MicroBio Chp: 3

Microscopy is

Use of a microscope to study objects

Light microscopes

Light passing through a lens or lens is used to produce a magnified image

Types

• bright field

• dark field

• phase contrast

• interference

• fluorescent

• confocal

What are the three principles involved in microscopy

Magnification

resolution

Contrast

Magnification

Creating a enlarged image

Resolution

Clarity of enlarged image, limit of resolution

Contrast

Special microscopes, application of stains

• change of color for background or item viewing to see difference

How is magnification achieved with a bright field microscope ?

By using the objective and ocular lenses to enlarge the image of the specimen.

how to increase contrast

using a Stain

Why are dyes applied to samples to be viewed with a light microscope

to increase contrast

what are the two categories of dyes used to stain specimens in microbiology

Basic dyes

Acidic dyes

Basic dyes

cationic, used in simple staining

these have positive charge

• adhere to a negatively charged cellular components

• Ex: Crystal violet, safranin, methylene blue

Acidic dyes, and Ex.

Anionic, used in negative staining

• Repelled by many cellular components

• Usually stain background than cell itself

• Ex; india ink, nigrosin

What is a differential stain?

Staining techniques that stain bacteria differently due to differences in chemical structure of component

How is it different from a simple stain?

Simple stain = one dye used to see cell shape, size, and arrangement.

Differential stain = multiple dyes used to distinguish different types of bacteria/cells based on how they react to the dyes.

What are examples of differential stains used in microbiology?

Gram stain

Acid fast stain

How do these staining protocols differentiate between microbes?

They use dyes and chemicals that react differently with different cell structures, causing microbes or their structures to appear different colors.

What are the 4 steps of the Gram stain, in order?

1. Crystal violet → stains all bacteria purple

2. Iodine → helps hold the crystal violet in the cell

3. Decolorizer (alcohol) → removes purple from Gram-negative cells

4. Safranin → counterstain Gram-negative cells pink/red

What is the function of crystal violet?

It is the primary stain that makes the bacteria purple.

What is the function of iodine in the Gram stain?

Iodine acts as a mordant, meaning it helps the crystal violet stick to the bacterial cell.

What does the decolorizer do?

It removes the crystal violet from Gram-negative bacteria, but Gram-positive bacteria keep the purple stain.

What is the function of safranin?

t is the counterstain that makes Gram-negative bacteria pink/red after they lose the purple stain.

Gram-positive outcomes

• 🧱 THICK peptidoglycan cell wall

• Alcohol dehydrates/shrinks the thick wall

• Traps the crystal violet–iodine complex

• Stays purple

Gram-negative outcomes

• 🧱 THIN peptidoglycan wall

• Has an outer membrane that is disrupted by alcohol

• Crystal violet can escape

• Becomes colorless

After a Gram stain, what color are Gram-positive and Gram-negative bacteria?

• Gram-positive = PURPLE

• Gram-negative = PINK/RED

Acid fast stain is used to detect what

the presence of the bacteria that causes tuberculosis

What bacterium causes tuberculosis?

Mycobacterium tuberculosis

Acid fast stain tell apart

Acid-fast bacteria = red/pink
Non-acid-fast bacteria = blue

Acid fast stain steps

Carbolfuchsin → stains bacteria red 🔴
↓
2. Heat → helps red dye enter waxy cell wall
↓
3. Acid-alcohol → removes red from non-acid-fast bacteria
↓
4. Counterstain → makes non-acid-fast bacteria blue 🔵

Common cell shapes of prokaryotes

Coccus - round

Rod - rod looking

Vibrio - macaroni look

Spirillum - spaghetti noodle look

Spirochete - slinky, up down up down, zig zag

Strepto

cells arranged in chains

sarcina arrangement of cells …

Cells form cubical packets, usually groups of 8 or more

Staphylo arrangement cell…

Cells form irregular clusters

Myxobacteria

swarm of cells that move as a pack

what are biofilms

A biofilm is a group of microorganisms that stick to a surface and to each other, forming a protective layer called a matrix

Why are biofilms of clinical significance?

They protect bacteria, making them harder for antibiotics and the immune system to attack, which can cause infections to persist.

• Very resistant

• A biofilm can contain one or more different types of microorganisms living together in a protective matrix.

what is quorum sensing ?

Bacteria communicating with each other.

