Unit 3.4 - Microbiology Flashcards
What is microbiology?
The study of microorganisms including prokaryotes (bacteria and archaea), protoctists, fungi and viruses
Which element of microbiology does the A level course mostly focus on?
Bactria
Examples of protoctists
Amoeba
Macroalga (seaweed)
Paramecium
Slime mole
(There’s a large variety)
What are amoeba and paramecium examples of?
Protoctists
How come fungi are involved in microbioloy?
Although they appear to behave like multicellular organisms in mushrooms and toadstools, the individual cells can live as single celled organisms
How do viruses reproduce?
By taking over host cells
What are viruses the borderline between?
Living organisms and complex chemicals like proteins
What can be though of as the borderline between living organisms and complex chemicals like proteins?
Viruses
Are viruses fully functioning living organisms?
No - more of a borderline between living organisms and complex chemicals (e.g - proteins)
Examples of prokaryotes
Bacteria
Archaea
Size of a prokaryotic cell
0.5 - 10 micrometres
Ribosomes in prokaryotic cells
70s
2 types of circular DNA in prokaryotic cells
Plasmids
Nucleoid
What does the cell wall of prokaryotic cells contain?
Peptidoglycan
Why do all prokaryotic cells have cell walls? Explain
They live in water-filled environments
The water potential of the cell is lower than the external water potential
Water diffuses in via osmosis
The cell wall avoids the lysis of the cell (stops it from bursting)
What would happen to prokaryotic cells if they didn’t have their peptidoglycan cell walls? Why?
They would burst (lyse) due to water diffusing in via osmosis from their environment
How do some antibiotics work because of the structure of prokaryotic cells?
They prevent the cell wall from working properly so that the cell cannot stand the pressure of the water diffusing in from its environment and bursts
What does the capsule of a prokaryotic cell do?
Protects the cell from chemicals and enzymes
What are the pilus of prokaryoties?
Tubular protein molecules
What do pili (more than 1 pilus) allow prokaryotic cells to do?
Allow cells next to each other to connect so that plasmids can move from 1 cell to the next
Plasmids
Circular DNA in prokaryotic cells that’s separate to the main chromosomal DNA
What can be transferred from one prokaryotic cell to another using pili?
Plasmids
Multiple pilus
Pili
What do plasmids allow prokaryotic cells to do?
Pass genes on plasmids from one cell to another without reproducing
How do prokaryotic cells pass on genes from one cell to another without reproducing?
Using plasmids
How are antibiotic resistance genes spread throughout a population of prokaryotic cells?
Using plasmids, which can be passed from one cell to another without reproducing
Mesosome
Infolding of the cell membrane
What are mesosomes in prokaryotic cells similar to in eukaryotic cells?
Similar to cristae in mitochondria
Where is the site of aerobic respiration in bacterial cells?
Mesosome
What is the mesosome of a prokaryotic cell useful for?
Is the site of aerobic respiration in bacterial cells
What does the flagellum of a prokaryotic cell do?
Rotates from a protein in the membrane to provide movement
Motile bacteria
Bacteria that can move on its own
Bacteria that can move on its own
Motile bacteria
Compare the differences between eukaryotic and prokaryotic cells
(Eukaryotic first each time)
Larger cells
80s larger ribosomes bound to the rough endoplasmic reticulum, 70s smaller ribosomes, free in the cytoplasm
Membrane bound organelles, no membrane bound organelles
DNA contained to the nucleus and is linear, DNA free in the cytoplasm and is circular
Nucleus has a double membrane, no nuclear envelope (double membrane(
No plasmids, plasmids
Cell wall (when present) is composed of cellulose or chitin, cell wall is composed of peptidoglycan (mucopolysaccharide)
Mitochondria are used for aerobic respiration (no mesosome), no mitochondria, uses a mesosome for aerobic respiration
DNA associated with histones, DNA not associated with histones
Ribosomes in eukaryotic cells
80s
Ribosomes in prokaryotic cells
70s
DNA in eukaryotic cells
Contained to the nucleus + linear
DNA in prokaryotic cells
Free in the cytoplasm + circular
Cell wall in eukaryotic cells
(When present) is composed of cellulose or chitin
Cell wall in prokaryotic cells
Composed of peptidoglycan (mucopolysaccharide)
What’s used for aerobic respiration in prokaryotic cells?
