Microbiology and pathogens

Step 1: Decide which organisms you want to culture, and obtain a culture of them.

Step 2: Provide the right nutrients for the microorganism to grow-require a good source of carbon and nitrogen. The nutrient medium can be in the form of:                                          

• Nutrient broth-nutrients in liquid form
• Nutrient agar-nutrients in solid form, sets at 50C but doesnt melt until 90C   
• Selective medium-contains a very specific mixture of nutrients, so only a particular type of microorganism will grow in it.    

Step 3: Introduce your microorganism, eg. by inoculating:

• Sterilise the inoculating loop by holding it in a Bunsen burner.
• Dip the sterilised loop into bacteria and streak the loop across the surface of the agar.
• Replace the petri dish lid, tape closed and turn upside down (make sure some air can still get in otherwise only anaerobic bacteria may survive).

General rules:

• Close windows and doors to reduce draughts
• Make transfers over a disinfected surface-ethanol disinfection
• All work must be done close to a Bunsen burner where air currents are drawn upwards
• On opening a test tube or bottle, the neck must be immediatelt warmed by flaming so that any movement of air is outwards from the vessel
• When using a petri dish, limit exposure of the sterile inner surfaces to contamination from the air.
• All items which come into contact with microorganisms must be sterilised before and after each such exposure
• Hold bottles at an angle to minimise the amount of airborne microbes that can fall into them
• Keep petri dish lids on to reduce contamination
• All agar plates are incubated upside down to reduce bacterial contamination and to reduce the possibility of water condensation that may be on the lid dripping onto the agar

Cell counts: The sample of nutrient broth is diluted and trypan blue dye is added to stain the dead cells blue, so that the living cells can be identified and counted using a microscope. A haemocytometer is also used and this contains a grid which enables you to calculate the number of microorganisms in a standard volume of broth.

Optical methods: As the number of bacterial cells in a culture increase it becomes more cloudy looking or turbid. A calorimeter can measure how much light passes through the sample and a calibration curve can be produced. The turbidity is measured and a cell count using a haemocytometer is made so that the number of microorganisms can be calculated.

Dilution plating: Used to find the total viable cell count. The original culture is diluted in stages until a point is reached when the colonies can be counted. The number of colonies can then be multiplied by the dilution factor and a total viable cell count for the original sample determined.

Area and mass of fungi: The diameter of the patches of mycellium can be measured and used to compare growth rates in different conditions. 

Bacterial growth graph:

• Lag phase-bacteria are adapting to new environment and not yet reproducing at maximum rate
• Log phase-rate of bacterial reproduction is doubling in a given time period and is close to or at its theoretical maximum
• Stationary phase-rate of reproduction is equal to rate of cell deaths
• Death phase-reproduction has almost stopped and death rate of cells is increasing. This may be because of a reduction in the amount of nutrients available or a build-up of waste products.

Endotoxins

• Endotoxins are lipopolysaccharides that are an integral part of the outer layer of the cell wall of Gram-negative bacteria. The lipid part of the polysaccharide acts as the toxin, while the polysaccharide stimulates an immune response.
  
• They have effects around the site of infection by the bacteria.
• The pathogenic effects of bacterial endotoxins tend to be symptoms such as fever, vomiting and diarrhoea.
• Antibiotic treatments that destroy the bacterial cells by lysis of the cell wall can also lead to further endotoxins release due to the lipopolysaccharide component of the cell wall.

Case study: Salmonella spp.

• The bacteria invade the lining of the intestine and the endotoxins cause inflammation. The cells no longer absorb water, so the faeces becomes liquid. The gut then goes into spasms of peristalsis that result in diarrhoea.
• Salmonella is spread by ingestion of food and water contaminated with infected faeces. Salmonella bacteria live in the gut of many animals and can easily survive if the meat is not cooked properly.

Exotoxins

• Exotoxins are usually soluble proteins that are produced and released into the body by bacteria as they metabolise and reproduce in the cells of their host.
• They are produced by both Gram-negative and positive bacteria and their effects can be more widespread than that of endotoxins. 
• There are many different types with specific efects. Some damage cell membranes causing cell breakdown or internal bleeding, some act as competitive inhibitors to neurotransmitters, while others directly poison cells.

Case study: Staphylococcus spp.

• Staphylococcus bacteria only cause disease if they get inside the tissues of the body, if the normal skin flora is changed, or if the person has a compromised immune system. 
• Staphylococcus spp. are Gram-positive bacteria and they produce exotoxins that can cause anything from mild skin diseases to rapid death.

