Summary

• Culture

  Microorganisms are provided with the nutrients, level of oxygen, pH and temperature they need to grow in large numbers so they can be observed and measured
• Aseptic technique


  Only introducing the desired bacteria into the medium, under sterile conditions, in order to prevent the growth of unwanted organisms
• Aseptic culture technique


  1. Decide on the microorganisms you want to culture and obtain the culture

  2. Provide microorganisms with appropriate nutrients in sterile nutrient medium (broth or agar)

  3. Inoculate the culture
• Selective medium


  • Medium containing a very specific balance of nutrients - this means only very specific bacteria will grow in it and mutant strains won't
• Inoculating agar


  1. Make a streak plate (sterilise inoculating loop by flaming, dip in culture, sterile plate, at least three streaks straight or zig-zag, turn, streak which must overlap with first streak, turn, streak to try to obtain single colonies)

  2. Make a spread plate (drop some on and use a sterile spreader to distribute it)
• Broth


  Can provide anoxic conditions as well as oxygen closer to the surface so can provide information about what kind of oxygen requirements the microbes have. They can also grow a much larger volume of bacteria. However, you can't get a single, discrete, pure colony from a broth to inoculate with/study
• Aseptic technique


  • All equipment, including agar and Petri dishes, should be sterile
  • Flaming equipment in a Bunsen flame ensures sterility
  • Inoculation should be done with a flamed instrument
  • Lids should be replaced as quickly as possible
• Growth curve of a microorganism in a closed culture

  1. Lag phase - the microorganisms are adapting to their environment and reproduction rate increases slowly

  2. Log phase - microorganisms grow at their maximum rate, as long as there are sufficient nutrients

  3. Stationary phase - death rate = reproduction rate (due to build up of waste products and lack of nutrients)

  4. Death phase - deaths exceed new cell population as conditions continue to deteriorate
• Cell Count


  Cells can be counted using a haemocytometer (a thick microscope slide engraved with a grid and a rectangular chamber that holds a standard volume of liquid (0.1 mm3)). The sample of broth is diluted 1:1 with trypan blue, which stains dead cells blue. You count the bacterial cells in each of the four sets of 16 squares and take a mean. The haemocytometer has been pre-calibrated so that the number of bacterial cells = number counted * 10^4 per cm3. This is repeated at regular intervals throughout growth.
• Turbidimetry


  A specialised form of colorimetry. As turbidity increases, transmission decreases and absorbance (measured in Au, arbitrary absorbance units) increases. This value can be linked to cell count by measuring absorbance of samples with a known cell count (via counting cells with a haemocytometer or using dilution plating), and using a calibration graph to obtain the cell count in an unknown sample.
• Dilution Plating


  Works on the principle that every colony is grown from a single, viable microorganism. Immediately after culturing, colonies cannot be counted because a single mass is often present. So that single colonies can be seen, the original culture is serially diluted, a lawn plate made and the colonies counted. This is then multiplied by the dilution factor to obtain a cell count.
• Endotoxins


  Lipopolysaccharides in the outer lipid membrane of Gram negative bacteria e.g. Salmonella. Endotoxins may be released from a bacterium if it breaks down, and have effects local to the site of infection.
• Exotoxins


  Soluble proteins produced and released by bacteria as they metabolise and reproduce e.g. Staphylococcus. Exotoxins are spread around the body in blood and body fluids.
• Tuberculosis (TB)


  1. First infection is symptomless. Infected phagocytes are sealed in tubercles as a result of an inflammatory response in the lungs.

  2. Bacteria lie dormant inside the tubercles. They are not destroyed by the immune system as the tubercles are covered with a thick waxy coat.

  3. When the immune system becomes weakened, the bacteria become active again, and slowly destroy the lung tissue, thus leading to breathing problems, coughing, weight loss, and fever.

