A Level Edexcel Biology B Topic 6

How do you produce an aseptic culture?

Provide organisms with appropriate nutrients

Use a sterile medium

Use an inoculating loop to transfer pathogen

Prevent contamination from air (convection current)

Pass bottle through flame

Only open lids partway

Why must aseptic techniques be used?

To prevent potentially harmful microorganisms escaping from culture to air

To prevent microorganisms from the air contaminating the culture

What are the different types of culture media?

Liquid (broth)

Solid (grows cultures with discrete colonies)

Non-selective (allows broad range of microorganisms to grow)

Selective (allows only a narrow range of microorganisms to grow)

How do you isolate a singular microorganism from a culture?

streak plating

What are the steps for streak plating

1. Flame inoculating loop

2. Dip loop in culture

3. Make three streaks on sterile plate

4. Turn the plate 90 degrees and streak again, overlapping last streaks

5. Turn and streak again

6. use isolated colonies to prepare a second plate

How do you make a serial dilution?

1. Start with 10cm^3 of undiluted culture

2. Add 1cm^3 of first tube into 9 cm^3 of sterile saline
3. Continue to add 1cm^3 from previous tubes to form serial dilutions

Why is an appropriate dilution a compromise when diluting a culture?

Must have a low enough number of microorganisms to count and a high enough number to provide a reliable sample of the culture

What are the two types of cell count?

Total count: All cells in the culture, dead or alive

Viable count: Only the living cells in the culture

What is the lag phase of bacterial growth?

First phase after inoculation when microorganisms are adjusting to their environment so the population is only increasing very slowly

What is the log phase of bacterial growth?

exponential growth

What is the stationary phase of bacterial growth?

population reaches maximum due to limiting nutrients and a build up of toxic substances

What is the death phase of bacterial growth?

A lack of nutrients and increase of toxic products cause death of microorganism

How is a direct cell count calculated?

A haemocytometer (a glass slide with a grid etched on) is placed on a slide

Bacteria cells=number counter x1064 per cm^3

Only counts viable cells

How is an indirect cell count used to measure dry mass of cells?

1. Filter a sample of known dilution through a pore
2. Remove water by evaporation
3. subtract final mass from initial mass (including filter paper) to calculate mass of dry microorganisms

How is an indirect cell count used to measure turbidity of a diluted culture?

The cloudiness of a sample can be calculated using a simple colorimeter to measure absorbance or transmission.

Use a haemocytometer to produce a calibration curve that relates to absorbance or transmission to the the actual number of cells.

What are the benefits and drawbacks of using serial dilutions to calculate cell count?

Benefits: cheap and simple

Drawbacks: Only counts viable cells; very slow due to incubation period and serial dilutions

What are the drawbacks of measuring turbidity?

Have to use a direct method to calculate cell count from calibration curve

Expensive

Assumes cell density is equal across culture

What is an endotoxin?

Associated with some Gram-negative bacteria. An endotoxin is a toxin that is a structural molecule of the bacteria found in the outer membrane of the cell wall.

What is an exotoxin?

A soluble protein produced and released by bacteria as they metabolise and reproduce

Found in both Gram-negative and Gram-positive bacteria

How do pathogens harm the host?

Invade and destroy host tissues

Produce toxins

How does Mycobacterium tuberculosis attack a cell?

By host tissue invasion

Lies dormant in tubercles and not destroyed by immune system (tubercles coated in thick waxy coat)

What is host tissue invasion?

When bacteria invades and damages cells

How does Salmonella attack a cell?

By endotoxins

How does Staphylococcus attack a cell?

By exotoxins (gram positive)

Can only cause disease if they get inside tissue; if the flora changes; or if the immune system is compromised

What are bactericidal antibiotics?

Antibiotics that kill bacteria by destroying cell walls causing bacteria to burst

Especially effective against Gram-positive as cell wall is so thick

(e.g. penicillin)

What are bacteriostatic antibiotics?

Antibiotics that inhibit the growth of bacteria by stopping protein synthesis and production of nucleic acids so bacteria cannot replicate

Especially effective against Gram-negative as lipid bilayer is damaged

(e.g. tetracycline)

What is primary resistance?

When the formation of the bacteria causes it to be unaffected by an antibiotic (e.g. Gram-negative bacteria is resistant to penicillin as they only have thin cell walls)

What is secondary resistance?

When bacteria that were once susceptible to antibiotics develop resistance

What causes antibiotic resistance to develop?

Natural Selection: a mutation will create an allele of a gene which changes the effect of the antibiotic. Antibiotic resistance will be passed on to offspring increasing its frequency

Why is HIV hard to treat?

It has a constantly changing protein coat that means it is not recognised by the immune system

How is MRSA prevented?

New patients screened, isolated and treated

Antibiotics only used if needed

Antibiotic courses completed

Strict hygiene regimes

How is the spread of antibiotic resistance controlled?

Isolating infected people

Hand washing and antibacterial gel

Gloves and aprons

Restricting use of antibiotics

Completing courses of antibiotics

Developing new antibiotics

How is influenza transmitted?

via droplets generated when infected persons cough or sneeze; animal waste; infected surfaces

What is the mode of infection of influenza?