Host tissue invasion

Case study: Myobacterium tuberculosis

• TB is most commonly caused by Myobacterium tuberculosis, spready by droplet infection.
• TB often affects the respiratory system, damaging and destroying lung tissure. It also suppresses the immune system, making the body less able to fight the disease. 
• Once the bacteria are inhaled, they invade the cells of the lungs and multiply slowly. This primary infection often causes no symptoms.
• If you have a healthy immune system there will be a localised inflammatory response forming a mass of tissue called a tubercle, containing dead bacteria and macrophages. In about 8 weeks the immune system controls the bacteria, the inflammation dies down and the lung tissue heals.
• Myobacterium tuberculosis, however, has an adaptation that enables it to avoid the immune system, allowing some bacteria to survive the primary infection stage. The bacteria produce a thick waxy layer that protects them from the enzymes of the macrophages.
• These bacteria remain deep in the tubercles in the lungs and only cause active tuberculosis when the person is malnourished, weakened or their immune system does not work well. Once they become active again they can grow and reproduce very rapidly.

Treating bacterial diseases

• All modern antimicrobial drugs work against microorganisms by the principle of selective toxicity. They interfere with the metabolism or function of the pathogen, with minimal damage to the cells of the host.
• When an antiobiotic is taken, it may have one of two different effects:                                             

1. It may be bacteriostatic, which means that the antiobiotic used completely inhibits the growth of the microorganism. Bacteriostatic antimicrobial actions include: antimetabolites, which interrupt metabolic pathways, and protein synthesis inhibitors which prevent or interrupt transcription or translation.

 2. It may be bactericidal, which means it will destroy alomst all of the pathogens present. Bactericidal antimicrobial actions include: cells wall agents, that prevent the formation of cross-linking in cell walls, so bacteria are killed by lysis and cell membrane agents, that damage the cell membrane.

Viral diseases 

• Viral infections are often specific to a particular tissue. This specificity is the result of the presence or absence of antigenic markers in the surface of host cells. 
• Each type of cell has its own markers, and each type of virus can only bind to particular antigens on the host cells that have a shape which is complementary to the proteins the virus uses to attach to the cell.
• The presence of absence of these antigens can affect the vulnerability of whole groups of living organisms to attack by viruses.

Influenza

Transmission:

• There are many modes of transmission of the flu largely linked to droplet infection. As well as this, it can be transmitted by direct contact with animal droppings or with virus-filled mucous from the nose.

Modes of infection:

• The flu virus infects ciliated epithelial cells of the respiratory system. The viral RNA reaches the nucleus of the host cell and takes over the biochemistry, producing new virus particles.
• Eventually the cell lyses, relasing more viruses and dying in the process. 
• The death of the ciliated epithelial cells of the trachea and bronchi leaves the airways open to infection, so many people die from secondary bacterial infections.

Treatment and control

• The flu virus is an RNA virus, which makes it more prone to mutation. This makes producing a vaccine difficult as a new one has to be produced each year. 

Puccinia graminis-stem rust fungus

Transmission:

• Puccinia graminis is a parasitic fungus that feeds on the living tissues of its host. It has two different hosts-cereal plants such as wheat and barley, and Berberis, a genus of shrubs.
• The disease is transmitted when spores from the infected plant are carried to young crop plants by the wind. 

Mode of infection:

• When spores land on the host plant they need water to germinate. Then a thread-like hypha emerges from the spore and penetrates one of the stomata of the leaves of stem.
• The hypha secretes enzymes, such as cellulases, which digest the plant cells and the nutrients are then absorbed into the fungus.
• The hyphae branch to form a mycelium that feeds and grows, hidden in the stem or leaves.
• The stem rust fungus grows best when it has hot days, mild nights and wet leaves from either rain, dew or irrigation. The spore needs this water to germinate.

Puccinia graminis-stem rust fungus

Pathogenic effect:

• The parasitic fungus affects the crop in several ways:                                                                    

1. It absorbs nutrients from the plant, reducing the yield of grain  

2. The pustules break the epidermis, making it more difficult to control transpiration.

3. The mycelium grows into the vascular tissue, absorbing water and nutrients.

4. It weakens the stems so the plants are more likely to fall over in heavy winds and rain.

Controlling stem rust in wheat:

There are a number of ways to control this disease:                                                                  

1. Bigger spaces between plants to increase distance for spore to travel                                              

2. Reduce use of fertilisers (high nitrate levels favour the fungus)                                                       

The malaria parasite

Transmission:

• Plasmodium spp, the malaria parasite, feeds on the living tissues of its host. The parasite is transmitted to a human host by a mosquito vector when it takes a blood meal.

