MICROBIOLOGY
MICROBIAL TECHNIQUES
Culturing microorganisms involves growing microorganisms so they can be measured or studied. Once you
decide which organism you want to cut, you obtain a culture of them. Liquid cultures of nutrient agar are used to
provide the right amount of nutrients, oxygen, pH and temperature. Risks we need to avoid:
❏ Entry of other microorganisms from air/ skin into the culture that will contaminate it.
❏ Mutant strain that may be pathogenic.
❏ Infecting other people with pathogens by accident.
aseptic techniques – prevent contamination from pathogens.
- sterilising equipment before starting the culture using bunsen
burner flame
- clean surfaces before and after with ethanol
- culture must not leave the lab
- dispose all the cultures safely by sealing them in plastic bags
and sterilising them
MEASURING GROWTH OF BACTERIAL COLONIES
CELL COUNTS – pipetting a small sample of the liquid culture onto a slide with a grid
on it (haemocytometer). Number of cells for given volume of sample can be
counted and multiplied up = number of cells in whole volume of culture.
MASS — known volume is sampled from a liquid culture, placed in a centrifuge and
spun till only the solid mass of cells remain. It is then weighted; the
mass then multiplied to find total mass of culture.
dilution plating – used to obtain a culture plate with a countable
number of bacterial colonies. number of colonies x dilution factor = total
viable cell count
turbidimetry – a spectrophotometer measures the amount of light
passing through, giving a measurement of size of the culture. Higher
concentration of bacterial cells = becomes more turbid = less light can
pass through.
,Bacteria and viruses
BACTERIA
all bacteria have:
- cell wall containing peptidoglycan
- a cell surface membrane
- a nucleoid (a single, circular strand of
DNA, the genetic material of bacterium)
- 70S ribosomes which are the site of
protein synthesis
some bacteria have (features of prokaryotes):
- pilli (thread-like projections from the
surface of the cell wall)
- flagella (long rapidly rotating whip-like
structures which move the bacteria)
- a capsule or slime layer (a thick slippery
substance around outside of cell wall)
- mesosomes (internal extensions of the
membrane which fold into the cytoplasm
and may be the site of cellular respiration)
- plasmids (small circles of additional DNA that
code for specific characteristics).
VIRUSES
Viruses are the smallest of the microorganisms, viruses are non-living.
all virus have:
- capsid, a protein coat used to protect the genetic material of viruses.
- nucleic acids (DNA or RNA) acting as genetic material. The genetic
material is used differently in the host cell to make new viruses depending
on which form it is in.
- specific proteins (antigens) aka virus attachment proteins (VAPs) which
specifically target proteins in the host cell surface membrane = infection.
some virus have:
- a lipid envelope, produced from the host cell membrane, covers the genetic
material and protein coat. It makes it easier for viruses to pass from cell to cell, but it does make them
vulnerable to substances that will dissolve the lipid membrane.
dna viruses - have DNA as their genetic material. The viral DNA acts directly as a template (model) for both new
viral DNA and mRNAs needed to introduce synthesis of viral proteins. e.g smallpox, lambda phage.
rna viruses - have RNA as genetic material. More likely to mutate. Don’t produce DNA as part of their life cycle.
The majority contain a single strand of RNA. e.g tobacco mosaic viruses, ebola fever, polio, measles.
retroviruses - a type of RNA virus. They have a protein capsid and a
lipid envelope. The single strand of viral RNA controls the synthesis
of the enzyme reverse transcriptase, used to convert their RNA into
DNA, which can be inserted into host cell’s genome so viral proteins
are transcribed. e.g human immunodeficiency virus and leukaemia.
, bacteria virus
- can reproduce in many places - can only reproduce inside a host cell
- contain ribosomes - don’t contain ribosomes
- living single-celled microorganisms - around 1 hundredth of the size of bacteria
- reproduce as binary fission - not living as they require other living cells to function
- can only replicate by invading host cells, which transcribe
and translate the viral genetic information into proteins
How virus reproduce
Viruses infect a host cell and use it to replicate themselves and produce new viral particles.
