Lecture 2 - The bacterial cell envelope- Gram positive versus Gram-negative

Question 1

Why is it necessary to dilute?

Answer 1

You have to dilute to a number that is countable on a petri dish. If you do not dilute, you will get a LAWN on the petri dish.

Question 2

What is the benefit of serial dilutions?

Answer 2

You can get a viable cell count meaning you are counting only the bacteria that are alive.

Question 3

When expressing dilution counts we must report in:

Answer 3

CELLS/ML

Question 4

Which will you have more of

Serial Dilution Count or DIRECT COUNTING METHOD?

Answer 4

You should always get more cells with the direct counting method (microscopy count) than with the serial dilution (viable cell count) because those are ONLY LIVE CELLS.

Question 5

How do you calculate % Viability

Answer 5

In order to calculate the % viability, you must be give 2 values:
the microscopy count (direct counting method) &
the viable cell count (serial dilution method)

You must set up an equation:
%Viability = (Viable cell count)/(Direct cell count) x 100

Question 6

If given that you observe 5x10^6 cells/mL with microscopy and 5x10^5 cells/mL viable. What would % viability be?

Answer 6

%Viability = (Viable cell count)/(Direct cell count) x 100

= (5x10^5)/(5x10^6) x 100
= (1/10) x 100
= 0.10 x 100 = 10%

Question 7

In 1884 _______ developed the Gram Stain

Answer 7

Hans Christian Gram

Question 8

Bacteria are either ______ or _____ and this has implications for how they respond to certain antibiotics. This process ___________________

Answer 8

GRAM POSITIVE
GRAM NEGATIVE

STAINS THE CELL ENVELOPE

Question 9

What is the process of Gram Staining?

Answer 9

1. FIX bacteria cells to a slide with HEAT
   (drop liquid on slide, run over flame briefly)
2. Add dye called CRYSTAL VIOLET
   (deep purple stain)
3. Treat with IODINE (brown) This complexes with crystal violet to form an insoluble complex - all cells look dark brown/purplish at this point
4. Decolorize with ALCOHOL
5. If bacteria is GRAM POSITIVE - it will still appear purple in color
   GRAM NEGATIVE bacteria - cells will become clear, because they lose the colored complex as a precipitate
6. Add SAFRANIN as a counterstain so that clear GRAM NEGATIVE cells can become visible as pinkish/RED
   Gram POSITIVE cells will remain a dark purple

Question 10

What is cell envelope in GRAM POSITIVE BACTERIA?

Answer 10

GRAM POSITIVE: Have a plasma membrane containing proteins
Outside of the plasma membrane is a THICK CELL WALL
Collectively, the membrane and the cell wall are called the CELL ENVELOPE

Question 11

What is the periplasmic space?

Answer 11

The area between the inner membrane and outer membrane of Gram NEGATIVE CELLS - you can say that the cell wall is contained between the periplasmic space.
Some proteins are periplasmic, while others are in the cytosol or the Inner Membrane.

Question 12

Explain why gram stain affects Gram positive and negative cells differently .

Answer 12

Gram positive stains bacteria have such a thick cell wall that the dye gets trapped in. The Gram negative bacteria have a thinner wall, unable to keep the crystal violet iodine complex - so the stain gets washed away with alcohol.
THEREFORE, GRAM STAINING TELLS YOU ABOUT THE STRUCTURE OF THE BACTERIAL CELL, SPECIFICALLY WHAT ITS CELL ENVELOPE COMPOSITION IS.

Question 13

CHARACTERISTICS were used before genetic sequences and cloning were available.
These are more specific than just GRAM STAIN alone …
The following characteristics were used to clinically and experimentally determine the identity of bacterial cells: (6)

Answer 13

1. TEMPERATURE OPTIMUM
2. PH OPTIMUM
3. GROWTH RATE
4. GAS REQUIREMENTS
5. NUTRITIONAL REQUIREMENTS / SUGAR UTILIZATION
6. GAS/ACID PRODUCTION

Question 14

What does gram staining mean in terms of cell envelope?

Answer 14

The reason there is a difference in staining is that gram positive cells and gram negative cells have a different structure in the cell envelope:
GRAM POSITIVE:
- Have a PLASMA MEMBRANE containing proteins
- Outside of the plasma membrane is a THICK CELL WALL
- Collectively, the membrane and the cell wall are called the CELL ENVELOPE

GRAM NEGATIVE:

• Have a plasma membrane ( aka INNER MEMBRANE)
• Outside of plasma membrane is a THIN CELL WALL (much thinner compared to cell wall of Gram + bacteria - if Gram + cell wall is 40 layers thick, Gram - cell wall may be only 2 layers)
• Outside of the cell wall, also have membrane called OUTER MEMBRANE. Because of this, plasma membrane may also be referred to as INNER MEMBRANE
• Collectively, all 3 layers (OM, Cell Wall, IM) are called CELL ENVELOPE

Question 15

What is cell envelope in GRAM NEGATIVE BACTERIA?