A mesosome
Similarities between prokaryotic cells and eukaryotic cells
Both contain ribosomes, cell membranes, DNA and genetic material
How can we identify different types of bacteria?
From their shapes
Rod shaped bacteria
Bacillus
Bacillus bacteria shape
Rod shapes
Examples of bacillus bacteria
Escherichia coli (E.Coli)
Salmonella
What can both of the bacillus bacteria E.Coli and Salmonella cause?
Food poisoning
Pathogen
An organism that can produce disease
Describe the bacillus bacteria salmonella
Pathogenic
Is all bacteria pathogenic? Give an example
No
Although E.Coli can be pathogenic, there’s already some in our intestine which isn’t pathogenic
Sphere shaped bacteria
Cocci
Cocci bacteria shape
Sphere shaped
Example of cocci bacteria
Staphylococcus aureus
Example of a staphylococcus aureus bacteria and explain why it’s problematic
MRSA
Is resistant to most of antibiotics, making it very difficult to treat (only 2 antibiotics are effective, and they have bad side effects)
What do the different arrangements of cocci depend on?
The species of the coccus bacteria
What are the 2 less common types of bacteria?
Vibrio
Spirillum
Comma shapes bacteria
Vibrio
Vibrio bacteria shape
Comma shaped
Example of vibrio bacteria
Vibrio cholerae
What is vibrio cholerae and how is it caused?
Cholera, caused by drinking contaminated drinking water
Spiral shapes bacteria
Spirillum
What shape is spirillum bacteria?
Spiral shaped
Example of a spirillum bacteria and what it causes
Leptospira, which causes Weil’s disease
How is Weil’s disease obtained?
By drinking water contaminated with rat urine
What is Gram’s stain?
A technique used in microbiology to help with the identification of bacteria
What does using the gram’s stain process to identify bacteria rely on?
The fact that bacteria have 2 basic cell wall structures, one of which retains a crystal violet stain and the other of which does not
What can bacteria be divided into using their basic cell wall structures?
Gram positive
Gram negatives
What colour do gram positives appear at the end of gram’s stain?
Purple
What colour do gram negatives appear at the end of gram’s stain?
Red/pink
Procedure for gram’s stain
1.) place the bacterial sample on a glass microscope slide
2.) stain with crystal violet (purple dye)
3.) apply iodine to fix the stain (iodine = a mordant)
4.) alcohol wash to decolorize
5.) stain with a a counter-stain —> safranin (red)
What is iodine?
A mordant
Purpose of the alcohol wash in the gram’s stain technique
To decolorize
Counterstain in the gram’s stain technique
Safranin
Colour of the safranin counterstain
Red
Cell walls of gram-positive bacteria
Simple cell walls, composed mainly of peptidoglycan
Cell walls of gram-negative bacteria
More complex cell walls which have an outer lipopolysaccharide membrane
What does crystal violet do to gram-positive bacteria?
Stains all cells purple
What does crystal violet do to gram-negative bacteria?
Stains all cells purple
What does iodine do with gram-positive bacteria?
Forms cross bridges with peptidoglycan and binds the crystal violet to the cell wall
What does iodine do with gram negative bacteria?
Doesn’t bond to the cell wall upon the addition of iodine. The lipopolysaccharide membrane prevents this.
What does alcohol do to gram-positive bacteria?
Doesn’t wash out the crystal violet as it’s binded to the cell wall
What does alcohol do to gram-negative bacteria?