● bacteriophages inject their genome into the hist bacterial cell but the main part of the virla material
remains outside the bacterium. The viral DNA forms a plasmid within the bacterium.
for animals:
● enter by endocytosis and the host cell then digests the capsid, releasing the viral genetic material
● or the viral envelope combines with the host cell surface, releasing the rest of the virus inside the
membrane
plant viruses:
● enter the plant cell using a vector (often an insect) to penetrate the cellulose cell wall.
Once a virus is in the host cell, there are two different routes of infection:
lytic cycle:
1. bacteriophage attaches to the host cell and injects its phage DNA
2. it brings about the synthesis of viral enzymes.
3. phage DNA inactivates the host DNA and takes over the cell biochemistry.
4. phage DNA is replicated.
5. new phage particles are assembled as new protein coats are formed around phage DNA. (enzyme
lysozyme is synthesised or released).
6. lysis - bacterial cell bursts due to lysozyme action and releases many phages to infect new bacteria.
lysogenic cycle:
1. bacteriophage attaches to the host cell.
2. DNA inserted into host DNA (provirus) so it's replicated everytime the host cell divides without causing
damage.
3. this also causes the synthesis of viral enzymes.
4. sometimes (in certain conditions), it changes into the lytic pathway because the viral DNA leaves the
main genome to form a plasmid which is only then translated and transcribed.
mRNA is not produced because one of the viral genes causes a repressor protein to be produced which
prevents the translation of the viral genetic material. Virus is latent (state of non-virulent virus) during this period
of lysogeny (period when a virus is part of the reproducing host cell but does not affect the host in a negative
way). Sometimes, if the host cell is damaged, viruses in the lysogenic state are activated so the amount of
repressor protein decreases and the viruses enters the lytic pathway become virulent.
MICROBIAL TECHNIQUES
Culturing microorganisms involves growing microorganisms so they can be measured or studied. Once you
decide which organism you want to cut, you obtain a culture of them. Liquid cultures of nutrient agar are used to
provide the right amount of nutrients, oxygen, pH and temperature. Risks we need to avoid:
❏ Entry of other microorganisms from air/ skin into the culture that will contaminate it.
❏ Mutant strain that may be pathogenic.
❏ Infecting other people with pathogens by accident.
aseptic techniques – prevent contamination from pathogens.
- sterilising equipment before starting the culture using bunsen
burner flame
- clean surfaces before and after with ethanol
- culture must not leave the lab
- dispose all the cultures safely by sealing them in plastic bags
and sterilising them
MEASURING GROWTH OF BACTERIAL COLONIES
CELL COUNTS – pipetting a small sample of the liquid culture onto a slide with a grid
on it (haemocytometer). Number of cells for given volume of sample can be
counted and multiplied up = number of cells in whole volume of culture.
MASS — known volume is sampled from a liquid culture, placed in a centrifuge and
spun till only the solid mass of cells remain. It is then weighted; the
mass then multiplied to find total mass of culture.
dilution plating – used to obtain a culture plate with a countable
number of bacterial colonies. number of colonies x dilution factor = total
viable cell count
turbidimetry – a spectrophotometer measures the amount of light
passing through, giving a measurement of size of the culture. Higher
concentration of bacterial cells = becomes more turbid = less light can
pass through.
,Bacteria and viruses
BACTERIA
all bacteria have:
- cell wall containing peptidoglycan
- a cell surface membrane
- a nucleoid (a single, circular strand of
DNA, the genetic material of bacterium)
- 70S ribosomes which are the site of
protein synthesis
some bacteria have (features of prokaryotes):
- pilli (thread-like projections from the
surface of the cell wall)
- flagella (long rapidly rotating whip-like
structures which move the bacteria)
- a capsule or slime layer (a thick slippery
substance around outside of cell wall)
- mesosomes (internal extensions of the
membrane which fold into the cytoplasm
and may be the site of cellular respiration)
- plasmids (small circles of additional DNA that
code for specific characteristics).
