M315 Final Flashcards
Describe the position of your hands when carrying the microscope to and from your laboratory bench.
Both hands should be used– one holding the bottom of the microscope and one holding the arm
Differentiate between the limit of resolution of the typical light microscope and that of the unaided human eye.
light microscope = 0.2 um
unaided human eye = 0.2 mm
What two adjustments can be made to the condenser?
height and diaphragm
What effect does adjusting the height of the condenser have?
When the condenser is raised, the amount of light on the slide is increased.
What effect does opening the diaphragm have?
When the diaphragm is open, the amount of light on the slide is increased.
Why are condenser adjustments preferred over the use of the light intensity control?
Condenser adjustments increase light without affecting the bulb light.
Why is it necessary to use oil in conjunction with the oil immersion lens and not with the other objectives?
The small working distance does not let enough let in alone and the oil is needed to direct more light into the lens. The oil has the same refractive index as glass.
What is the relationship between the working distance of an objective lens and its magnification power?
When the power of the objective lens increases, the working distance decreases.
Objective lens provides that provides the highest magnification
oil immersion
Objective lens that provides the second-highest magnification
high-dry (40x)
Objective lens that provides the lowest magnification
low power (5x)
This objective lens has the shortest working distance
oil immersion
The coarse focus knob should be adjusted only when using this objective lens
low power
This lens collects and focuses light from the lamp onto the specimen on the slide
condenser
This lens, also known as the eyepiece, often comes in pairs
ocular
Diopter adjustments can be made to this lens
ocular
A diaphragm is used to regulate light passing through this lens
condenser
TRUE or FALSE: Acetone is the safest solvent for cleaning an objective lens
FALSE– acetone is a powerful solvent that could possibly dissolve the lens mounting cement if used too liberally
TRUE or FALSE: only lint-free, optically safe tissue should be used to wipe off microscope lenses
TRUE
TRUE or FALSE: once focus in achieved at one magnification, a higher-power objective lens can be rotated into position without fear of striking the slide
TRUE
The resolving power of a microscope is a function of…
The magnifying power of the lenses and the numerical aperture of the lenses
The coarse and fine focus knobs adjust the distance between…
The stage and the objective lens
A microscope that maintains focus when the objective magnification is increased is called…
parfocal–hence why the microscope does not lose focus when increasing magnification
The total magnification achieved when using a 100 x oil immersion lens with 10 x binocular eyepieces is…
1000 x
The most useful adjustment for increasing image contrast in low-power magnification is…
closing down the diaphragm
Before the oil immersion lens is rotated into place, you should…
Center the object of interest in the preceding lens and place a drop of oil on the slide
In what ways do the macroscopic features of bacterial colonies differ from those of molds?
Bacteria colonies look smooth and small while mold is large and fuzzy.
Why is the level of contamination measured as number of colonies rather than size of colonies?
Colony size indicates growth rate. Number of colonies indicates number of cells present.
Should one be concerned to find bacteria on the skin? How about molds?
There are bacteria that normally grow on skin so that should not be a concern but mold can a sign of contamination from the environment.
How can microbial levels be controlled on the skin, surfaces in the environment, and in the air?
Skin: hand-washing
Surfaces: disinfectants, such as bleach
Air: filtration systems
Compare size of bacteria and eukaryotes.
Bacteria: .5-10 um
Eukaryotes: ~1000x bigger
Compare the organization of genetic material in bacteria and eukaryotes.
Bacteria: DNA is in the cytoplasm
Eukaryotes: DNA is in the nucleus
Compare the ribosomes of bacteria and eukaryotes.
Bacteria: 70S
Eukaryotes: 80S
Compare the cell wall of bacteria and eukaryotes.
Bacteria: cell wall made of peptidoglycan
Eukaryotes: cell wall made of chitin/cellulose
Compare the respiration and photosynthesis processes of bacteria and eukaryotes.
Bacteria: lack mitochondria and chloroplasts but can still carry out respiration and photosynthesis
Eukaryotes: respiration and photosynthesis occurs in mitochondria or chloroplast
Compare motility systems of bacteria and eukaryotes.
Both bacteria and eukaryotes have flagella for motility, but the bacterial flagella are simpler in structure and more numerous.
Staining of cells is often performed to enhance images acquired by brightfield microscopy. Phase-contrast microscopy does not require cell staining. Why is this advantageous?
Cell staining kills the cell. That does not happen in the phase-contrast microscopy so cell activity and movement can be monitored.
As light passes through a transparent object, how are direct and diffracted light rays produced? How much phase shift occurs?