Answer 15

GRAM NEGATIVE:

• Have a plasma membrane ( aka INNER MEMBRANE)
• Outside of plasma membrane is a THIN CELL WALL (much thinner compared to cell wall of Gram + bacteria - if Gram + cell wall is 40 layers thick, Gram - cell wall may be only 2 layers)
• Outside of the cell wall, also have membrane called OUTER MEMBRANE. Because of this, plasma membrane may also be referred to as INNER MEMBRANE
• Collectively, all 3 layers (OM, Cell Wall, IM) are called CELL ENVELOPE

Question 16

TEMPERATURE OPTIMUM to differentiate bacteria:

Answer 16

The temperature the bacteria grow well at
Thermophile - High temp bacteria
Psychrophile - Low temp bacteria

Question 17

GROWTH RATE to differentiate bacteria:

Answer 17

There are slow growing bacteria and fast growing bacteria

Question 18

GAS REQUIREMENTS to differentiate bacteria:

Answer 18

Bacteria respire and need to use gases as fart of their electron transport chains. We use oxygen as a final electron acceptor. Some bacteria use oxygen but some can use phosphates, nitrates, and a variety of compounds and gases.
Ex: Aerobes (Facultative & Strict)
Anaerobes (Facultative & Obligate/Strict)

Question 19

NUTRITIONAL REQUIREMENT/ SUGAR UTILIZATION to differentiate bacteria:

Answer 19

Sugar utilization is important. All bacteria should be able to use glucose but they may vary in what other sugars they can metabolize.
S mutans. can use sucrose, while other bacteria may be able to

Question 20

PH Optimum to differentiate bacteria:

Answer 20

Does the bacteria grow in acidic PH or alkaline?

Ex: Acidophile (low pH optimum)

Question 21

GAS/ACID PRODUCTION to differentiate bacteria:

Answer 21

Some bacteria produce nitrogen, CO2 or other gases.
Some bacteria produce acetate or other acids. This is especially important when considering oral bacteria like S. Mutans which can produce acids that will decay teeth.

Question 22

If you know what a colony looks like, in addition to other such characteristic factors as: 
Optimum temperature, 
Ph, 
Gas requirements, 
gas/acid production, , 
nutrition/sugar requirements, 
growth rate, 
it helps to what?

Answer 22

Suggest which species the bacteria is. Even this is not definitive though because other bacteria may have similar properties.

Question 23

What is a definite way to Identify bacteria? Why?

Answer 23

16 s Ribosomal RNA sequence analysis

It is highly conserved within a group so it allows you to identify organisms as part of a certain kingdom or even as part of a certain species. The sequence is highly indicative of the organism in which it resides.
THE TREE OF LIFE WAS DETERMINED USING 16 S SEQUENCING!

Question 24

What is 16 S rRNA?

Answer 24

A signature because all organisms have it and it changes little over time (HIGHLY CONSERVED)
Certain 16 S rRNA sequences are found in one group but not others.

EX:
sequence CACYYG @ Position 315 is found in > 95 % of all bacteria but is not found in the kingdom Archaea or Eukarya
sequence CACACACCG @ position 1400 is found in 100% of the organisms in the kingdom archaea but not in an y bacteria or eukaryotes
sequenceALL HUMANS HAVE THE SAME
CLOSELY RELATED BACTERIA HAVE MORE SIMILAR 16 S Sequences
SIGNATURE 16 S Sequence!!!!

Question 25

Why is 16S rRNA highly conserved?

Answer 25

Since it is an essential part of a cell. Ribosomes are made up of protein and RNA. There is this 16 sequence which is present in EVERY living organism. There is a gene that encodes the 16 S rRNA fragment.

Question 26

The more similar two organisms 16 S Sequences are,

Answer 26

the more closely related they are

Question 27

Does the 16 S rRNA sequence stay the same?

Answer 27

It does not but it changes little over time

Question 28

Every living organism has a DNA sequence encoding for

Answer 28

the 16 S fragment, and within it are slight changes, or SIGNATURE SEQUENCES that help us to distinguish between kingdoms, groups, species, etc …

Question 29

What is the function of the membrane
Cytosolic membrane in GRAM POSITIVE
Inner membrane in GRAM NEGATIVE

Answer 29

1. Permeability baRRier
   allows cell to keep certain things in and others out
2. Protein anchor
   membrane proteins can be localized to allow for transport proteins or to create the electron transport chain
3. Energy conservation
   proton pump generates an electromotive force at the membrane (generates division to create a proton-motive force for ATP synthesis)

Question 30

___ Prevents the bacterial cell from bursting and maintains specific shapes (rods, etc.) it is made up of ____

Answer 30

CELL WALL

PEPTIDOGLYCAN

Question 31

What is structure of peptidoglycan

Answer 31

It contains peptides and glycans (sugars)
- the sugars in the cell consist of several repeating disaccharides of
NAG (N-acetylglucosamine) and
NAM (N-acetylmuranic acid)
which are linked by BETA 1-4 bonds.
- chains of these disaccharides are connected in layers by PEPTIDE CROSS LINKS.

