Lecture 2 - The bacterial cell envelope- Gram positive versus Gram-negative Flashcards
Why is it necessary to dilute?
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.
What is the benefit of serial dilutions?
You can get a viable cell count meaning you are counting only the bacteria that are alive.
When expressing dilution counts we must report in:
CELLS/ML
Which will you have more of
Serial Dilution Count or DIRECT COUNTING METHOD?
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.
How do you calculate % Viability
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
If given that you observe 5x10^6 cells/mL with microscopy and 5x10^5 cells/mL viable. What would % viability be?
%Viability = (Viable cell count)/(Direct cell count) x 100
= (5x10^5)/(5x10^6) x 100
= (1/10) x 100
= 0.10 x 100 = 10%
In 1884 _______ developed the Gram Stain
Hans Christian Gram
Bacteria are either ______ or _____ and this has implications for how they respond to certain antibiotics. This process ___________________
GRAM POSITIVE
GRAM NEGATIVE
STAINS THE CELL ENVELOPE
What is the process of Gram Staining?
- FIX bacteria cells to a slide with HEAT
(drop liquid on slide, run over flame briefly) - Add dye called CRYSTAL VIOLET
(deep purple stain) - Treat with IODINE (brown) This complexes with crystal violet to form an insoluble complex - all cells look dark brown/purplish at this point
- Decolorize with ALCOHOL
- 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 - 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
What is cell envelope in GRAM POSITIVE BACTERIA?
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
What is the periplasmic space?
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.
Explain why gram stain affects Gram positive and negative cells differently .
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.
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)
- TEMPERATURE OPTIMUM
- PH OPTIMUM
- GROWTH RATE
- GAS REQUIREMENTS
- NUTRITIONAL REQUIREMENTS / SUGAR UTILIZATION
- GAS/ACID PRODUCTION
What does gram staining mean in terms of cell envelope?
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
What is cell envelope in GRAM NEGATIVE BACTERIA?
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
TEMPERATURE OPTIMUM to differentiate bacteria:
The temperature the bacteria grow well at
Thermophile - High temp bacteria
Psychrophile - Low temp bacteria
GROWTH RATE to differentiate bacteria:
There are slow growing bacteria and fast growing bacteria
GAS REQUIREMENTS to differentiate bacteria:
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)
NUTRITIONAL REQUIREMENT/ SUGAR UTILIZATION to differentiate bacteria:
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
PH Optimum to differentiate bacteria:
Does the bacteria grow in acidic PH or alkaline?
Ex: Acidophile (low pH optimum)
GAS/ACID PRODUCTION to differentiate bacteria:
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.
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?
Suggest which species the bacteria is. Even this is not definitive though because other bacteria may have similar properties.
What is a definite way to Identify bacteria? Why?
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!
What is 16 S rRNA?
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!!!!
Why is 16S rRNA highly conserved?
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.
The more similar two organisms 16 S Sequences are,
the more closely related they are
Does the 16 S rRNA sequence stay the same?
It does not but it changes little over time
Every living organism has a DNA sequence encoding for
the 16 S fragment, and within it are slight changes, or SIGNATURE SEQUENCES that help us to distinguish between kingdoms, groups, species, etc …
What is the function of the membrane
Cytosolic membrane in GRAM POSITIVE
Inner membrane in GRAM NEGATIVE
- Permeability baRRier
allows cell to keep certain things in and others out - Protein anchor
membrane proteins can be localized to allow for transport proteins or to create the electron transport chain - Energy conservation
proton pump generates an electromotive force at the membrane (generates division to create a proton-motive force for ATP synthesis)
___ Prevents the bacterial cell from bursting and maintains specific shapes (rods, etc.) it is made up of ____
CELL WALL
PEPTIDOGLYCAN
What is structure of peptidoglycan
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
How are chains of NAM and NAG connected?
By Peptide cross links
Every bacterial cell has a plasma membrane.
In Gram Positive it is called:
In Gram Negative it is called:
In Gram Positive it is called: CYTOSOLIC MEMBRANE
In Gram Negative it is called: INNER MEMBRANE
What is the structure of the membrane?
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.
What are functions of the CELL WALL? PEPTIDOGLYCAN?
- 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 - provides a platform for surface appendgages
- the flagella, fimbrae, pilli
- the cell wall in bacteria is PEPTIDOGLYCAN
What is the enzyme that makes peptide cross links ?
PEPTIDOGLYCAN TRANSPEPTIDASE
What is the process of creating the cross links in peptidoglycan
TRANSPEPTIDATION
The outer membrane in GRAM NEGATIVE BACTERIA STRUCTURE
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)
What is LPS
Lipopolysaccharide
An important component of bacterial cells responsible for its immunogenicity and toxic shock syndrome
What is the Structure of LPS?
- Lipid A
- Oligosaccharide Core
- O-Specific Polysaccharide (antigen)
What is the Lipid A of LPS?
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
What is the Oligosaccharide Core?
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!
What is KDO?
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!
What is the O-antigen or O-specific Polysaccharide
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
What are Serotypes?
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
E coli O157 H7 is what?
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