Direct: produced when light passes through a transparent medium without a phase change
Diffracted: produced when light is bent with a phase shift of 1/4 the wavelength
How do coincidence and interference of light rays differ?
Coincidence: when direct and diffracted waves are brought into phase with each other which creates a brighter image
Interference: when two waves of the same amplitude are in reverse phase and cancel each other out, producing a dark image
Which two items can be used to check the alignment of the annulus and phase ring?
Centering telescope and optovar
A phase-contrast microscope differs from a brightfield microscope by having…
a diaphragm with an annular stop and a phase plate in the objective lens
What is the purpose of the annular stop?
To allow a hollow cone of light rays to pass through the condenser to the specimen.
Amplitude summation occurs in phase-contrast optics when both direct and diffracted rays are…
in phase
How is air contamination prevented when an inoculating loop is used to introduce or take a bacterial sample to/from an agar plate?
By holding the lid over the top of the plate when using the inoculating loop.
Where should a label be written on an agar plate?
On the bottom of the plate.
How should agar plates be incubated and why?
They should be incubated upside down in order to prevent moisture from condensing on the agar surface and spreading the inoculated organisms.
Against which organisms are disinfectants effective?
They are effective against vegetative cells and viruses.
Against which organism may disinfectants not be that effective?
Not very effective against bacterial endospores.
What disinfectants do we use in lab?
Ethanol
In regard to bacterial growth on solid media, define the term “colony.”
A single pure colony is the identical progeny derived from a single cell. One colony is equal to approximately one billion cells.
Why is dilution a necessary part of pure culture preparation?
Dilution is needed in order to isolate colonies.
What advantage(s) does the streak-plate method have over the pour-plate method?
Streak-plate method is more economical in materials and time.
What advantage(s) does the pour-plate method have over the streak-plate method?
Pour-plate method requires less skill.
Why is it better to make each of the four sets of streaks use approximately the same amount of the plate for the quadrant streak?
Isolated colonies may occur in any one of the four sets of streaks.
How many times should your loop be sterilized when using the quadrant streak isolation method?
6 times
Before inoculating and pouring molten nutrient agar into a plate, why must the agar first be cooled to 50 C?
To avoid condensation of moisture on the cover.
Describe the structure of a flagellum.
Composed of a filament, hook, and basal body
Lophotrichous flagella
Tuft of flagella at one end of the cell
Monotrichous flagella
One flagellum at one end of cell
Amphitrichous flagella
One flagellum at each end of cell
Peritrichous flagella
Flagella all over the cell
How do flagella generate cell motility?
There is a motor in the plasma membrane where it converts energy from the proton motive force.
What cell types do motility occur in?
Rod-shaped and spirochetes but almost never cocci.
Directional motility
Rapid swimming and directional change
Brownian movement
Jiggling motions without vectorial movement
Water current movement
Sweeping motion
Between wet mount and hanging drop slide preparations, which is more resistant to evaporation?
Hanging drop
Between wet mount and hanging drop slide preparations, which works best with phase-contrast microscopy?
Wet mount
What concentration of agar is used in a semisolid medium for motility determination and how does this compare to a typical solid medium?
Semisolid: 0.4%
Typical solid: 1.5
Why are semisolid media sometimes preferred over slide techniques for evaluating bacterial motility?
Exposure to pathogens is more likely with wet slides than semisolid and there are fewer false positives because there is no Brownian movement or water current.
Semisoft media is preferred over microscopic techniques for determining the motility of…
Pathogenic bacteria
Why is it important to limit quantity of cells used to prepare a smear?
Too many cells will result in large clumps, making it difficult to distinguish individual cells.
For preparation of a smear on a slide, what is the purpose of heat fixation?
Kills the cells and causes them to adhere to the glass so they do not get washed off during staining.
What problems can arise when the slide (during staining) is heated in a flame?
Overheating can damage the cells and cause them to distort in shape.
Why is the Gram stain considered a differential stain?
It differentiates two types of bacteria based on the composition of their cell walls.
How do gram-positive and gram-negative bacteria differ in cellular structure, and how does this contribute to their differential staining properties?
Gram-positive has a thick cell wall and retains the dye better from decolorizer than gram-negative which has a thin wall.
How does the age of a culture affect the Gram stain reaction?
Old cultures of Gram-positive may not retain the stain as well as younger cultures and could give false negative results.
What is an optimum age for a valid Gram reaction?
18-24 hours old
Which step in the Gram stain procedure is most prone to error?
The decolorizer step
What is the function of a mordant and which reagent serves this purpose in the Gram stain procedure?
A mordant combines with crystal violet and forms an insoluble complex in the Gram-positive cells. Iodine is used as the mordant.