Mode of infection:

• When the malaria parasite enters the blood of the human host it travels to the liver. It remains in the liver for a time, before releasing the next stage of its life cycle into the blood.
• At this point the parasite invades the red blood cells and reproduces asexually. At regular intervals they burst out of the rbc, destroying them and moving on to infect more rbc.
• The malarial parasite is then transmitted to mosquito taking a blood meal where they go thorugh more life cycle stages before being transmitted to another person.

Treatment:

• The parasite spends most of its time hidden from the immune system in the body cells and the antigens on the surface change frequently. This makes a vaccine difficult to develop.

Cell recognition

• Antigens are molecules (usually proteins) found on the surface of cells. When a pathogen invades the body, the antigens on its surface are recognised as foreign, which activates cells in the immune system/

Inflammation

• Mast cells are found in connective tissue below the skin and around blood vessles. When this tissue is damaged, these mast cells and basophils release chemicals called histamines.
• Histamines cause the blood vessles in the area to dilate, causing local heat and redness. This raised temp reduces the effectiveness of pathogen reproduction in the area.
• The histamines also make the walls of the capillaries leaky which results in fluid, containing leucocytes and antibodies, to be forced out of capillaries causing swelling.
• The antibodies disable the pathogens and the macrophages and neutrophils destory them by phagocytosis.

Fevers

• When a pathogen infects the body it causes the hypothalamus to reset to a higher body temperature. This helps the body combat infection in two ways:                                              

1. Many pathogens reproduce most quickly at 37°C or lower. Therefore, a raised temperature will reduce the ability of many pathogens to reproduce effectively.                                                      

2. The specific immune response works better at higher temperatures.                                         

• In a bacterial infection the temperature rises steadily and remains fairly high until treatment is successful or the body overcomes the infection.
• In a viral infection the temperature tends to 'spike', shooting up high everytime viruses burst out of the cells and then dropping down towards normal again. 
• If your body temperature rises above 40°C, the denaturation of some enzymes takes place and you may suffer permanent tissue damage. 

Phagocytosis

• There are two main types of phagocytes:                                                                                     

1. Neutrophils are granulocytes and each neutrophil can only ingest a few pathogens before it dies. They also cannot renew their lysosomes so once the enzymes are used up, the cell cannot break down any more pathogens.

2. Macrophages have an enormous capacity for ingesting pathogens because they can renew their lysosomes so they last much longer. 

• A phagocyte recognises the antigens on a pathogen and the cytoplasm of the phagocyte moves around the pathogen, engulfing it.
• The pathogen is now contained in a phagocytic vacuole called a phagosome, which then fuses with a lysosome. The enzymes in the lysosome break down the pathogen.
• The phagocyte then presents the pathogens antigens to activate other immune system cells, it is now called an antigen-presenting cell (APC).
• The phagocyte also produces chemicals called cytokines that are effective cell signalling molecules that stimulate other phagocytes to move to the site of infection. 
• Opsonins bind to pathogens making them more easily recognised by phagocytes. 

DIfferent kinds of lymphocytes

B cells

• B cells are produced in the bone marrow and once matured are found in the lymph glands and free in the body.
• B cells have membrane-bound globular receptor proteins on their cell surface membrane that are identical to the antibodies they will later produce. 
• When a B cell binds to an antigen, the following types of cells are produced:

1. B effector cells-these cells divide to form plasma cell clones

2. Plasma cells-these produce antibodies to particular antigens 

3. B memory cells-these provide the immunological memory to a specific antigen, allowing the body to resond very rapidly if you encounter a pathogen carrying the same antigen again.

T cells

• T cells are produced in the bone marrow but mature and become active in the thymus gland.
• The surface of each t cell displays thousand of identical T-cell receptors.
• T-cell receptors bind to antigens on infected body cells and then several further types of T-cells are produced and play different roles in the immune repsons:

1. T killer cells-these produce chemicals to destroy infected body cells

2. T helper cells-these activate plasma cells to produce antibodies against the antigen on a particular pathogen and also secrete opsonins to 'label' the pathogen for phagocytosis by other white blood cells.

3. T memory cells-these are very long loved cells that make up the immunological memory. When they meet a pathogen for the second time, they divide rapidly. This forms a large clone of T killer cells which then quickly destroy the pathogen.

• The workings of many of these cells depend on special proteins known as major histocompatibility complex (MHC) proteins that display antigens on their surface membrane.

