Microbiology

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Study Guide for Exam 1
Chapter 1:

1. What is a “microbe”? What groups of organisms are included in this category?
Microbes (microorganisms) are minute living things that individually are too small to be seen with the unaided eye the group includes bacteria, fungi, protozoa, and microscopic algae, viruses
Only minority of microorganisms are pathogenic(disease-producing)

2. What are some of the benefits provided by microbes?
Decompose organic waste, producers in the ecosystem by photosynthesis, produce industrial chemicals such as ethanol and acetone, produce fermented goods such as cheese, vinegar, and bread, produce products used in manufacturing (ex. Cellulose) and treatment (ex. Insulin)

3. Review scientific nomenclature and rules for properly writing scientific names.
Linnaeus established the system of scientific nomenclature
Each organism has 2 names: the genus(first name; always capitalized) & the specific epithet(species name; not capitalized; follows the genus)
The organism is referred to by both the genus and the specific epithet, and both names are underlined or italicized
After a scientific name has been mentioned once, it can be abbreviated with the initial of the genus followed by the specific epithet
EX) Escherichia coli and Staphylococcus aureus are found in the human body. E. coli is found in the large intestine, and S. aureus is on skin
Scientific names can describe an organism, honor a researcher, or identify the habitat of a species
EX) Staphylococcus aureus - staphylo- describes the clustered arrangement of the cells; coccus indicates that they are shaped like spheres; aureus describes the golden-colored colonies
EX) Escherichia coli – honors the discoverer Theodor Escherich, specific epithet coli describes the bacterium’s habitat- the large intestine, or colon

4. Draw a flow chart that identifies the three domains used for classifying organisms, what groups of organisms are in each domain, and how these classifications relate to prokaryote/eukaryote designations.

Bacteria
Cell walls contain a protein-carbohydrate complex called peptidoglycan
Unicellular
Prokaryotes – no nucleus
Bacillus, coccus, and spiral
Divide by binary fission(divide into 2 equal cells)
Possess flagella
Use chemical substances for nutrition

Archea
Prokaryotic cells – lack peptidoglycan in cell walls
Live in extreme environments- include methanogens, extreme halophiles, and extreme thermophiles
Eukarya
Protists(slime molds, protozoa, and algae)
Protozoa - move by pseudopods, flagella, or cilia; obtain nutrients by absorption or ingestion through specialized structures
Algae- either unicellular or multicellular that obtain nutrients by photosynthesis; cellulose cell walls; produce molecular oxygen or organic compounds
Virus- noncellular entities that are parasites of cells; consist of either DNA or RNA surrounded by a protein coat; replicated only when they are in a living host cell
Fungi(unicellular yeasts, multicellular molds, mushrooms) - multicellular - eukaryotic cells – true nucleus - obtain nutrients by absorbing organic material from environment
Plants(mosses, ferns, conifers, and flowering plants)
Animals(sponges, insects, worms, and vertebrates) – parasitic flatworms and roundworms are called helminths

5. Be familiar with the names and accomplishments/claims-to-fame of the following historical scientists/physicians: Hooke, von Leeuwenhoek, Needham, Pasteur, Lister, Koch, Jenner, Erlich, Fleming, Lancefield, and Berg.

Hooke
Observed that cork was composed of “little boxes”
Introduced the term “cell”
Hooke’s observations laid the groundwork for development of the cell theory – all living things are composed of cells and come from preexisting cells
Von Leeuwenhoek
Using a simple microscope, made the first detailed study of microorganisms, which he called “animalcules” - first to observe microorganisms
Needham
Claimed that microorganisms could arise spontaneously from heated nutrient broth
Pasteur
Microorganisms are in the air everywhere and offered proof of biogenesis
His discoveries led to the development of aseptic techniques used in laboratory and medical procedures to prevent contamination by microorganisms
Found that yeast ferment sugars to alcohol and that bacteria can oxidize the alcohol to acetic acid
Disproved spontaneous generation - life did not arise spontaneously from nonliving matter
Lister
Introduced the use of a disinfectant to clean surgical wounds in order to control infections in humans
Koch
Proved that microorganisms cause disease
Used Koch’s postulates ,that are used today to prove that a particular microorganism causes a particular disease
Jenner
Demonstrated that inoculation with cowpox material provides humans with immunity to smallpox
Erlich
syphilis
Fleming
penicillin
Lancefield
Classified streptococci according to serotypes(varients within a species)
Berg
Genetic engineering – showed that fragments of human or animal DNA that code for important proteins can be attached to bacterial DNA
6. Explain the concept of spontaneous generation and how it was eventually shown to be wrong. In particular, review again Pasteur’s experiment in which this idea was finally abolished: what aspect(s) of the experiment was/were different from earlier experiments?
Spontaneous generation (abiogenesis)=some forms of life could arise spontaneously from nonliving matter