List the reagents of the Gram stain technique in order and their general role in the staining process.
- Crystal violet: primary stain
- Grams iodine: mordant
- Alcohol: decolorizer
- Safranin: counterstain
In what type of cell would you find lipopolysaccharide in its cell wall?
Gram-negative
What is the role of oxygen for cellular respiration?
It is the final electron acceptor for the electron transport chain.
What type of metabolism occurs in the absence of oxygen?
Anaerobic
Obligate (strict) aerobes
Bacteria that needs to grow in oxygen because their metabolism requires oxygen. Carry out respiration in which oxygen is utilized as the terminal electron acceptor in the electron transport chain. (Psuedomonas, Micrococcus, Bacillus)
Microaerophiles
Aerobic bacteria that prefer to grow in oxygen concentrations of 2-10% rather than the 20% that is found in the atmosphere. Lower concentrations of oxygen is necessary for their respiratory metabolism. (Helicobacter pylori)
Facultative anaerobes
These bacteria grow very well aerobically but can also grow anaerobically if oxygen is not present. Their metabolism is flexible in that they undergo respiration if oxygen is present but they will undergo fermentation if oxygen is not present. (E. coli)
Aerotolerant anaerobes
These bacteria can tolerate oxygen and even grow in its presence, but they do not require oxygen for energy production, relying strictly on fermentation. (Streptococci that produce food products by fermentation, Enterococcus faecalis, Streptococcus pyogenes)
Obligate (strict) anaerobes
These bacteria cannot tolerate oxygen and must be cultured under conditions in which oxygen is completely eliminated, or they will be harmed or killed by its presence. Only found among the prokaryotes and some protozoa, in soil, rumen of cattle, and anaerobic sewage digesters. (Clostridium, Methanococcus, Bacteroides)
What does Brewer’s Anaerobic Agar contain?
Thioglycollate (a reducing agent) and Resazurin (an oxidation/reduction indicator)
Why is a GasPak anaerobic jar necessary for the culture of anaerobes on plates of Brewer’s Anaerobic Agar but not in tubes of FTM?
Because FTM is placed in a tube and can facilitate growth for both anaerobes and aerobes. Anaerobes will grow at the bottom of the tube and aerobes wil grow at the top, due to oxygen being present closer to the surface. Also contains a small amount of agar which helps to localize the organisms and favors anaerobiasis on the bottom of the tube.
FTM
Fluid Thioglycollate Medium
A rich liquid medium that supports the growth of both aerobic and anaerobic bacteria. Contains glucose, cystine, sodium thioglycollate and reazurin
TGYA shake
Solid medium that is used to prepare “shake tubes” used to determine the oxygen requirements of different bacteria. It is inoculated in the liquefied state, shaken to mix the organisms, and allowed to solidify. Oxygen requirements are determined based on where the growth occurs in the tube.
Psychrophiles
Bacteria that grow between -5C to 20C and are typically found in the supercooled waters of the Arctic and Antarctica.
Mesophiles
Bacteria that grow between 20C and 50C, which is what most bacteria grow in, including pathogens.
Thermophiles
Bacteria that grow between 50C to 80C.
Hyperthermophiles
Bacteria that grow in temperatures above 80C. Have been isolated from thermal vents deep within the ocean floor and from volcanic heated hot springs.
Differentiate between psychrophiles and psychrotrophs.
The difference is that psychrophiles have an optimum temperature range in which they specifically grow in cold temperatures, whereas psychotrophs grow outside of their optimum temperatures. For example, bacteria like Proteus are mesophiles but can grow at 4C.
Why are psychrotrophic bacteria of concern to those in the food-service industry?
They can grow at low temperatures, like refrigerator temperatures, but can also tolerate higher temperatures, so they can spoil food.
What is the optimum growth temperature for most human pathogens and why?
37C– this is the temperature of the human body.
How are enyzmes influenced by changes in temperature?
Each enzyme has its own optimum temperature range and if temperatures are above the maximum, enzymes begin to denature and lose activity. If they are below the minimum, chemical activity slows down and some denaturation occurs.
How is cellular transport influenced by changes in temperature?
As temperature decreases, transport of nutrients into the cell decreases due to fluidity changes in the membrane.
How are membrane lipids influenced by changes in temperature?
If the temperature increases above an organism’s maximum, membrane lipids can be destroyed, resulting in serious damage of the membrane and death of the organism.
How are ribosomes influenced by changes in temperature?
If extremes of temperature occur, ribosomes will cease to function adequately.
How does pH negatively affect the metabolism of microorganisms?
