Chapter 3  Microscopy and Cell Structure

 

Overview

Microscopes are the basic tools for the study of microscopic form and structure. This chapter introduces

the various kinds of microscopes and staining techniques used by microbiologists to study both

prokaryotic and eukaryotic microorganisms. The prokaryotic cell is presented with special emphasis on

the cell wall, external structures, cell membrane and internal components of the cell. The eukaryotic cell

is also presented with a focus on the components that are involved in microbial infection and resistance.

The two kinds of cells are compared and contrasted.

 

Learning Objectives

After studying the material in this chapter, you should be able to:

Microscopy

1.        List and describe the kinds of microscopes used in microbiology.

2.        Define and contrast:

  Magnification

  Resolution

3.   Describe the preparation of a smear used in staining procedures.

4.   List the two kinds of differential staining procedures; give specific examples and uses of each type.

5.   List the three kinds of special staining procedures; give specific examples and uses of each type.

6.        Describe the Gram stain procedure and explain the function of each step.

7.        Interpret a Gram stain.

Prokaryotic Cells

1.   Describe the different morphological forms and arrangements of prokaryotic cells and give specific

examples of each.

2.   List the characteristics of prokaryotic cells.

3.   Describe the structure of a prokaryotic cell and give the specific function of each of the major

components.

4.   Describe the fluid mosaic model of the cytoplasmic membrane.

5.   Describe the major roles of the cytoplasmic membrane.

6.   Describe the role of the cytoplasmic membrane in energy transformation.

7.   Describe the processes by which substances are transported across cell membranes by:

  Passive transport mechanisms

  Active transport mechanisms

8.   Define:

  Simple diffusion

  Osmosis

  Osmotic pressure

9.   Describe what will occur when a bacterial cell with a cell wall is place into a:

  Hypertonic solution (higher concentration of solute outside of the cell than within of the cell)

  Hypotonic solution (higher concentration of solute inside of the cell than outside of the cell)

10. Define:

  Transport protein

   Facilitated diffusion

  Active transport

  Proton motive force

  ABC transport systems

  Group translocation

11. Describe the structure of the bacterial cell wall. Differentiate between Gram-negative and Gram-

positive cell walls.

12. Describe a typical bacterial chromosome.

13. Describe a plasmid and explain what it does.

14. Describe a bacterial ribosome.

15. Give the function of endospores (bacterial spores) and name the two genera in which endospores are

generally found.

 

Key Concepts

1.  Microscopes are the basic tools that make it possible to observe the microscopic world.

2.  The functions of microscopes are to enlarge (magnify) and to make visible as separate objects

(resolve) two objects that are close together.

3.  The major differences between the different types of microscopes are the types of lenses and how the

specimen is illuminated.

4.   Staining techniques make it possible to study more clearly the structure of both prokaryotic and

eukaryotic cells.

5.   Prokaryotic cells are designed to carry on the basic functions of life and they have the appropriate

structures to do so.

6.  The structure of the bacterial cell wall is an important determinant in many of the properties of

bacteria including differential staining characteristics, susceptibility to antibiotics such as penicillin

and susceptibility to changes in extracellular osmotic concentrations.

7.  The cytoplasmic membrane is the major determinant as to what enters and leaves a cell.

8.  Prokaryotic cells have developed cellular modifications such as the glycocalyx, sheaths, flagella and

pili that enable them to compete successfully.

9.  The bacterial chromosome consists of a single, double-stranded DNA molecule that resides in the

cytoplasm of the cell rather than in a nucleus.

10. Extrachromosomal DNA called plasmids are sometimes found in prokaryotic cells and may be

advantageous, but not essential, to the cell.

11. The structure of ribosomes differs in prokaryotic and eukaryotic cells making the prokaryotic

ribosomes useful targets for antimicrobial agents.

12. Endospores are dormant forms within the life cycle of some bacterial species that enable the microbe

to withstand difficult environmental conditions.

