Microbiology test 1
Quiz yourself by thinking what should be in
each of the black spaces below before clicking
on it to display the answer.
Help!
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| Communicable disease | when an infected host can transmit the infectious agent to another host and establish infection in that host
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| contagious. | Highly communicable diseases
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| Non-communicable infectious diseases | do not arise through transmission from host to host.;occurs primarily when a compromised person is invaded by his or her own normal microflora;Can occur through direct contact with organism in natural, non-living reservoir
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| how many of nosocomial infections could be prevented. | More than 1/3rd; 2 to 4 million cases/year in U.S. with approximately 90,000 deaths
Most commonly involve urinary tract, respiratory tract, and surgical incisions
Most common organisms involved: Gram-negative intestinal flora;E. coli, Pseudomonas, Staph
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| Hand sanitizer | is a non-water-based hand hygiene agent.
Active ingredient is usually isopropanol or ethanol
Needs to contain at least 60% alcohol to be effective
Added benefit of viral inactivation
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| Antibacterial soap | most Triclosan-based (antibacterial compound) based:
Many Triclosan resistant bacteria can be found in the environment
Triclosan has no effect upon viruses
Antimicrobial effects are concentration dependent
High – biocide – multiple cytoplasmic and mem
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| emulsification. | allows water to remove normally-insoluble matter
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| Soaps Anionic surfactants | have both a hydrophilic end, which dissolves in water, as well as a hydrophobic end, which is able to dissolve nonpolar molecules like grease; emulsification
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| Joseph Lister | father of antisepsis; cleaned using carbolic acid solution; stiches; sterile instruments could be left in patients; got idea from Pasteur,
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| Ignaz Semmelweis | handwashing in Vienna General in 1846; puerperal fever in babies;
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| Normal microbiotia or normal flora | More or less permanent residents of your body
Actual numbers and types may vary over time
Different people will have different distributions
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| Transient microbiota or transient flora | Present only for days or weeks in/on the body
Some of these are pathogens; i.e. bacteria that could colonize and cause an infection – resulting in disease
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| Oceans | 1 billion of microbial cells per liter of sea water
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| Soil | There are more microbes in a gram of soil than there are humans alive today.
There are 10,000 different species found in one gram of soil
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| Humans | There are 100 trillion (1014) microbial cells in the gut. Ten times the number of human cells in an adult body
Over 1,000 different species; prevalence and type vary from individual to individual
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| Robert Hooke | reported that living things were composed of little boxes or cells
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| Antonie van Leeuwenhoek | construction of microscopes which could magnify up to 30 - 100X
Described microorganisms that he observed in teeth scrapings & rain water
“animalcules”
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| Germ Theory of disease | The theory that proposes that microorganisms are the cause of many diseases.; Pasteur and Koch
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| Louis Pasteur | Disproved theory of spontaneous generation
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| Robert Koch | Developed postulates that validated the germ theory of disease
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| Spallanzani – 1700s | Experiment effectively disproved spontaneous generation
Previous experiments performed in this manner were refuted in that “the boiling kills the life force”
How did Spallanzani disprove this?
Critics then argued “air” was required for spontaneous gen
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| Why was Koch successful? | Developed techniques for culturing microorganisms
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| Agar | Not degradable by most microorganisms
Liquefies at 100°C
Solidifies at temps under 40°C
Once solidified, can be incubated at temps up to 100°C
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| Streak plate | Most common
Determines the “what” is in the culture
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| Serial dilution and plating | Determines the “how much” is in a culture
Used in conjunction with spread plating
Expressed in terms of cfu/ml
Colony forming units per milliliter of culture
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| Pipetman | Dual stop: should go down to 1st stop to take up sample and should go to 2nd stop to expel sample
P-1000
100 – 1000 µl sample transfer
P – 200
20-200 µl sample transfer
P- 20
2-20 µL sample transfer
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| cfu/ml | Colony counts are expressed from serial dilutions;Colony forming units/ml
Each colony represents outgrowth from a single cell, but an individual colony is made up of millions of bacterial cells!
