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Micro Lab midterm
Terms in this set (170)
Colony morphology (6): round, smooth, flat, opaque, shiny, cream colored
Cellular morphology: (shape) bacillus, (arrangement) irregular
Colony morphology: round, smooth, flat, opaque, shiny, white
Cellular morphology: (shape) coccus, (arrangement) staphylo
Colony morphology: round, smooth, umbonate, opaque, dull, yellow
Cellular morphology: (shape) coccus, (arrangement) micrococcus
Colony morphology: rhizoid, lobate, flat, opaque, dull, orange yellow
Cellular morphology: (shape) bacillus (arrangement) streptobacillus
Colony morphology: round, smooth, flat, opaque, dull, white
Cellular morphology: (shape) bacillus, (arrangement) filamentous
Colony morphology: round, smooth, umbonate, opaque, shiny, white
Colony morphology: irregular, lobate, flat, opaque, dull, white
Colony morphology: round, smooth, convex, opaque, dull, red
Cellular morphology: (shape) bacillus (arrangement) single bacillus
Cellular morphology: (shape) spiral (arrangement) random
(shape) coccus, (arrangement) single coccus
contains capsules used by bacteria to adhere to tooth enamel
central endospore forming
(shape) bacillus, (arrangement) streptobacillus
(shape) coccus, (arrangement) micrococcus
What are 6 common streak plate mistakes?
1) gauging agar
2) streaks not close together
3) confluent growth (thick growth in all quadrants)
4) growth in only 1 quadrant
5) thick growth in only 1 quadrant, very few growth in others
6) poor isolation
discard slides and broken glass in
red plastic box labeled "broken glass disposal"
discard petri dishes in
orange/red biohazard bags
discard tips/swabs/tongue depressors in
the specific container on bench top
paper trash that is not contaminated goes in
contaminated paper trash
in biohazard bag
discard contaminated glassware in
bottom of the cart
discard used gloves
in appropriate used glove container
microorganisms grow in culture mediums that contain nutrients, such as carbon and nitrogen
composed of digests of chemically undefined substances such as yeast and meat extract
a precise chemical composition is known
liquid culture medium
solid culture medium
contains a solidifying agent (usually agar).
ex: slant, deep, plate
what is agar extracted from?
Rhodophyta- red algae
-after being heated to 100 degrees C, it will solidify at 40-42 degrees C and will not melt again until 80-90 degrees C
What grows on the surface of plates
used for isolation and growth of microorganisms
What grows in deeps
What grows in slants
maintenance of stock cultures of microorganisms
general-purpose, pH= 6.8
Trypticase soy agar
general-purpose, pH= 7.3
Sabouraud dextrose agar
fungi or yeasts, pH=5.6
the killing or removal of all living organisms and their viruses from growth medium
sealed device- entrance of steam under pressure
-moist heat facilitates killing of all microorganisms which are heat-resistant
-heated to 121 degrees C at 15 psi for 15 min
-for liquid and heat resistant media
dry heat sterilization
kills by oxidation effects
hot air sterilization
items are placed in an oven and heated to 170 degrees C for 2 hours
-useful for glassware
-used for heat-sensitive liquids or gases
-filter allows for passage of the liquid or gass but not the microorganisms
gaseous chemosterilizers (cold-sterilization)
-used for petri dishes
-gas used is ethylene oxide
-this is released into a tiny sealed chamber where it circulates for up to 4 hours with the items sterilized
-this is called cold sterilization because it does not use heat so it will not melt the item
used to sterilize many substances by using rays such as Ultraviolet radiation
-this causes damage to DNA leading to death of the organism
-UV can not penetrate solid, opaque, and light absorbing surfaces so it is useful to sterilize exposed surfaces
causes ions and other reactive molecules to be produced and they degrade or alter biopolymers such as DNA and proteins
-useful in the food industry and medical supplies
liquid media sterilized?; heat-sensitive?
how could a bandage be sterilized?
what temp is the incubator for plates?