1. Francisco Redi demonstrated that maggots appear on decaying meat only when flies are able to lay eggs on the meat(1668)  against spontaneous generation
2. John Needham claimed that microorganisms could spontaneously arise from heated nutrient broth(1745)
3. Lazzaro Spallanzani repeated Needham’s experiments and suggested that Needham’s results were due to microorganisms in the air entering his broth
4. Rudolf Virchow introduced the concept of biogenesis: living cells can arise only from preexisting cells(1858)
5. Louis Pasteur demonstrated that microorganisms are in the air everywhere and offered proof of biogenesis(1861)
6. Pasteur’s discoveries led to the development of aseptic techniques used in laboratory and medical procedures to prevent contamination by microorganisms
Pasteur’s 1st experiment:
Pasteur demonstrated that microorganisms are present in the air and can contaminate sterile solutions, but that air itself does not create microbes
Pasteur filled several short-necked flasks with beef broth and then boiled their contents- some were then left open and allowed to cool – in a few days, there flasks were found to be contaminated with microbes- the other flasks, sealed after boiling, were free of microorganisms - Pasteur reasoned that microbes in the air were responsible for contaminating nonliving matter such as the broths in Needham’s flasks
Pasteur’s 2nd experiment
Pasteur heated the neck of the flask and bent it into an S-shaped curve, then boiled the broth for several minutes
Microorganisms did not appear in the cooled solution, even after long periods- bc the curved neck trapped any airborne microorganisms that might contaminate the broth
Conclusion
Abolished the idea of spontaneous generation
Microbial life can be destroyed by heat and that methods can be devised to block the access of airborne microorganisms to nutrient environments
Microorganisms cannot originate from mystical forces present in nonliving materials – rather, any “spontaneous” life in nonliving solutions can be due to microorganisms that were already present in the air or in the fluids themselves
Pasteur’s discoveries led to the development of aseptic techniques used in laboratory and medical procedures to prevent contamination by microorganisms

7. Why was the era between 1857 and 1914 called the “golden age of microbiology”? Be familiar with the most significant discoveries that occurred in this period of time.
Called “golden age of microbiology” bc the science of microbiology advanced rapidly btw 1857 – 1914
Discoveries during these years included both the agents of many diseases and the role of immunity in preventing and curing disease

8. What do the terms “fermentation” and “pasteurization” mean?
Fermentation – conversion of sugar to alcohol to make beer and wine
Microbial growth is responsible for spoilage of food
Bacteria use alcohol and produce acetic acid spoil wine by turning it to vinegar(acetic acid) – In the presence of air, bacteria change the alcohol in the beverage into vinegarb
Pasteurization –application of a high heat for a short time – used to kill bacteria in some alcoholic beverages and milk – Pasteur’s solution to the spoilage problem

9. What are the four steps of “Koch’s Postulates”? Why are they significant?
Pp 405
Microbiologists use these steps to identify the cause of emerging diseases
1) Some microbes are present in every case of the disease
2) These microbes can be isolated from the tissues of an infected individual and cultured
3) When these pure cultures are inoculated into a healthy animal, the same disease results
4) The disease is reproduced in a laboratory animal; microorganisms are identical microbes can be isolated and re-cultured
Proved that a specific microbe causes a specific disease

10. What is an antibiotic and how does it differ from other types of chemotherapy? What was the first antibiotic to be discovered? What were some of the first synthetic drugs to be used as antimicrobials?