If pH values exceed the optimum, the solubility of charged molecules can be adversely affected and molecules can precipitate out of solution. pH can directly affect the charge on AAs in proteins and result in denaturation and loss of enzyme activity.
Neutrophiles
Bacteria that grow at or near neutral pH– most bacteria.
Acidophiles
Bacteria that grow at acidic pH.
Alkaliphiles
Bacteria that grow at alkaline pH.
How would the pH of a culture medium be influenced by sugar fermentation?
Fermentation of foods can yield acids such as lactic acid and acetic acids, which LOWER the pH of the food and prevent the growth of many microorganisms and the spoilage of food.
How would the pH of a culture medium be influence by urea hydrolysis?
Urea hydrolysis degrade urea to produce ammonia that INCREASES the pH.
Why are bacteria generally resistant to hypotonic environments, whereas animal cells are not?
Bacteria have cell walls, whereas animal cells do not. The rigid cell wall protects the membrane from being damage by the osmotic pressure exerted against it and prevents the membrane from disrupting when water diffuses across the cell membrane into the cytoplasm.
Hypotonic
A medium where solute concentrations on the outside of the cell are lower than the cytoplasm.
Isotonic
Environments where the solute concentration is the same inside and outside the cell. Animal cells require this.
Hypertonic
Environments where the solute concentration is greater on the outside of the cell relative to the cytoplasm and this causes water to diffuse out of the cytoplasm.
How do hypertonic environments negatively affect most bacterial cells?
PLASMOLYSIS: water will leave the cell, causing the membrane to pull away from the cell wall, disrupting the membrane.
Halophiles
Bacteria that require high concentrations of NaCl inorder to grow.
Halotolerant
Bacteria that are capable of growth in moderate concentrations of salt.
Osmophiles
Bacteria that are able to grow in environments where sugar concentrations are excessive.
Why are staphylococci well suited for the colonization of skin?
They are halotolerant and can tolerate the salt that builds on the skin.
Which type of bacteria would be more likely to cause spoilage of jams and jellies?
Osmophiles– they grow in environments where sugar concentrations are excessive.
When performing a standard plate count, why are the counts reported as colony forming units (CFUs)?
There is uncertainty in how many actual cells form a colony.
How would you inoculate a plate to get a 1:10 dilution?
For a 1:10 dilution, take 1 mL of the full strength solution, and dilute it by a factor of 9 and then plate it.
Why is it necessary to perform a plate count in conjunction with the turbidimetry procedure?
Turbidimetry gives a quick count of biomass but a plate count is more accurate regarding the number of cells that are actually alive.
Coliforms
Indicator bacteria that are found in the intestines of humans and warm-blooded animals and ferment lactose in order to produce acid and gas. The presence of these bacteria in water suggests the potential for disease.
Most Probable Number (MPN)
Determining the number of bacteria in a sample by the relationship of some growth parameter to statistical probability. A series of tubes with lactose is inoculated with water samples and the pattern of tubes showing acid and gas is compared to statistical tables that give the probable numbers of coliforms present. Gives a VIABLE count.
Microscopic Counts
A sample can be diluted and the cells in the sample can be counted with the aid of a microscope and a special slide with a grid on it. Milk samples are usually counted this way. Gives a TOTAL count.
Standard Plate Count (SPC)
A sample is diluted in a series of dilution blanks and aliquots of the dilution are plated onto media and the numbers of colonies are counted after incubation for 24-48 hours. The number of bacteria in the original smaple is determined by multiplying the number of colonies by the dilution factor. Gives a VIABLE count.
Turbidity Counting
As cells divide (grow), the culture will become more turbid, and the turbidity of the culture can be determined and related to growth. Can be measured in a spectrophotometer, which measures the absorbance or optical density of a culture. Gives a TOTAL count.
Which type of food has the highest bacterial count?
Ground meat due to it being fresh and not frozen.
Which type of food has the lowest bacterial count?
Dried fruit due to a lack of water that is available to the bacteria.
Why might one expect to find coliforms in samples of meat?
Fecal matter from the animal’s intestines contaminates meat during butchering.
High coliform counts in food indicate the potential for finding which intestinal pathogens?
Salmonella, shigella, vibrio cholera, intestinal viruses.
In terms of food safety, why is it suggested to cook hamburgers medium-well to well-done, whereas steaks can be cooked rare?
Pathogens invade hamburger during grinding and processing– and the inside of steak is not processed.
What considerations should be made to safely thaw frozen foods for later consumption?
When thawing frozen food, it should either be placed in a refrigerator overnight or in the microwave. It should not be thawed over long periods of time at room temperature or in warm water.