37 degrees C
minimum amount of microorganisms or viruses sufficient to establish an infection
normal or resident microbiota (flora)
establish themselves on or in the body but do not produce disease under normal conditions
-can survive and multiply on the body
-can become pathogenic if the opportunity arises
transient microbiota (flora)
may be present on the body temporarily but do not become firmly entrenched
-unable to multiply on skin and die
pH of the skin
associated with apocrine and sebaceous glands in the skin that provide moisture and nutrients
why could there be more bacteria after hand washing?
sloughs off dead outermost layers and may expose normal flora
population of cells which arise from a single cell growing on a solid medium
what often differs between microorganisms
what are the 6 categories of colony morphology
whole colony shape, margin shape (from the side), elevation (from the side), optical properties, surface characteristics, pigmentation
whole colony shape
round, irregular, rhizoid
smooth, lobate, filimentous
convex, umbonate, flat
can genetically DIFFERENT organisms share the same colony morphology? Is the morphology dependent upon the medium?
yes, yes- can influence size and shape
culture containing a single kind of organism
-dilution technique used to isolate a pure culture
-one visible colony will be re-streaked to obtain a pure culture
for a streak plate
start in the new quadrant and streak back a few times in the previous quadrant (start at edge of the plate) keeping the streaks close but not touching
(streak quadrants 2-4 should be parallel to the quadrant line)
Why must the loop be flamed between each quadrant?
-kills any bacteria on the look
-ensures dilution takes place
contains 2 or more species
overnight coliforms may reach how many cells?
what are the 3 types of counts?
direct microscopic count, standard plate count, turbidimetric assay
direct microscopic count
uses a hemocytometer
-a broth culture of the bacterium is placed on the counting chamber
-the number of cells counted are then multiplied by a dilution correction corresponding to the area utilized
-includes all cells both live and dead
Pros: rapid, no selections against certain organisms
cons: counts all cells live and dead, requires more skill
Standard plate count
also known as a viable count
-based on the assumption that one living cell will give rise to one colony
-a colony may arise from more than one cell so quantifies as colony-forming units per milliliter
Pros: measures only living cells
cons: needs time for incubation, selects agains certain organisms that can't grow
Standard plate count
(a) spread plate
-aliquots of diluted bacteria are spread onto the surface of the appropriate medium in petri dishes with a sterilized glass rod called a "hockey stick"
-organisms that require oxygen for growth
-colonies on surface
pros: simple to perform
cons: need good spread technique
Standard plate count
(b) pour plate
aliquots are placed in an empty sterile petri dish and the appropriate melted and tempered medium is added and mixed with the bacterium
-used for separating a series of organisms
-colonies on surface and within
pros: no alcohol and flames involved
cons: trickier than spread plate, need melted agar deeps
what happens after incubation?
a countable plate is selected
between 30-300 colonies
few than 30 are not significant and greater are not valid because they may not have provided adequate nutrients for all cells to grow to form colonies
-this number is multiplied by the dilution correction to obtain the cfu/ml in the original broth
cloudiness caused by microbial growth in broth
a spectrophotometer measures the amount of light that passes through a liquid
-standard curve is established to determine the cfu/ml
pros: rapid (no incubation)
cons: can't use for microorganisms that grow in clumps
what is the first step in making a simple or differential stain?
preparing a smear
-it is dried then heat fixed
size of prokaryotic cells
why are cells difficult to see even under magnification
cells are mostly water (90% by weight) so there is a lack of contrast
what does staining do
makes the cell more visible
what is heat fixation
kills the cells, destroying autolytic enzymes, and causes the cells to adhere to the slide
-adhesion of the cells to the slide is essential or the cells will be washed from the slide during staining
what is the temp of the slide warmer?
