Cells and Methods to Observe Them Ch3

Microscopes

most important tool for studying microorganisms

different types

• light microscope

• electron microscope

• atomic force microscope

Compound light microscopes

use visible light to observe objects

compound - 2 sets of magnifying lenses

• ocular lens (5x, 10x)

• objective lenses - 4x, 10x, 40x, 100x

microscope parts and their function (8)

ocular lens: eye piece

• can see the image, magnifies the image, 10x

specimen stage

• stage clip holds slide in place

condenser lens

• focuses the light up to the specimen, collects all the light, shines it up to the stage to the specimen

iris diaphragm lever

• controls the amount of light that enters the objective lens, works like a shutter on the camera, helps control and adjust the amount of light focused on the specimen

objective lens

• has a opening on the bottom, with 10x its pretty big and then inc gets smaller

• a selection of lens options provides different magnifications. the total magnification is the product of the magnifying power of the ocular lens and the objective lens

light source

rheostat

• controls the brightness of the light

side circle

• course and fine adjustment focus nob, used for low power → doesn’t let you run into the slide and instead slowly go into it

principles of light microscopy

visible light passes through specimen and then series of magnifying lenses, illuminator provides light

How good an image is, depends on: magnification, resolution, contrast

Magnification

apparent increase in size

• two lens: objective and ocular

• total magnification TM = power of ocular lens x power of objective lens (10x * 100x) = 1,000x

• Bacteria - only seen with 100x (oil immersion) objective lens

  • always start at lowest objective lens

Resolution

resolving power

• ability to distinguish 2 objects that are very close together (how sharp the image is)

• depends on quality and type of lens, wavelength of light (visible light, uv rays, beams), and specimen preparation, the shorter the wavelength the higher the resolution

• maximum resolving power of light microscope is 0.2 micrometer (using this resolving power, if u have 2 things that are 0.2 microns apart, you’ll see them as separate, if they’re closer, they’ll blend together and the image isn’t as sharp)

  • minimum distance between 2 points at which those points can be observed as separate

• 100x lens - need immersion oil to enhance resolution, wont be able to get in focus without it ever

  • immersion oil: reduces light refraction, has nearly same refractive index as glass

  • as magnification inc, the objective lens opening becomes smaller, so fewer light rays can enter the lens (but we want a lot), air has a lower refractive index than glass, so light bends as it passes from the glass slide → air → objective lens, causing some light rays to be lost, immersion oil has a refractive index similar to glass so there is less bending of light between the slide and OL, this allows more light rays to enter the objective lens → improves resolution → produces a clearer image

  • refractive index: a measure of how much a material slows down and bends light as light passes thru it

    • low refractive index: light bends MORE when entering/leaving another material

    • similar refractive index: light bends less

      • glass + immersion oil = similar RI

Contrast

determines how easily cells can be seen against background

• live bacteria lack contrast bcuz they have no color, difficult to see against colorless background

• increase contrast by:

  • staining: increases contrast but kills microbes

  • using phase-contrast and darkfield microscopy, creates areas of different color between the organism and background

Light Microscope

uses visible light, magnifies images approx 1,000x

3 types of light microscopy for different uses: brightfield, darkfield, phase-contrast

• Brightfield: used to view colored or stained specimen, most common type of microscopy, the background is bright/white, red perimysium

• Darkfield: increases contrast of live specimen, cells appear as bright objects against dark background, used to view transparent live organisms

• Phase-contrast Microscope: Uses special optics in the condenser and objective lens to enhance differences in refractive index within a specimen. It amplifies differences between the cell's structures and the surrounding medium, creating contrast. Denser areas of the cell generally appear darker, while less dense areas appear lighter/brighter. Allows you to see internal structures of living, unstained cells.

  • Unlike dark-field microscopy, which mainly shows a bright specimen against a dark background, phase-contrast microscopy can show both lighter and darker areas within the cell.

Electron Microscopes

Uses electron beams, a lot shorter in wavelength and bcuz of this we can achieve greater resolving power

Resolving power of ~0.3nm (know mm, nm etc)

• magnify images up to 100,000x

Two types:

• SEM - used to observe surface details, scanning electron microscope, produces 3d like image for texture and surface details

• TEM - used to view internal details, transmission electron microscope, we take our bacteria and embed it in a plastic block and then slice the block into tiny pieces, pic- new cell wall and membrane revealing that its about to divide

Scanning Probe Microscopes

uses metal probe

detailed images of surfaces

• ex. atomic force microscope

• resolving power much greater than that of EM

• sharp probe moves across sample’s surface

  • feels bumps, valleys of atoms

• pic: a protein, enzyme that’s a dna or rna structure

• yellow: virus, surface of the cell, the bumps are where the virus that has infected the cell is gonna come from

Preparing Specimens for Light Microscopy

Wet mount uses a drop of liquid specimen

• live bacteria - hard to see due to lack of contrast

• higher contrast is achieved by

  • using different types of microscopies

  • specimen staining

Prepare a smear

• smear: a thin layer of cells dried and fixed onto a slide before staining

  • fixing: in the lab using heat from a bunsen burner, thru the flame a couple times → first it will kill the bacteria and preserve it…, then it denatures the protein on the surface of bacteria, then attaches bacteria on the slide