VIRUSES
Viruses are the smallest of the microorganisms, viruses are non-living.
all virus have:
- capsid, a protein coat used to protect the genetic material of viruses.
- nucleic acids (DNA or RNA) acting as genetic material. The genetic
material is used differently in the host cell to make new viruses depending
on which form it is in.
- specific proteins (antigens) aka virus attachment proteins (VAPs) which
specifically target proteins in the host cell surface membrane = infection.
some virus have:
- a lipid envelope, produced from the host cell membrane, covers the genetic
material and protein coat. It makes it easier for viruses to pass from cell to cell, but it does make them
vulnerable to substances that will dissolve the lipid membrane.
dna viruses - have DNA as their genetic material. The viral DNA acts directly as a template (model) for both new
viral DNA and mRNAs needed to introduce synthesis of viral proteins. e.g smallpox, lambda phage.
rna viruses - have RNA as genetic material. More likely to mutate. Don’t produce DNA as part of their life cycle.
The majority contain a single strand of RNA. e.g tobacco mosaic viruses, ebola fever, polio, measles.
retroviruses - a type of RNA virus. They have a protein capsid and a
lipid envelope. The single strand of viral RNA controls the synthesis
of the enzyme reverse transcriptase, used to convert their RNA into
DNA, which can be inserted into host cell’s genome so viral proteins
are transcribed. e.g human immunodeficiency virus and leukaemia.
, bacteria virus
- can reproduce in many places - can only reproduce inside a host cell
- contain ribosomes - don’t contain ribosomes
- living single-celled microorganisms - around 1 hundredth of the size of bacteria
- reproduce as binary fission - not living as they require other living cells to function
- can only replicate by invading host cells, which transcribe
and translate the viral genetic information into proteins
How virus reproduce
Viruses infect a host cell and use it to replicate themselves and produce new viral particles.
● bacteriophages inject their genome into the hist bacterial cell but the main part of the virla material
remains outside the bacterium. The viral DNA forms a plasmid within the bacterium.
for animals:
● enter by endocytosis and the host cell then digests the capsid, releasing the viral genetic material
● or the viral envelope combines with the host cell surface, releasing the rest of the virus inside the
membrane
plant viruses:
● enter the plant cell using a vector (often an insect) to penetrate the cellulose cell wall.
Once a virus is in the host cell, there are two different routes of infection:
lytic cycle:
1. bacteriophage attaches to the host cell and injects its phage DNA
2. it brings about the synthesis of viral enzymes.
3. phage DNA inactivates the host DNA and takes over the cell biochemistry.
4. phage DNA is replicated.
5. new phage particles are assembled as new protein coats are formed around phage DNA. (enzyme
lysozyme is synthesised or released).
6. lysis - bacterial cell bursts due to lysozyme action and releases many phages to infect new bacteria.
lysogenic cycle:
1. bacteriophage attaches to the host cell.
2. DNA inserted into host DNA (provirus) so it's replicated everytime the host cell divides without causing
damage.
3. this also causes the synthesis of viral enzymes.
4. sometimes (in certain conditions), it changes into the lytic pathway because the viral DNA leaves the
main genome to form a plasmid which is only then translated and transcribed.
mRNA is not produced because one of the viral genes causes a repressor protein to be produced which
prevents the translation of the viral genetic material. Virus is latent (state of non-virulent virus) during this period
of lysogeny (period when a virus is part of the reproducing host cell but does not affect the host in a negative
way). Sometimes, if the host cell is damaged, viruses in the lysogenic state are activated so the amount of
repressor protein decreases and the viruses enters the lytic pathway become virulent.