55 degrees C for 5 min
what does staining allow?
cellular morphology to be determined
what are the 6 cell shapes?
coccus, bacillus, spirillum, spirochete, vibrio, pleomorphic
what are the 5 arrangements for coccus?
single coccus, diplococcus, staphylococcus, streptococcus, micrococcus
what are the 3 arrangements for bacillus?
single bacillus, streptobacillus, filamentous
why doesn't staphylobacilli exist?
bacilli only divide along their axis making clusters impossible
another name for filaments
-form mycelium which is a network of hyphae
the suffix "myces" refers to?
fungal-like cell morphology
what is a simple stain?
an aqueous or alcohol solution of a single basic dye (stain the slide then rinse it with water)
what kind of microscope do we use?
-compound (consists of series of ocular and objective lenses)
where are the objective lenses?
on a revolving nosepiece above the stage of the microscope
for each eye there is an ocular containing a magnifying lens
ocular + objective magnifications
what is magnification limited by?
the closest spacing between two points at which the points can still be seen as separate entities
-depends on wavelength/2(NA)
-the max is about 0.2 micrometers
reduces the amount of light lost and increases resolution
where slide with smear is placed
verticle and horizontal adjustment knobs
course and fine focusing knobs
raise and lower stage
light intensity knob
set on 3-5
lens that collects and concentrates light onto the object on the stage (must be fully raised)
on the condenser- can be opened or closed to allow more or less light through
on the revolving nosepiece, 10x, 40x, 100x
eyepiece for viewing
light passes up through this tube and through tht ocular lens
never use course focus with what?
what to clean lens with
lens paper only
what is a gram stain?
a differential stain
-tells the difference between bacteria
-uses a combination of dyes to demonstrate a chemical or structural component of a cell
Gram should be written...
what do bacterial cell walls contain?
-consists of repeating disaccharides attached by a polypeptide to form a lattice
-surrounds and protects the cell
-cell walls contain many layers of peptidoglycan which make the structure rigid.
-also contain teichoic acid
contain few layers of peptidoglycan surrounded by an outer membrane
-the outer membrane consists of lipopolysaccharides (LPS), lipoproteins, and phospholipids
-do NOT contain teichoic acid
For Gram stain, what is the order of applications?
primary stain, mordant, decolorizing agent, counterstain
what is the primary stain?
what is the mordant?
color of Gram-positive?
color of Gram-negative?
explain the theory of Gram stain
-when the crystal violet is added to the smear, all cells will appear purple because the dye enters all the cells
-the mordant iodine is added and complexes with the crystal violet inside the cells
-this complex is larger than the crystal violet molecule and when the decolorizer is applied, it will not was out through the THICK (+) peptidoglycan layer
-the Gram + cells remain purple because the dye remains
-the decolorizer disrupts the lipopolysaccharide layers of Gram - cells and the complex of crystal violet and iodine is washed out of the thin petidoglycan layer
-at this point, the Gram - cells are colorless so a counterstain is added (Safranin a red dye) so the Gram - cells appear red
error of a Gram stain
many Gram + cells begin to lose the ability to retain the primary stain and will appear Gram -
Procedure of the Gram stain
-make a smear
-pass quickly through the flame with close pin
-cool on stain rack
-crystal violet for 1 minute
-rinse gently with water until you no longer see the crystal violet running
-iodine for 1 minute
-rinse gently with water
-briefly drop ethanol on the smear until the crystal violet no longer runs from the smear (should take a few seconds and a few drops)
-rinse gently with water
-Safranin for 1 minute
-rinse gently with water
-BLOT gently with bibulous paper
If the mordant was not added when performing the stain, what would be the Gram reaction of all cells?
red- no complex for the purple to stay
what would be the color if the decolorizer were not added?
purple because the crystal violet will stay
*if over decolorized then red
incorrect gram stain outcome without omitting steps?
the cells are probably dying
a specialized dormant structure that is commonly found in certain Gram positive rods such as those of Clostridium and Bacillus
when will an endospore form?
