Lab 3 test

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What does EMB stand for?

Eosin Methylene Blue

Is this media selective, differential, or both?

Selective and differential

It is selective for what bacteria? (what grows?)

G (-)'s

It is selective against what bacteria? (what doesn't grow?)

G (+)'s

What compounds in the medium inhibit certain bacteria (what makes it selective)?

tolerence of bile salts and methylene blue & eosin byes

How is this medium differential? Based on...

lactose fermentation

This media differentiates between what 2 groups of bacteria?

lactose fermenters & lactose nonfermenters

Specifically, how do E. coli colonies appear on this media?

Greenish metallic sheen

MacConkey test

Is this media selective, differential, or both?

Differential & selective

It is selective for what bacteria? (what grows?)

G (-)'s

It is selective against what organisms? (what doesn't grow?)

G (+)'s

What compounds in the medium inhibit certain bacteria (what makes it selective)?

tolerance of crystal violet dye and bile salts

How is this medium differential? Based on..

lactose fermentation

This medium differentiates between what 2 groups of bacteria?

lactose fermenters and lactose nonfermenters

What is the name of the pH indicator in this medium?

neutral red

This indicator turns _______when the pH is acidic.

Hot pink colonies

How do lactose-fermenting colonies appear on this medium?

Hot pink colonies

How do nonlactose-fermenting colonies appear on this medium?

colorless colonies

Why is MacConkey agar preferred to EMB?

Because it inhibits the swarming of Proteus

Who are the lactose fermenters?

Escherichia coli; Enterobacter cloacae; Klebsiella pneumoniae (EEK)

Who are the lactose nonfermenters?

Proteus vulgaris; Pseudomonas aeruginosa; Salmonella typhimurium; Shigella flexneri (P2S2)

Phenol Red Broth - Carbohydrate Fermentation

Is this medium selective, differential, or both?

Differential

How is it differential? Based on....

sugar fermentation

Is fermentation anaerobic or aerobic?

Anaerobic

What 2 end products of fermentation are we testing for with this medium?

acid & carbon dioxide gas

Name the 3 sugars used in this lab exercise?

Sucrose, glucose, lactose

What is the purpose of the Durham tube?

To trap gas

What is the name of the pH indicator in this medium?

Phenol Red

This pH indicator is _______color when neutral and _______(color) when acidic.

Red;
yellow

Phenol Red broth results -

Pseudomonas aeruginosa: Red (-), Red (-), Red (-) = no sugars fermented
Shigella flexneri: Red (-), Yellow (+), Red (-) = 1 sugar fermented (Glucose)
Salmonella typhimurium: Red (-), yellow (+), Red (-) = 1 sugar (Glucose)
Proteus vulgaris: Yellow (+), Yellow (+), Red (-) = 2 sugars fermented (Sucrose & Glucose)
Escherichia coli: Red (-), Yellow (+), Yellow (+) = 2 sugars fermented (Glucose & Lactose)

What does TSI stand for?

Triple Sugar Iron

Is this media selective, differential, or both?

differential

How is this medium differential? (2 ways)

1.) Based on Sugar fermentation
2.) Based on Sulfer reduction (H2S production)

What are the 3 sugars used in this medium? Why this instead of phenol red broth?

Lactose, sucrose, glucose
(Much more efficient than using 3 seperate tubes as in Phenol Red Broth tests)

This medium is used to differentiate what group of bacteria?

Lactose Nonfermenters (P2S2)
Pseudomonas aeruginosa, Proteus vulgaris, Salmonella typhimurium, Shigella flexneri

What is the name of the pH indicator in this medium?

Phenol Red

How is sugar fermentation observed (what do you look for in the tube?)

Yellow in the tube (If 2 or more sugars are fermented then butt and slant turn yellow, if it's only glucose then butt turns yellow & slant reverts red

Some bacteria reduce sulfates in the process of anaerobic respiration (remember sulfates being the final electron acceptor in electron transport chain?); we call them 'sulfur reducers." In this process sulfate is reduced to hydrogen sulfide (H2S). H2S then combines with an __________ indicator in the medium to produce a __________ (color) precipitate in the ___________(butt or slant) of the tube.

phenol red;
black;
butt

Why are we looking here for this precipitate?