Why is refrigeration not always an effective means for preventing food spoilage?
Some microbes are able to grow and reproduce at refrigerator temperatures.
Why are dried fruits somewhat resistant to spoilage?
Water content is not high enough to support most types of microbial growth. Also, they typically have a high sugar content that will suppress growth to an extent.
Is the presence of microorganisms in food always an indication that food is spoiled or that it has the potential to cause disease?
No, some foods can have high counts because microorganims are used in their production, such as in the cases of yogurt, sauerkraut, and summer sausage. Processes like pasteurization or smoking can greatly reduce the number of bacteria, though.
Thermoduric bacteria
While pasteurization kills the majority of bacteria in milk, some bacteria can still remain and this could cause the spoilage of milk. These are called thermoduric.
Saprophytes
Organisms that live in decaying plant and animal material and are harmless.
Presumptive test
15 tubes of lactose broth are inoculated with measured amounts of water to see if the water contains any lactose-fermenting bacteria that produce gas. If gas is seen in any of the broths, it is presumed that coliforms are present in the water sample. Also used to determine the MPN of coliforms present per 100 mL of water.
Does a positive presumptive test mean that water is absolutely unsafe to drink?
No– the bacteria could be harmless saprophytes or some other type of bacteria that does not cause disease in humans
What might cause a false positive presumptive test?
There could be two or more non-coliform bacteria that are working together to produce sufficient gas in the tube.
Characteristics of a good sewage indicator
Easy to identify, present only in sewage, lives slightly longer than pathogens in water
The fermentation of what disaccharide is the basis for determining the presence of coliforms?
Lactose
Why don’t health departments routinely test for pathogens instead of using a sewage indicator?
It would be tedious and difficult to specifically test for each of the pathogens. Also, they are often fastidious and they might be overgrown by other bacteria in the water if we tried to culture and test for them. It is much easier to demonstrate the presence of some indicator bacterium (E. coli)
Describe the appearance of coliforms on Levine EMB agar.
Nucleated colonies with a dark center that may have a metallic green sheen.
Confirmed Test
Plates of Levine EMB agar or Endo agar are inoculated from gas-producing tubes to see if the organisms that are producing the gas are gram-negative. Both media inhibit gram-positive growth and cause colonies of coliforms to be distinguishable from non-coliforms.
Describe the appearance of coliforms on Endo agar.
Reddish colonies.
What does the presence of coliform-like colonies confirm?
The presence of a lactose-fermenting, gram-negative bacterium.
Characteristics of coliform bacteria.
Gram-negative, facultative anaerobes, non-endospore forming, lactose-fermenting, acid & gas producing
Completed Test
This test is used to determine if the isolate from the agar plates actually matches the definition of coliform, using a nutrient agar slant and a Durham tube of lactose broth. If gas is produced in the lactose tube and the slide reveals that we have a gram-negative, non-spore-forming rod, we know we have a coliform.
For which types of specimens is darkfield microscopy preferred over brightfield?
Delicate transparent living organisms.
What advantage does a cardioid condenser have over a star diaphragm?
You have to use high-dry or oil immersion with the star diaphragm and the oblique rays are not as carefully metered as is necessary for the higher magnifications.
What are chromophores?
Color-bearing ionic groups
Why do acidic dyes not stain bacterial cells?
They have anionic chromophores that are electrostatically repelled by the negatively charged bacterial cell.
Crystal violet is an example of what type of stain?
Simple dye
Rod-shapedRod-shaped
Rods or bacilli can have rounded, fat, or tapered ends.
Fusiform bacteria
Rods with tapered ends that are prevalent in the human mouth.
Cocci-shaped
Round balls that may occur singly, in chains, in tetrads, or in irregular masses.
Spiral-shaped
Can exist as spirochaetes, as a spirillum, or as a comma-shaped, curved rod.
Pleomorphism
Pertaining to irregularity of form; demonstrating several different shapes.
Metachromatic granuless
Distinct reddish-purple granules, that are actually masses of volutin, within cells that show up when the organisms are stained with methylene blue.
Palisade arrangement
“Picket fence” arrangement– parallel arrangement of rod-shaped cells.
What are negative stains useful for?
characterizing
Chromophores of negative stains
Because negative stains are acidic, they have negatively charged chromophores that do not penetrate the cell but rather are repelled by the similarly charged bacterial cell.
Examples of negative stain
India ink, nigrosin
Negative stain procedure
Mixing the organism with a small amount of stain and spreading a very thin film over the surface of a microscope slide. Cells are not usually heat-fixed prior to the application. Can also be mixed with positive staining for better demonstration of structures, like capsules.
