T6

topic 6 microbiology and pathogens edexcel B

aseptic culture techniques

aseptic culture techniques

1. disinfect surfaces

2. work near a Bunsen flame (convection current)

3. Heat wire loop + allow to heat

4. Flame neck of tube

5. Open petri lid slightly

types of nutrient medium

Broth – mixture of distilled water, nutrients and a bacterial culture

Agar plate – petri dish containing agar jelly

Selective media – inhibits growth of certain organisms whilst encouraging growth of others

How can you measure growth of microorganisms?

1. Cell counts

2. Optical methods

3. Dilution plating

4. Area and mass of fungi

Cell counts

1. Haemocytometer (microscope slide with grid lines which holds standard volume of liquid - 0.1mm³)

2. dilute sample w half vol =culture, half= trypan blue (stains dead cells)

Pros	Cons
Counts only viable	Slow
Accurate	Expensive equipment

Optical methods

Turbidity (type of colorimetry)

As number of cells increase, turbidity/opacity increases = lower % of light transmitted

compared against calibration curve made with cell count

Pros	Cons
Quick	Expensive equipment
Can be done in field	Includes non-viable cells
	Requires calibration curve
	Assumes equal density

Dilution plating

total viable cell count= (num of colonies isolated) x (dilution factor)

Each colony grows from a single cell

• Too many colonies in original concentration so its diluted until singular colonies are observed

Pros	Cons
Counts only viable	Slow incubation period
Direct count	Takes time to do serial dilutions
No expensive equipment

Area and mass of fungi

Mass

1. Liquid growth medium

2. Samples of broth removed at regular intervals and fungi separated by centrifuge/filtering

3. Dried thoroughly - when no more loss of mass recorded (Eg. oven overnight 100ºC)

Conditions that produce greatest dry mass = optimum for growth

Area: growth of same num of colonies and same conditions in diff temps, biggest incr in area= closest to optm cond

Generation time

Time between bacterial divisions

Number of bacteria in population equation

Nt = No x 2^kt

Nt = number of organisms at time t

No = number of organisms at time 0

k = exponential growth rate constant

t = time colony has been growing

Growth rate constant equation

k = log₁₀Nt - log₁₀No
log₁₀2 x t

Phases of bacterial growth

Lag - Bacteria adapting

Log - Rate of bacterial reproduction is close/at theoretical maximum

Stationary - Rate of reproduction = rate of death

Death - Reproduction stops and cells die

exponential growth end causes

• Less nutrients available (unless more added)

• accumulation of toxic waste products (pH/CO₂)

ways in which bacteria causes symptoms of disease

1. endotoxins (Gram -ve)

2. exotoxins  (Gram +ve & -ve)

3. by invading host tissue + damaging cells (response of host org to cell damage = most of symptoms of disease)

endotoxins characteristics

example bacteria that works by endotoxins + mode of action

salmonella Spp

spread by ingestion of food/ water contaminated w infected faeces (bac survives stomach acid)

1. bacteria invade lining of the (large) intestine

2. endotoxins cause inflammation 

3. cells can no longer absorb water=> faeces become liquid

4. gut goes into spasms of peristalsis: result in diarrhoea  

exotoxins characteristics

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example bacteria that works by exotoxins

staphylococcus spp (gram +ve)

1. can be v varied + dangerous

example bacteria that causes symptoms of disease by invading host tissue + damaging cells 

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action of antibiotics

work on selective toxicity: disrupt function/ metabolism bac, minimal damage host cells

1. bacteriostatic: inhibit growth + repr of bac ( bac kept in Stationary phase of growth, no cell div)

   1. usually efficient: comb w immune cyst, ensures path destroyed

2. bactericidal: destroys most pathogens present. 