  4. TB can be fatal.
• Bactericidal antibiotics


  Kill bacteria by destroying their cell wall thus causing them to burst. An example is Penicillin.
• Bacteriostatic antibiotics


  Inhibit the growth and reproduction of bacteria by stopping protein synthesis and production of nucleic acids so the bacteria can't grow and divide. An example is Tetracycline, which interferes with protein synthesis in 70S ribosomes.
• Antibiotic resistance

  Bacteria become resistant to antibiotics as a result of natural selection. The bacteria which are not killed by the antibiotic possess a selective advantage - resistance which enables them to survive and reproduce. Therefore, the allele for antibiotic resistance is passed onto their offspring, thus creating a resistant strain.
• There is an ongoing evolutionary race between organisms and pathogens as pathogens evolve adaptations which enable them to survive and reproduce. For instance, the constantly changing protein coat (antigen coat) of HIV means that the virus is not recognised and destroyed by the immune system.
• Controlling the spread of antibiotic resistant infections in hospitals


  1. New patients are screened at arrival, isolated and treated if they are infected to prevent the spread of bacteria between patients.

  2. Antibiotics are only used when needed and the entire course of antibiotics is completed to ensure that all the bacteria are destroyed and to minimise the selection pressure on bacteria to prevent resistant strains from forming.

  3. All staff must follow the code of practice which includes strict hygiene regimes such as washing hands with alcohol-based antibacterial gels and wearing suitable clothing which minimises the transmission of resistant bacteria.
• Influenza


  • Transmission: droplet infection, direct contact with virus-filled mucus, direct contact with animal waste (zoonotic infection), direct contact with infected surfaces.
  • Mode of infection: infects ciliated epithelial cells of the lungs (antigen fits into receptor on host cell, injects viral RNA), viral RNA takes over cell biochemistry, cell produces new virus particles, cell lyses, many virus particles released.
  • Pathogenic Effects: headache, sore throat, coughing, sneezing, muscular/joint pain, vomiting, fever etc. lasting about 5-7 days.
  • Treatment/Control: antiviral medication, antibiotics for secondary bacterial infections, treatment of symptoms e.g. painkillers.
• Puccinia Graminis (Stem Rust Fungus)


  Transmission: wind carries spores
• Hygiene regimes


  • Washing hands with alcohol-based antibacterial gels
  • Wearing suitable clothing which minimises the transmission of resistant bacteria
• Other Pathogenic Agents and Problems of Controlling Endemic Diseases
• Virus: Influenza


  • Transmission: droplet infection, direct contact with virus-filled mucus, direct contact with animal waste (zoonotic infection), direct contact with infected surfaces
  • Mode of infection: infects ciliated epithelial cells of the lungs (antigen fits into receptor on host cell, injects viral RNA), viral RNA takes over cell biochemistry, cell produces new virus particles, cell lyses, many virus particles released
  • Pathogenic Effects: headache, sore throat, coughing, sneezing, muscular/joint pain, vomiting, fever etc. lasting about 5-7 days
  • Treatment/Control: antiviral medication, antibiotics for secondary bacterial infections, treatment of symptoms e.g. painkillers
• Fungus: Puccinia Graminis (Stem Rust Fungus)


  • Transmission: wind carries spores from infected plants, infected fragments left in soil from the two hosts – cereal crops and Berberis
  • Mode of Infection: spore germinates in water on plant, produces hyphae which enter the plant through the stomata, hyphae grow into mycelium, surround all tissues in the plant, produce enzymes e.g. cellulase to digest the plant and nutrients are absorbed into the fungus
  • Pathogenic Effects: nutrients lost to the fungus, weakened stem, water loss as the plant can't control transpiration (reduced photosynthesis), pustules on epidermis which eventually burst to release more spores
• Protozoan: Plasmodium spp. Malaria

  • Transmission: transmitted through the vector of the female Anopheles mosquito when she feeds to get protein to lay eggs
  • Mode of Infection: parasite transmitted via mosquito, travels to liver, infects red blood cells, reproduces asexually inside erythrocytes and causes lysis
  • Pathogenic Effects: Malaria bursts out of red blood cells every 2-3 days, causing paroxysm, sweating, shaking, muscle pains, headaches, liver damage, anaemia
  • Plasmodium spp reproduce sexually in mosquitoes and asexually in red blood cells in humans
  • Treatment/Control: mosquito nets (especially LLINS –50% more effective), insect repellent, pesticides, mosquito screens, more clothing, avoiding standing water, treating standing water with pesticides to remove mosquito larvae, proper disposal of sewage, introducing predators for mosquitoes. Quinine, chloroquinine and artemisinin are antimalarial drugs that work best in combination. Accurate diagnosis via observation with a microscope
• Controlling endemic diseases


  • The disease is often widespread
  • Difficult to remove all sources of infection
  • Expensive to provide treatment
• Preventing mosquito bites