• secrete homoserine lactone

• telling other bacteria to build and form larger

Prokaryotic Cell membrane composition

Phospholipid Bilayer

Proteins ½ mass

Prokaryotic cell Functions

Barrier- selectively permeable

Energy transformation ( Make and use energy)

Selective transport ( controls what enters and exits)

What group of prokaryotes includes sterols in their cell membranes?

Mycoplasma and Ureaplasma.

• Archea and bacteria

Why is the cell membrane able to act as a barrier?

because it is made of a phospholipid bilayer

What is meant by energy transformation?

changing energy from one form into another form.

• 🍎 Food (chemical energy)
  ⬇
  ⚙ Cell breaks it down
  ⬇
  🔋 ATP is produced
  ⬇
  💪 Cell uses ATP to do work

Permeability of cell membranes

Water, gases, small hydrophobic molecules

Simple Diffusion

Movement of molecules across a membrane driven by differences in concentrations

• no energy needed

What causes diffusion to occur?

particles naturally move from an area of high concentration to an area of low concentration.

What is osmosis ?

Flow of water across Semi-permeable membrane from an area of low solute concentration to an area of high solute concentration

What causes Osmosis to occur

there is a difference in the concentration of water (or solutes) on the two sides of a selectively permeable membrane.

What is a hypotonic solution?

has more solute in the cell than outside

💧 Water moves into the cell → the cell swells.

What can happen to a cell placed in a hypotonic solution?

hypotonic solution can swell and possibly burst because water moves into the cell.

What is a hypertonic solution

the solution has more solute and less water compared with the cell.

💧 Water moves out of the cell → the cell shrinks.

What can happen to a cell placed in a hypertonic solution?

hypertonic solution can shrink because water moves out of the cell.

What is plasmolysis?

cell loses water and the cell membrane pulls away from the cell wall.

What is proton motive force?

Proton motive force is the stored energy created when hydrogen ions (H⁺) build up on one side of a membrane.

Super simple:

1. ⚡ Energy is used to pump H⁺ (protons) across the membrane.

2. H⁺ builds up on one side → creating a gradient.

3. H⁺ wants to flow back across the membrane.

4. As H⁺ flows back through ATP synthase, the energy is used to make ATP. 🔋

What creates proton motive force?

created when H⁺ (protons) are actively transported across a cell membrane.

⚡ Active transport pumps H⁺ → H⁺ builds up on one side → proton motive force is created.

What is proton motive force used for?

Flagellar movement, ATP synthesis

active transport ?

Transport systems required to..

bring most molecules across cell membrane

and utilize a cell membrane spanning protein

What is the difference between facilitated diffusion and active

transport?

Facilitated diffusion: 🟢 High → Low, no energy, Moves with gradient
Active transport: 🔴 Low → High, needs energy, MOves against gradient

What are the characteristics of facilitated diffusion?

• No energy (ATP) is required ❌🔋

• Moves substances from high → low concentration

• Uses a membrane protein to help substances cross

• Moves substances down their concentration gradient

• Helps substances that cannot easily pass through the phospholipid bilayer on their own

What are the two sources of energy for active transport?

Proton motive - energy stored in a H⁺ gradient is used to drive transport.

ATP - energy directly from ATP is used to move substances across the membrane.

What classes of transport proteins uses these sources of energy?

PMF - transport some nutrient , efflux waste

ATP - ATP binding cassette

what is secretioin

the process of transporting proteins out of the cell

what do proteins destined to be

secreted contain a signal sequence

what is a signal sequence made of

about 20 hydrophobic amino acids

What is the purpose of a signal sequence

it directs the protein to the pathway for secretion out of cell

does sectarian require energy

yes ATP

What happens to ATP during secretion

ATP → ADP → Pi, realising energy

Signal sequence

address label for a protein

Where is the cell wall located in relation to the cell membrane?

is located outside the cell membrane.

Inside → Cell membrane → Cell wall → Outside

What are the characteristics of the prokaryotic cell wall?

• Present in most bacteria

• Rigid: rovides structural support

• unique structures

• Cell wall type used to group bacteria

Gram positive, Gram negative, Acid fast

Why is knowledge of the structure of the prokaryotic cell wall of clinical significance?

the structure of a bacterial cell wall can help doctors identify bacteria and choose treatments.

• 🔬 How they are identified, such as with a Gram stain

• 💊 Which antibiotics work against them

• 🛡 How the bacteria respond to the immune system

What is the structure of peptidoglycan?