Why is avoiding heat fixation in negative staining important?
If the capsule surrounding the cell is to be observed, heat fixation will severely shrink the structure, making it very difficult to see.
Capsule
The extracellular gel-like layer occurring outside the cell wall that is distinct and gelatinous.
Slime layer
The extracellular gel-like layer occurring outside the cell wall that is diffuse and irregular.
Glycocalyx
If a capsule or slime layer is made of polysaccharides, it is known as this.
Functions of the capsule/slime layer
Protect the cell from being engulfed and destroyed by pathogens and also help the cell attach to solid surfaces in the environment.
Endospores
When species of bacteria of Bacillus or Clostridia exhaust nutrients, they can produce endospores that will allow these bacteria to survive environmental conditions that are not favorable for growth.
Characteristics of endospores
Very dehydrated structures that are not actively metabolizing and are resistant to heat, radiation, acids, and many chemicals, such as disinfectants, that normally harm or kill vegetative cells.
Exosporium
The protein coat that forms a protective barrier around an endospore.
How is an endospore heat resistant?
The higher the water content in an endospore, the less heat resistant it will be. During sporulation, the water content of the endospore is reduced to 10-30% of that of a vegetative cell.
What is the mordant in endospore staining?
Heat
What makes Mycobacterium resistant to staining?
It has mycolic acid in its cell wall, which is a waxy lipid in the cell wall.
Which bacterial genuses are acid-fast?
Mycobacteria and Nocardia
What is the primary stain in the acid-fast stain?
Carbol fuchsin
What is the mordant in the Ziehl-Neelsen acid-fast stain?
Heat
What is the color of Mycobacterium after the addition of the secondary stain?
Pink
Which stain would be most useful in staining the leprosy bacillus?
Acid-fast
What is the advantage of the Kinyoun staining procedure over the Ziehl-Neelsen method?
It does not require heat so phenol fumes are not generated.
Complex medium
The exact composition and amounts of the individual amino acids, vitamins, growth factors, and other components that make up the medium are not exactly known.
Defined medium
The specific chemical composition is known and the individual components are weighed out exactly.
Heterotrophs
Obtain their carbons from organic sources.
Autotrophs
Obtain their carbons from fixing carbon dioxide.
Chemoorganotrophs
Derive their energy needs from the breakdown of organic molecules by fermentation or respiration.
Chemolithotrophs
Oxidize inorganic ions to obtain energy to fix carbon dioxide.
Photoautotrophs
Contain photosynthetic pigments that convert solar energy into chemical energy by the process of photosynthesis.
Photoheterotrophs
Derive their energy needs from photosynthesis, but their carbon needs come from growth on organic molecules.
Nutritional requirements of bacteria
Carbon, energy, nitrogen, minerals, vitamins and growth factors, water
Selective medium
Medium in which components will allow the growth of some bacteria but inhibit the growth of others.
Differential medium
Contains substances that cause some bacteria to take on an appearance that distinguishes them from other bacteria.
Agar
A complex polysaccharide that is isolated from seaweed and is added in a concentration of 1.5% to solidify liquid media.
Color of gram-positive cell stain
Purple (due to retaining crystal violet)
Color of gram-negative cell stain
sPink (due to losing crystal violet and retaining safranin)
How do viruses differ from bacteria in terms of the microscope?
Viruses are much smaller than bacteria so they are below the resolution of the light microscope. The smallest virus is one million times smaller than a typical euk. cell.
Viruses
Obligate intracellular parasites that require a host cell in order to replicate and reproduce, and hence they cannot be grown on lab media, but we can detect their presence by the effects that they have on their host cells.
Composition of viruses
Composed of RNA or DNA (never both) and a protein coat, or caspid, that surrounds the nucleic acid.
Why are viruses dependent on cells?
Due to their lack of metabolic machinery necessary for the synthesis of viral components. By invading a host cell, they can utilize the metabolic systems of the host cells to achieve their replication.
Viruses that infect bacterial cells
Bacteriophages–diverse in morphology and size; some even just having ss DNA
Composition of phages
Head, sheath, and tail fibers
What is the caspid made of?
May be round, oval, or polyhedral and is composed of individual protein subunits called caspomeres.
What does the caspid do?
Forms a protective covering around the viral genome.
What does the tail structure or sheath of a phage do?
Composed of a contractile protein that surrounds a hollow core, which is a conduit for the delivery of viral nucleic acid into the host cell.
What do the tail fibers of a phage do?
