Lab Exam Flashcards
What consists of the frame of microscope
-arm and base
-arm for carrying
-base for support
How should the microscope be set when u are packing it away (thus when u receive it)
-cord wrapped around base or around cord caddy
-x4 objective lens should be in placed
Stage
Stage:
-between upper lens system and lower devices
-has a light that shines through hole
-mechanical stage holds light in place
-adjustment knobs that move stage vertically and horizontally
Use of stage
only the x4 or x10 objective lens should be used when placing or removing a slide on the stage
-dont need to move the stage out of focus since lots of space between x4 and x10
Use of ocular head
-dont apply pressure when turning head
-adjust the eyepieces to fit ur eyes
-to adjust eyepiece focus; focus right eye and right eyepiece on specimen then focus left
-focus on same spot using focussing ring
Name of objective lenses
4x: scanning
10x: low power
40x: high-dry
100x: oil immersion
-all are on a nose piece that rotates
what does the 100x require that the others dont
OIL
-needs immersion oil to enable the object to collect light at angles that would normally fall outside its collection window
What objective does the ocular lens piece have
-10x
How to calculate magnification
ocular lens x objective lens
ex: 40x high dry lens used (objective)
ocular is always 10x
40x10 =400
Condenser and Iris diaphram
light from lightbulb is directed to here
-placed under central opening of stage
-CONDENSER collects and concentrates light, directing up THROUGH any object on stage and can be adjusted by opening or closing iris diaphram
Raise condenser all the way then turn 1/4 down for best results (with condenser adjustment knob)
If too much light is sent through what happens?
-contrast decreases and depth of field becomes less that desired
what does lowering condenser do
decreases the amount of light that reaches the object
FOR 100x, make sure condenser up akk the way
Iris Diaphragm
-regulates the amount of light that passes through the condenser
-higher the objection, higher the intensity of light required
Start by having iris diaphram in
half position, if lacks contrast, adjust
What is used for critical focussing
fine adjustment knob
when stsrting to use microscope, what do u do to iris
half open (black adjustment with numbers)
Basic rule of focus
FOCUS AWAY FROM THE SPECIMEN
-bring specimen as close to lens as possible using fine adjustment (look not thorugh eyepiece)
-THEN look through eyepiece and bring it away from ituntil in foxus (turn knob other way)
-adjust light to comfortable level
-unfold or fold eyepieces to make one circle
Most bacteria exists in what shapes
Rods (bacillus), circle (coccus), corkscrew, spiral (spirochette or spirillium)
Stalked forms (caulobacter), club-shaped (corynebacterium), comma-shaped (vibrio)
Variations due to…
-age
-composition of culture medium
What are the characteristics
check table
How to calculate magnification of specimen in drawing
size of specimen in drawing (um) / actual size of image (um)
WHo was gram stain created by
Danish scholar Christian gram in 1884
What type of stain is a gram stain and who was it made by
Differential stain: used to differentiate bacteria types based on their ability to retain stain
Different from simple stain since that just helps u see bacteria, doesnt differentiate them
Christian gram in 1884
What are the stains used in gram stain
Primary stain: Crystal violet
Mordant( sticks stain to cells): Gram’s Iodine
Decolourizing agent: 95% ethyl alcohol
Counter-stain: Safranin
What are the results of gram stain
General:
Gram Pos: Purple bc wont be decolourized
Gram-neg: Pink bc will be decolourized and stained by safranin
What affects culture staining
1) Age of culture
2) PH of medium used to grow bacteria
gram pos may look gram neg or gram variable due to acid medium or old medium
gram neg may look gram variable due to same thing
Iodine Mordant
-acts as a glue to stick crystal violet to the nucleic acid of cells to form a complex which is not readily remov ed from gram pos in decolourzing step
Acid Stain
-differential stains
-used primarily for mycobacterium, turbucolosis bacillus and turbo leprae
-used bc these bac have waxy cell walls due to high levels of liposodal material (mycolic acids), lipid rich so imppermable to certain stains
Ziel-nielson Acid fast technique
-smear with carbolfuschin (dark red, with 5% phenol), heat used to penetrate dye
-decolourized using acid alcohol
-counterstain of methylene blue used
non-acid fast will be decolourized and will be blue
acid fast will resist decolourizing by acid alcohol so they will be red
Kinyon acid fast
-cold stain
-concentrations of phenol and carbolfuschin increased and detergent is added so that heating is not needed
Are all acid fast gram positive
Yes, all acid fast are gram postiive but NOT ALL GRAM POSITIVE ARE ACID
Results of Acid-fast
Staphyloccocus epidermidis: BLUE
Mycobacterium: RED
(spiral)
What are spores, why?
