Lab Practical 1 Additional Review Flashcards
Culture
a medium that contains living microbes
What are some general practices you should adhere to when performing an aseptic transfer? (7)
- Minimize potential contamination (do not do transfers over your books or bags)
- Be organized (arrange all media in advance and clearly label with your name, date, the medium, and the inoculum)
- Place all media tubes in a test tube rack when not in use whether they are sterile or not.
- Take your time (work efficiently, but do not hurry)
- Never hold a tube culture by its cap.
- Hold the inoculating loop or needle like a pencil in your dominant hand and relax.
- Adjust your Bunsen burner so its flame has an inner and outer cone.
Sterilization of inoculating instruments is done in what part of the Bunsen burner flame?
The hottest part of the flame - the tip of the inner cone
Heat-fixing bacterial smears on slides and incinerating the mouths of open glassware items are done in what part of the Bunsen burner flame?
the outer cone of the flame
When are broths generally used?
to grow microbes when fresh cultures or large numbers of cells are required.
What are agar slants generally used for?
to grow stock cultures that can be refrigerated after incubation and maintained for several weeks
What are plated media typically used for?
obtaining isolation of species, differential testing, and quantifying bacterial densities
What are the steps to transfer a broth culture to a sterile broth?
- Label the sterile broth tube (name, date, medium, and organism)
- While holding the inoculation loop in your dominant hand, flame the loop and wire from base to tip until uniformly orange-hot
- With your free hand pick up the culture tube and suspend the organisms by gently mixing.
- Remove the tube’s cap with the little finger of your loop hand and, holding it at an angle, flame the tube’s lip by passing it quickly through the flame a few times.
- move the tube under the inoculating loop and place the cooled inoculating loop into the broth and remove it being careful not to catch it on the tube’s lip (there should be a film of broth in the loop)
- Again flame the tube’s lip, replace the cap, and return the tube to the test tube rack.
- With your free hand pick up the sterile broth tube and remove the cap with the little finger of your loop hand.
- Hold the sterile broth tube at an angle and flame the lip.
- Move the tube under the inoculating loop and place the inoculating loop with the film of broth on it into the sterile medium and mix.
- Withdraw the loop from the broth and while still in the tube tap the film out of the loop before removing it completely.
- Again flame the tube’s lip, replace the cap, and return the tube to the tube rack.
- Flame the wire and loop again as before
- Incubate the inoculated culture at the assigned temperature for the assigned time.
How is transferring from an agar slant different than from a broth?
- no need to suspend the organisms as they are in a more solid form
- when flaming the tube at a slant you need to make sure the agar surface is facing upwards
- instead of dipping the inoculating loop into a liquid broth you carefully place it in the tube over some growth and then touch it to the agar surface in order to pick up a very small amount of the growth.
4 when inoculating your sterile agar slant you move the tube up the inoculating loop wire until the loop is at the base of the agar and slowly remove the tube from over the wire making a zigzag pattern in the agar with your loop.
What are the steps when transferring from a plate culture to a sterile broth or agar slant?
- label the sterile tube with your name, date, medium, and organism
- While holding the inoculation loop in your dominant hand, flame the loop and wire from base to tip until uniformly orange-hot
- leave the plate on the table and lift the lid OR with the plate inverted on the table, remove the base and hold it at an angle with the agar up.
- Touch the loop to the growth and obtain a small amount.
- Keeping the loop still, replace the plate lid
- Continue on with the normal inoculation procedure for a broth or slant.
What is a semi solid deep agar used for?
Used to grow bacteria that need less oxygen than is present on the surface of a medium. Also determines motility.
Does growth occur at different levels in agar deep?
yes
Consider agar slant, deep and broth cultures. Which can you see if it’s pure by just visually inspecting it?
Only agar slant, because the bacteria grows on the top and is all visible
Describe the growth pattern of a motile organism in a semi-solid deep.
There will be lateral growth of a motile organism. Growth will move away from the point of inoculation and will look like an inverted pine tree.
How will the arrangement of the staphylococcis epi. in broth differ from the arrangement in the slant culture?
broth will show turbidity, pellide or nocculent (or sedirrent) when arrangement in slant culture will show colonies and shape/ arrangement/ individual colonies
Why do you flame a loop?
Aseptic technique is used to insure that only your “microorganism” of choice is propagated.
Pathogen
A microorganism that resides on or in another species and causes damage to their host (causes disease)
Nonpathogenic
“free-living” - a microorganism that does not reside on or in a specific plant or animal host and are not known to cause disease
Commensal
microbes benefit from but have no significant effect on their host
Mutualistic
microbes benefit their host rather than damage them
Opportunistic pathogens
microbes that are capable of producing a disease state if introduced into a suitable part of the body.