Bind to chemical groups on the surface of the bacterial cell and are responsible for recognition. Lysozymes associated with this part will erode and weaken the cell wall of the host cell, facilitating the injection of the viral nucleic acid by the sheath contracting and forcing the hollow core through the weakened area in the cell wall.
Two outcomes of viral infection
The lytic cycle and the lysogenic cycle
Lytic cycle
Involves virulent phages that cause lysis and death of the host cell. The virus assumes control of the cell metabolism and uses the cell’s metabolic machinery to manufacture phage components. Mature phage particles are assembled and released from the cell where they can invade new host cells. Almost always results in death of host cell.
Lysogenic cycle
Involves temperate phages, which can either lyse the host or integrate their DNA into host cell DNA and alter the genetics of the host cell. The viral DNA of the temperate virus is integrated into host DNA and no mature phages are made. Cells grow normally and are immune to further infections by the same phage. No visible evidence to even indicate that a virus is in the cell. In some cases, the virus can carry genes that confer new genetic capabilities on the virally infected cell.
Lysogen
Virally infected cell in the lysogenic cycle
What happens when Corynebacterium diptheria is infected with a lysogenic phage?
The phage carries a toxin gene in its genome so the host cells begin to produce a potent toxin responsible for the symptoms of diptheria.
Lysogenic conversion
When a virus carries genes that confer new genetic capabilities on the virally infected cell. Responsible for some of the toxins produced by various pathogens.
Coliphages
Bacteriophages that infect E. coli, such as T-even phages and lambda phage. Readily isolated from raw sewage and coprophagous insects (like flies).
Phases of lytic cycle
- Absorption: Phage is absorbed to specific receptor site on the bacterial cell surface.
- Eclipse Stage: Phage DNA enters cell and bacterial DNA is degraded in mins.
- Phage caspids, tails, and DNA begin to appear in cell as phage reorients cell metabolism to its own fabrication processes.
- Components of phage are assembled into mature infective virions.
- Cell wall opens up due to enzymatic action to release mature virions
Burst size
Number of mature virions released by the cell during the last stage of the lytic cycle.
What does it mean that bacteriophages are obligate intracellular parasites?
They must invade a host cell in order to replicate and reproduce. This is because they are made of only a single kind of nucleic acid.
Virus specificity
Viruses exhibit specificity for their host. A certain bacteriophage may only infect a specific strain of a bacterium. Resides in differences in receptors.
Virion
A single virus or phage particle
Examples of virus receptors
Pili and lipopolysaccharide in gram negative; teichoic acids in gram positive.
Productive infection
Results from lytic cycle; new phages are made and released from the cell.
How do the parts of phage components assemble?
Once all the parts have been synthesized in the host cell, they will come together by the process of self-assembly to form mature phage particles
Prophage/pro-virus
Integrated phage genome in lysogens
Why aren’t mature phages typically produced in the lysogenic cycle?
The phage genome can integrate into host DNA to become a stable genetic element in the host cell genome. The genes for the synthesis of viral components are not usually expressed, so mature phages are not produced in the cells. But every time the cell’s DNA is replicated, so is the phage’s DNA.
When can mature phages be made in the the lysogenic cycle>
Occasionally, the viral DNA in a lysogenic infection can excise from the host DNA, which results in the expression of viral genes responsible for the synthesis of phage components. Will then be made by the processes of the lytic cycle.
One phage particle infecting a single host cell can produce as many as…
infecting a single host cell can produce as many as…
…200 virions
Number is called burst size
Confluent lawn
When bacterial cells are mixed with bacteriophage in soft agar, this is what the bacteria will first grow.
Plaques
Clear areas in the confluent lawn that is from phages infecting cells and causing them to undergo lysis. Each plaque is formed by the progeny of a single virion that has replicated and lysed the cells.
Plaque-forming units
Like CFUs, can be counted to determine the number of viral particles in a suspension of phage.
Three groups of gram-negative rods
The enterics, the nonfermenters, and the coccobacillary
Enterics
Gram negative, nonsporeforming, facultativce anaerobic rods that ferment glucose with the production of acid and gas and can grow in intestinal tracts of warm-blooded animals. Largest group of gram negative rods.
Enteric genera
Escherichia, salmonella, shigella, citrobacter, klebsiella, enterobacter, serratia, and proteus.
Escherichia
Number one cause of UTIs in women. Can cause septicemia, an infection of the blood stream. Can cause spinal meningitis in newborns. Present in abdominal and wound infections.
Only pathogenic organisms
Salmonella, shigella, and vibrio; all others can cause an infection if enter body at some point other than intestinal tract.
Salmonella
Causes irritation of mucous membranes, resulting in typhoid fever, gastroenteritis (food poisoning), and osteomyelitis, which is an infection in the bone.
