A ____ consists of one or more species of microorganisms growing in a nutrient growth medium often at specific temp
A culture consisting of ONLY 1 species of microorganism is called a what?
The composition of nutrients that all organisms require to grow and survive is called a ___ medium.
A liquid solution in which microbes, especially microbes & protozoa, will grow is known as a ___ ___.
A broth supplemented with a solidifying agent that produces a relatively solid medium on which bacteria and fungi easily grow is called what?
Agar is a polymer of what extracted from what?
Galactose extracted from cell walls of red algae
Agar liquifies at ___o C and solidifies at ___oC.
100 and 40
Describe a "circular" form of a colony.
Circular w/smooth edges like and "O"
What type of microscope utilizes visible light to magnify and resolve objects?
Compound light microscope
Which staining technique uses 2 contrasting colored stains to allow bacteria to be separated into 1 of 2 grps and also visualize structures such as endospores and capsules?
What are the 3 purposes of simple staining?
Measure cell size
Determine cell shape
Determine cell arrangements
What are the 3 main purposes of differential staining?
Cell size, shape and arrangements
Separate Gram-pos & Gram-neg
Visualize cell structures
Define working distance as it relates to microscopy.
Proximity of the slide to the bottom of the objective lens
What aspect of microscopy pertains to the light-bending ability of glass, oil and air media thru which light must pass during image formation?
What are 3 types of basic stains?
Basic stains carry a ___ electrical charge.
The ___ stain technique allows us to see unstained bacteria on a stained background and determine their morphology.
____ stains such as nigrosin, congo red & india ink carry a neg charge & are therefore what?
Repelled by negatively charged bacteria
What are the steps in preparing a bacterial smear?
Asceptically place bacteria on glass slide
Air dry and heat fix
Rinse, dry and view
Define a bacterial smear.
Thin layer of bacteria placed on a slide for staining
If agar slants are used when making a bacterial smear then a what should be added to the slide as well?
A loopful of water
What are the 3 things accomplished by heat fixing?
Kills any bacteria that may still be alive
Facilitates stain penetration
Fixes cells to the slide so they do not wash off when stained
Simple staining is an easy way to determine what 3 things about bacterial cells?
Name 3 basic stains we discussed?
The ___ ___ technique permits one to see unstained bacteria on a stained background & determine their morphology.
Nigrosin, congo red & India ink are ___ stains that carry a ___ charge and are therefore repelled by ____ charged bacteria.
What form of bacteria found in dental plaque are best stained in a negative manner and why?
Spirochetes b/c they do not stain well with other techniques
The use of excessive stain when utilizing the negative stain technique causes what problem?
It makes the organisms difficult to locate
Negative stain must not be allowed to become contaminated because. . . .
It will support bacterial growth
What is the quick procedure for the negative stain technique?
Add loopful of bacteria at end of slide
Place sm drop of acidic dye at end w/bacteria
Pull dye/bacteria mix across face of slide w/ second slide
Air dry and observe
What are 2 advantages to the negative stain technique?
It does not distort bacterial cells
Some cells such as spirochetes dont stain well w/other techniques
T or F. Negatively stained slides are air dried and heat fixed.
False - Air dried ONLY
When the negative stain technique is used what are we looking for in the microscope?
White or clear cells on a colored background
When labeling a diagram of a slide what should we note?
Size, shape, arrangement
Full binomial name
What is a second method for preparing a negative stain?
Prepare air dried smear
Pass a felt tipped marking pen several times over the smear
Ink will stick to slide & bacteria will appear white against colored background
Why do we not heat fix bacterial smears iN negative staining?
B/c it will disrupt the bacterial cells
What is aseptic transfer?
Sterile, microbe free mvmt or transfer of microorganisms fr one medium to another.
Why do we use aseptic technique?
In order to maintain pure cultures & create subcultures
What is the red pigment produced by S. marcescens called?
What are 3 advantages of the streak plate technique over the pour plate technique?
