Lab Exam 2

15-

1. TNTC is a plate w/ >_____

2. TFTC is a plate w/ <_____

1. 300

2. 30

What is the DF after two 1:10 (10^-1) dilutions and then a 1:100 (10^-2) dilution? Draw out the dilution steps

15- serial dilution equation

(#CFU/mL) = (#Colonies) x (DF) / (mL)

15- Verbally:

1. 1E6 = “_____”

2. 1E7 = ____E6 = “ _____”

3. 1E9 = “_____”

4. 9.9E11 = ____E9 = “_____”

1. one million

2. 10 ; ten million

3. one billion

4. 990 ; nine hundred ninety billion

16- LBS Agar:

1. what experiment?

2. What is it used for?

3. carbon source?

4. nitrogen source?

5. fatty acid/lipid source?

6. why selective?

1. kefir

2. isolation and enumeration of lactobacilli from foods

3. glucose

4. ammonium salt

5. polysorbate 80

6. ammonium citrate lowers pH 5.5 to select for lactobacilli

17- Starch Plate:

1. visualize what?

2. what is it/reaction?

3. microbe that is + ?

1. starch hydrolysis

2. amylase breaks glycosidic bonds in extracellular starch

3. B. cereus = amylase+

17- amylase:

1. what is it?

2. reaction drawn out?

3. 2 steps how test for?

4. what microbe is +?

1. exoenzyme that breaks glycosidic bonds in extracellular starch

2. starch -(amylase)→ glucose

3. Starch agar + flood w/ iodine

4. B. cereus

17- Tributyrin Agar:

1. visualize what?

2. what enzyme and what bond is it breaking

3. reaction?

4. microbe that is +

1. lipid hydrolysis

2. lipase breaks ester bond in extracellular lipids

3. tributyrin -(lipase)→ glycerol + fatty acids

4. B cereus+

17- lipase:

1. what is it?

2. reaction drawn out?

3. what agar?

4. what microbe is +?

1. lipase breaks ester bond in extracellular lipids

2. triglyceride -(lipase)→ glycerol + fatty acids

3. Tributyrin agar

4. B cereus+,

17- Milk Agar:

1. visualize what?

2. what is it?

3. microbe that is +

1. casein hydrolysis

2. Caseinase + proteinase breaks a peptide bond in extracellular casein + protein

3. B cereus+

17- caseinease:

1. what is it?

2. reaction drawn out?

3. what agar?

4. what microbe is +?

1. breaks peptide bonds in extracellular casein

2. casein peptide bond -(caseinase)→ amino acids

3. Milk agar

4. B cereus+

17- Gelatin Agar:

1. visualize what?

2. what is it?

3. microbe that is +

4. 3 enzymes in total

1. gelatin hydrolysis

2. gelatinase breaks peptide bonds in extracellular gelatin

3. B cereus+

4. gelatinase, lipase, bromelain

17: gelatinase:

1. what is it?

2. reaction drawn out?

3. what agar?

4. what microbe is +?

1. gelatinase breaks peptide bonds in extracellular gelatin

2. gelatin peptide bond -(gelatinase)→ amino acids

3. Gelatin Agar

4. B cereus+

17: DNA Agar:

1. visualize what?

2. what is it?

3. indicator?

4. microbe that is +

1. DNA hydrolysis

2. DNase breaks phosphodiester bonds in DNA

3. methyl green

4. S. marcescens

17: DNase:

1. what is it?

2. what agar?

3. what microbe is +?