Dyes and stains (coloring agents)

made of organic salts ( + or - charge) → bacteria has a slight neg charge

• basic dyes - carry pos charge, colored ion has a +

  • bond to cell

  • commonly stain the cell, bcuz its attracted to the neg charged bacteria

  • more commonly used than acidic dyes

  • include:

    • methylene blue, crystal violet, safranin (red), malachite green

• acidic dyes - carry neg charge

  • repelled by cell, bcuz its repelled by the neg charged bacteria

  • commonly stain the background

  • nigrosin - black dye

• Both used in different staining procedures: simple, differential, special

Simple staining procedure

Uses one stain to stain cell, background or bacterial cell

• increases contrast → size (can observe the different types if there are different ones), shape, arrangement of cells

• all cells are stained same color

  • no differentiation between cell types

  • but we can see that its safranin rod-shaped bacteria and they are connected together in a string (strepto)

Differential Staining procedure

used to distinguish different types of bacteria

uses a series of reagents

2 most common differential stains: gram stain, acid-fast stain

Gram Stain** tested on it in the lab

most widely used staining procedure for bacteria - differential

developed by Dr. Hans Christian Gram,

a series of reagents and stains and separates bacteria into 2 major groups based on cell wall structure and chemistry

does not work for all bacteria

• one of the reasons why acid fast stain was developed

purple - positive & red/pink - negative

• can tell difference cuz when mixed its separate

Gram Stain Procedure and Result

Gram - Positive (has nothing to do with charge but whether they retain the primary dye or they dont)

• stained purple (retains primary dye)

Gram - Negative

• stained red or pink (loses primary dye)

Between each step have to rinse with ample amount of water for 1 min until there’s no more dye

1st step: make a smear and heat fix it

1st stain used is CV; binds to both types of bacteria

2nd is iodine for 1 min, helps to enhance the purple color by creating the CV Iodine complex, binds to CV in bacteria to form CVI complex which is more purple than the crystal alone (intensifies it) → both cells will be purple

3rd: most important step, the differentiating step, add enough but not too much, allows us to differntiate between g+ and g- cells, alcohol wash, decolorizing step. when alcohol is added it removes the dye from gram - cells and become colorless, gram + should still be purple if done carefully bcuz of their cell wall structure

4th: stains the gram - cells that were colorless, g+ already have purple so won’t pick up red from safranin

Acid-fast Stain

Used to detect members of genus Mycobacterium (doesnt stain well with gram stain)

• includes causative agents of TB and leprosy - these are caused by mycobacterium

• cell wall contains high concentrations of mycolic acid

  • waxy fatty acid that prevents uptake of dyes, dyes don’t penetrate and stain well

  • harsher methods needed - once stained difficult to decolorize

• used for presumptive identification of agents (those pathogens) in clinical specimens for diagnosis

  • somebody thought to have TB, will take the sputum and do an acid fast stain, if it comes back with a acid fast bacteria, they will be given an antibiotic right away

Acid-fast stain procedure

requires multiple steps

• Primary dye (carbol fuchsin)

  • colors all bacteria red

• decolorizer (acid alcohol)

  • removes red stains from non acid-fast bacteria

• counter stain (methylene blue)

  • colors non acid-fast bacteria blue, so acid fast is red

Capsule Stain

capsule: gel like polysaccharide layer surrounding cell

• stains poorly, negative stain often used, use acidic dyes

• allows capsule to stand out around organism

Endospore stain

endospore: resistant, dormant structure formed by species of bacillus Clostridium, resists gram stain, often appears as clear object

endospore stain: uses heat to facilitate uptake of the primary dye malachite green by endospore, counterstain (usually safranin) used to visualize vegetative cells

Flagella stain

flagella: used for prokaryotic motility, too thin to be seen with light microscope, presence and distribution can help in identification

flagella stain: dyes and coats flagella to thicken and make visible

bacterial shapes

…

binary fission

most prokaryotes divide by bf, cells may stick together following division, form characteristic groupings or arrangements depending on plane of division

planes

plane

structures external to cell wall

gel like layer outside cell wall

• capsule: organized, attached to cell wall

• slime layer: unorganized, loose

• composed of sugar (glycocalyx) and/or polypeptides

  • excreted by organism, not always present

• function

  • protection from host defenses (phagocytosis)

  • attachment to surfaces

    • both increase virulence of pathogens**

  • protection against drying (desiccation)

  • reserve of nutrients

capsules

dental plaques: oral streptococci use capsular slime to adhere to surfaces of teeth and gums —> biofilm

Pili

protein appendage, not essential, but give advantage

shorter, thinner than flagella; found only on gram negative bacteria

fimbriae:

• hair-like, facilitates attachment

• contributes to pathogenicity

• Neisseria gonorrhoeae

sx pilus

• attachment to another cell

• transfer of dna between cells during conjugation

Flagella

protein appendage, not essential, but give advantage

long protein structure for motility

typically, on rod-shaped bacteria

some important in bacterial pathogenesis

• helicobacter pylori penetration through mucous coat

has three basic parts:

