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PHAR2812 L2: Handling and visualizing microorganisms -

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what is microbiology important o microorganisms cause infectious diseases, and are humanity’s oldest enemies o Today, microbes still cause much suffering and death o Vast majority of microbes are not pathogens  Play essential roles:  Are part of our normal flora and fight against pathogenic microbes  Microbes have antibiotics  Weakened strains of microbes can be used vaccines Microbial nutrition o They are made of same biochemical as plants and animals and therefore need the same elements o Only need a little bit of trace elements because it can be poisonous

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media used for growing microbes o liquid media: broth  contains nutrients required for growth  shaken to mix cells, nutrients, oxygen  homogenous: all cells in same physiological state  useful for growing large numbers of microbes  how it done: example  use a flask that is sterile  need a cotton wool stopper: which allows entry of air but not microbes  all the bacteria grown in a broth culture is homogenous or the cells are in the same physiological state o solid media: agar plates  the agar has the same ingredients as the broth  allows for single colonies  for growing colonies of microbes theory: a single colony arises from a single cell  heretergenous: cells in different part of a colony are in different physiological states  useful for growing small number of cells o complex media  ingredients derived from organisms, exact composition unknown eg yeast extract in lysed yeast cells  can grow a large number of microbes o defined media  ingredients a pure compounds for example sugar (sucrose) or inorganic slats  downside is that they don't support the growth of wide range of microbes however defined media you can predict the growth/compostion general purpose, selective and differential media o general purpose  b allows growth of many microbial types eg nutrient broth  good for starting off when you want to just grow tham and then isolate them o selective  favors growth of one microbial type eg media containing antibiotics  this is done by adding or removing ingredients o differential  different microbial types give different colour/visual reactions eg ph indicator  for example distinguishing between whether the microbe can break down lactose, the ones that can break down lactose will generate acids  turns the agar yellow  can differentiate salmonella from ecoli (one pathogen the other isn’t) pure vs mixed cultures o natural microbial communities are mixed cultures  complex, different interacting species o pure cultures contain only a single species  this is unnatural but useful for research  they are useful because it allows us to relate microbial identity and function  limitatiolns

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 many microbial types cannot be isolated in oure culture  pure cultures are not representative of microbial diversity  pure cultures behave differently to microbial mixtuures  microbial interactions can only be tested in mixtures aspectic technique: the core skill of microbiology o prevents contamination of cultures, worker, environment o use of sterile equipment  inoculating loop, media, pipettes, flasks o clean work environment  disinfected surfaces, biosafety hood, Bunsen flame o careful handling  be aware of ubiquity of microbes  avoid contact with hands, dust, surfaces and non-sterile items isolating of pure cultures: streak plate o seperates cell by streaking sample on agar plate, diluting the mixture until single cells are obtained o single cells grow into a single colony of one cell type o using a liquid broth culture you can isolated colonies by diluting broth

microscopy: magnification and resolution o microbial populations (colonies on agar) are visible to the nakend eye, but study of individual cells need microscopy o need to magnify the object and resolve fine details o total magnification=mag (objective) x mag (ocular) o say you use 40x objective lense then you times with the ocular lense which is 10x so your toal magnification is 400x o resolution  is the smallest distance between two points that can be seen as separate

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o 10x: used for finding cells on slide before switching to higher magnification. Resoljution is=1.1um o 40x: for examination of large micfrobial cells: fungi, algae, protists. Resolution 0.4um o 100x: for examination of smaller microbial cells: bacteria. The resolution is=0.2um fixing and staining of microbial samples o samoles are first fixed to the slide by heat or chemicals: this kills and imbolises cells to visulation o cells then stained to give contrast (most bacterial cells are transparent and invisible unless stained ) o most biological stains are basic (positive charged)and these bind to the acidic group (negative change) in the proteins, DNA, cell surface gram stains o most widely used stain for bacteria: differentiates them into gram negative (pink) or gram positive(purple) o gram positive wall is thicker than gram negative; reaminsl crystal violet dye after an alcholhol rince. Gram negative counterstained with safarnin o separation into gram negative and positive biologically meaningful correlates with natural evolutionary grouping of bacterial

phase contrast microscopy o allows visualization of live, unfixed, unstained samples  Determined of motility  can see natural morphology  can see some internal structures without staining o how does it work  phase rings in microscope difference in refractive index into difference in contrast electron microscopy: SEM and TEM o Resolution of the light microscope is limited by the wavelength of light – eg. viruses (~100 nm) are smaller than visible light wavelengths (~400-700 nm)