-when environmental conditions are harsh (ex, lack of important nutrients), to permit further vegetative growth, certain bacteria condense their vital cellular components into ENDOSPORES
-vegetative cell slows down, loses moisture, and withdraws its substance into one area which it surrounds with thick impermeable wall.
-slowly original cell reminants falls apart and the spore that is highly resistant to environmental conditions remains
-What is it highly resistant to?
-high heat, ionizing radiations, dessication, and chemicals
-Bacillus and clostridium genus do this
Spore staining
-spores resist staining of ordinary dyes such as meth blue, carbofuschin etc so specific dyes must be used (malachite green)
-decolourizing will also be resisted by spores
SO
Malachite green is the primary stain used, heat is used to drive it into it
then safranin is used to differentiate the vegetative cells (actively growing) from the spores
Size and placement of endospores in cell is a characteristic of spore forming bacteria
Schaeffer-fulton method: uses steam to drive malachite green into spore
Cold methed (Bartholomew and mittwer): increases concentratoon of malachite green and uses detergent to replace heating to drive stain into cells
Results of Spore Stain
Bacillus brevis: Yes, central endospores
Clostridium sporogenes: Yes,
-live in environments that cause them to need ot have a way to live, like digestive tract.Live in ocean sediment, high temp, high pressure
Negative Stain
-Another simple stain
-Uses Negatively charged dye (negatively charged chromophore) instead of positively charged dye like the other simple staining technique used
-Nigrosin is the dye used
-it repells the net negative charge of the cell surface
-chromophore repells cell surface so cell remains undyed and there is a deposit that forms around the cells that is clear, background dark
Advantages:
-doesnt use heat so cells cant be distorted
-natural size and shape of cells is maintained
-also used to visual bacteria that are hard to stain like mycobacterium
Result of negative stain
Bacillus brevis
-rod
Negative stain procedure
Place a dot of dye and place a loopful of bacteria on top, mix them gently
-if a solid culture of bac used, add water to nigrosin with the solid bac
-gently slide a second slide on top, creating a thin layer
-let it dry
Capsule Stain
-envelope of muciliaginous substances which can be referred to as a capsule, slime layer or glycocalyx
-consists of polysaccharides, peptides, and carbohydrate material which accumulate on the cell surface, giving structure an enlarged appearance.
-Alcian blue is the dye that is water soluble in nature, it forms linkages with acidic groups of mucopolysacchariedes.
-can aslo be identified by negative stain since polysacharides are water-soluble so normal simple staining dyes will not stick to it, this technique allows for the cell and background to be stained
-most staining techniques stain the cell and the background, leaving only the capsule unstained
Capsules info
-size various sizes dependent on species and strain of species
-more virulent bacteria have thicker (heavy capsule) as these bacteria are harder to destroy by phagocytic cells
-identification of things such as pneumonia is therefore used by capsule stain
Capsule stain results
Klebsiella pneumonaie: purple background, purple cell, qhite halo like capsule around cell
most common ranges for micropipettes
0.5 to 10ul
10-100 ul (P100)
100 to 1000 ul (P1000)
p20 also used for 2-20ul
-they must be used within their range to have accucuracy achieved
Tips for the micropipettes are
autoclaved and sterile
How to eject tip
either push ejector button or push to third stop on plunger
p100 settings
100|0 is 100
050|0 is 50
p20 settings
05|0 is 5
10|0 is 10
20|0 is 20 ul
Culture medium
material in which or on which bacteria are grown
-used for cultivation of microorganism
-can be liquid or solid or semi-solid
-materials in medium can be limited to well-defined inorganic or organic compounds