Colony morphology: shape
- round (circular)
- irregular
- punctiform (tiny pinpoint)
Colony morphology: margin
- entire (smooth with no irregularities)
- undulate (wavy)
- lobate (lobed)
- filamentous (unbranched strands)
- rhizoid (branched like roots)
Colony morphology: elevation
- flat
- raised
- convex
- pulvinate (very convex)
- umbonate (raised in the center)
Colony morphology: texture
- moist
- mucoid (sticky)
- butyrous (buttery)
- dry
Colony morphology: pigment production
- color
- optical properties (opaque, translucent)
Colony morphology: surface
- smooth
- rough
- wrinkled (rugose)
- shiny
- dull
What are the advantages and disadvantages of using a slant?
The advantages of agar slants include providing bacterial storage over extended periods with a minimal risk of contamination or desiccation while disadvantages involve the organisms being less observable and accessible than those in petri dishes. Agar slants also lend themselves to the identification of bacteria by characteristic patterns of movement.
What terms are used to describe colony morphology? (7)
- Size
- Shape
- Margin
- Surface
- Texture
- Elevations
- pigment production
What terms are used to describe growth patterns on slants?
- Filiform
- pigmented
- friable
- spreading edge
- translucent/transparent
Filiform
growth that is dense and opaque with a smooth edge
Pigmented
colored
Friable
growth that is dry and crusty
Spreading Edge
growth that spreads at the edge (spreading margin)
What terms are used in application to broths?
- pellicle
- sediment
- uniform fine turbidity
- flocculent
flocculent
big chunks of bacteria
pellicle
a surface membrane produced when organisms float on top of the medium.
sediment
organisms sink to the bottom
uniform fine turbidity
organism concentrates light and makes illumination of specimen more uniform
Autotrophs
organisms able to survive with carbon dioxide as the only carbon source
heterotrophs
organisms requiring organic carbon
defined media
one in which the amount and identity of every ingredient are known, which means it must be formulated from purified chemicals.
undefined media
composed of extracts from, or digests of, plant or animal matter and are usually quite rich in nutrients; exact composition of the medium and the amount of each ingredient are unknown.
What are the three elements of major importance in regards to microbial growth?
carbon, nitrogen, and oxygen
What are the two elements that organisms ultimately need?
energy and carbon
What are the four biochemical families that all living things are composed of?
- proteins
- carbohydrates
- lipids
- nucleic acids
Which organisms require the least “assistance” from the environment to grow?
autotrophs
Which organisms require preformed organic compounds from the environment?
heterotrophs
What is fluid thioglycollate medium used for?
generally used to cultivate anaerobic and microaerophilic bacteria. (This medium can support growth of a broad variety of aerobic and anaerobic, fastidious and nonfastidious organisms.)
aerotolerance
a microorganisms ability or inability to live in the presence of oxygen
aerobic
the gradient of oxygen concentration nearest the oxygen source
anaerobic
the gradient of oxygen concentration furthest from the oxygen source
obligate
organisms that require oxygen for aerobic respiration
facultative
grow in the presence or absence of oxygen
Aerotolerant anaerobes
organisms that don’t require oxygen and are not adversely affected by it
Obligate aerobes
organisms that require oxygen
Microaerophiles
organisms that survive only in environments containing lower than atmospheric levels of oxygen.
Fastidious organism
any organism that has a complex nutritional requirement. In other words, a fastidious organism will only grow when specific nutrients are included in its diet.
On a bacterial growth curve, what is lag phase?
“adjusting” - very little to no bacterial growth
On a bacterial growth curve, what is log phase?
“healthy; rapidly dividing” - the number of bacterial cells doubles at a constant, exponential rate.
On a bacterial growth curve, what is stationary phase?
“amount replicated = dying bacteria” - population growth levels off as the rate of cell death equals the rate of cell division.
On a bacterial growth curve, what is death phase?
more cells die than will grow and divide; characterized by a steady decline in population numbers from starvation and/or high toxic concentrations
What is the generation time?
the time it takes one cell to become two (ex. if we start with one Staph. aureus cell, in 30 minutes there will be two. In another 30 minutes, there should be four, and so on)
What apparatus is used in generating a bacterial growth curve? What info does it gather and how?
A spectophotometer is used to monitor bacterial growth by measuring the turbidity or Optical density which is the measure of the amount of light absorbed by a bacterial suspension.
What is turbidity?
the cloudiness or haziness of a fluid caused by large numbers of individual particles that are generally invisible to the naked eye, similar to smoke in air.
How does measuring turbidity provide information on bacterial growth?
The degree of turbidity in the broth culture is directly related to the number of microorganism present, either viable or dead cells, and is a convenient and rapid method of measuring cell growth rate of an organism.
When generating a growth curve what info is your x axis and which is your y?
X (horizontal) - time in minutes (0, 30, 60, etc)
Y (vertical) - absorbance at 600nm (0.1, 0.2, 0.3, etc)
What effect does less than optimal temperatures have on microbe growth?
it can slow microbe growth via protein denaturation and/or disruption of membrane potentials.