To form the mesh of peptidoglycan, NAM cross links other NAM molecules via side chain residues. these cross links, are formed via a process called trans-peptidation. This is done by enzyme called PEPTIDOGLYCAN TRANSPEPTIDASE

Question 32

How are chains of NAM and NAG connected?

Answer 32

By Peptide cross links

Question 33

Every bacterial cell has a plasma membrane.
In Gram Positive it is called:
In Gram Negative it is called:

Answer 33

In Gram Positive it is called: CYTOSOLIC MEMBRANE

In Gram Negative it is called: INNER MEMBRANE

Question 34

What is the structure of the membrane?

Answer 34

it is a phospholipid bilayer with hydrophilic region on the outside and the hydrophobic fatty acids on the inside. It is a fluid membrane. Proteins can move laterally through it.

Question 35

What are functions of the CELL WALL? PEPTIDOGLYCAN?

Answer 35

1. prevents the cell from bursting
   - if you do an experiment to digest the cell wall, it will become a sphere, because it no longer maintains its shape.
   - the main purpose of the cell wall is structural
2. provides a platform for surface appendgages
   - the flagella, fimbrae, pilli
   - the cell wall in bacteria is PEPTIDOGLYCAN

Question 36

What is the enzyme that makes peptide cross links ?

Answer 36

PEPTIDOGLYCAN TRANSPEPTIDASE

Question 37

What is the process of creating the cross links in peptidoglycan

Answer 37

TRANSPEPTIDATION

Question 38

The outer membrane in GRAM NEGATIVE BACTERIA STRUCTURE

Answer 38

Gram negative bacteria have an outer membrane that is not like the inner membrane which is a phospholipid bilayer.

INNER LEAFLET OF THE OUTER MEMBRANE IS A PHOSPHOLIPID MONOLAYER

OUTER LEAFLET OF OUTER MEMBRANE IS LPS
(LIPOPOLYSACCHARIDE)

Question 39

What is LPS

Answer 39

Lipopolysaccharide

An important component of bacterial cells responsible for its immunogenicity and toxic shock syndrome

Question 40

What is the Structure of LPS?

Answer 40

1. Lipid A
2. Oligosaccharide Core
3. O-Specific Polysaccharide (antigen)

Question 41

What is the Lipid A of LPS?

Answer 41

Lipid A is the fatty acid portion of the LPS.
- Anchors the LPS to the inner leaflet and is connected to the core polysaccharide of LPS
- Also known as ENDOTOXIN - this is the highly toxic part of the LPS that induces the releases of cytokines and can cause a hyperactive immune response - that overwhelms the body (endotoxin toxic shock syndrome). Infection in the blood can cause bacteremia and shock.
Someimtes LPS as a WHOLE IS REFERRED TO AS ENDOTOXIN

Question 42

What is the Oligosaccharide Core?

Answer 42

Also called the Polysaccharide core
Central part of the LPS
- Consists of several sugars, the most important of which is KDO-Keto-deoxyoctulosonate. KDO is unique to LPS and is connected to other sugars like galactose, and glucose, that together form the oligosaccharide core.
- Each bacteria may have different polysaccharides BUT THEY ALWAYS HAVE KDO!

Question 43

What is KDO?

Answer 43

Keto-deoxyoctulosonate

Consists of several sugars, the most important of which is KDO-Keto-deoxyoctulosonate. KDO is unique to LPS and is connected to other sugars like galactose, and glucose, that together form the oligosaccharide core.

Each bacteria may have different polysaccharides BUT THEY ALWAYS HAVE KDO!

Question 44

What is the O-antigen or O-specific Polysaccharide

Answer 44

Antigen

• The oligosaccharide core is connected to more sugars called O-specific polysaccharide which si also called the antigen.
• This repeating unit is a 3-5 sugar pattern that can be repeated 5-50 times. This is the longest part of the LPS structure
• The O-antigen is the immunogenic part of LPS because it is on the outside and it is what the immune system can see. Antibodies are often developed against different O-antigens.

All E Coli cells have the same 16 S but different strains have many different o-antigens - these are called SEROTYPES

Question 45

What are Serotypes?

Answer 45

Determined by the different antibodies to make them. They are different O-antigens

Some bacteria can have 100-200 serotypes - though they are the same species, they differ in their O-antigen structure

Question 46

E coli O157 H7 is what?

Answer 46

the food poisoning strain of E. Coli.
Other E. coli don’t cause food poisoning strain. - - - O157 means it is the 157th structure of the O antigen discovered for E. coli
- H7 refers to the flagella type

SEROTYPE IS DIAGNOSTIC FOR THE STRAIN