Chemotherapy is the treatment of disease by using chemical substances
Antibiotics are the chemicals produced naturally by bacteria and fungi to act against other microorganisms
Synthetic drugs- chemotherapeutic agents prepared from chemicals in the laboratory
First synthetic drugs : 1) salvarsan(Paul Ehrlich) – arsenic-containing chemical against syphilis; 2) quinine-extract from the bark of a South American tree, used by Spanish conquistadors to treat malasia; 3) sulfonamides
First antibiotics: penicillin founded by Alexander Fleming; penicillin is an antibiotic produced by a fungus

11. What is an example of how microbes can be used in biotechnology?

Recombinant DNA technology – a new technique for biotechnology, enables bacteria and fungi to produce a variety of proteins including vaccines and enzymes

Gene therapy – inserting a missing gene or replacing a defective one in human cells – this technique uses a harmless virus to carry the missing or new gene into certain host cells,where the gene is picked up and inserted into the appropriate patients with ADA deficiency

12. What is meant by the term “normal microbiota” and what are some of the benefits we receive from them?

Normal microbiota(or flora) – microbes that normally present in and on the human body
Benefits of normal microbiota: prevent growth of pathogens, produce growth factors such as folic acid, vitamin K, and some B vitamins

13. Why are biofilms of particular concern in either industrial or medical applications?
Biofilm : a complex aggregation of microbes ; bacterial communities that form slimy layers on surfaces
Bacteria in biofilms are often resistant to antibiotics bc the biofilm offers a protective barriers
Biofilms can clog water pipes, and on medical implants such as joint prostheses and catheters
They can cause infections such as endocarditis(inflammation of the heart)

14. What are some of the problems with over-using antimicrobial products?

Overuse of these products can create environments in which antibiotic-resistant bacteria thrive
Random mutations in bacterial genes can make a bacterium resistant to an antibiotic

Chapter 3: (Not covered: Confocal Microscopy, Two-Photon Microscopy, and Scanning Acoustic Microscopy, pp. 62-63; Scanned-Probe Microscopy, pg. 65)

15. Review relationships of primary units used in microscopy (mm, um, nm). How big is a typical bacterium? How does that size compare to a typical eukaryotic cell? (Review Fig. 3.2)

1um (micrometer)=10^-6m=0.000001m=10^-3mm
1nm (nanometer)=10^-9m=0.000000001m=10^-6mm
1000nm=0.001m=10^-3m=1um
0.001um=0.000001m=1nm

16. How is total magnification of a compound light microscope calculated? What are the magnification limits of light and electron microscopes?

Total magnification = ocular lens magnification X objective lens magnification
The light microscope can magnify objects up to 1000x which allows us to view all bacteria and some large viruses
The electron microscope can magnify objects up to 1 million times which allows us to view all viruses and some large macromolecules

17. What does the term “resolution” mean? Why is oil used at higher magnification levels?

Resolution is the ability of the lenses to distinguish fine detail and structure- specifically, it is the ability of the lenses to distinguish two points a specified distance apart
We use oil to preserve the direction of light rays at the highest magnification – the immersion oil has the same refractive index as glass, so the oil becomes part of the optics of the glass of the microscope
The oil has the same effect as increasing the objective lens diameter and that it improves the resolving power of the lenses
If oil is not used with an oil immersion objective lens, the image becomes fuzzy, with poor resolution
Refraction in the compound microscope using an oil immersion objective lens – because the refractive indexes of the glass microscope slide and immersion oil are the same, the light rays do not refract when passing from one to the other when an oil immersion objective lens is used.

18. What are the terms “staining,” “smear,” and “fixing” a smear mean? What is a positive stain? What is a negative stain?