PHAR2812 o Solution: use electrons (wavelength ~0.1 nm) instead of light toview samples à electron microscope. o Scanning electron microscope (SEM) bounces electrons off sample surface; transmission electron microscope (TEM) sends electrons through a thin section. o Sample preparation for EM is intensive (disadvantage): fix, dehydrate, coat with heavy metal atoms to increase contrast.

L3 Major Infectious Diseases 10/3/17 -

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Major infectious disease: HIV/AIDS, Malaria, TB and influenza o The first 3 disease were named the big 3, responsible for 6 million deaths in 2004 o Influenza; because eof its potential for really big pandemic o Each have a major on human life over a significant part of the world Human Immunodeficiency Virus (HIV) o Genome: RNA virus (retrovirus) o Important enzyme  reverse transcriptase (RNA-dependent RNA polymerase) o Target: human’s immune cells  CD4 cells are also called T cells; a type of white blood cells that a play a major role in protecting body from infection o Transmission  Virus is present in blood, saliva. Semen and vaginal secretions o It's a retrovirus, which are single stranded RNA viruses o The virus particle has membrane envelope HIV VIRUS replication-1 o First step when HIV gets into the body/blood system is to bind to the CD4 cells o The virus particle has a protein in its virus membrane which binds to a receptor protein called CD$ on the host membrane (T cell) o Virus binds to the CD4 cells, a set of cells in the immune system. This biding is followed by fusion of the 2 membrane o The synethisis of a double strand DNA is as follows:  A reverse transcriptase using the single strand RNA molecules are as template for the first DNA strand  Using that DNA strand as template to make double strand DNA  The double stranded DNA is then transferred to the nucleus and incorporated into the host genome.  This integrated HIV genome is then part of the host cell genome for as long as that cell or its descendants live  Once in the nucleus the DNA is used for making new RN, that can be used for either making new virus particles or as mRN for synthesis of viral proteins. Incorporation into the host genome is essential for viral replication -

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origins of HIV o The virus HIV occurs in 2 forms, HIV-1 and HIV-2, both related to SIV, Simian Immunodeficiency Virus, found in other primates. o SIV seems not to have symptoms in its original monkey hosts. o The first indications of HIV came in the USA in 1981, when clustered cases of rare diseases were observed in homosexual men. There were also cases of Kaposi’s sarcoma, a generally very rare cancer. This was shown to be due to a deficiency in immunity. o The distribution of the diseases suggested that this immunodeficiency might be infectious, and when found in intravenous drug users it became the best hypothesis, and is now well established. Relationship of HIV and SIV o The African origin was confirmed by discovery of SIV in African primates, and it is now generally accepted that SIV crossed the species barrier at least twice, probably from Chimpanzee to give HIV-1, and perhaps from the Sooty Mangabey, to give HIV-2, as that has the closest relative of HIV-2. o The most popular estimates are about 1930 for HIV-1, and 1940 for HIV-2. o HIV/AIDS appeared from the start to be a new disease in central Africa that was discovered independently, it was suspected that it had an African origin. However while the viruses were the same, propagation in Africa was independent of homosexuality

PHAR2812 and intravenous drug use, and seemed to be related to heterosexuality and generally spread by female prostitutes. -

