Quiz 1 Flashcards
What is Treponema pallidum and what does it cause?
It is a spirochete seen using a negative stain and it causes syphilis
How do you calculate total magnification?
Objective x ocular = total
What are the 4 types of lenses and what are their magnifications?
1) Scanning - 4x lens
2) Low power - 10x
3) High dry - 40x
4) Oil immersion - 100x
What are the basic steps of how to use a microscope?
1) put slide on the stage and move slide to center letter
2) move nosepiece and put smallest 4x objective in place
3) use coarse focus to move stage all the way up
4) adjust width of eye piece
5) use coarse focus, lower stage until you see the letter
6) use the fine focus to get into clear focus
What is the general function of the nosepiece?
rotating disc that holds/switches the objective lenses
What happens to the orientation of the letter e as you view through the microscope?
E is upside down and backwards
What do we call the microscope and what should we do once we go up in magnification?
Parfocal: microscope stays approximately in focus when switching objectives
Only use the fine focus after going up in magnification
As magnification increases, what happens to the working distance?
Working distance decreases
As magnification increases, what happens to the light intensity and size of the field of view?
They decrease
What are different ways to adjust the light?
Rheostat (aka light intensity knob): like a dimmer switch, controls the light
Condenser: gathers and focus light to illuminate the specimen
Iris Diaphragm: controls the amount of light that exits the condenser
What is the purpose of immersion oil?
has an index of refraction that is similar to glass that helps to focus light through the lens
What should you never do when going up in magnification?
Don’t adjust the coarse focus
What should you never do once you put oil on the slide?
Do not go back to the 40x lens
What is a stain?
Colored molecule in a solvent
What is a chromogen and solvent?
a chromogen is a colored molecule
a solvent is usually water or an alcohol such as ethanol
What is a auxochrome?
charged portion of the chromogen
What is a chromophore
colored portion of the chromogen
What kind of stain is a simple stain?
It is a basic stain that accepts hydrogen ions
carries positive charge
Why do we use a basic stain?
cell is negatively charged so the positive charge on the chromogen will be attracted to the positive charge on the cell
What are 4 examples of a basic stain?
1) methylene blue
2) crystal violet
3) safranin (reddish pink color)
4) malachite green (teal green)
Why did we do the simple stain in the lab?
We did it to visualize cheek cells
What is aseptic technique
to transfer bacteria from one place to another without contaminating ourselves or the culture
What does it mean to inoculate?
to introduce microbes into a new environment
Why do we need the inner cone of the flame when doing aseptic technique?
It is the hottest part of the flame
What are the three types of media and what are their differences?
a) broth (liquid) - inoculated with a loop
b) slant (solid) - inoculated with a loop usually (except when you need to transfer only a small amount, like for a citrate slant)
c) agar deep (semi-solid) - stab inoculated with a needle
Know difference between a needle and a loop
loop is used for broth and slant while needle is used for agar deep
What are some key points when working with aseptic technique?
1) work out in front of you
2) work close to the flame
3) hold the loop like a pencil towards the back of the loop
What are the steps of aseptic technique?
1) Label tube
2) flame loop starting at base and work towards loop
3) do not put test tube cap on desk
4) flame opening of tube
5) reflame tube after taking sample out of tube
6) zig zag inoculation with slant test
What is the purpose of the streak plate?
to isolate individual colonies of bacteria from large mixed populations
What are some key methods of streak plating?
a) label the agar side of the plate
b) always incubate agar side up so that condensation ends up in lid and not on sample
c) work with plate by flame
d) open lid like a clamshell
e) understand the importance of the loop angle (keep loop at approximately a 45 degree angle
f) also go side to side when doing streak
g) vortex the tube before taking sample
What are the steps of plate streaking?
a) deposit culture in a line across area in 1st quadrant, then streak 1st quadrant
b) flame loop, make 6-8 directional streaks
c) flame loop, make 6-8 directional streaks
d) make zig zag streaks without flaming loop
What is agar?
a) complex polysaccharide that is used as a solidifying agent for culture media
b) generally not metabolized by microbes (sugar that can’t be digested)
At what temperature does agar liquify and solidify?
liquefies at 100 degrees Celsius and solidifies at 40 degrees Celsius
What is chemically defined media and what is it used for?
a) exact chemical composition in known
b) it is used for fastidious organisms
What are fastidious organisms?
Those that require many growth factors provided in chemically defined media
What is the purpose of a negative stain?
to visualize bacterial cells and determine morphology, arrangement, and accurate cell size
What stains require heat fixation and air dry?
simple stains and gram stains
Why do we not use heat fixation on negative stains?
the heat may cause cells to shrink and fragile cell types such as spirochetes may become damaged
Know the morphology of bacteria and their arrangement
Morphology
a) coccus = spherical
b) bacillus = rod
c) vibrio = curved rod
d) spirillum = rigid spiral
Arrangements
a) diplo = two
b) staphylo = grape like clusters
c) strepto = chains
Which morphology and arrangement pair does not exist?
staphylobacillus
What is one bacteria you would look at on a negative stain?
Treponema pallidum - an STI that causes syphilis
Why do we only use a half drop of congo red when doing a negative stain?
a full drop would allow for too much of the dye to come out
What is the purpose of a capsule stain?
Differentiate between capsule producing cells and unencapsulated cells
What is the function of a capsule?
a) acts as an adherence factor (sticky)
b) protects the bacteria from dehydration, nutrition loss, and phagocytosis (anti-phagocytic)
What is the composition of the capsule?
a) mucoid polysaccharide (carbohydrate)
b) OR polypeptide (less common but used in bacillus anthracis)
What is the cell stained with in a negative stain?
Safranin (basic stain)
What is the background stained with in a capsule stain?
Congo red (negative stain)
What is the purpose of a gram stain?
To stain and differentiate gram positive bacteria from gram negative bacteria
What dyes do we use to stain the cell in a gram stain?
Basic dyes - crystal violet and safranin
What colors do gram positive and gram negative bacteria appear?
gram positive = purple
gram negative = pinkish red
What is the difference between gram positive and gram negative?
a) gram positive will retain the crystal violet color because it has a thick layer of peptidoglycan
b) gram negative has an outer membrane and they have a thin layer of peptidoglycan which makes the crystal violet come out
What do we use to transfer bacteria in a gram stain?
a needle
What is the purpose of heat fixing?
a) kill the cells
b) adheres bacteria to the slide
c) proteins denature and coagulate
What are the steps of a gram stain?
a) Primary stain: crystal violet for 1 minute and rinse
b) mordant: iodine for 1 minute and rinse
c) (MOST IMPORTANT) decolorization: acetone/alcohol until solution runs clear and rinse
d) secondary stain: safranin for 1 minute and then rinse
e) use kim wipe to blot off top
What is the purpose of the iodine in the gram stain?
forms a crystal violet iodine complex that helps to retain crystal violet