Staining means coloring the microorganisms with a dye that emphasizes certain structures
Fixing uses heat or alcohol to kill and attach microorganisms to a slide –also preserves various parts of microbes in their natural state with only minimal distortion
Smear is a thin film of material used for microscopic examination
Positive stain includes crystal violet, methylene blue,malachite green, and safranin –bacteria are negatively charged, and the colored positive ion of a basic dye will stain bacterial cells
Negative stain is produced when the colored negative ion of an acidic dye stains the background of a bacterial smear – procedure that results in colorless bacteria against a stained background – valuable for observing overall cell shapes, sizes, and capsules bc cells are made visible against a contrasting dark background – ex) eosin, acid fuchsin, and nigrosin

19. Know the steps of the Gram stain and what the results are of each step for Gram-positive and Gram-negative bacteria.
The Gram stain procedure uses a purple stain(crystal violet), iodine as a mordant, an alcohol decolorizer, and a red counterstain (ex. Safranin)
1)A heat-fixed smear is covered with a basic purple dye, usually crystal violet
2) After a short time, the purple dye is washed off, and the smear is covered with iodine, a mordant. When the iodine is washed off, both gram-positive and gram-neg. bacteria appear dark violet or purple
3) Next, the slide is washed with alcohol-acetone solution(decolorizing agent) – removes the purple from the cells of some species but not from others
4) The alcohol is rinsed off, and the slide is then stained with safranin, a basic red dye
Gram-positive bacteria retain the purple stain after the decolorization step
Gram-negative bacteria do not retain the purple stain after decolorization, and thus appear pink from the counterstain – bc gram-neg bacteria are colorless after the alcohol wash, they are no longer visible. This is why the basic dye safranin is applied and it turns the gram-neg bacteria pink

20. What types of special stains are there?
Special stains are used to color and isolate specific parts of microorganisms, such as endospores and flagella, and to reveal the presence of capsules
Negative staining is used to make microbial capsules visible
Endospore stain and flagella stain are special stains that color only certain parts of bacteria

21. Know the basics of the different types of microscopy we discussed: What type of microscopy is good for viewing live cells? What type provides 3-D images of specimens? Why would you use immunofluorescence?

Phase-Contrast microscopy good for viewing internal structures in living microorganisms
Based on diffraction of the light that passes through a specimen
The specimen is illuminated by light passing through an annular diaphragm. Direct light ray (unaltered by the specimen) travel a different path than light rays that are reflected or diffracted as they pass through the specimen. These two sets of rays are combined at the eye

Fluoresence microscopy
Takes advantage of fluorescence, the ability of substances to absorb short wavelengths of light(ultra-violet) and give off light at a longer wavelength(visible)
Dyes called fluorochromes absorb UV light and re-emit light at a lower(visible) wavelength – when microorganisms stained with a fluorochrome are examined under a fluorescence microscope with un ultraviolet light source, they appear as bright objects against a dark background
Immunofluoresence – combine fluorence with an antibody; antibody binds to specific component of cell
Principle of immunofluorescence: a type of fluorochrome is combined with antibodies against a specific type of bacterium. When the preparation is added to bacterial cells on a microscope slide, the antibodies attach to the bacterial cells, and the cells fluoresce when illuminated with ultraviolet light

Electron Microscopy
Examine objects smaller than about 0.2 um, such as viruses or internal structures of cells
Use a beam of electrons focused by magnetic lenses instead of light
Resolving power of the electron microscope is far greater than other microscopes, due to shorter wavelengths of electrons
Images produced by electron microscopes are always black and white
Instead of glass lenses, uses electromagnetic lenses to focus a beam of electrons onto a specimen
2 types : transmission electron microscope &scanning electron microscope
Disadvantage of killing the organism

Transmission Electron Microscope(TEM)
RP(resolving power) = 0.2 nm
Magnification = 1 million times
A finely focused beam of electrons from an electron gun passes through a ultrathin section of the specimen
Film or screen absorbs electrons to create a picture
Thin sections of organisms can be seen in an electron micrograph produced using a transmission electron microscope

Scanning Electron Microscope(SEM)
3 dimensional quality to image
Specimen is coated with a heavy metal to provide contrast
Electrons are defracted off the surface of the specimen
Electrons are collected by computer to make an image
Useful in studying the surface structures of intact cells and viruses

Chapter 4: (Not covered: Atypical Cell Walls, pp. 87-88; Damage to Cell Wall, pp. 88-89; Movement of Materials Across Membranes, pp. 91-94; Metachromatic Granules, p. 95; Carboxysomes and Gas Vacuoles, p. 96)