AIDS o The immunodeficiency that is characteristic of AIDS is because virus infection reduces the number of CD4+ cells. o Completely shuts down the immune system so easily can get sick o Presumably this doesn’t happen to any great extent in natural hosts, as SIV for example does not cause disease in its normal chimpanzee or monkey host. o The infections and cancers that first alerted the world to HIV are the major cause of death from AIDS. Many pathogens are commonly found in AIDS patients, but rarely found other than in immunocompromised people; and many non-pathogens also invade. - Current knowledge of HIV o HIV infection requires virus to get into the bloodstream. Spread, unprotected sex, sharing needles for injection and by blood transfusion is easily understood. o It is now detectable by DNA or antibody techniques and has been essentially eliminated from blood transfusion, at least in the more developed world. o The virus is present in most secretions including saliva and semen, but can be spread by any body fluids. Symptoms usually appear years after infection and this facilitates spread of HIV.I o It has proved very difficult to contain the virus, particularly in countries that do not like to publicise use of condoms, or do not have facilities for diagnosis during the long latent period. Malaria o Caused by a single cell parasite o Plasmodium o It is a single-cell eukarypte- a protest o Mosquito-borne infectious disease o The mosquito bite introduces the parasites from the mosquito’s saliva into a person’s blood o It is curable with appropriate drugs and has been driven from many areas by a combination of killing mosquitoes and such drugs. But in many tropical contries it is still gaining ground o Caused about 700 000 in 2010 compared to 1 million in 2000 - Several plasmodium species o There are several species of plasmodium that can infect humans. The life cycle is similar but the severity and frequency of bouts of fever can vary o Four well-recongised species infect humans they have much in common  P.vivax  P.malarie  P.ovale  P.flaciparium: this causes the most severe disease, but some of the other are more persistent - Plasmodium life style

PHAR2812 o The plasmodium vivz life style involves replication in the mosquito and human host and it has to alternate the hosts to complete its life cycle o In the human it infects both liver and blood cells in sequential steps o Infection is often persistent but fever only occurs intermittently o It appears that there is a complex interactiton between parasite and the immune system as the plasmodium is at very low levels between bouts of fever o Injected into the human blood by the salivary glands of the mosqutoesInfect the liver infects blood cells explodes the blood cells gets released  really bad feedback (really bad fevers symptom) - Who fact sheet o Nearly half of the worlds population is at risk of malaria o Increased preventon and control measures have led to a reduction in malaria death rates (death per million in at risk areas) by 65% globally since 2000 - Control of malaria immunity and vaccines o Malaria is unusual in that people can be infected many times o The immune response is complex and this has hindered vaccine development, despite major attempts around the world. It is difficult to control - Summary of malaria as a major disease o Plasmodium is a very successful parasite, but does not survive where treatment is readily available o Its continuing success is the great difficult is breaking the cycle. In countries that are functional economically and have stable government, it is a realistic option o Its current success is due to its ability to re-infect after a cure and the development of drug resistance by plasmodium and insecticide resistance by mosquitoes, making local elimination expensive o It is realistic to expect an effective vaccine to arrive, with several undergoing field trials o A much more encouraging prognosis than for HIV/AIDS Tuberculosis - Background o bacterial disease caused by Mycobacterium tuberculosis o thick lipid-rich cell wall  chronic bacterial infection (life-long infection is some cases) causing high disease morbidity and mortality world-wide o is an airborne disease usually transmitted only after prolonged exposure to someone with active-disease o known to have prevent 3000 years ago, 1.8 million death in 2015 o TB usually infects the lungs but other organs can be involved in later stages of infection - Mycobacterium tuberculosis o It is a complex relatively impermeable cell wall and its very slow growing o Also very persistent, but it can be cured and has been eliminated in many countries, but it can be difficult to detect in early infections and it has a latent stage there will always be a few cases brought in by travelers or immigrant o Tubercle  An infected area is often surrounded by host cells and the bacteria ceases growing but survives in these tubercles-they can be detected by X-ray