1. DNase breaks phosphodiester bonds in DNA

2. DNA agar

3. S. marcescens

17- Optimal Growth Temps:

1. psychrophiles =

   1. optimal =

2. mesophiles =

   1. optimal =

3. facultative thermophiles=

   1. optimal =

4. obligate thermophiles =

   1. optimal =

5. Serratia pigmentation =

6. fungi/yeast =

1. psychrophiles = -10-25

   1. optimal = 0-5

2. mesophiles = 20-45

   1. 35-40 optimal

3. facultative thermophiles = 35-60

   1. 45-60 optimal

4. obligate thermophiles =50+

   1. 60+ optimal

5. Serratia pigmentation = 23 (room temp)

6. fungi/yeast = 30C

1. Serratia pigmentation =

2. fungi/yeast =

1. Serratia pigmentation = 23 (room temp)

2. fungi/yeast = 30C

1. obligate thermophiles =

   1. optimal =

1. obligate thermophiles =50+

   1. 60+ optimal

1. facultative thermophiles=

   1. optimal =

1. facultative thermophiles = 35-60

   1. 45-60 optimal

1. psychrophiles =

   1. optimal =

1. psychrophiles = -10-25

   1. optimal = 0-5

17- General optimal growth pHs:

1. acidophile = 3

2. neutrophile = 7

3. alkaliphile = 9

4. fungi = 5

19-Carbohydrate Fermentation:

1. what 2 broths?

2. extra equipment?

3. results?

1. Phenol Red Broth: Yellow = fermentation,, red = none

2. Dextrose (glucose) Broth: Yellow = fermentation,, red = none

3. Air bubble in Durham tube = CO2 product

19- Phenol Red Broth:

1. test for what?

2. based on what?

3. results?

1. Lactose/dextrose Fermentation based on pH from acid production: Yellow = (+), Red = (-)

19- IMViC Tests:

1. what is the purpose of these tests?

2. what are the four tests?

3. what question does each test answer?

1. How to distinguish Enterics (Human GI bacteria in Enterobacteriaceae)

2. Tests:

   1. Indole Test

      1. degrade tryptophan into indole?

   2. Methyl Red (MR) Test

      1. ferment glucose into organic acids?

   3. Voges-Proskauer (VP) Test

      1. ferment glucose into nonacidic or neutral products?

   4. Citrate Test

      1. import citrate?

19- Indole Test:

1. what does it test for?

2. 2 steps?

   1. Results?

3. Drawn out reaction?

4. microbe that is +?

1. degrades tryptophan into indole?

2. culture in SIM medum (deep) for 2 days. Add Kovac’s reagent

   1. Red at top = +

3. tryptophan -(tryptophanase)→ indole

4. e. coli

“e. col = indol”

19- Kovac’s reagent:

1. what test uses it

2. what does it do?

1. indole test

2. indicator that turns red if indole is present

19- tryptophanase:

1. what test uses it

2. what reaction

1. indole test

2. tryptophan -(trypophanase)→ indole + pyruvate + ammonia

19- Methyl Red (MR) Test

1. what does it test for?

2. how is the test done?

   1. Results?

3. Indicator?

4. what other test is it paired w/ and why?

1. ferment glucose into organic acids (Lactic)?

2. culture in MR-VP broth

   1. red color = (+), yellow = (-)

3. MR pH indicator

4. paired w/ VP test bc both start w/ incubating in MR-VP broth

19- Voges-Proskauer (VP) Test:

1. what does it test for?

2. 2 products?

3. 3 steps

   1. what does each step indicate?

4. what other test is it paired w/ and why?

1. ferment glucose into neutral products (acetylmethylcarbinol “ABC” (buttery odor) + 2-3 butanediol)?

2. Steps

   1. culture in MR-VP broth

   2. Add Barritt’s Solution (a-napthol + 40% potassium hydroxide)

   3. Add Guanidine group

19- products being tested for:

1. MR Test

2. VP Test

1. MR = organic acids

2. VP test = neutral

   1. 2,3-butanediol

   2. acetylmethylcarbinol “AMC” (buttery odor)

19- Citrate Test:

1. what’s it test for?

2. what agar?

   1. carbon source

   2. nitrogen source?

   3. indicator, what does it detect?

3. results?

1. citrate permease (transporter protein)

2. citrate agar

   1. C = sodium citrate

   2. N = ammonium salts

   3. indicator = bromothymol blue (detects basic)

3. blue = (+), green = (-)

“bro, mo citrus please”

19- Hydrogen Sulfide Test:

1. what agar?

2. 3 things it is testing for?