Shigella
Grows and multiples in the large intestine, producing toxins that can result in bacillary dysentery, which is diarrhea containing white blood cells or pus.
Citrobacter
Abdominal and wound infections.
Klebsiella
Can cause severe enteritis in children, as well as pneumonia and upper respiratory infections. Also cause nosocomial (hospital acquired) UTIs.
Enterobacter
Abdominal and wound infections.
Serratia
Thought to be innocuous, but now implicated in pulmonary infections and septicemia.
Proteus
The number one cause of UTIs in males; abdominal and wound infections.
Oxidase test–enterics
Enterics are oxidase negative
Vibrio
Oxidase positive, so not enterics.
MacConkey agar
Selective medium since specimen is almost always polymicrobial. Selective due to bile salts and CV, differential due to pH indicator, neutral red. Tests for lactose fermentation and indicates organism if gram negative rod.
TSI
Contains 3 sugars, with 10 x more lactose and sucrose than glucose, sodium thiosulfate, which detects organism that can produce H2S, and phenol red. Red indicates alkaline and yellow indicates acid.
Urease
Several enterics (not pathogenic ones) can split urea to ammonia in presence of urease. Ammonia cause the pH of the medium to rise above 8.4 where phenol red, the pH indicator, is a deeper red.
Indole
Organisms that possess the enzyme tryptophanase can cleave tryptophan to indole, whose presence can be detected by Kovac’s Reagant, which produces a cherry red color. (E. Coli)
Citrate
Some enterics can use citrate as sole source of carbon and energy for growth. Contains ammonium phosphate, which will release ammonium ions and increase pH. pH indicator, Brom Thymol Blue, changes from green to blue above 7.6. Part of IMViC. (Kleb)
Non-fermenters
Pseudomonas, alcaligenes, acinetobacter, and moraxella. Truly opportunistic pathogens, but patient has to be debilitated for them to be pathogenic. Environmental organisms.
Pseudomonas
Can be cultured form every water faucet, drinking fountain, or water line in any hospital; extremely resistant to antibiotics. Causes UTIs, septicemia, upper and lower respiratory infections, and colonizes wound and burn infections.
Acinetobacter
Isolated less frequently than psuedomonas but can cause the same type of infections. Not as resistant to antibiotics.
Psuedomonas aeruginosa
Most common nonfermenter. If grown on light-colored media will produce a diffusible, water soluble, green pigment called pyocyanin. Produces grape-like odor. Oxidase positive.
Oxidase test
Tests for presence of cytochrome C. Cytochromes are part of the ETC so all organisms that can grow in presence of air have cytochromes.
MR-VP medium
A glucose broth that is buffered with peptone and dipotassium phosphate.
If an organism is Gram positive rods, what should the next test be?
Catalase: if positive, test for sporeforming. If negative, test for anaerobic or facultative sporeforming.
If an organism is Gram positive coccus, what should the next test be?
Catalase: if negative, strep. If positive, staph.
Sporeforming test
Sporeforming: bacillus
Nonsporeforming: Corynebacterium
If an organism is Gram negative coccus, what should the next test be?
Oxidase: if positive, neisseria.
If an organism is Gram negative rods, what should the next test be?
Facultative/aerobic oxidase: if postive, psuedomonas. If negative, do lactose fermenting test.
If an organism is facultative Gram negative rods, which next should be done?
Lactose: if positive, do the IMViC test. If negative, do the Urease test.
IMViC test results
++–: Escherichia
–++: Enterobacter
Urease test results
Positive: proteus
Negative: serratia
The chalky appearance on the outside of grapes is due to..
Yeast
Who is considered the father of medical microbiology?
Robert Koch
What does the catalase test do?
Degrades hydrogen peroxide into oxygen and water
What did Robert Koch use to find something to grow and culture bacteria on?
Pieces of potatoes, meat broths, pieces of carrots, meat broth plus gelatin.
Who identified contaminated water as a “causative agent” of disease during the 1840 cholera outbreak in London?
John Snow
How does the motility test media differ from a plate media?
Less agar content
Serratia marcescens produces a red pigment (prodigosin) at…
Room temperature
What is best suited for sterilization of a liquid, heat-labile preparation of antibiotics?
A filter
What compound did we use to test the effect of osmotic pressure on bacterial growth?
NaCl
Yogurt, sauerkraut, and summer sausage have high bacterial counts during production because…
They are made by microbial fermentation
What is the major difference between Gram positive and Gram negative bacteria?
Gram negative bacteria have an outer membrane above a thin peptidoglycan layer.