Washes out any unbound crystal violet from the cell walls
What does the safranin counter-stain do to gram-positive bacteria?
Has no effect
What does the safranin counter-stain do to gram-negative bacteria?
Unstained cells are stained red/pink by the safranin
What’s different about the cell walls of gram-negative bacteria to gram-positive bacteria?
Gram-positive —> composed mainly of peptidoglycan
Gram-negative —> an outer lipopolysaccharide membrane
Why doesn’t crystal violet bond to the cell wall upon the addition of iodine in gram-negative bacteria?
The lipopolysaccharide membrane prevents it
What can the lipopolysaccharide layer in cells do?
Protect it from antibiotics
Culturing
Growing bacteria in a laboratory to form a bacterial culture
What do we used for culturing micro organisms?
Culture media
Solid culture medium
Agar plates
What are liquid culture media in?
In flasks
How do we prepare agar plates?
1.) Heat until liquid
2.) Mix in nutrients that bacteria needs
3.) pour into petri dished when hot
What is agar derived from?
Seaweed
How do we grow bacteria on agar?
1.) spread sample on surface
2.) dots appear after incubation
3.) form colonies
What does a liquid culture medium include?
The nutrients that bacteria needs and water
Where does bacteria grow in a liquid culture medium?
In the liquid
What doesn’t happen in liquid culture media?
Colonies aren’t formed
Which factors need to be kept at optimum by microorganisms for growth?
Nutrients
Growth factors
Temperature
pH
Why do bacteria need nutrients?
They’re heterotrophic = needs inorganic compounds already formed
Where are nutrients supplied to bacteria in laboratories?
In the nutrient media
What’s a liquid culture medium often called?
Nutrient broth
What, as well as nutrients, do culture media supply?
Water
Which nutrients are required for bacteria?
Carbon
Nitrogen
Where does the carbon and energy source for bacteria usually come from?
Glucose
Why do bacteria need nitrogen?
For amino acid synthesis
How do bacteria obtain nitrogen?
In organic molecules
In inorganic form, such as nitrate ions
What are bacteria good at synthesising and from what?
Their own amino acids from carbon and nitrogen
At which concentrations are the growth factors of bacteria?
Low concentrations
Growth factors needed by bacteria
Vitamins
Mineral salts
Example of a vitamin needed by bacteria
Biotin
Examples of mineral salts needed by bacteria
Sodium
Magnesium
Chloride
Sulphate
Phosphate
Why do bacteria need sulphate salts?
For some amino acids
Why do mineral salts need phosphate salts?
To synthesise phospholipids and nucleic acids
Nucleic acids synthesised with phosphate
DNA
RNA
ATP
How do we keep bacteria, when culturing it, at the right temperature?
Incubate it
What is bacterial metabolism regulated by?
Enzymes
Which temperature is suitable for most bacteria and why?
25 - 45 degrees Celsius
As bacterial metabolism is regulated by enzymes, and this is optimum for them
Optimal temperature for mammalian pathogens
37 degrees Celsius
Which bacteria have an optimum temperature of 37 degrees Celsius and why?
Mammalian pathogens
Human body temperature
Name for bacteria that have about 37 degrees Celsius as their optimum temperature
Mesophiles
Optimum temperatures for psychrophiles
Very low temperatures, even below 0 degrees Celsius
What type of bacteria have an optimum temperature that can be below zero degrees?
Psychrophiles
Where could psychrophiles live?
In deep oceans
Optimum temperature of hyperthermophiles
Very high
What type of bacteria have very high optimal temperatures?
Hyperthermophiles
Where could hyperthermophiles live?
Volcanic springs
List 4 biological problems which Psychrophiles must overcome to carry out cellular processes
Transport proteins inactive
Inflexibility of cellular membranes
Freezing of cell contents
Less energy for enzyme activity
Which pH conditions do most bacteria grow best in?
Slightly alkaline conditions (pH 7.4)
Which pH conditions do fungi grow best in?