22. What are the major differences between prokaryotic and eukaryotic cells?
Prokaryotes
Eukaryotes
No DNA
No defined nucleus
No membrane-bound organelles
Cell walls contain polysaccharide peptidoglycan
Divide by binary fission(DNA is copied and the cell splits into 2 cells)
Respiration occurs in plasma membrane rather than mitochondria
Ribosomes are smaller

True nucleus-presence of DNA
Membrane-enclosed organelles, including mitochondria, endoplasmic reticulum, Golgi complex, lysosomes, and sometimes chloroplasts
Cell division involves mitosis- chromosomes replicate and identical set is distributed into each of 2 nuclei

23. What are the basic prokaryotic morphologies? In what aspect would you find the most prokaryotic diversity?

1) Coccus
Spherical
Diplococcus: two
Streptococcus: chain
Tetrad: four
Sarcina: eight
Staphylococcus: cluster

2) Bacillus: rod-shaped
Diplobacillus: two
Streptobacillus: chain
Coccobacillus: a more rounded bacillus

3) spiral vibrio: comma shaped – curved rods spirillum: rigid – helical shape, rigid bodies spirochete: flexible –use flagella to move

- Other shapes: square, star-shaped, triangular
- Pleomorphic: bacteria can have variable shape
- Most bacteria are monomorphic – maintain a single shape

24. What are the functions of bacterial flagella, pili, and fimbriae?

Bacterial flagella
- Long appendages that propel bacteria
- made of chains of flagellum
- attached to a protein hook
- anchored to the wall and membrane by the basal body
Pili
Appendages that are longer than flagella & number only 1 or 2 per cell
Mating (transfer of DNA)
Twitching motility

fimbriae
- Adherence: fimbriae help cells adhere to each other and to surfaces
-aggregation: form biofilms and aggregations on the surfaces of liquids, glass, and rocks

25. What is the glycocalyx? Explain how a capsule helps protect a bacterium.

Glycocalyx a sugar coat that surrounds cells viscous(sticky) slime layer – unorganized and loosely attached to cell wall capsule – organized and firmly attached to cell wall
Extracellular polysaccharide(EPS) allows cells to attach
Cell wall composed of polysaccharide and polypeptide
Capsule
- protect pathogens from phagocytosis
- adhere to surfaces, prevent desiccation, and may provide nutrients

26. What is meant by the term “fluid mosaic model” in describing a plasma membrane, and what are the plasma membrane’s primary functions?
Plasma membrane encloses the cytoplasm and is a lipid bilayer with peripheral and integral proteins (the fluid mosaic model)
The plasma membrane is selectively permeable
Plasma membranes contain enzymes for metabolic reactions, such as nutrient breakdown, energy production, and photogynthesis
Mesosomes, irregular folds of the plasma membrane, are artifacts, not true cell structures
Plasma membranes can be destroyed by alcohols and antibiotics, such as polymyxins

Fluid mosaic model
Dynamic arrangement of phospholipids and proteins comprising the plasma membrane a phospholipid layer with integral membrane proteins (peripheral membrane proteins on one or other face of the bilayer)
No cholesterol
Hopanoids are structurally similar to cholesterol and play a role in stabilizing the membrane

Functions of Plasma membrane
Serve as a selective barrier through which materials enter and exit the cell ( selective permeability)  certain molecules and ions pass through the membrane, but others are prevented from passing through it
Break down nutrients and produce energy  plasma membranes of bacteria contain enzymes capable of catalyzing the chemical reactions that break down nutrients and produce ATP

27. How is a cell wall different from a plasma membrane?
Plasma membrane
Inner membrane
Thin structure lying inside the cell wall and enclosing he cytoplasm of the cell
Consist of phospholipids and proteins
Prokaryotic plasma membranes are less rigid than eukaryotic membranes
Selective transport of materials into and out of the cell
Oxidative phosphorylation(respiration) – enzymes and chromatophores are integral membrane proteins

Cell wall
Protects the cell from osmotic lysis(destruction caused by rupture of the plasma membrane and theloss of cytoplasm)
Gives shape and rigidity to cell
Composed of peptidoglycan, which consists of a repeating disaccharide(NAG and NAM)
Alternating NAG and NAM molecules are linked to form a carbohydrate “backbone”
Adjacent rows are linked by polypeptides