No time constraints due to agar cooling
Less likelihood of contamination
Easier access to bacterial colonies
Why do we incubate plates in the inverted position?
So when condensation forms on the lid it doesn't fall into the cultures
In 1884 ___ ___, a Danish physician, discovered certain bacteria aft being stained w/crystal violet & iodine, would lose their color on subsequent tx w/alcohol while other bacteria would retain the color.
When Gram staining bacteria that retain crystal violet stain are known as ___-___ & those that do not are ___-___.
Gram staining is classified as a ____ staining technique.
What 2 types of bacteria do not respond to Gram staining?
Spirochetes & Mycobacteria
___ ___ & ____ form a complex that exits easily fr Gram neg bacteria but not fr Gram pos when ___ is applied.
What is it in the cell walls of Gram positive bacteria that trap the crystal violet/iodine complex?
What is it that makes Gram pos cell walls retain the crystal violet/iodine complex and causes Gram neg walls not too?
Gram pos bacteria have larger amts of peptidoglycan in their cell walls that traps the complex whereas Gram neg walls have less & fail to retain it
The Gram stain technique is a ___ part procedure in which 2 dyes, a ____(iodine) & a ___ agent are used.
Other than a culture what supplies are needed for a Gram stain procedure?
Crystal violet stain
95% ethyl alcohol
What are the steps in a Gram stain?
Prepare air dried, heat fixed smear
Apply crystal violet & set for 1 min
Flood w/Grams iodine & set for 1 min
Decolorize w/95% ethyl alcohol for 15 sec, x2
Counterstain for 30 sec
Blot dry w/bibulous paper
The iodine in a Gram stain acts as a mordant which means what?
It increases the affinity of dye to the cell by forming a complex w/the stain
As a general rule how long should decolorization go on for?
Until the ethyl alcohol running off is no longer purple
What is the possible result of excessive decolorization in Gram staining?
Gram pos bacteria may lose their color and appear Gram neg
What is the possible result of insufficient decolorization when performing a Gram stain?
Gram neg bacteria may retain purple color and appear Gram pos
Prior to counterstaining in the Gram technique what would the slide look like under a microscope?
Gram pos bacteria would appear purple & Gram neg bacteria would appear transparent
What effect will safranin have on Gram pos bacteria?
After decolorization, flood smear w/____ for ___ secs.
Gram pos bacteria will appear ___ to ___ & Gram neg will appear ____ to ____.
Blue to purple
Orange to Red
When observing Gram stained slides, be sure to confine your work to areas containing thin smears of bacteria b/c why?
Thicker areas may resist decolorization due to heavy concentration of bacteria
Endospores are formed by members of several gram-___ bacterial genera such as ____ &____.
Bacillus and Clostridium
What are 3 characteristics of endospores?
Resist boiling for 2 hrs or more
Contain little water
Exhibit few chem reactions
What happens to spores when the chem environment are favorable?
Protective layers break down & vegetative cells emerge to grow & reproduce
Name 4 notable diseases caused by sporeformers.
What name is given to a layer of polysaccharides & proteins secreted by certain bacteria including pathogens?
What functions does a capsule perform?
Buffer between cell & its external environment
Protects against dehydration
Traps nutrients fr surrounding environment
Contributes to est of disease
Helps resist phagocytosis
A thin flowing capsule isnt really a capsule at all. It is called a ___ ___.
The term ____ refers to both capsule and slime layer
____ are protein appendages that facilitate motion of bacteria?
Many species of bacterial ___ & ___ & a few species of ___ possess flagella
Rods & spirilla
Why is it that flagella are so long but cannot be seen with a light microscope?
B/c although they are many times longer than a cell they are extremely thin
What is the purpose of the spore stain technique?
To contrast vegetative cells fr endospores
Why is it that bacterial endospores cannot be penetrated easily by stain using simple or Gram stain techniques?
B/c they contain numerous protective layers
What do we do to assist with stain penetration when staining bacterial endospores?
What stain do we use as the primary stain for spore stain?