Neural or slightly acidic
What grow best in neutral or slightly acidic conditions?
Fungi
How do we maintain the pH of a growth medium?
Use buffers
3 classifications of bacteria in terms of oxygen requirements
Obligate aerobes
Obligate anaerobes
Facultative anaerobes
Obligate aerobes
Will only grow in the presence of oxygen, respire aerobically
Bacteria that will only grow in the presence of oxygen and respire aerobically
Obligate aerobes
Obligate anaerobes
Will only grow when oxygen is absent, respire anaerobically
Bacteria that will only grow when oxygen is absent and respire anaerobically
Obligate anaerobes
Facultative anaerobes
Will grow in the absence of oxygen, although they do grow best in aerobic conditions with oxygen
Bacteria that will grow in the absence of oxygen, although they do grow best in aerobic conditions with oxygen
Facultative anaerobes
What is oxygen to obligate anaerobes?
Toxic
2 examples of obligate anaerobes
Clostridium tetani
Clostridium botulinum
What does clostridium tetani cause?
Tetanus
What causes tetani?
Anything that causes a deep penetrating wound (e.g - dog bites)
What happens during tetanus and why?
Can grow deep in the tissue where there’s low oxygen levels
Releases a powerful neurotoxin that causes paralysis of the body muscles = all contract at the same time
Deadly pathogen since is can paralyse breathing muscles
What are both clostridium tetani and clostridium botulinum?
Deadly pathogens
What does clostridium botulinum cause?
Botulism food poisoning
What causes botulism food poisoning?
Canned foods that haven’t been properly treated
For example, air got in or it wasn’t cooked at a high enough temperature
What happens during botulism food poisoning and why?
Clostridium botulinum produces a powerful neurotoxin
Causes all muscles to relax as the toxin blocks the nerve impulse form crossing the synapses = can’t move
Can be fatal is breathing muscles all relax
Which less dangerous thing is C.botulinum used in? Why?
Botox
In small amounts, it can relax the muscles in the skin that cause wrinkles
Liquid culture name
Nutrient broth
Which 2 methods can be used to count bacteria in liquid culture?
Directly (by counting each cell)
Indirectly (by measuring turbidity - cloudiness - of the culture medium with a colorimeter)
Turbidity of a culture medium
Cloudiness
What do we measure the turbidity of a culture medium with?
A colourimeter
2 types of direct counts for counting bacteria
Total counts
Viable counts
Total counts of bacteria
Include both living and dead cells
Viable counts of bacteria
Only count living or actively growing cells
= underestimates population size
2 examples of total counts methods for counting bacteria
Direct counting with a counting chamber
Indirect counting by measuring bacteria numbers by optical density
What type of total counts counting is using a counting chamber?
Direct counting
What type of total counts counting is measuring bacteria numbers by optical density?
Indirect counting
Which device is used to measure optical density by total counts?
Spectrophotometer
What happens with a spectrophotometer with a bacterial suspension with low bacterial numbers?
High transmission and high level of light detected
What can we do with the data collected with a spectrophotometer when completing a total count of bacteria and what does this tell us?
Produce a calibration curve
Can work out a certain number of cells from the light transmission
How can we work out the number of bacterial cell in a suspension when measuring by optical density?
Produce a calibration curve
On what are viable counts completed and what are we counting?
On agar plates
Count cells which are able to grow into visible colonies
Procedure of completing viable counts on an agar plate
1.) put sample of culture onto agar plate
2.) incubate at optimum temperature
3.) count the number of colonies growing on the plate
We assume that each colony grow from a single cell
What do we assume when doing viable counts of bacteria?
That each colony grows from a single cell
When would a dilution technique be used on a bacterial sample?
If the population density of the sample is too high to count
What do we do if the population density of a sample of bacteria is too high to count?