The humoral response

• The humoral response reacts to antigens found outside the body cells including antigens on pathogens and antigen-producing body cells.
• The repsonse results in production of antobodies which are not attached to cells but are carried around the body in the tissue fluid and blood.

T helper activation:

• When a macrophage engulfs a pathogen, it separated off its antigens and combines them with the major histocompatibility complex. The macrophage with these antigen/MHC protein complexes now displayed on the cell surface is known as an antigen-presenting cell (APC).
• T cells have receptors on their outer membrane that bind to the specific antigen of the antigen/MHC complex on the APC.
• This binding with the APC triggers the T cell to reproduce and form a clone of cells.
• Most of these cloned cells become active T helper cells, which are then used in the rest of the immune system.
• The remainder of the cloned cells form inactive T memory cells, which remain in the body and rapidly become active if the same antigen is encountered again.

The humoral response

The effector stage 1:

• Some B cells will have immunoglobulins on their surface that are specific for the antigen presented by the pathogen and will bind to it.
• The B cell then engulfs the whole pathogen by endocytosis. Enzymes from a lysosome break down the pathogen to leave fragments of processed antigen. These fragments become attached to MHC proteins within the cell and the MHC/antigen complex is transported to the cell surfce membrane of the B cell where the antigen is displayed.
• A T helper cell from the active clone produced in the T helper activation stage recognises the specific antigen displayed on the MHC complex on the B cell and binds to it. This triggers the release of cytokines from the T helper cell which stimulate the B cells to divide and form clones of identical cells-clonal selection. 
• New clones of B effector and B memory cells are produced. The B effector cells differentiate to form plasma cells.

The humoral response

T helper activation diagram:

The humoral response

The effector stage 2:

• Plasma cells can produced up to 2000 antibody molcules per second. They have extensive endoplasmic reticulum and many ribosomes, allowing them to produce such large quantities.
• The plasma cells produce large amounts of antibodies that are identical to the immunoglobulins of the original parent B cell. An antibody will bind to a specific antigen on the particular pathogen that has triggered the immune response, causing its destruction in one of several ways:

1. Agglutination: When antibodies bind to the antigens on pathogens, the microorganisms agglutinate or clump together. This helps to prevent them spreading through the body and makes it easier for them to be engulfed by phagocytes.

2. Opponisation: The antobody acts as an opsonin, a chemical which makes an antigen or pathogen more easily recognised by phagocytes.

3. Neutralisation: Antibodies neutralise the effects of bacterial toxins by binding to them.

The humoral response

The effector stage diagram:

The cell-mediated response

• This response happens when the pathogen is inside the host cell.
• When a body cell is infected by a bacterium or virus, the pathogen is digested and the surface antigens become bound to an MHC. This results in an APC however, the body cell is still infected by the pathogen.
• T killer cells bind to the matching antigen/MHC complex on the surface of the body cell. If the T cells are then exposed to cytokines from an active T helper cell, produced thorugh T helper cell activation, they undergo a rapid series of cell divisions to form a clone of identical T killer cells which can all bind to the infected body cells.
• The T killer cells release enzymes that make pores form in the membrane of the infected cells. This allows the free entry of water and ions, so the cells swell and burst.
• Any pathogens that are released intact are labelled with antibodies produced by the plasma cells, and then destroyed. 
• T killer memory cells are also produced.

Primary and secondary responses

• The primary immune response involves the production of antibodies by the plasma cells produced from the B effector cells and the activation of T killer cells, and is extremely effective. 
• However, it can take days or even weeks for the primary immune response to become fully active against a particular pathogen. This is why we get symptoms of disease-we feel ill when pathogens are rapidly reproducing inside our bodies.
• We also have a secondary immune response which is quicker, greater and longer lasting. When the B-cell APC divides, it also produces B memory cells. 
• When you encounter the disease again, the B memory cells help you produce the antibodies against it so rapidly that it is destroyed before the symptoms of the disease develop.

Different types of immunity

• Natural active immunity: This is when you become immune after catching a disease and your body actively makes antibodies to destroy the pathogen.
• Natural passive immunity: This is when a baby becomes immune due to the antibodies it receives from its mother, through the placenta and in breast milk. It is passive because your body doesn't make the antibodies and is quite short-lived as the antibodies arn't replaced.
• Artificial active immunity: This is when you become immune after you've been given a vaccine containing a harmless does of antigen.
• Artificial passive immunity: This is when antibodies formed in one individual are extracted and injected into another individual. This is also quite short-lived as the antibodies are gradually broken down and not replaced.
• Herd immunity: This occurs when a significant proportion of the population is vaccinated against a disease.