PHAR2812 o Usually transmitted only after prolonged exposure to someone with active disease o It is very persistent, and in most people becomes latent- in tubercules o Many cases of sickness are due to reactivation - Epidemiology o In 2015, 10.4 million people around the world fell ill with TB and 1.8 million deaths worldwide o TB is a leading killer of people who are HIV infected (35% of HIV deaths were due to TB) o But death rate fell 47% between 1990 and 2015 o Reemergence of TV is due o drug resistance and the soft target of immunodeficient people with HIV and AIDs o Sprad of AIDS has been a significant factor as AIDs aufferers are very susceptible to TB o Drug treatment for TB may be prolonged, gibing good opportunity for drug resistant forms to appear Influenza - background o viral disease o genome: ssRNA (single strand RNA) o flue virus sprads around the world in the winter seasons of the 2 hemisphere o there are several different forms of which group A is the most important for humans. The group A virus has 8 short segments of ssRNA (single strand RNA), equivalent in some ways to chromosomes but much more smaller, and mostly each codes for only one protein o the influenza virus enters a host cell, where its nucleic acid genome (RNA in this case, its replicated) - influenza proteins o antibody against haemagglutinin (HA) and Neuramidindase (NA) is protective. This generates strong selection for change to escape the immunity o the result is that new mutations accumulate each year and over time lead to enormous variation in the sequence of the genes o the human hosts respond with new antibodies . this is called antigenic drift and even afte4r 1 year the changes allow significant reinfection o occasion ally there is a re-assorment of the segmets and HA or NA segment from a related influenza virus replaces the previous one, giving a much greater antigenic change- called angtigenic shift o there are several HA and NA forms recognized; major pandemic strain was H1N1 in 1918 and in the last 30 years its H2N2 L4 – Prokaryote Structure: General Features Microbes:  Treatment of microbial infections vary, depending on the microbe responsible for the symptoms  I.e. differentiating between a virus, bacterium or protest  The context in which you come into contact with a microbe will affect the outcome  An important issue with treating microbial infections is to selectively target microbes

PHAR2812 o A broad spectrum of eradication may remove good microbes Infectious disease and Context  There is often a competition between internal and external microbes  Thus, when classifying microbes, we must ensure the system is applicable at all scales 

On the global scale, external factors will have a direct influence on microbial population o In the global environment, microbes are the predominant organisms present o Essential biological processes are exclusive to microbes o Mass – bacteria and archaea are the largest component of the biosphere o Number of individuals – viruses vastly outnumber any other microorganism o Biochemical diversity – bacterial viruses are the most genetically diverse

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Regional scale: o Also dominated by microbes o Microbes are often used to decompose organic matter to aid in waste disposal o Microbes determine soil and water health

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Personal scale: o There is a spectrum of microbial influence  Pathogens ---> Parasites ---> Commensals ---> Co-operators ---> Mutualists  The majority of microbes which come into contact with humans do not lead to pathogenesis (i.e. commensals)  Many microbes can provide beneficial effects (i.e. co-operators and mutualists)

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Gut microbiota o Microbes aren’t essential, but we aren’t “normal” without them o Lifestyle diseases (cancer, heart disease, diabetes, obesity) are due to microbial dysbiosis:  i.e. our lifestyle is affecting our internal microbial population

Properties of Microbes: (LEARNING OUTCOME)  Microbes are microscopic biological entities that can autonomously replicate or can subvert another organism to their own replication (e.g. viruses and intracellular parasites)  Small size = few resources required to autonomously replicate  Small size = high surface area to volume ratio o Very efficient at competing for resources in their local environment  Much faster and more efficient replication than macro-organisms

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Uncontrolled growth of microbes can result in death of the organism

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Replicative properties of microbes: o Prions, plasmids and viruses are acellular (not consisting of cells) and retain SOME properties of living organisms. o They ARE NOT capable of autonomously replicating, but they DO HAVE autocatalytic properties, whereby they can self-propagate IF found in other cells o Prions (e.g. mad cow) are just protein o Plasmids are typically dsDNA molecules which replicate separately from the chromosome o Viruses are combinations of packaged nucleic acid and proteins which can infect other cells and replicate themselves

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Cellular microorganisms o Cells are surrounded by an envelope which includes at least one lipid membrane o They are energised and dynamic (constantly changing via chemical processes and producing metabolites) o In prokaryotes, there is coupled transcription and translation o In eukaryotes, the nucleus is membrane-bound, which separates transcription and translation o This distinction has allowed for the classification of microbes:

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