A dilution technique is used
Name for the process of diluting bacterial samples
Serial dilution or dilution plating
Method for serial dilution
1.) fill 5 test tubes with 9cm^3 sterile water using a sterile pipette
2.) add 1cm^3 of your sample to the first tube. This is a 1 in 10 dilution or 10^-1
3.) mix the 10^-1 dilution thoroughly and pipette 1ml into the second test tube. This is a 1 in 100 or 10^-2 dilution.
4.) repeat this process until you reach a 1 in 10,000 dilution (10^-4)
5.) plate each dilution on nutrient agar using aseptic techniques
6.) incubate the plates at 25 degrees Celsius for 48 hours
Why is serial dilution done?
When placing a sample that isn’t very diluted onto an agar plate, all of the individual colonies could be merged together. Therefore, we can’t count them.
When it’s diluted, the colonies are clearly separate and are easier to count.
Which dilution factor are colonies the most clearly separated?
At about 10^-5
What happens after the dilusion factors of about 10^-5 during serial dilution?
The number of colonies can become too small to be statistically significant
What happens if we don’t have enough colonies when counting on a bacterial sample?
Unreliable estimate
What provides the most reliable results when counting the number of colonies in a bacterial sample?
When there’s enough colonies but they’re still easy to count
What happens if we have too many colonies to count in a bacterial sample?
Too many to count reliably
What’s the problem when colonies merge when trying to count them?
Impossible to distinguish individual colonies, so it’s impossible to count them
Equation for estimating bacterial numbers in a culture - total viable count
Number of cells in 1cm^3 of original sample =
Number of colonies x 1/dilution factor
—————————————————-
Sample volume
Weaknesses of the total viable count method
1.) number is usually an underestimate since it doesn’t include dead or non-viable bacteria
2.) cannot be sure that each colony as grown from a single bacterium (bacterial cells may have been clumped together)
3.) Takes time and skill
4.) Lots of steps where contamination can happen
When are aseptic techniques used?
When transferring bacteria from 1container to another
What type of techniques are used when transferring bacteria from 1 container to another?
Aseptic techniques
What are aseptic techniques used to prevent?
- Contamination of the environment (and ourselves) by the microorganisms being handled
- contamination of the bacterial cultures by unwanted microorganisms from the environment (and ourselves)
What must all equipment and growth media which come into contact with microorganisms bing cultured (grown) be?
Sterile
5 examples of sterilisation
- use pre-sterilised Petri dishes (sterilised with UV light when manufactured)
- passing metal transfer tools (such an inoculating loops) through a roaring/blue Bunsen flame until they glow red
- sterilising any glassware used under high pressure and high temperature (121 degrees) in a special piece of apparatus called an autoclave for 15 minutes
-heating the nutrient agar used for the plates in an autoclave to sterilise it before pouring into the plates and letting it set
-disinfect bench
How are pre-sterilised Petri dishes pre-sterilised?
Using UV light when manufactured
Where are glassware and nutrient agar sterilised?
In an autoclave
How long does glassware go into an autoclave for?
15 minutes
Temperature of autoclave
121 degrees Celsius
Process of using an autoclave
1.) place the glassware and water in\
2.) seal the lid
3.) put on heat (121 degrees Celsius)
4.) water heats up and forms a steam
5.) steam builds up pressure
6.) high pressure = boiling point of water goes up = bacterial spores are killed
How does a liquid culture medium look initially when we produce it?
Clear
What do we do when initially producing a liquid culture medium? Why?
Heat sterilise
To ensure there isn’t anything already living there
Why do we heat sterilise culture media before introducing the bacteria?
To ensure there isn’t anything already living there
What happens to a liquid culture medium when bacteria is introduced?
Turns cloudy
How much of a bacterial sample is usually placed on an agar plate?
0.1cm^3
What is meant by bacteria “growing”?
Cell division
What is the unit of the number of cells in 1cm^3 of a bacterial sample?
Cellscm^3
Are clumping and merging of bacteria the same?
No
What does the microbial growth curve involve?