   1. used for more severe infections/ when immune sys suppressed (transplant)

effectiveness depends on: conc of dose, pH, whether antibiotic destroyed by path/ host, can drug reach infected area…

penicillin action:

BACTERICIDAL

type of cell wall agents

• prevents formation of cross-linking in cell walls (weaker)

• so bac killed by lysis (during div)

some also: cell membrane agents

• damage cell membrane

• metabolites leak out/ water moves in

tetracycline action

BACTERIOSTATIC 

protein synthesis inhibitors

• interrupt/ prevent transcription and/ or translation of bac genes

• protein production affected 

(can just use mRNA, enzymes + proteins it alr has)

explain how drug resistant organisms evolve, developments and spread of antibiotic R (link t3)

1. variation: some organisms resistant due to (random) mutation (eg. in gene for production of enzymes / proteins that make drug ineffective)

2. mutated cell more likely to survive and reproduce

3. drug/treatment acts as selection pressure: means some organisms with resistant alleles are better adapted to survive= selective advantage

4. therefore pass on resistant gene / allele to offspring

5. resulting in an incr in resistant allele freq in population

If no antibiotic present = no selection pressure, mutated strain may die out due to comp

If present, mutated organisms have selective advantage = only mutants survive, reproduce rapidly w/o comp

• original mutation is a random event, not caused by presence of antibiotic

• but the spread of resistant allele is due to selection by an antibiotic-rich environment

  .: widespread of use of antibiotics accelerates this process, incrs selection pressure for evolution of bac that are resistant 

Explain why doctors have been advised to limit the prescription of antibiotics (2)

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explain the development of drug R HCAI (healthcare associated infections)

• antibiotic R bac commonly found in healthcare centres: bc antibiotic use at its highest, selective pressure

• also bc very weak/ immune sys: act as opportunistic infections

difficulties in preventing development of antibiotic R in bac

1. balance between use few to have some in reserve if all else fails + variety

2. nums of antibiotic developed falling

methods of controlling spread of drug R bac

1. reduce use, only used when absolutely necessary (incl. antibiotics only prescribed if illness caused by bacteria)

2. complete each course: if taken full prescribed duration, kill most bac. if not, immune system cannot destroy all bac. those that survive have some R. // no bac exposed to sub-lethal doses of antibiotic, which promote development of R mechs.

3. use as few antibiotics as possible

4. vary antibiotics used 

5. hygiene measures: wash hands using alcohol based gels (readily available), disinfect w antiseptic, laundry linen, clothing for doctors

6. isolation of patients w R bac, minimise spread

7. prevent drug R entering hospitals: screening patients. can be treated immediately and isolated, prevents spread to others (particularly weak immunes system)

   1. visitors not bringing in disease

viruses: main way in which cause symptoms

: when cause lysis of host cells

• protein coat of viruses can act as toxin, causing disease

• sometimes when virus takes over metabolisms of cell synthesised toxins

• often specific to particular tissues

  • this is due to the presence/ absence of antigenic markers on cell host surface

influenza

infection that crosses species barrier:

 zoonotic

puccinia graminis

plasmodium spp transmission

MALARIAL PARASITE (feeds on living tissue of host), found in blood

life cycle involves 2 hosts

transmission 

• parasite transmitted to a human host by mosquito vector: Anopheles vector

• transmission to/ from human host occurs when mosquito takes blood meal

  • female mosquitoes need 2 blood blood meals for protein for developing eggs

  • saliva contains anticoagulants

  • (blood stream) person ←→mosquito (saliva)

  • different stage of life cycle in each   

plasmodium spp: mode of infection

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plasmodium spp pathogenic effect

• when burst out of RBC: cause flu like symptoms

• due to response of body to lysis of cells

• also: long term damage to liver

• steady reduction in num of RBC: weakness, severe anaemia, eventually death

adaptations for survival of malarial parasite (diff to cure + vaccine):

• can survive in dormant form w/o needing to move to new host

• in humans: inside liver cells/ RBC: hidden from immune system

• takes a long time to kill human host: many opps for it to be passed into female mosquito

• antigens on its surface change frequently

vector def

organisms/ environmental factors that transmit infection from one host to another

endemic disease

active disease / pathogen is constantly present in a particular country/ area

general problems of controlling endemic disease

disease often WIDESPREAD

1. AREA: eradication problem covers large area

2. ENVIRON: widespread in environ, diff to track down/ remove sources

3. PEOPLE: reqs large pop nums to eradicate

= very expensive

controlling endemic disease + limitations

implications of control methods of malaria

role of sci community in control of spread of malaria

analyse cost of treatments compared to effectiveness, provide helpful guidelines

1. diagnosis use microscopes to see protozoa: avoids treating diseases w similar symptoms w expensive antimalarial drugs

