Lab

Question 1

resolving power = ____

Answer 1

light wavelength/ (2 x NA-numerical aperture)

Question 2

microscope steps

Answer 2

1) turn on - 3 blue LED lights
2) push iris lever to left -> allow the most light
3) raise stage up on 4x lens
4) raise condenser up
5) close iris diaphragm until we see only a point of light
6) center the light in middle with screws
7) lower condenser until light is crisp
8) lower condenser slightly until under stage
9) reopen iris diaphragm

Question 3

microscope clean up checklist

Answer 3

-clean 100x lens
-light turned off
-stage lowered
-4x lens
-eyepieces wiped with kimwipe & ethanol
-unplug

Question 4

gram stain slide steps

Answer 4

1) large circle on underside of slide with grease pencil
2) flame loop
3) broth: one loop of broth; plate: 1 loop of H2O + part of small colony
4) flame loop
5) dry smears
6) heat fix by passing through flame

Question 5

gram staining steps

Answer 5

1) wear gloves
2)1-2 drop of crystal violet for 1 min
3) wash with H2O
4) 1-2 drops of iodine for 1 min
5) wash with H2O
6) wash ethanol (8-10 sec)
7) wash with H2O gently
8) 1-2 drops of safarinin for 1 min
9) wash with H2O & pat dry & remove gloves

Question 6

catalase test

Answer 6

-determines presence of catalase (protects cell from H2O2); bubbles formed (+)
-aseptically place heavy colony in H2O2

Question 7

oxidase test

Answer 7

-determines presence of cytochrome c oxidase (part if ETC); TMPD is e- donor -> when oxidized it is blue/purple
-aseptically place heavy colony in paper + 2-3 drops of TMPD

Question 8

ONPG test

Answer 8

-determines ability to hydrolyze lactose; uses ONPG (similar to lactose) as substrate of B-galactosidase -> product is yellow
-aseptically place heavy colony in ONPG, incubate @ 37C for 1 hour max

Question 9

urease test

Answer 9

-determines presence of urease; urea hydrolyzed to produce ammonia -> rise in pH, indicator turns cerise
-aseptically place heavy colony in urea, incubate @ 37C for 1 hour max

Question 10

DMCA test

Answer 10

-determines presence of tryptophanase; DMCA lyses cells to release indole (made during tryptophan metabolism) -> indole reacts with DMCA -> blue-green colour
-aseptically place heavy colony in paper + 2-3 drops of DMCA

Question 11

EMB Agar (can you ferment lactose?)

Answer 11

mixed acid fermentation - blue/metallic green (lactose +)
butanediol - fish eye (lactose +)
lactose non-fermenters - pale/colourless/ pale pink/red (lactose -)

Question 12

MAC Agar (can you ferment lactose?)

Answer 12

bright pink (lactose fermenter)
pinkish tan gelatinous (lactose fermenter)
small circular white/ very pale pink (lactose non-fermenter)

Question 13

(gram -) oxidase -, onpg -, urease +, dmca +

Answer 13

proteus vulgaris

Question 14

(gram -) oxidase -, onpg +, urease -, dmca +

Answer 14

escherichia coli

Question 15

(gram -) oxidase -, onpg +, urease -, dmca -

Answer 15

klebsiella aerogenes

Question 16

(gram -) oxidase +, onpg -

Answer 16

pseudomonas

Question 17

bile-esculin test

Answer 17

-grow presence of bile & hydrolyze esculin ; hydrolysis produces glucose -> agar turn brown-black
-aseptically streak heavy colony on slant, incubate @ 37C for 1 hour max

Question 18

BAP test (blood hemolysis)

Answer 18

-gram + cocci produce hemolysins (lyse (sheep) RBC)
-streak on BAP + parafilm + incubate 37C
-a hemolysis (green zones around colonies)
-B hemolysis (clearing or lysing of RBC)
-y hemolysis (no clearing of RBC)

Question 19

MSA (can you ferment mannitol)

Answer 19

-NaCl, mannitol, pH indicator phenol red
-mannitol ferment. -> acidic products -> red to yellow media
-non-path staph doesn’t ferment mannitol -> media remained red
-

Question 20

( gram +) catalase +, B-hemolysis (clearing)

Answer 20

Staphylococcus aureus

Question 21

( gram +) catalase +, Y hemolysis (no clearing)

Answer 21

Staphylococcus epidermis

Question 22

( gram +) catalase -, bile esculin -, B-hemolysis (clearing)

Answer 22

Streptococcus pyogenes

Question 23

( gram +) catalase -, bile esculin +, a/Y-hemolysis (greening/no clearing)

Answer 23

Enterococcus faecalis

Question 24

(gram +) MSA yellow medium

Answer 24

Staphylococcus aureus, Enterococcus faecalis

Question 25

(gram +) MSA red medium

Answer 25

Staphylococcus epidermis

Question 26

enrichment broth for E. faecalis

Answer 26

2X azide dextrose borth

Question 27

E. coli presumptive test? confirmed test? completed test?

Answer 27

-acid + gas in 2x lactose broth, green colonies in EMB, gram - rods

Question 28

E. faecalis presumptive test? confirmed test? completed test?

Answer 28

-2x azide dextrose broth, red colonies on KF, gram + cocci in chains, catalase -