Proteins, amino acids and enzymes IMViC tests

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1

IMViC test

Indole
Methyl red
Voges proskauer
Citrate

2

Methyl red test

Identifies bacterial ability to produce stable acid end products by mixed acid fermentation of glucose

3

Methyl red test and voges proskauer test medium

MR-VP

4

MR-VP

Soln with onky peptone, glucose and phosphate buffer

5

What do peptone and glucose provide

Protien and fermentatble carb

6

What is the phosphate buffer

Potassium phosphate resists pH changes in medium

7

MR test

Methyl red test
Detects organisms capable of performing mised acid ferm which overcomes phosphate buffer and lowers pH, acids produced are stable

8

How is mixed acid fermentation verified

Addition of methyl red indicator dye
Red at pH 4.4
Yellow at pH 6.2
Between two pH is orange

9

Red color

Only true positive result
Orange is negative or inconclusive
Yellow is negative

10

Mixed acid fermenter

E. coli p 82

11

Voges proskauer test

Identifies organisms able to produce acetoin from degradation of glucose during 2,3 butanediol fermentation

12

What was voges proskauer test designed for

Organisms that are able to ferment glucose but quickly convert acid end products to acetoin and 2,3 butanediol

13

Reagent in vp test

Barritts reagent
40% KOH

14

What happens when reagents added

Oxidizes acetoin (if present) to diacetyl which reacts with guanidine nuclei from peptone in medium and produces red color (positive result)

15

Copper color

Negative vp resulting from interactions between reagents
P 98

16

Citrate test

Used to determine organisms ability to use citrate as sole carbon source

17

Where is citrate used

Citrate (citric acid) is produced as acetyl coa (from oxidation of pyruvate or b oxidation of fatty acids) reacts with oxaloactetate at entry to Krebs cycle
Citrate then converted back to oxaloactetate beginning cycle again

18

What happens in medium containing citrate as only carbon source

Bacteria that have citrate permease can transport the molecules into the cell and enzymatic ally convert it to pyruvate
Pyruvate then converted to a variety of products depending on pH of enviornment

19

Citrate chemistry

P 64 draw image

20

If acidic pH what is formed

Pyruvate decarboxylated to form acetoin, acetate and lactate

21

If alkaline pH what is formed

Pyruvate converted to acetate and formate

22

Simmons citrate agar

Defined medium
Contains sodium citrate as only carbon source
Ammonium phosphate only nitrogen source

23

Bromthymol blue dye

Citrate test reagent
Green at pH 6.9
Blue at pH 7.6

24

What happens to bacteria that survive in bromthymol bkue

Utilize the citrate and a,so convert the ammonium phosphate to ammonia and ammonium hydroxide...both alkalinize the agar
As pH rises medium changes from green to blue

25

Positive citrate test

Conversion of medium to blue

26

No color change

Sometimes citrate positive organism will grow in citrate slant without producing color change bc too short incubation
No color change but growth on slant is positive citrate test

27

Image of citrate results

65

28

What is component in SIM deeps that makes medium suitable to detect production of indole by bacteria

Tryptophan

29

What organic molecule is necessary to detect mixed acid fermentation by bacteria

Glucose

30

Why do you shake the MR-VP CULTURE

to oxygenate the culture

31

Can a bacteria that ferments using the 2,3 butanediol path also use the mixed acid route?

Yes the use of butanediol path decreases acid formation and causes acetoin production

32

Why is chemically defined medium necessary for detection of citrate utilization by bacteria

Because citrate is onky carbon source


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