1. Ciliated epithelial cells are infected
2. Viral RNA takes over biochemistry
3. Cell produces new virus particles
4. Cell lyses
5. Many viral particles are released
6. Virus taken into cells lining bronchi and bronchioles by endocytosis

What are the pathogenic effects of influenza?

Lysis of cells and release of toxins cause coughing, headache, vomiting and other symptoms

How is Stem Rust Fungus Transmitted?

Airborne spores of fungus

What is the the mode of infection of Stem Rust Fungus?

1. Spore germinates in water
2. Produces hyphae which enters host by stomata
3. Fungus grows into mycelium
4. Fungus surrounds all tissues in the plant

What is the pathogenic effect of Stem Rust Fungus?

Enzymes are produced to digest the plant and absorb its nutrients which causes stem to be shortened and weakened

How is malaria transmitted?

Parasite enters the body in the saliva of a female mosquito when it bites

What is the mode of infection of Malaria?

1. Parasite transmitted via mosquito
2. Parasite travels to liver
3. Parasite infects red blood cells
4. Parasite reproduces asexually inside red blood cells and causes lysis

What is the pathogenic effect of Malaria?

Lysis of red blood cells causes sweating, shaking, anaemia and liver damage

How can malaria be prevented?

Mosquito nets, insect repellent, pesticides, proper disposal of sewage, drain wetlands where mosquitoes breed, vaccination

What are the issues with malaria prevention

Not possible to drain wetlands that people use

Insecticides could have unexpected effects to food chains

Expensive to develop vaccines

What is an antigen?

A macromolecule that cause an immune response by lymphocytes.

What are physical barriers to infection?

Skin

Stomach acid (kills bacteria)

Gut and skin flora (natural bacteria flora competes with pathogens for food and space)

What is phagocytosis?

When white blood cells engulf foreign cells and digest them

What does a neutrophil do?

Ingests bacteria cells, kills it then digests it

What do macrophages do?

Ingest not only foreign cells but also body cells that are dead or dying

Display antigen of digested bacteria on surface membranes

Releases cytokines to stimulate other immune responses

What is an Antigen-presenting cell?

A cell that isolates the antigen from a pathogen and places it on its surface membrane so that it can be recognised by other cells in the immune system

What are cytokines?

chemical messengers produced in response to a stimulus

What happens when a lymphocyte binds to an antigen on an antigen presenting cell?

The lymphocyte becomes sensitised and divides repeatedly to produce a clone of cells

Most become activated lymphocytes

What is clonal selection?

The repeated mitotic divisions of a sensitised lymphocyte to produce a large number of genetically identical cells

What are B cells?

Lymphocytes that provide a humoral immune response against antigens

What are T cells?

lymphocytes responsible for the cell-mediated immune response (T Killer cells) against abnormal cells and pathogens within cells

What is the cell-mediated immune response?

1. A pathogen invades a host cell and the host cell becomes an Antigen-Presenting Cell (APC)
2. An inactive T Killer cell binds with a complementary receptor binds to the antigen on the APC.
3. Active T helper cells secrete cytokines which stimulate T killer cells to divide into active T killer and T killer memory cells.
4. An active T killer cells binds to the receptor on the APC and secretes perforin which causes pores to form on the cell membrane of the pathogen leading to its death.

What is the humoral immune response?

1. A pathogen is engulfed by a macrophage and the cell becomes an Antigen-Presenting Cell (APC)
2. The antigen of the APC binds to a T helper cell with a complementary receptor and the T helper cell becomes activated so divides into active T helper and T memory cells.
3. Antigens from APCs that are complementary are taken in by endocytosis by B cell.

4. The B cell becomes an APC and binds to an activated T helper cell which causes it to release cytokines
4. The cytokines stimulate the B cells to divide and form B memory cells and B effector cells
5. B effector cells differentiate into plasma cells which can produce antibodies which attack the pathogen.

Antibodies attack via agglutination- causes pathogens to clump together.
Opsonisation- causes the pathogen’s antigen to be more visible.
Neutralisation- reduces the toxicity of the pathogens toxin by making them insoluble.
Lysis- causes the pathogen to burst.

What is natural immunity?

Active: natural contact with antigen through infection (e.g. recovery from chickenpox)

Passive: Baby gets antibodies from crossing of mother’s antibodies via the placenta or from breast milk

What is artificial immunity?

Active: Vaccination which stimulate immune system and lead to antibody production

Passive: Injection of antibodies

What is a secondary immune response?

The response to infection by a pathogen carrying antigen to which there are memory cells already present in the host's body

What is herd immunity?

When 80-90% of population are vaccinated to make transmission of a disease unlikely to the immunocompromised

What are some non-specific responses to infection?

Inflammation: Histamines released by damaged white blood vessels and cause vasodilation to increase speed of delivery of antibodies

Fever: Hypothalamus increases body temperature to decrease speed of pathogen reproduction

Lysozyme action: Lysozyme (enzyme) kills bacterial cells by damaging the cell wall