magnification lens

• immersion oil is used to keep light from bending

microscope resolution OR resolving power

resolution is the ability of a lens to separate or distinguish small objects that are close together

-microscopic resolution is the shortest distance b/w 2 points in a microscope’s field of view that can still be distinguished as 2 distinct objects

• wavelength of light is major factor in resolution as shorter wavelength gives greater resolution

• white light has long wavelength and cannot resolve structures less than 0.18micrometers apart

• resolution of leeuwenhoek’s microscope was 1 micrometer

• resolving power = wavelength of light / 2 x numerical aperture

property magnification

scanning: 4x

low power: 10x

high power: 40-45x

oil immersion: 90-100x

illuminator

the light source

condenser

has lenses that direct rays through the specimen

objective lens

lens closet to the specimen and used to magnify (make object appear enlarged) the image; different powers such as 4X, 10X, 100X

ocular lens or eyepiece

magnifies the image & also used to view the image, magnification power is 10X

light microscope

any kind of microscope that uses visible light to observe specimen

includes:

• bright field

• dark field

• phase contrast

• fluorescence

-these are compound microscopes, image formed by action of > or equal to 2 lenses

-microscopes used by leeuwenhoek was a simple microscope, 1 lens.

bright-field microscope

produces a dark image against a brighter background

• has several objectives lenses these are parfocal microscopes (remain in focus when objective lenses switched)

• total magnification: product of the magnifications of the ocular lens and the objective lens (ocular lens magnification x objective lens magnification)

dark field microscopy

used to study living microorganisms, dark field condenser is used that contains an opaque disc. it will block light from directly entering the objective lens. only the light reflected from the specimen enters the objective lens,

• specimen appears light against black background. very thin spirochete as treponema pallidum (causes syphilis) is examined

electron microscopy

used to examine objects smaller than 0.2 micrometers like viruses or internal cellular structures, a beam of electrons is used instead of light

• better resolution bcs of shorter wavelength of electron (100,000X shorter than light)

• electromagnetic lenses (not glass) are used to focus beam of electrons onto a specimen

• images are always black/white but may be colored artificially

two types:

-transmission electron microscope (TEM)
-scanning electron microscope (SEM)

transmission electron microscope

electrons pass through the specimen and are scattered. magnetic lenses focus on image onto a fluorescent screen or photographic plate. (right) this colorized transmission electron micrograph. (TEM) shows a thin slice of paramecium. in this type of microscopy. the internal structures present in the slice can be seen.

scanning electron microscope

an electron gun produces a beam of electrons that scans the surface of an entire specimen. secondary electrons emitted from the specimen produce a 3 dimensional image. primary electrons sweep across the specimen & knock electrons from its surface, secondary electrons are picked up by collector, amplified, and transmitted onto viewing screen or photographic plate. In this colorized scanning electron micrograph, the surface structures of paramecium are seen. see the 3 dimensional appearanceo of cell, in contrast of 2 dimensional in transmission electron.

TEM electron microscopy

• ultra thin section required

• objects magnified 10,000 to 100,000X

• resolution about 2.5 nm

• internal structures seen

• electron beam passes through the specimen and produces image

• image can be viewed thru eye piece

SEM electron microscopy

• no sectioning required

• objects magnified 1,000 to 10,000X

• resolution abt 20nm

• external structures seen

• electron beam removes electron from surface of specimen

• image is viewed on the viewing screen

smear prep: the smear

a bacterial smear is a fried preparation of bacterial cells on a glass slide, requires only a small amount of the microbial culture. if smear is thick, light may not pass thru smear making it hard to see the morphology of microbial cell

good smears is one in which:

• microbes are evenly spread on the surface of slide

• microbes are not washed away during staining

• microbial forms are not distorted

smear prep: heat fixation

done by passing the air-dried smear several times over flame having smear side up OR by using the blow dryer from the underside of the slide (smear side up)

• smear is fixed (attached) on the slide by heat otherwise smear will be washed away during the staining procedure

• heat fixation coagulate bacterial proteins so bacteria stick to the slide surface

• heat fixation also kills bacteria

chemistry of a stain

chemically, a stain is an organic compound, 3 parts of a stain:

• benzene: organic color solvent

• chromophore: chemical that is color (it gives color to the organic solvent)

• auxochrome: ionizes the chromogen so that it can bind with the cells, fiber, or tissues

• chromogen = benzene + chromophore. a colored compound not a stain.

staining

microbes are generally colorless, so it is difficult to visualize them. staining the color the microbes (or background) with a dye that:

• creates a contrast b/w the bacteria & the background

• emphasizes certain microbial structures

good for study of microbial properties & to group microbes in specific groups for diagnosis

stains are salts composed of a positive & negative ion, one of which is colored & is known as the chromophore

-in a basic dye the chromophore is a CATION (ex. crystal violet, methylene blue, malachite green, safranin)
-in an acidic dye the chromophore is an ANION (ex. esoin, acid fuchsin, nigrosin)

as bacteria are slightly negatively charged, acidic dyes are repelled by most of the bacteria, a dye stains the background (negative staning, heat fixation is not required)

negative staining is used to observe the overall shape, size and capsules (if present)

what are the 3 kinds of staining procedures?