When doing a spore stain we should allow the slide to remain over ___ for ___ mins, continually adding stain.
Steam for 3 mins
Heat during spore stain does what?
Forces stain into endospores & vegetative cells
When rinsing spore stained slides vegetative cells will ___ their color while endospores will what?
When spore staining we counterstain w/___ for __ min. This will have what effect?
Safranin for 1 min
this will stain the vegetative cells but have no effect on the spores
When spores are found within vegetative cells, their position could be ___, ___ or ___.
Central, subterminal, terminal
Young cultures often contain spores within ___ ___ while older cultures contain more free ___ & fewer ___ ___.
When utilizing the alternate spore stain technique, we cover the smears w/___ ___ ___ for how long?
7 1/2% malachite green
Visualization of the bacterial capsule by staining is a 2-step procedure involving both ___ & ___ staining.
Negative & simple
Why is it that water or heat should not be used in any step of capsule staining?
Capsules are easily destroyed by both
Why is it helpful to use milk cultures of organisms when capsule staining?
Media containing milk encourage capsule production
What are the quick steps to produce a capsule stain?
Prepare neg stain of bacterial smear
Stain w/crystal violet for 1 min
Rinse w/ SALINE SOLUTION, dry, observe
When viewing capsule stain, what are we looking for?
Purple cells surrounded by capsules which appear as white halos
What are 2 methods for determining motility?
Hanging drop technique
Utilizing motility agar
What characteristics of motility should we not when viewing our slide?
Patterns of motion
Where cells accumulate
Relative shapes & sizes
Speed of mvmt
Erratic vibrations of cells in place & lack of directed mvmt demonstrates a phenomena called what?
What causes Brownian mvmt?
Molecules striking organisms & displacing them briefly
What is motility test agar?
Semisolid growth medium containing a reduced amt of agar
What is special about semisolid motility agar?
The semisolid state allows bacteria to move freely throughout the medium
How will motile organisms present in test agar in comparison to non-motile organisms?
Motile organisms will spread away fr the inoculation line & est broad zone of growth where nonmotile organisms will grow only along the line of inoculation
Name 3 characteristics that are used to help ID an organism.
Size, Shape, Arrangement
Response to staining
Presence of spores, capsules, flagella
Why is it necessary to invert plates when incubating them?
B/c condensation develops on the lid of the plate & we don't want it to drip on the culture
In ____ colonies, bacteria form many hairlike filaments as they grow outward from the center of the colony.
____ colonies grow outward in fewer and thicker filaments, like the roots of a miniature tree.
____ colonies bulge up from the plate in a pronounced semicircle.
____ colonies look like raised colonies, but they have a small projection in the middle.
____ colonies resemble raised colonies, but they have a tiny depression like a dimple in the middle.
The ___ describes the edge of a bacterial colony.
A colony where the edge is lumpy and irregular is considered to be what shape?
Some margins are ____, with tiny filamentous strands of bacteria growing out from a central mass.
____ margins have a wavelike edge, while ____ margins have fingerlike projections growing outward from the edges of the colony.
Cloudyness in a broth culture is described as being ____.
A broth culture that contains small particles is said to be what?
A broth culture that contains small masses is said to be ____.
Broth cultures that contain large particles are described as ____.
Sediment at the bottom of a tube that clumps together is said to be ____.
Aerobic growth is located where in a thioglycollate medium tube?
Growth at the top of the tube
Growth at the bottom of a thioglycollate tube indicate what type of growth?
Growth throughout a thioglycollate tube indicates what type of growth?
Why is it said that biochemical characteristics serve as fingerprints for a bacterial species?
B/c the results of a series of biochemical tests can help to ID an unk organism & potentially ID a disease
What is contained in Bergey's Manual of Systematic Bacteriology?
Std results for the biochemical tests we performed
___ is a type of microbial metabolism which an organic intermediary molecule serves as an electron acceptor
What are the two products of fermentation that our tests looked for?
Carbon dioxide gas production
What is the media used in fermentation testing consist of?