Glycan chain composed of subunits consisting of N-acetyl gluscosamine ( NAG) and N-acytel muramic acid ( NAM)

• Forms a polysacharide

Peptidoglycan is present in both ..

Gram positive and Gram negative cell walls

How are polymers of peptidoglycan crosslinked?

Short peptide chains link neighboring NAM sugar chains together.

Peptide chains connect the NAM molecules of neighboring sugar chains, creating a strong, mesh-like cell wall.

In what prokaryotic cell walls is peptidoglycan found?

Peptidoglycan is found in the cell walls of Bacteria

How does lysozyme and penicillin affect peptidoglycan?

Lysozyme

• Breaks the bonds between the NAG and NAM sugars.

• This breaks apart the sugar backbone of peptidoglycan.

Breaks the sugars apart

Penicillin

• Prevents the peptide cross-links from forming.

• This makes the cell wall weak.

• The bacterium can eventually burst (lyse).

Dont let prevents the links from forming

Which of these agents can break existing covalent bonds in peptidoglycan and which prevents the formation of crosslinks?

Lysozyme = cuts what is already there
Penicillin = prevents new links

What components are found in the Gram positive cell wall?

• Contains up to 30 layers of peptidoglycan

• Layers are all cross-linked

• Fully permeable

• Teichoic acids

• Lipoteichoic acids

What

is the major component of the Gram positive cell wall?

A thick layer of peptidoglycan.

What is the structure of the Gram negative cell wall?

Only contains a thin layer of peptidoglycan

• 
  

What are the components of the Gram negative cell wall and where are they located in relation to each other?

• outer membrane ( located outside the peptidoglycan layer)

• Lipid Bilayer ( outer leaflet composed of lipqplysachrides ( very toxic))

connected to peptidoglycan with lipoproteins

Porin proteins imbedded in outer membrane ( allow to move molecules through membrane)

What component of the Gram negative cell wall is known as endotoxin?

is LPS (lipopolysaccharide) in the outer membrane of Gram-negative bacteria.

What is the structure of endotoxin?

found in the outer membrane of Gram-negative bacteria.

lipid A connected to a O antigen

toxic component of LPS (endotoxin) is

toxic component of LPS (endotoxin) is Lipid A

What is the function of porin proteins in the Gram negative cell wall?

tiny channels or holes in the outer membrane of Gram-negative bacteria.

Do all bacteria have cell walls?

No

which groups of bacteria lack cell walls?

mycoplasma, ureaplasma

Sterols in cell membrane…

• increased strength of membrane

Cell walls of Archaea

More diverse in structure and composition

Do not contain peptidoglycan

Some contain pseudopeptidoglycan

Where is the glycocalyx located in relation to the cell wall?

is located outside the cell wall.

What is the composition of most glycocalyxes?

Most glycocalyxes are made of polysaccharides (sugars).

What are the two types of glycocalyxes?

Capsule

Slime layer

What is the difference between capsule and slime layer

• Capsule: Firmly attached to the cell surface; does not easily wash off.

• Slime layer: Loosely attached; can easily be removed or washed away.

What are the functions associated with the glycocalyx?

• Protection → helps protect the cell from drying out and harmful substances.

• Helps bacteria stick to surfaces → allows them to attach to cells, tissues, or other surfaces.

• Biofilm formation → helps bacteria stick together and form biofilms.

• Helps avoid the immune system → a capsule can make it harder for immune cells to engulf the bacteria (phagocytosis).

• good at interacting with outside cells

Of what clinical significance is the glycocalyx?

because it can help bacteria cause and maintain infections.

what are the major components of bacteria flagella

Filamentous protein

appendages

flagella

basal body ( anchors) ( motor)

Rigid: spin like propellers

• can be polar or peritrichous

peritrichous

Flagella are spread all over the surface of the bacterium.

How does the bacterial flagellum function?

A bacterial flagellum works like a tiny propeller.

• The motor at the base spins.

• This makes the flagellum rotate.

• The spinning pushes the bacterium through liquid.

• This allows the bacterium to move from place to place.A bacterial flagellum works like a tiny propeller.

What are the arrangements of bacterial flagella on the surface of

bacteria?

Monotrichous, lophotrichous, amphitrichous, and peritrichous.

peritrichous

has multiple tails all over

amphitrichous

Has two tails on either side

lophotrichous

a truff of tails on either side