 

Summary Outline

I.        Microscopic techniques: the instruments

A.   Light Microscopes

1.     The bright-field microscope: Visible light passes through the specimen

2.     Phase contrast microscope: Amplifies differences in refraction

3.     Dark-field microscope: Directs light toward a specimen at an angle

4.     The fluorescent microscope: used to observe cells that have been stained with

fluorescent dyes

5.     The confocal scanning laser microscope: Used to construct a three-dimensional image

of a thick structure and to provide detailed sectional views of the interior of an intact cell

B.   Electron Microscopes

1.      Use electromagnetic lenses, electrons, and phosphorus screens to produce a magnified

image

a)    Transmission electron microscopes (TEM) transmit electrons through a

specimen that has been prepared by thin-sectioning, freeze-fracturing, or freeze-etching

b)    Scanning electron microscopes scan a beam of electrons back and forth over the

surface of a specimen, producing a three-dimensional effect

2.        Scanning probe microscopes: Maps the bumps and valleys of a surface on an atomic scale

C.   Dyes and staining

1.      Differential stains

a)    The Gram Stain

(1)  Gram-positive bacteria stain purple

(2)  Gram-negative bacteria stain pink

b)    The acid-fast stain: Stains organisms such as mycobacteria, which do not take

up stains readily; acid-fast organisms stain pink and all other organisms stain

blue

2.     Special stains to observe cell structures

a)    Capsule stain colors the background, allowing the capsule to stand out as a halo

around an organism

b)    Spore stain stains endospores

c)    Flagella stain stains flagella

3.     Fluorescent dyes and tags: Some fluorescent dyes bind compounds that characterize

all cells, others bind to compounds specific to only certain cell types

D.   Morphology of prokaryotic cells

1.      Shapes

a)    Cocci

b)    Rods

c)    Coccobacilli

d)    Vibrios

e)    Spirilla

f)     Spirochetes

g)    Pleomorphic bacteria have variable shapes

2.     Groupings: Cells adhering to one another following division form a characteristic

arrangement that depends on the plane in which the bacteria divide

3.     Multicellular associations

a)    Associations containing multiple cells, such as myxobacteria

b)    Biofilms often alter their activities when a critical number of cells are present

II.        The Structure of the Prokaryotic Cell

A.   The cytoplasmic membrane

1.      Structure and chemistry of the cytoplasmic membrane

a)    Phospholipid bilayer embedded with a variety of different proteins

b)    Differential barrier between the cell and the surrounding environment

c)    Membrane proteins function in transport or provide a mechanism by which

cells can sense and adjust to their surroundings

2.     Permeability of the cytoplasmic membrane

a)    Selectively permeable

b)   Inflow of water into the cell exerts more osmotic pressure on the cytoplasmic

membrane than it can generally withstand

3.     The role of the cytoplasmic membrane is involved in energy generation: Electron

transport chain within the membrane expels protons, generating an electrochemical

gradient, which contains a form of energy called proton motive force

B.   Directed movement of materials across the cytoplasmic membrane

1.    Transport systems

a)    Facilitated diffusion: Moves impermeable compounds from one side of the

membrane to the .other by exploiting the concentration gradient

b)    Active transport mechanisms use energy to accumulate compounds against a

concentration gradient

c)    Members of the major facilitator superfamily use proton motive force for energy

(1)   Symporters

(2)   Antiporters

(3)   Uniporters

d)    ABC transport systems require ATP for energy

e)    Group translocation chemically modifies a molecule during its passage through

the cytoplasmic membrane

2.    Secretion: The general secretory pathway is the primary mechanism used to secrete

proteins

C.   Cell Wall

1.      Peptidoglycan

a)    Found only in bacteria and provides rigidity to the cell wall

b)    Composed of peptidoglycan which contains alternating subunits of N-

acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) interconnected

via the tetrapeptide chains on NAM

2.     The Gram-positive cell wall

a)    Relatively thick layer of peptidoglycan

b)   Teichoic acids and lipoteichoic acids stick out of the peptidoglycan molecule