  • Insect repellents, mosquito nets with insecticides, screens on doors and windows, clothing to cover skin
• Controlling mosquito numbers


  • Avoid standing water and sewage, water treatment to kill larvae, biological control by introducing predators
• Implications of control methods

  • Ethical
  • Social
  • Economic
• Ethical implications


  • Informed consent may be difficult
  • Spraying mosquitoes with insecticides will affect other organisms
  • Money spent on vaccines could instead be spent on education/preventing famine
• Social implications


  • Vaccines need to become accepted
  • Social changes to reduce infection are difficult to bring about
• Economic implications

  • Treatment, control and prevention of endemic diseases is very expensive
  • Malnutrition may be more of a threat to human life than malaria
• Physical barriers to infection

  • Skin is a tough physical barrier consisting of keratin
  • Stomach Acid (hydrochloric acid) which kills bacteria
  • Gut and skin flora – natural bacterial flora competes with pathogens for food and space
• Non-specific responses of the body to infection


  • Inflammation – histamines released by white blood cells cause vasodilation, which increases the flow of blood to the infected area and increases permeability of blood vessels. As a result, antibodies, white blood cells and plasma leak out into the infected tissue and destroy the pathogen
  • Fever – the hypothalamus sets the body temperature higher. This decreases speed of pathogen reproduction and increases rate of specific immune response
  • Lysozyme action – lysozyme is an enzyme found in secretions such as tears and mucus which kills bacterial cells by damaging their cell wall
  • Phagocytosis is a process in which white blood cells engulf pathogens and destroy them by fusing a pathogen such as bacteria enclosed in a phagocytic vesicle (phagosome) with a lysosome
• Specific immune response


  • Antigen specific and produces responses specific to one type of pathogen only. Relies on lymphocytes produced in the bone marrow
  • B cells mature in the bone marrow and are involved in the humoral immune response
  • T cells move from the bone marrow to the thymus gland where they mature, and are involved in the cell mediated immune response
• Cell-Mediated Response


  1. Pathogen invades a host cell

  2. The host cell displays the antigens on its Major Histocompatibility Complexes and becomes an Antigen-Presenting Cell

  3. T Killer cell with complementary receptor proteins binds to the APC

  4. Cytokines secreted by active T Helper cells stimulate the T Killer cell to divide by mitosis

  5. T Killer cell divides to form active T Killer cells and T Killer Memory cells

  6. Active T Killer cells bind to APCs and secrete chemicals which cause pores to form in the cell membrane

  7. The infected cell dies
• Humoral Response


  1. T Helper Activation: Bacterium is engulfed by a macrophage. Surface antigens are passed along the endoplasmic reticulum into a vesicle which are transported to the cell surface membrane

  2. Macrophage acts as an APC and presents antigens on MHCs

  3. Macrophage APC binds to T Helper cell with complementary receptor proteins

  4. The T Helper cell is 'activated' and divides by mitosis to form T memory cells and active T helper cells

  5. Effector Stage: Antigens from APCs that are complementary to the antibodies on B cells bind and are taken in by endocytosis

  6. The B cell acts as an APC and presents antigens on MHCs

  7. An activated T helper cell with a complementary receptor protein to the antigens binds to the APC. It produces cytokines

  8. Cytokines stimulate the B cell to divide by mitosis and form B memory cells and B effector cells

  9. B effector cells differentiate into plasma cells

  10. Plasma cells synthesise antibodies

  11. Effects of antibodies: Agglutination, Lysis, Opsonisation, Precipitation/Neutralisation

  12. T Suppressor cells stop the immune response
• Immunity


  • Active immunity results from the production of antibodies by the immune system in response to the presence of an antigen
  • Passive immunity results from the introduction of antibodies from another person or animal
  • Natural active immunity arises from being exposed to an antigen/getting the disease
  • Natural passive immunity is the result of crossing of mother's antibodies through the placenta and their presence in breast milk
  • Active artificial immunity is acquired through vaccinations which stimulate the immune system and lead to production of antibodies
  • Passive artificial immunity is where antibodies are injected into the body
• Herd Immunity


  • Enough people have been vaccinated to make transmission of a disease very unlikely. Requires 80-90% vaccination
  • Immunisation is the process of protecting people from infection with passive/active artificial immunity – vaccination is the process by which this is achieved through the use of attenuated antigens