   1. draw reactions for both?

3. indicator

   1. reaction for indicator

4. results?

1. SIM Agar

2. Tests

   1. reduces cysteine?

      1. cysteine -(cysteine desulfurase)→ pyruvate + H₂S

   2. reduces inorganic sulfur?

      1. thiosulfate -(thiosulfate reductase)→ sulfite + H₂S

   3. agar also shows motility

3. Ferrous sulfate indicates H₂S

   1. Ferrous sulfate + H₂S = FeS (black precipitate)

4. Black precipitate = (+), colorless = (-)

19- SIM Agar:

1. used in what 2 tests

1. indole and

2. hydrogen sulfide tests

19- In chemistry, what is LEO GER?

Loss of e- = Oxidation

Gain e- = Reduction

20- Urease Test:

1. identifies what microbe?

2. reaction?

3. indicator?

4. results

1. Proteus Vulgaris

2. urea -(urease)→ Ammonia NH₃

3. phenol red pH indicator

4. ammonia = alkaline = deep pink color

“peenol red”

20- Catalase Test:

1. what testing for?

2. reaction?

1. catalase

2. H2O2 -(catalase)→ H20 + O2

20-

1. what are SOD and catalase needed for?

2. 2 coupled reactions?

1. life in the presence of O2

2. ROS -(SOD)→ H2O2 + O2

3. H2O2 -(catalase)→ water + O2

20- Decarboxylation Test:

1. what’s it testing for/under what condition?

   1. how condition met?

2. what agar?

3. reaction?

4. indicator/what type?

5. results?

1. removal of carboxyl group (COO-) under anaerobic conditions

   1. layer of glycerol added to block O2

2. Moeller’s decarboxylase broth

3. lysine -(lysine decarboxylase)→ cadaverine + CO2

4. brom cresol blue (acid pH turns yellow, neutral/basic = purple)

5. yellow at 24h → purple at 48h = (+)

20- decarboxylase:

1. what type of enzyme?

2. what does that mean for this enzyme?

3. 2 steps + timing + results?)

1. decarboxylases are inducible enzymes

2. enzyme won’t activate until acidic 5pH reached

   1. microbe must use glucose to make media acidic

      1. first 24H, (starts purple → yellow)

   2. once acidic, decarboxylase is induced and can convert lysine into cadaverine + CO2

      1. second 24 hours (yellow → back to purple)

1. alternative decarboxylation enzyme/reaction (not lysine →cadaverine)

2. what type of enzyme?

1. omithine -(omithine decarboxylase)→ putrescine

2. decarboxylases are inducible enzymes

20- Phenylalanine deaminase Test:

1. what testing for?

2. reaction?

3. indicator?

4. results?

1. deamination of phenylalanine

2. phenylalanine -(phenylalanine deaminase)→ keto acid (phenylpyruvic acid) + ammonia

3. Ferric chloride FeCl3 detects keto acid

4. green = (+)

“dee-am and kee-ac” “phenyl = ferric”

20- ONPG Test

1. enzyme ?

2. natural reaction?

3. reaction w/ ONPG?

   1. results

1. b-galactosidase

2. Lactose →glucose + galactose

3. ONPG → b-D-galactose + a-nitrophenol

   1. nitriophenol is yellow = (+)

e. coli, shigella, proteus, and Citrobacter are + for what 3 tests?

indole, MR, VP

What 4 tests distinguish b/t e. coli, shigella, proteus, and Citrobacter?

Citrate, H2S, Urease, swarming

citrate test for e. coli, shigella, proteus, and Citrobacter:

1. who is +?

2. what result?

1. Proteus = +

2. + = blue

H2S test for e. coli, shigella, proteus, and Citrobacter:

1. who is +?

2. what result?

1. Citrobacter and Proteus are +

2. + = black

Urease test for e. coli, shigella, proteus, and Citrobacter:

1. who is +?

2. what result?

1. Citrobacter and Proteus are +

2. + = deep pink

how to distinguish b/t citrobacter and proteus?

proteus has swarming motility