Lab 9: Media Demonstrating Unique Enzymes

Question 1

1) Are the SM110 (staphylococcus medium) and MS (mitis salivarius) media liquid or solid?
2) What color was the MS medium?
3) What was done with the SM110 and MS media?

Answer 1

1) Solid
2) Blue
3) Streaked for isolated colonies

Question 2

1) Were the NO3 (nitrate), TSB (coagulase), and UREA (urease) media solid or liquid?
2) What was done with them?

Answer 2

1) Liquid
2) Loopful into broth

Question 3

1) Were the DNase and Starch (amylase) media solid or liquid?
2) What was done with them?

Answer 3

1) Solid
2) A line inoculation

Question 4

1) Were the GEL (gelatinase) and SIM (sulfide indole motility) media solid or liquid?
2) What was done with them?

Answer 4

1) Solid
2) A stab inoculation using a plain piece of wire; all the way to bottom of tube

Question 5

1) Was the PHE (phenylamine deaminase) media solid or liquid?
2) What was done with it?

Answer 5

1) Solid
2) Streaked the surface of the slant

Question 6

1) Was the LYS (lysine decarboxylase) media solid or liquid?
2) What was done with it?

Answer 6

1) Liquid
2) Loopful into broth; add 1ml of mineral oil on top

Question 7

1) Was the TSA (catalase) media solid or liquid?
2) What was done with this media?

Answer 7

1) Solid
2) Streak for isolated colonies

Question 8

SM-110 test:
1) What is this test selective for? How?
2) What is this test differential for?
3) What does the mannitol do?
4) What enzyme is being observed and what’s added to see it?
5) What indicates a positive result?
6) What has yellow-orange colonies?

Answer 8

1) Selective for G+ bacteria
2) Differential for staph
3) Differentiate between SA and SE (SA+ coagulase; it can use fructose-phosphate from mannitol)
4) Gelatin allows gelatinase to be observed by adding ammonium sulfate
5) A halo around the colonies indicates (+)
6) Sa has yellow-orange colonies

Question 9

Nutrient gelatin test:
1) What does it test for?
2) What needs to be done to the tubes during this test?
3) What is a positive result and what is a negative result?

Answer 9

1) The degradation of gelatin by gelatinase
2) Place tubes in the fridge after incubation
3) Stays liquid = (+).
Solidifies if = (-)

Question 10

MS (mitis salivarius) test:
1) What is this test selective for?
2) What is this test differential for?
3) What are this test’s ingredients and which is toxic to G-?
4) What species’ colonies look like gumdrops on this media?
5) What species has small dark blue colonies on this media?
6) What inhibits other G+ from growing on this media?

Answer 10

1) Selective for G+
2) Differential for strep (mannitol)
3) 5% sucrose, CV, and tellurite. Tellurite is toxic to G-
4) Salivaris looks like gumdrops
5) E. faecalis colonies are small and dark blue
6) Crystal violet

Question 11

SIM (sulfide indole motility) test:
1) What is this test differential for?
2) What is this test media made of?
3) Which reacts with H2S and what does this create?
4) What does a clean stab in this test allow for?
5) How can you test for indole production with this media? (what is added, and what indicates + or -?)

Answer 11

1) Differential for G- (EC+; EA-)
2) Casein peptone, meat peptone, sodium thiosulfate, ferric ammonium citrate, agar
3) Ferrous iron reacts with H2S to make FeS (black).
4) Interpretation of motility
5) Add 8-10 drops of Korac’s solution (1m):
a) Tannish yellow = (-).
b) Reddish purple = (+), indicates tryptophan has broken down into indole

Question 12

NO3 (nitrate) test:
1) What are its three ingredients?
2) Which two of its ingredients react with NO2 to make the red color?
3) What should be added if there’s no red color after adding the primary ingredients? Why?
4) What are the 3 possible results?