-in a genetically capable cell when essential nutrients are depleted or when water is unavailable
-they are highly durable dehydrated structures
one that can grow and divide under optimal conditions
what are endospores, in contrast, said to be?
cryptobiotic state- no metabolic activities are occurring
-forms when nutrients and water is depleted
-they are survival structures
one endospore will later germinate to form?
a vegetative cell
the process of endospore formation within a vegetative cell
first stage of endospore formation
-the replicated chromosome and a small amount of cytoplasm are isolated by a spore septum (ingrowth of plasma membrane)
-this becomes a double layered plasma membrane surrounding the genetic material and at this stage it is termed a forespore
-peptidoglycan will be laid down between the two plasma membranes and this layer is called the cortex
-spore proteins form a spore coat around the outside of the structure.
-the spore coat is responsible for the resistance of the endospore to harsh chemicals
-the peptidoglycan and spore coat each form thick layers
consists of the usual cell wall, cytoplasmic membrane, cytoplasm, and nucleoid
either in the central, terminal, or subterminal position in the vegetative cell
-or the swollen sporangium
vegetative cell -> formation of endospore -> vegetative cell dies -> free endospore -> germination
When viewing your endospore stain, the free endospores stain __________________, endospores within vegetative cells stain __________________, and vegetative cells stain _____________________.
green; green; red
what does the endospore contain?
DNA, RNA, ribosomes, enzymes and few other important small molecules
-dipicolinic acid is found in all endospores and is located in the core
-contain high levels of calcium complexed with the dipicolinic acid
-this complex plays a role in protecting spore DNA from damage
visible swelling due to water uptake and synthesis of DNA, RNA, and bacterial proteins
-the cell will then emerge from the spore coat and is again termed a vegetative cell
bacterial endospores of Bacillus stearothermophilus
used to indicate the effectiveness of autoclaves
-the endospores can survive high heat so if they die in the autoclave it is pretty effective
Clostridium botulinum- botulism
spore-forming anaerobic bacillus
what are the 2 types of bacteria in the endospore exp.
Bacillus megaterium (central) and Bacillus sphaericus (terminal)
when writing a bacteria, the genus is?
the genus is capitalized, species lowercase and underline the entire thing!
useful for quickly determining if Mycobacterium is present, providing a rapid presumptive diagnosis so that treatment of the infected individual can begin
-aerobic, non-endospore forming, nonmotile, rod-shaped bacteria that possess unique lipid components called mycolic acids
-the lipid component makes it difficult to stain
-the resist decolorization
heat is useful for
driving stain into mycobacterium
Explain the Ziehl-Neelson Acid-fast Stain
-Primary stain- carbol-fuchsin (red) is used
-phenol and heating enhance penetration of the fuchsin into the lipid of the cell
-decolorization with acid-alcohol
-counterstain with methylene blue
-cells that are not acid-fast have lost the primary stain during decolorization and will take on the counterstain and appear blue
Decolorization with acid alcohol removes the carbolfuchsin from the non-acid-fast cells.
why are the Mycobacterium colonies wrinkled
why is egg albumin used?
surface lipids in the Mycobacterium causes the cells to form tight aggregates that are hard to disperse in aqueous solutions
-egg albumin has a high protein content and is used to make the smear
what are 2 diseases cause by members of Mycobacterium
-consists of layers of polysaccharide or polypeptide-containing material which surrounds the cell wall of bacteria
Azotobacter vinelandii= polysaccharide capsule
Bacillus antbracis= polypeptide capsule
what is the principle component of capsule?
-may prevent desiccation (drying out)
if capsule enhances the organism's ability to cause disease
when are capsules formed?
in presence of excess nutrients
-serves as a reserve energy store
acid stain in capsule stain
Congo Red (stains the background)
basic stain in capsule stain
Maneval's stain (stains the cells)
background= dark purple-red
cells= reddish brown
what are 4 possible functions of the capsule?
1) protects from certain chemicals
2) aids in adherence
3) serves as nutrition during starvation
4) reduces likelihood of phagocytosis
oval shaped stained cell with white halo ring around each cell
which pipet can accurately measure 20-200 microliters?
gold button that reads .20/200
which pipet can accurately measure 200-1000 microliters?
blue button that reads .100/1000
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