What is a psychrophile?
an organism that only grows below 20 degrees Celsius
common in ocean and arctic/antarctic habitats where the temperature remains cold with little or no fluctuation
What is a psychrotroph?
organisms adapted to to cold habitats that fluctuate from about 0 degrees Celsius to above 30 degrees Celsius
What is a mesophile?
bacteria adapted to temperatures between 15 and 45 degrees Celsius (most bacterial residents in the human body, as well as numerous human pathogens are mesophiles)
What is a thermophile?
organisms adapted to temperatures above 40 degrees Celsius.
What is Sorensen’s formula for the calculation of pH?
pH = -log[H+]
What is the log scale range and how is it divided in regards to acidity and alkalinity?
Range from 0 - 14
0-6 being acidic
7 being neutral
8-14 being alkaline
Why does pH have such a profound effect on the growth of organisms?
Bacteria live in habitats throughout the pH spectrum but range of most individual species is small. Changes to pH outside an organisms range may destroy necessary membrane potential and damage vital enzymes beyond repair.
Acidophiles
organisms adapted to grow well in environments below about pH 5.5
Neutrophiles
organisms that prefer pH levels between 5.5 and 8.5
Alkialiphiles
organisms that live above pH 8.5
What pH do most bacteria grow between?
pH 6.5 - 7.5
What pH range is typical for life in general?
pH 6-8
What pH do molds and yeast grow between?
pH 5-6
Osmosis
net movement of water across a semipermeable membrane (high to low concentration)
Diffusion
the net passive movement of particles (atoms, ions or molecules) from a region in which they are in higher concentration to regions of lower concentration. It continues until the concentration of substances is uniform throughout.
Osmotic pressure
the pressure needed to stop the movement of water across the membrane
Isomotic
Having an osmolality equal to another fluid -no net movement of water
hyposmotic
Having an osmolality less than another fluid - in a hyposmotic solution there will be a net movement of water into the cell
hyperosmotic
Having an osmolality greater than another fluid - in a hyperosmotic solution there will be a net movement of water out of the cell
What is UV radiation?
Ultraviolet radiation is a type of electromagnetic energy that travels in waves and is distinguishable from all others by its wavelength.
Why are the lower wavelengths of light more threatening to living organisms?
it results in irreparable DNA damage and death of the organism (UV-C: wavelengths ranging from 100-280nm)
Why are spore forming organisms better able to survive exposure to UV radiation?
endospores are non dividing cells and are not subject to the same effects as reproducing cells - (Spore-forming bacteria such as Bacillus are able to survive much higher doses of UV and other types of radiation. Spores can survive very harsh conditions (high/low temperature, low water, radiation, antibiotics, osmotic pressure, etc.) for extended periods of time.
UV-A
the longest wavelengths, ranging from 315 to 400 nm
UV-B
wavelengths between 280-315 nm
UV-C
wavelengths ranging from 100-280nm
What cell morphologies are associated with single cell arrangement?
Cocci, bacilli, spirilla, spirochetes, and vibrios
What cell morphologies are associated with paired cell (diplo) arrangement?
Cocci and bacilli
What cell morphologies are associated with chained cell (strepto) arrangement?
Cocci and bacilli
What cell morphologies are associated with tetrad cell arrangement?
Cocci
What cell morphologies are associated with cube (sarina) cell arrangement?
Cocci
What cell morphologies are associated with irregular cluster (staphylo) cell arrangement?
Cocci
What cell morphologies are associated with palisade and angular cell arrangement?
Bacilli
What is the three domain system?
divides organisms up into three domains based on rRNA sequencing results and other molecular comparisons (abandons the term prokaryote)
Three domains: Bacteria, Archaea, Eukarya
Amoeba
a single-celled animal that catches food and moves about by extending fingerlike projections of protoplasm. Amoebas are either free-living in damp environments or parasitic.
Paramecium
Paramecia are single-celled protists that are naturally found in aquatic habitats. They are typically oblong or slipper-shaped and are covered with short hairy structures called cilia.
Giardia
a genus of anaerobic flagellated protozoan parasites that colonize and reproduce in the small intestines of several vertebrates, causing giardiasis (diarrhea).
Plasmodium
a genus of parasitic protozoa, many of which cause malaria in their hosts.
Volvox
a Chlorophyte, or green alga. It exists as a grand spherical colony.
Saccharomyces cerevisiae
a species of yeast. It has been instrumental to winemaking, baking, and brewing since ancient times.
Rhizopus
a genus of common saprophytic fungi on plants and specialized parasites on animals. They are found on a wide variety of organic substrates, including “mature fruits and vegetables”, jellies, syrups, leather, bread, peanuts, and tobacco.
Aspergillus
a fungus of the genus Aspergillus, and is one of the most common Aspergillus species to cause disease in individuals with an immunodeficiency.
What are the possible cell arrangements that go with cell morphologies?
- Single cells
- pairs of cells (diplo)
- Chains of cells (strepto)
- tetrads
- cube (sarina)
- Irregular cluster (staphylo)
- Palisade and angular
