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Preparing specimens for optical microscopes
1. Generally prepared by mounting a sample on a glass slide
2. How the slide is prepared depends on
a. The condition of the specimen (living or preserved)
b. The aims of the examiner (to observe overall structure, identify microorganisms, or see movement)
c. The type of microscopy available
How the slide is prepared depends on (3)
1. The condition of the specimen (living or preserved)
2. The aims of the examiner (to observe overall structure, identify microorganisms, or see movement)
3. The type of microscopy available
1. Wet mounts or hanging drop mounts
2. Wet mount:
Cells suspended in fluid, a drop or two of the culture is then placed on a slide and overlaid with a cover glass
Cover glass can damage larger cells and might dry or contaminate the observer's fingers
Hanging drop mount:
Uses a depression slide, Vaseline, and coverslip
The sample is (33) suspended____ from the coverslip
Wet mount (2)
1. Cells suspended in fluid, a drop or two of the culture is then placed on a slide and overlaid with a cover glass
2. Cover glass can damage larger cells and might dry or contaminate the observer's fingers
Hanging drop mount (2)
1. Uses a depression slide, Vaseline, and coverslip
2. The sample is (33)__suspended___ from the coverslip
You can also put in well
Fixed, stained smears
1. Smear technique developed by Robert Koch
a. Spread a thin film made from a liquid suspension of cells and air-drying it
b. Heat the dried smear by a process called heat fixation. HEAT DISTORTS bacterial morphology. If you need to determine bacteria morphology DO NOT use heat fixation.
c. Some cells are fixed using chemicals
Fixed, stained smears
1. (34)_Staining_____ creates contrast and allows features of the cells to stand out
a. Applies colored chemicals to specimens
b. Dyes become affixed to the cells through a chemical reaction
c. Dyes are classified as basic (cationic) dyes, or acidic (anionic) dyes.
Positive staining: the dye sticks to the specimen to give it color
Work with base = color + /OH- (hydroxide ion) cationic
1. Negative staining: The dye does not stick to the specimen, instead settles around its boundaries, creating a silhouette.
a. (35) NIGROSIN___ and INDIA INK commonly used
b. HEAT FIXATION not required, so there is less shrinkage or distortion of cells. Gives you true morphology.
c. Also used to accentuate the CAPSULE surrounding certain bacteria and yeasts
1. Require only a single dye. Just for contrast.
a. Examples include malachite green, crystal violet, basic fuchsin, and safranin
b. All cells appear the same color but can reveal shape, size, and arrangement
1. Use two (at least) differently colored dyes, the primary dye and the (36) _counterstain___(we have used safranin)_____
a. Distinguishes between cell types or parts
b. Examples include Gram (positive and negative), acid-fast, and endospore stains
Gram staining (2)
1. The most universal diagnostic (ex. infection) staining technique for bacteria
2. Differentiation of microbes as gram positive(_purple__) or gram negative (__pink___)
Acid-Fast staining (4)
1. Important diagnostic stain
2. Differentiates acid-fast bacteria (__pink_) (CARBOL FUSION - penetrates by heating it up) from non-acid-fast bacteria (_blue__) or specimen cells
3. Important in medical microbiology
4. What acid is in the membrane of acid-fast bacteria that is preventing other stains from penetrating? (41) MYCOLIC ACID
Endospore stain (3)
1. Dye is (42)__forced__ by heat into resistant bodies called spores or endospores
2. Distinguishes between the spores and the cells they come from (the vegetative cells - performing normal functions)
3. Significant in medical microbiology because spores are hard to kill and require no nutrients to survive
1. Used to emphasize certain cell parts that aren't revealed by conventional staining methods
Examples: capsule staining, flagellar staining
Acid = color - / H+ (aka proton)
acid stains don't stain bacteria. reflect off cell wall of bacteria.
Anionic = negative
are cells negative or positive?
cell walls are negative
negative around the outside of proton, so acid (color -) will be attracted
1. crystal violet = simple stain = basic (purple)
2. gram iodine = held together with crystal violet. stays purple
3. decolorization = changes cell wall. Differential step. Clear = negative. Purple = positive
4. Safranin - enters gram positive & gram negative cells. Gram reaction = is positive & negative.
positive staining - appearance & background
staining bacteria cells
appearance of cell = colored by dye
background = not stained, generally white
positive staining - dyes employed (4)
Crystal violet (simple stain)
Methylene blue (counterstain)
safranin (simple stain & counterstain)
Malachite green (simple stain)
Positive staining - subtypes of stains (3)
1. Simple stains
2. Differential stains
a. Gram stain
b. Acid-fast stain
c. spore stain
3. Special stains
e. Nucleic acid
Positive staining - types of special stains (5)
e. Nucleic acid
Acid-Fast staining - Mycolic acid
mycobacteria -----Mycolic acid (in membrane - waxy like substance that doesn't like substances to pass thru)
*1. primary stain (pink) CARBOL FUSION
*2. rinse = acid alcohol - will take away anything that hasn't been stained.
* counterstain methyline blue
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