28. What are the key differences in cell wall structure between Gram-positive and Gram-negative bacteria?

Gram-positive cell walls
Cell walls consist of many peptidoglycan
Contain teichoic acids(alcohol) and phosphate
Teichoic acids: lipoteichoic acid links to plasma membrane; wall teichoic acid links to peptidoglycan
May regulate movement of cations(positive ions) into and out of the cell
Polysaccharides provide antigenic variation
Role in cell growth – prevent wall breakdown and possible cell lysis

< The structure of peptidoglycan in gram-positive bacteria>
Together the carbohydrate backbone and tetrapeptide side chains make up peptidoglycan

Gram-negative cell walls
Cell walls consist of a thin layer of peptidoglycan
Outer membrane consists of lipopolysaccharides(LPS), lipoproteins, phospholipids
Do not contain teichoic acids
Since cell walls contain only a small amount of peptidoglycan, they are more susceptible to mechanical breakage
Forms the periplasm between the outer membrane and the plasma membrane

Gram-positive cell wall
Gram-negative cell wall
Thick peptidoglycan
Teichoic acids
Thin peptidoglycan
Outer membrane
Periplasmic space

How do these differences explain different reactions from Gram stain reagents?
- Crystal violet stains both gram-positive and gram-negative cells purple bc the dye enters the cytoplasm of both

- When iodine(mordant) is applied, it forms large crystals with the dye that are too large to escape through the cell wall
- the application of alcohol dehydrates the peptidoglycan of gram-positive cells to make it more impermeable to the crystal violet-iodine

Alcohol dissolves the outer membrane of gram-negative cells and even leaves small holes in the thin peptidoglycan layer through which crystal violet-iodine diffuse
Bc gram-negative bacteria are colorless after the alcohol wash, the addition of safranin(counterstain) turns the cells pink – safranin(pink) provides a contrasting color to the primary stain(crystal violet)

29. What types of different flagella arrangements are possible?

30. What are inclusion bodies? What is an example of a type of material contained within an inclusion body?
Inclusion bodies
Reserve deposits
Cells accumulate certain nutrients when they are plentiful and use them when the environment is deficient
Granules containing a variety of organic and inorganic compounds
Some are bounded by membranes while others are not
Organic bodies: glycogen – large carbohydrate used to store energy
Inorganic bodies: polyphosphate granules, sulfur granules
Polysaccharide granules – consist of glycogen and starch
Lipid inclusions – a common lipid-storage material is the polymer poly-B-hydroxybutyric acid
Sulfur granules – sulfur and sulfur-containing compounds
Magnetosomes –iron oxide(Fe^3O^4)

31. How is an endospore different from a reproductive spore?

Endospores are resting structures formed by some bacteria; they allow survival during adverse environmental conditions
Process of endospore formation is called sporulation; the return of an endospore to its vegetative state(breaking of spore’s dormant state) is called germination
Bc one vegetative cell forms a single endospore(which after germination remains one cell) sporulation in bacteria is not a means of reproduction- does not increase the number of cells (pp97)

32. What are the functions of the following eukaryotic cellular structures: ER, Golgi apparatus, mitochondrion?

ER (Endoplasmic Reticulum) extensive network of flattened membranous sacs called cisterns – The ER network is continuous with the nuclear envelope

Provides a surface for chemical reactions
Serves as a transporting network
Stores synthesized molecules
Protein synthesis and transport occur on rough ER- bc the outer surface of rough ER is studded with ribosomes
Lipid synthesis occurs on smooth ER – Smooth ER synthesizes fats and steroids

Golgi apparatus(Golgi Complex)
Consists of flattened sacs called cisterns
Membrane formation
Protein secretion

Mitochondrion
Spherical, rod-shaped organelles
Primary sites of ATP production
Multiply by binary fission
Contain 70S ribosomes and DNA
“powerhouses of the cell”
Contain machinery necessary to replicate, transcribe, and translate the information encoded by their DNA

33. Why was penicillin regarded as a “wonder drug”?

Penicillin was regarded as a “wonder drug” or “miracle drug” bc it doesn’t harm human cells – Eukaryotic cells do not contain peptidoglycan – antibiotics such as penicillin act against peptidoglycan and therefore do not affect human eukaryotic cells