4 stages of growth
4 stages of growth of the microbial growth curve
Lag phase
Exponential (log) phase
Stationary phase
Death phase
How does inoculation occur at the beginning of the microbial growth curve?
Inoculate a sterile culture medium with bacteria (e.g - inoculating loop + aseptically transfer them)
What happens to the number of bacteria during the lag phase?
No change
Why is there no change in bacterial numbers during the lag phase?
Bacteria are becoming accustomed to the new growth medium
How do bacteria become accustomed to a new growth medium?
Prepare for cell division + growth by…
Synthesising enzymes
Synthesising proteins
Replicating DNA
Exponential growth
Doubling per unit time
During which phase of the microbial growth curve does exponential growth occur?
Exponential (log) phase
Describe the rate of cell division against the rate of cell death during the exponential (log) phase
Rate of cell division»rate of cell death
Why is the rate of cell division much higher than the rate of cell death during the exponential (log) phase?
No limiting factors = plenty of nutrients and no competition = grow quickly
Which stage of the microbial growth curve does bacteria stay in the longest and how long is this?
Stationary phase
A number of hours
What does bacteria start doing during the stationary phase of the microbial growth curve and why?
Compete
Is comes to the point where there’s so much bacteria that’s grown that the finite number of nutrients won’t be enough for them all
What limits the rate of growth in the microbial growth curve and at which phase?
The concentration of nutrients
Stationary phase
Describe the rate of cell division against the rate of cell death during the stationary phase
Rate of cell division = rate of cell death
What happens to the absolute number of cells during the stationary phase?
Stays constant
When has bacteria reached the death phase on the microbial growth curve?
Once the numbers of bacteria start to decrease
Describe the rate of cell division against the rate of cell death during the death phase of the microbial growth curve
Rate of cell division<rate of cell death
What happens to the population of the growth culture during the death phase and why?
Decreases
The build up of toxic metabolic waste
Nutrients becoming scarce
Why do we plot log numbers instead of actual numbers on the microbial growth curve?
If we plotted number of cells against time, the line would eventually go vertical due to exponential growth
It’s increasing in steepness due to the rate of growth increasing
This is meaningless, so we plot lot numbers instead of absolute numbers
Why would plotting number of cells against time for the microbial growth curve increase in steepness until it becomes a vertical line?
The rate of growth is increasing due to exponential growth
How do you calculate the % increase in population size of a bacterial culture?
Increase (highest value - lowest value)
Answer/lowest value x100
How do we know whether a sample has been contaminated?
See different types of bacteria
How do we confirm bacterial numbers in a sample?
Repeat and calculate a mean
How many colonies of bacteria is too few counted to be valid?
Less than 30
What do we need to ensure we mention when describing serial dilution?
Aseptic techniques
Different ways to describe culture media
Defined medium
Undefined medium
Selective medium
Complete medium
Defined medium
Contains only known ingredients
Undefined medium
Contains components that are not all known because they include, for example, yeast extract or beer peptone
Selective medium
Only allows certain bacteria to grow
Complete medium
Contains all the chemicals needed to support growth
Disadvantage of total counts method
Can’t identify the death phase
Describe the peptidoglycan in the cell walls of gram positive bacteria
Thick layer
When do bacteria grow fastest?
At optimum temperature
What does bacteria do at optimum temperature?
Grows fastest
What does bacteria do to nutrients in the lag phase?
Absorbs them
What has been reached in the stationary phase of the microbial growth curve?
Carrying capacity
What has increased by the stationary phase of the microbial growth curve?
Environmental resistance
Environmental conditions that could affect the rate of bacterial cell division
Amount of nutrients
Accumulation of waste products
Temperature
pH
Oxygen
Total viable count
Total number of bacteria in a known volume of liquid
What do we need to use when plotting a graph after a total viable count and why?
Logs
Large numbers that are difficult to plot
Why is it difficult to count large colony numbers of bacteria?
Clumping