3.     The Gram-negative cell wall

a)    Thin layer of peptidoglycan sandwiched between the cytoplasmic membrane

and an outer membrane

b)    Periplasm contains a variety of proteins

c)    The outer membrane contains lipopolysaccharides. The Lipid A portion of the

lipopolysaccharide molecule is toxic, which is why LPS is called endotoxin

d)    Porins form small channels that permit small molecules to pass through the outei

membrane

4.     Antibacterial compounds that target peptidoglycan

a)    Penicillin binds to proteins involved in cell wall synthesis

b)    Lysozyme breaks the bond that links alternating NAG and NAM molecules

5.     Characteristics of bacteria that lack a cell wall

a)    Because mycoplasmas do not have a cell wall, they are extremely variable in

shape and are not effected by lysozyme or penicillin

D.   Surface layers external to the cell wall

1.      Glycocalyx: Enable bacteria to adhere to surfaces; some capsules allow disease-

causing microorganisms to thwart the innate defense system

a)    Capsule: A distinct and gelatinous layer made of polysaccharide

b)    Slime layer: Diffase and irregular layer of polysaccharide

2.     Sheath: A tube that holds a linear chain of cells; it is thought to protect the enclosed

organisms from disruption

E.    Filamentous protein appendages

1.      Flagella

a)    Long protein structures responsible for most types of bacterial motility

b)    Chemotaxis is the directed movement toward an attractant or away from a

repellant

2.     Pili

a)    Many types of pili (fimbriae) enable attachment of cells to specific surfaces

b)    Some pili play a role in specific types of motility

c)     Sex pili are involved in conjugation, which enables DNA to be transferred from

one cell to another

F.     Internal structures

1.      Chromosome

a)    The chromosome of prokaryotes resides in the nucleoid

b)    The typical chromosome is a single, double-stranded DNA molecule that

contains all the genetic information required by a cell

2.     Plasmids

a)    Plasmids are circular, double-stranded DNA molecules that typically encode

genetic information that may be advantageous, but not required by the cell

b)    Populations of cells can gain and lose plasmids, depending on the relative

advantages

3.     Ribosomes

a)    Ribosomes facilitate the joining of amino acids

b)    The 70S bacterial ribosome is composed of a 50S and a 30S subunit

4.     Storage granules: Dense accumulations of high molecular weight polymers, which are synthesized from a nutrient that a cell has in relative excess

5.     Gas vesicles are gas-permeable, water-impermeable rigid structures that provide

buoyancy to aquatic cells

6.     Endospores

a)    Dormant stage produced by members of the genera Bacillus and Clostridium,

they can germinate to become a vegetative cell

b)    Resistant to conditions such as heat, desiccation, toxic chemicals, and UV

irradiation

c)    Sporulation is an eight-hour process initiated when cells are grown under

nutrient-limiting conditions

d)    Germination is the process by which an endospore leaves its dormant state

 

Terms You Should Know

 

Microscopy and Cell

Morphology

Bacillus

Binary fission

Capsule

Coccobacillus

Coccus

Compound microscope

Condenser lens

Contrast

Decolorizing agent

Diplococcus

Electron photomicrograph

Endospore

Fluorescent dyes

Immunofluorescence

Mordant

Objective lens

Ocular lens

Pleomorphic

Refraction

Resolution

Spirillum

Spirochete

Prokaryotic Cell

Active transport

Antiporter

Capsule

Cell wall

Chemotaxis

Chromosome

Cytoplasm

Cytoplasmic membrane

Electrochemical gradient

Endospore

Endotoxin

Facilitated diffusion

Flagella

Fluid mosaic model

Gas vesicles

Germination

Glycocalyx

Group translocation

Lipid A

Lipopolysaccharide layer (LPS)

N-acetylglucosamine (NAG)

N-acetylmuramic acid (NAM)

Nucleoid

O polysaccharide

Osmosis

Osmotic pressure

Passive transport

Peptidoglycan

Pili (fimbriae)

Plasmid

Forms

Proton motive force

Ribosome

Selectively permeable

Sex pili

Sheath

Simple diffusion

Slime layer

Sporulation

Storage granules

Symporter

Teichoic acids

Transport proteins (permeases or carriers)

Uniporter