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| Spot plating: TNTC | too numerous to count
More than 30 colonies per spot
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| Spot plating: TFTC | too few to count
Less than 5 colonies per spot
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| Spread plating | more sensitive than spot plating; more expensive as you need more plates
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| Spread plates: TNTC | more than 250
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| Spread: TFTC | less than 25
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| Spread plate | Formula: #colonies x reciprocal of dilution x (1000/amt plated)This accounts for your µL to mL conversion
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| Spot Plate | Formula: #colonies (average of the 3 spots)x reciprocal of dilution x (1000/amt plated)This accounts for your µL to mL conversion
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| UTIs | only considered an active infection if plated cfu/mls exceed 105
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| Cfu/ml numbers can be used in the following: |
Estimate the amount of protein and or DNA based upon viable counts in a population for research purposes
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| most compound light microscopes | have 3 or 4 objectives mounted on the nosepiece directly over the stage
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| longer the lens... | greater the mag
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| Magnification is | limited by resolution
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| Resolution | Ability to see two objects as separate and distinct
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| resolution depends upon | refraction (scattering) of light
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| Refraction can be limited by: | Light wavelength
Shorter wavelengths = better resolution
most scopes employ a blue filter (short wavelenght)
Numerical aperture (NA) of the lens
The ability of the objective to capture scattered light
Related to curvature of the lens
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| Glass and air | different refractive indexes;- Some light is “lost” before it makes it to the objective
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| Glass and oil | almost identical refractive indexes;-More light makes it to the objective = less refraction, therefore better resolution
Use of oil immersion increases magnification approximately 2.5-fold
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| oil immersion lens | only fine adjustment; should be able to see seal
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| As you switch from low to high power | the field of view becomes darker.
To deal with this the iris diaphragm needs to be opened to allow more light into the condenser.
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| As you switch from low to high power | the field of view becomes smaller.
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| Parfocal | in focus with one lens – in focus with all (in theory) – higher magnifications may require slight adjustments
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| Brightfield microscopy | Objects are dark and the “field” is light
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| Stains | Are chromophores – charged ions
Acidic (-) or basic (+)
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| Direct/simple | stains organism; uses a basic stain
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| Negative/indirect | stains field or background; uses an acidic stain
Based upon the inherent properties of the negatively charged bacterial membrane and chromosome
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| What do we stain? | smear
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| what do we need? | clean slide
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| Why do you fix organism to slide? | to increase adherence; to increase stain uptake; kill bacteria
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| Negative Staining | Stains background not bacteria.
Ionic Repulsion – like charges repel
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| Pros of Negative Staining: | Bacteria less distorted- does not require heat fixation
Can display cell morphology and size more clearly than other staining.
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| Primary stain | Imparts color to all cells
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| Decolorizing agent | May or may not remove the primary stain from the entire cell or certain cell structures
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| Counterstain | Provides color contrast to primary stain
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| Gram positive | peptidoglycan 90% of cell wall;
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| Gram negative | peptidoglycan in 10% of cell wall; cells have outer lipolysaccharide layer (LPS)
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| Primary stain | Crystal Violet (1˚) dissociates into CV+ and Cl- ions
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| Iodine | (mordant) makes CV-I complexes
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| . Alcohol (decolorizing agent) | washes away outer layer of Gram- along with CV-I. CV-I in gram+ cells stays in layer of peptidoglycan so stays PURPLE.
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| Safranin (2˚) | gives gram- its PINK color.
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| Gram Positive characteristics | thick layer cell wall peptidoglycan; low lipid (1-4%); quite susceptible to penicillin; complex for many species (pathogens); more resistant to physical disruption
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| Gram Negative characteristcs | Think layer cell wall peptidoglycan; high lipid (11-22%); less susceptible to penicillin; simple; less resistant to physical disruption
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| Problems with gram staining | Some bacteria will not Gram stain (Mycobacterium, Mycoplasma, Nocardia spp. to name a few examples)
Accurate staining is often dependent upon culture age (18-24 hr cultures give best results)
Some bacteria give variable results – (Gram – v)
Stressed ba
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| some bacteria contain what which makes them impermeable to staining procedures | mycolic acids
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| Acid fast bacteria | Resist decolorization with acid alcohol
Carbol fuschin contains phenol which solubilizes traditional cell walls
Methylene blue serves as a counter stain
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| Steam heat | is used to facilitate the uptake of the carbol fuchsin by the acid fast bacteria
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| go over slides in the powerpoints | slides 24-28
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