1. simple

2. differential

3. special

simple staining

an aqueous or alcohol solution of a single basic dye, creates a contrast between bacteria & the background

-highlights the entire microorganism, so used to study the cell shape, size, and arrangement

-simple & ez to use

during simple staining:

-smear is heat fixed, simple stain is applied for a certain length of time & washed off, then the slide is dried & studied (ex. methylene blue, crystal violet, carbolfuchsin, safranin)
-sometimes a mordant is also used to hold the stain or coat the specimen to enlarge it.

differential staining

requires the use of at least 3 chemical reagents, that are applied sequentially to a heat fixed smear.

1. primary stain: imparts its color to all cell

2. decolorizing agent: based on chemical composition of cellular components, the decolorizing agent may or may not remove the primary stain from the entire cell or only from certain cell structures

3. counterstain: has contrasting color to that of the primary stain

   -following decolorization, if the primary stain is not washed out, the counterstain is not absorbed and the cell or its components will retain the color of the primary stain

gram staining the procedure

developed by hans christian gram in 1884

classify bacteria in 2 groups: gram positive & gram negative

4 steps:

1. primary stain Crystal violet (basic purple dye) applied to a heat fixed smear

2. after washing off the primary stain, iodine (mordant) is applied. it will make crystal violet iodine complex (CV-I complex). then mordant is washed too. all bacteria now is dark violet or purple.

3. alcohol or an acetone solution is used as a decolorizing agent. which removes the purple color (CV-I complex) from gram negative bacteria, but gram POS bacteria retain the PURple. most important step. over decolorizing will have loss of primary stain, causing gram pos to appear gram neg but under-decolorizing will cause gram neg to appear as gram pos.

4. alcohol is rinsed off, and the slide is now stained with counterstain Safranin (basic red dye), smear washed again, blotted dry & examined microscopically

• gram pos bacteria have thick layer of peptidoglycan in cell wall. cv-I complex is not washed off by alcohol so they will retain the complex and stay PURPLE

• gram neg bacteria have thin layer of peptidoglycan and also have a layer of lipopolysaccharide as part of their cell wall. alcohol wash disrupts the lipopolysaccharide layer and cv-l complex is washed through the thin layers. so gram neg will stay colorless unless stained with counterstain safranin, after which they are pink.

gram reaction of a bacterium is clinically important as gram pos and gram neg respond to diff antibiotics. beta lactams (ex. pencillins & cephalosporins) are more active against gram pos bacteria and less active against gram negs as they cannot penetrate the lipopolysaccharide later. some bacteria stain poorly or not at all with gram staining (myobacteria)

acid-fast staining

used to distinguish myobacterium species & some species of nocardia. acid fast stain binds strongly to a waxy material in the cell wall.

in the procedure:

• carbol fuchsin (red dye) is applied to a fixed smear. heating enhances the penetration of this dye

• after cooling & washing w/ water, smear is washed with acid alcohol (decolorizer). non acid fast bacteria lose the primary stain. acid-fast bacteria retain the dye because it is more soluble in the cell wall lipids than in the acid alcohol.

• counter stain methylene blue is used which stain non acid-fast bacteria as blue

special staining

used to color specific parts of microorganism

• endospore (special resistant, dormant structure)

• flagella (used for locomotion)

• capsule (gelatinous covering)

special staining: endospore staining

formed by only a few genera

simple stain & gram stain dyes do not penetrate the wall of endospore

• schaeffer-fulton endospore stain is used

  -heat fixed smear prepared

  -primary stain malachite green is applied & heated to steaming for about 5 mins (to help stain penetrate with endospore wall)

  -washed w water to remove extra malachite green

  -counterstain safranin is applied to stain other parts of the cell as pink

special staining: capsules staining

presence of capsule determine a microbe’s virulence, capsular material is soluble in water, so washing can remove this gelatinous covering

• india ink or nigrosin is used to stain background dark

• then simple stain like safranin is used to stain cells

• capsules do not accept most dye likes safranin

• capsules look like halos around stained cells

special staining: flagella staining

flagella are very thin!

• a mordant is used to build up the diameter of flagellum & when stained with carbolfuchsin it becomes more visible under the light microscope

• # and location of flagella is used in diagnosis