• filament: extends to exterior, made of proteins called flagellin

• hook: connects filament to cell

• basal body: anchors flagellum into cell wall

Flagellar arrangement

numbers and arrangements vary and help with characterization

motility

movement is a series of runs and tumbles

chemotaxis: bacteria sense chemicals and move accordingly

• nutrient - acts as attractant

• toxic compound - acts as repellent

cell wall

strong, rigid structure

functions: maintain shape of bacteria, protects cell from osmotic lysis (bursting)

major site of actions for most antibiotics

unique chemical structure: distinguishes gram positive from gram negative bacteria

differences in cell wall account for differences in staining characteristics

PTG - peptidoglycan

cell wall made of peptidoglycan (PTG), found only in bacteria

basic structure of PTG: polymer of NAG and NAM, cross-linked by tetrapeptide chains

a good target of antimicrobials since unique to bacteria

• interferes with synthesis of or break down of PTG → cell lysis

• examples include

  • penicillin: prevents cross-linking of adjacent glycan chains??

  • lysozyme: breaks bonds linking glycan chain

gram positive cell wall

has thick layer of PTG (as many as 30)

teichoic acid

no outer membrane

small periplasm

gram + bacterium:

• retains CV-I complex after decolorization

• decolorizer dehydrates thick layer of PTG

gram negative cell wall

more complex than gram +

has thin layer of PTG

no teichoic acids

has large periplasmic space with degradative enzymes

has outer membrane: lipopolysaccharide layer (LPS)

• portions medically significant

  • o-specific polysaccharide side chain → antigenic

    • used to identify species or stains

    • E. coli O157:H7

  • lipid A → endotoxin

    • gram negative infection of bloodstream

gram - bacterium:

• loses CV-I complex

• decolorizer damages outer membrane; thin layer of PTG

bacteria that lack a cell wall

mycoplasma species:

• have extremely variable shape

• antimicrobial directed towards cell wall ineffective: penicillin, lysozyme

• sterols in membrane give strength to membrane

other types of prokaryotic cell wall

primary difference between members of domain bacteria and domain archaea

• domain archaea do not contain PTG but rather pseudopeptidoglycan (lack NAM)

structures internal to cell wall

the cytoplasmic membrane

defines boundary of cell

• phospholipid bilayer embedded with proteins

• functions:

  • ATP production: ETC

  • serves as a selective barrier between cell and external environment

simple diffusion

molecules move freely across cytoplasmic membrane

• moves from area of high concentration to area of low concentration until equilibrium is reached

• no energy required

• water (osmosis), certain gases, small hydrophobic molecules

facilitated diffusion

moves molecules across membranes with help of transporter proteins

movement down concentration gradient - no energy required

active transport

moves molecules against a concentration gradient using a transporter protein

requires energy

group transport

transport mechanism that chemically alters molecule during passage

• requires energy

• phosphorylation of glucose

cytoplasm

substance inside plasma membrane

about 80% water

internal components - chromosome

single, circular, double stranded DNA

contains all genetic information

packed tightly - forms the nucleoid

internal components - plasmids

extrachromosomal, circular, dsDNA

• independently replicating

encode characteristic potentially enhancing survival

• antimicrobial resistance

internal components - ribosome

involved in protein synthesis

composed of 2 subunits:

• prokaryotic

  • large = 50s and small = 30s, total = 70s??

• eukaryotic - bigger

  • large = 60s and small = 40s, total = 80s

• target of many antibiotics due to this difference

internal components - cytoskeleton

internal protein framework

bacterial protein analogous to those in eukaryotic cytoskeleton

• actin (MreB)

• tubulin (FtsZ)

• intermediate filaments (crescentin)

function:

• controls cell shape

• involved in cell division

internal components - storage granules vs gas vesicles

SG: store excess nutrient (carbon, energy sources)

• ex. glycogen granules (glucose), poly-B-hydroxybutyrate

GV: small protein compartments containing gas, provides buoyancy to cell

internal components - endospores

unique dormant* structures, produced by members of bacillus clostridium, thick coat resisting damage conditions

• heat, desiccation, chemicals, uv light

found at different positions in cell

• central, terminal, subterminal

sporulation: endospore formation

germination: return to vegetative state

differences between prokaryotes and eukaryotes

P

• one circular chromosome, not in a nucleus

• no organelles

• PTG cell walls

• reproduce by binary fission

E

• multiple paired chromosomes, in nuclear membrane

• membrane bound organelles

• no cell wall except plants (polysaccharide)

• reproduce by meiosis and mitotic spindle

origins of mitochondria and chloroplasts

endosymbiotic theory: mitochondria and chloroplasts were derived from bacteria

• over billions of years each partner became indispensable (abs necessary) to the other

key supportive evidence:

• similar in size and shape to bacteria

• a circular chromosome

• 70s ribosomes

• divide by binary fission

• surrounded by a double membrane