UNE MICRO LAB FINAL QUESTIONS AND CORRECT
ANSWERS
kirby bauer diffusion test - Answers - most common method of assessing bacteria
antibiotic resistance
filter paper disks containing antibiotics are placed on agar plate with bacteria, antibitouc
diffuses aaway from disk, as bac grow they will be inhibited in regions where the
antibiotic concentration is above what the microorganisms can handle
areas where antibiotic concentration is low will allow for bac growth - growth will lead to
growth of lawn with zone around each disk called zone of inhibition
kirby bauer diffusion test interpretation - Answers - zone of inhibition
- measured in millimeters
- dependent on bac sensitivity to antibiotic and physical antibiotic properties
staining techniques - Answers - simple stain: one dye reveals shape, size, and
arrangement
direct stain: type of simple stain, only bacterial cell is colored leaving background clear
and colorless
negative stain: type of simple stain, cell is unstained and appears bright against dark-
stained environment
differential stain: 2 or more dyes used, allows to view cell morp and flagelle, capsulres,
and endospores
Gram stain procedure - Answers - 1. Primary stain (Crystal violet- a basic stain, purple
dye)
2. Mordant (Gram's iodine- I2 + KI)
(something that helps fix dye on or in a cell but does not itself contribute color)
3. decolorizing agent (Ethanol or acetone) (dehydrates gram pos cell, traps in dye,
washes dye from gram neg)
4. Counterstain (Safranin- a basic stain) ( no effect on gram pos, causes gram neg to be
pink)
3 main types of microscopes - Answers - optical or light microscopes: uses reflected
light and series of two or more convex lenses to magnify and focus (compound)
electron
, scanning probe
parts of microscope - Answers - ocular lens: short, look through, eye piece (10x or 15x
power)
objective lens: closest to stage, held by turret or revolving nose piece (4x, 10, 40x) -
100x is oil immersion lens
condensor: sit below stage, focuses light from illuminator onto slide
diaphragm: sits below stage and condensor, lets little light through, varies the field for
viewing of specimen
depth of field - Answers - distance range in focus at a given time (dependent on total
mag factor)
proper aseptic technique - Answers - techniques to prevent the accidental or inadvertent
spread of microorganisms beyond the intended work surface (maintain safety, isolate
pure cultures, prevent spread of unwanted microbes)
1. wash hands
2. sterilize with 10% bleach solution or bleach based cleaner
3. sterilize equipment (open flame or isopropyl alc)
4. incubate plates inverted or agar side up
streak plate isolation - Answers - Bacteria transferred from plate-to-plate or liquid-to-
plate using heat-sterilized metal loop.
ISOLATES 1 COLONY
FOV (field of view) calculation - Answers - measure diameter of length of field from one
of the circle to the other
record FOV in mm
convert to micrometers (1 mm = 1000 micrometers)
High power lens calc
low power fov x (TM of low power/TM of high power)
total magnification factor - Answers - ocular lens mag x objective lens mag (higher total
mag factor = smaller FOV)
viable plate count calculation or cf/ml cal - Answers - viable plate contains 30-300 CFU's
(CFu: each colony on a plate)
ANSWERS
kirby bauer diffusion test - Answers - most common method of assessing bacteria
antibiotic resistance
filter paper disks containing antibiotics are placed on agar plate with bacteria, antibitouc
diffuses aaway from disk, as bac grow they will be inhibited in regions where the
antibiotic concentration is above what the microorganisms can handle
areas where antibiotic concentration is low will allow for bac growth - growth will lead to
growth of lawn with zone around each disk called zone of inhibition
kirby bauer diffusion test interpretation - Answers - zone of inhibition
- measured in millimeters
- dependent on bac sensitivity to antibiotic and physical antibiotic properties
staining techniques - Answers - simple stain: one dye reveals shape, size, and
arrangement
direct stain: type of simple stain, only bacterial cell is colored leaving background clear
and colorless
negative stain: type of simple stain, cell is unstained and appears bright against dark-
stained environment
differential stain: 2 or more dyes used, allows to view cell morp and flagelle, capsulres,
and endospores
Gram stain procedure - Answers - 1. Primary stain (Crystal violet- a basic stain, purple
dye)
2. Mordant (Gram's iodine- I2 + KI)
(something that helps fix dye on or in a cell but does not itself contribute color)
3. decolorizing agent (Ethanol or acetone) (dehydrates gram pos cell, traps in dye,
washes dye from gram neg)
4. Counterstain (Safranin- a basic stain) ( no effect on gram pos, causes gram neg to be
pink)
3 main types of microscopes - Answers - optical or light microscopes: uses reflected
light and series of two or more convex lenses to magnify and focus (compound)
electron
, scanning probe
parts of microscope - Answers - ocular lens: short, look through, eye piece (10x or 15x
power)
objective lens: closest to stage, held by turret or revolving nose piece (4x, 10, 40x) -
100x is oil immersion lens
condensor: sit below stage, focuses light from illuminator onto slide
diaphragm: sits below stage and condensor, lets little light through, varies the field for
viewing of specimen
depth of field - Answers - distance range in focus at a given time (dependent on total
mag factor)
proper aseptic technique - Answers - techniques to prevent the accidental or inadvertent
spread of microorganisms beyond the intended work surface (maintain safety, isolate
pure cultures, prevent spread of unwanted microbes)
1. wash hands
2. sterilize with 10% bleach solution or bleach based cleaner
3. sterilize equipment (open flame or isopropyl alc)
4. incubate plates inverted or agar side up
streak plate isolation - Answers - Bacteria transferred from plate-to-plate or liquid-to-
plate using heat-sterilized metal loop.
ISOLATES 1 COLONY
FOV (field of view) calculation - Answers - measure diameter of length of field from one
of the circle to the other
record FOV in mm
convert to micrometers (1 mm = 1000 micrometers)
High power lens calc
low power fov x (TM of low power/TM of high power)
total magnification factor - Answers - ocular lens mag x objective lens mag (higher total
mag factor = smaller FOV)
viable plate count calculation or cf/ml cal - Answers - viable plate contains 30-300 CFU's
(CFu: each colony on a plate)