Answer 12

1) Nitrate A, nitrate B, and zinc
2) Nitrate A and nitrate B react with NO2 and turn red (nitrate reduction)
3) Add zinc if there’s no red color after nitrate A&B; will react with any NO3- left and turn bright red (shows that nothing happened).
4) -Red color before zinc: nitrate reductase (now NO2-)
-Red color after zinc: nothing (it’s still NO3)
-Colorlessness after zinc: denitrification (now N2)

Question 13

Urea (urease) test:
1) What reaction occurs during this test? Does it increase or decrease pH?
2) What is the pH indicator in this test?
3) What indicates a positive result?
4) What indicates a negative result?

Answer 13

1) Urea is broken down by urease and becomes NH4 and CO2. Increases pH.
2) Phenol red
3) Pink/ purple = positive (+)
4) Yellow = negative (-)

Question 14

LYS (lysine decarboxylase) test:
1) A bacteria must be what in order to do this test?
2) Why is only a little 0.1% glucose used in this test?
3) What reaction occurs during this test? What does this do to pH?
4) What is this test’s pH indicator?
5) What indicates a positive result in this test?
6) What indicates a negative result?

Answer 14

1) Must be a glucose fermenter
2) So it gets used up and forces bacteria to use lysine
3) Lysine is broken down by lysine decarboxylase into CO2, which raises pH.
4) Bromocresol purple is the pH indicator
5) Purple/black = (+), lysine degradation
6) Yellow = (-)

Question 15

TSB (coagulase) tests:
1) What is this test selective for?
2) What two things are added into a tube before incubating at 37 for an hour?
3) What are you looking to observe after incubation?

Answer 15

1) Selective for G+ cocci
2) 0.5ml culture and 0.5ml rabbit plasma
3) Looking for a clot in the tube

Question 16

DNase test:
1) What question does this test answer?
2) What does this test contain?
3) What can be added to make this cloudy? What does a clear zone near cloudiness mean?
4) What is a positive result and what is a negative result?

Answer 16

1) Does bacteria degrade DNA (does it have DNase?)
2) DNA and methyl green indicator
3) 0.1M HCl can be used to precipitate DNA; becomes cloudy near DNA. A clear zone near cloudiness indicates DNase is there (+).
4) Becomes clear = positive (+). Stays green = negative (-)

Question 17

Nutrient starch test:
1) What question is being answered with this test?
2) What are the ingredients of this test?
3) Is there a built-in indicator?
4) What indicates a positive result?
5) What indicates a negative result?

Answer 17

1) Does the bacteria have amylase?
2) Beef extract, soluble starch, agar, grams iodine (turns purple)
3) No built-in indicator
4) Becomes clear = positive (+), has amylase.
5) Stays purple from iodine = negative (-).

Question 18

PHE (phenylalanine deaminase) test:
1) What question is being answered by this test?
2) What reaction takes place during this test?
3) What reacts with PPA? What color is does this turn PPA?
4) What indicates a positive result?
5) What indicates a negative result?

Answer 18

1) Does the bacteria have phenylalanine deaminase?
2) Phenylalanine is broken down into NH4 and PPA.
3) Adding 10% FeCl3 reacts with PPA. PPA reacts with FeCl3 to become dark green.
4) Turns dark green = positive (+), has phenylalanine deaminase
5) Stays orange/ tan = negative (-)

Question 19

TSA (catalase) test:
1) What idea does this test use?
2) What is added onto colonies?
3) What indicates a positive result?

Answer 19

1) The idea that aerobic bacteria must degrade toxic H2O2 (hydrogen peroxide) with the enzyme catalase
2) Hydrogen peroxide is dropped onto colonies
3) Bubbling = positive (+), catalase is present

Question 20

Give 3 examples of extracellular enzymes

Answer 20

1) DNase
2) Gelatinase
3) Amylase

Question 21

Give examples of intracellular enzymes

Answer 21

1) Phenylalanine deaminase
2) Nitrogenase