2. prevention: insecticide-impregnated mosquito nets most effective. also means mosquitoes feld less, reduces mosquito population, benefiting those who do not sleep under mosquito nets too

3. treatment: combine w other drugs prevents resistance of parasites to drugs used. expensive, so only used on properly diagnosed cases, avoids wasting medicine

non-specific responses to infection

1. inflammation

2. fevers

3. phagocytes

inflammation

fevers

1. raised temp reduces ability path repr effectively: causes less damage

2. immune syst works better at higher temps/ incrs metabolism of cells: more successful in combating infection

• if too high: damage denaturation, permanent tissue damage. sweat→ dehydrated

phagocytes

opsonins

chems which bind to paths and label them, more easily recognised by phagocytes (freq antibodies)

t cells

activate in thymus gland 

bind to antigens on infected body cells

• t killer

• t helper: activate plasma cells, secrete opsonism

• t memory: long lived, immunological memory, 2nd time meet path, div rapidly

lymphocytes: B +T cells

MHC

MHC: major histocompatibility complexes, display antigens on cell surface membrane

MHCI: found on all other body cells, put own stuff

MHC II: B-cells + macrophages: put bits of pathogen 

immune response

specific response to body to invasion by pathogens

• cell recognition: distinguish self and non cell, using proteins on surface membrane, antigens

• diverse: recognises many antigens

• immunological memory

humoural response

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CD4

CD4 receptors on surface membrane of helper T cells bind to MHC II molecules on surface of APCs, such as macrophages and B cells, presenting antigens to the T cell receptor. activates them

effect of antibodies

1. agglutination - clumps together antigens/ path: prevents spread thru body, phagocytosis easier. antibodies flexible, able to bind to many antigens at once. 

2. can cause lysis

3. opsonization: antibodies label microbes: marked for phagocytosis, enhances it 

4. neutralisation: soluble toxins made insoluble, neutralise their effects by binding to them/ block binding sites of pathogen (can’t enter more cells)

plasma cells characteristics

• lots of RER: 

  • vesicles for exocytosis

  • many ribosomes for secreting many copies of antibody 

cell mediated response

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role of R and B memory cells in secondary immune response

memory cells can divide rapidly by mitosis into plasma (b) and killer and helper cells (t) if same antigen detected in blood

lots of antibodies produced very quickly - before pathogen can cause any symptoms 

very long lived, decades

types of immunity

active: contact with antigen, stimulates primary response

passive: immunity granted via antibodies, temporary, body cannot restore them

natural: infection, breast milk

artificial: vaccination (exposed to inactivated/ attenuated antigens)

herd immunity: when large proportion of population is immune to a pathogen, preventing spread of diseases, lowering risk of infection to all, incl those who are not vaccinated (less likely to encounter path)

Describe how vaccination enables active artificial immunity

1. vaccine contains those antibodies

2. digested and presented on antigen presenting cells/ MHCs

3. APC binds to CD4 receptors on T cell

4. this stimulates production of T memory cells

5. activated T cells release cytokinins and stimulate B cells

6. which produce B memory cells

7. in the secondary immune response, pathogen destroyed before symptoms/ damage

8. plasma cells release antibodies

Explain why the vaccine for one virus will not protect from infection from other pathogens.

• immune response is specific: has diff antigen

• so on infection with new virus there will not be memory cells present specific for it

• an other primary immune response will be initiate

• new virus will not be destroyed before it causes disease

vaccines advantages

• protects people from being infected 

• virus less likely to get passed on 

• virus destroyed before infections caused

• greater chance of developing herd immunity 

• therefore fewer infected people to pass virus onto uninfected people

• help to protect those who cant be vaccinated/ infected: allergic, immuno suppressed, immunodeficient 

explain potential issues in populations where a proportion choose not to vaccinate.

1. Decreased Herd Immunity: leaves vulnerable individuals at higher risk of contracting disease

2. Resurgence of Vaccine-Preventable Diseases: leading to outbreaks and increased mortality.

3. Healthcare Strain

State ways in which the skin flora can help to protect a person from infection by pathogenic bacteria.

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Suggest why treatment with antibiotics may not be effective against the dormant bacteria in the tubercles.

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Describe the events that take place resulting in T helper cell activation, following the formation of pseudopodia by the macrophages

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