this is where anaerobic bacteria grow

Results of TSI

Detail of results

1.) Control
2.) Pseudomonas aeruginosa (red slant/ red butt - no sugars fermented or sulfur reduction)
3.) Shigella flexneri (red slant/ yellow butt - 1 sugar: Glucose, no sulfur reduction)
4.) Escherichia coli (yellow slant/ yellow butt - 2 sugars: Glucose & lactose, no sulfur reduction)
5.) Proteus vulgaris (yellow slant/ yellow butt - 2 sugars: sucrose & glucose, sulfur reducer)
6.) Salmonella typhimurium (red slant/ yellow butt - 1 sugar: glucose, sulfur reducer)

How are Salmonella & Proteus different from the rest of the group (Pseudomonas, Shigella, E. coli)?

They are both sulfur reducers

List the characteristics shared by Proteus & Salmonella

Proteus - yellow butt, yellow slant - ferments 2 sugars (sucrose & glucose), black precipitate - sulfur reducer
Salmonella - yellow butt, red slant - ferments 1 sugar (glucose), black precipitate - sulfur reducer

Normally TSI is not run on lactose fermenters (EEK group). So why did we include them in this lab activity?

To help you see how they are different & show what a wrong turn or contamination would look like (yellow, yellow & no black butt)

IMViC tests - What does the acronym IMViC stand for?

I = indole, M = Methyl Red, V = Voges - Proskauer tests (MR - VP), C = Citrate (test)

How many tests are included in the series?

4 tests

Is this test selective, differential or both?

differential

This test is helpful in differentiating between what 3 bacteria? (give species names)

EEK = Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae

Knnow the results (positive or negative) in order for the 2 groups of organisms for each of the tests

E. coli: + + - -
Enterobacter & Klebsiella: - - + +

Indole test (uses Tryptone broth)

(same test you do with SIM's medium, so we wont use SIM's)

Bacteria metabolize tryptophan into____________

Indole and pyruvic acid

What kind of molecule is tryptophan?

amino acid

What reagent is added after incubation?

Kovac's reagent

What indicates a positive result?

presence of indole/ red ring at top
(+ Escherichia coli) (- Enterobacter cloacae & Klebsiella pneumoniae)

Methyl Red test (MR) Why is it used?

This test is differential based on the fermentation of glucose into multiple acids.

What is the name of the pH indicator added after incubation?

Methyl red

In this test, the pH indicator turns _________ in the presence of acid.

red

What color indicates a positive result?

Red
(+ Escherichia coli) (- Enterobacter cloacae & Klebsiella pneumoniae)

Voges-Proskauer test (VP) Why is this test used?

This test is differential based on a type of alcoholic fermentation

What 2 reagents are added after incubation?

Barritt's A & Barritt's B

What color indicates a positive result?

Rose/red color
(+ Enterobacter cloacae & Klebsiella pneumoniae) (- Escherichia coli)

VP results should always be ___________ (opposite of or the same as) MR results

same as

Citrate test - Why is it used?

This test is differential based on the utilization of citrate as the sole source of carbon.

What color is the medium to begin with?

green

What is the name of the pH indicator in the medium?

bromthymol blue

This indicator turns _________in the presence of a base, indicating a positive result.

blue
(+ Enterobacter cloacae & Klebsiella pneumoniae) (- Escherichia coli)

Results of IMViC test

Escherichia coli
I + M + Vi - C -
Enterobacter cloacae & Klebsiella pneumoniae
I - M - Vi + C +

Urea Hydrolysis Confirmation

Is this medium selective, differential, or both?

differential

This test is helpful in confirming what 2 bacteria? (give species names)

Proteus vulgaris & Salmonella typhimurium

How is it differential? Based on....

production of urease & hydrolysis of urea

What 6 characteristics are shared by the 2 organisms listed above?

G (-)'s, lactose nonfermenters, Glucose fermenters, sulfur reducers/H2S production, Rod shaped (bacillus)

What is the chemical reaction for urea hydrolysis?

Urea ----} ammonia + CO2

What end product of urea hydrolysis are we testing for?

Ammonia (bad smell)

Is this end product an acid or a base?

base pH (went up)

What is the name of the enzyme produces by the bacteria to hydrolyze the urea?

Urease

What is name of the pH indicator in this medium?

phenol red

This pH indicator is _______ (color) when neutral, __________ (color) when acidic, & ___________(color) when basic or alkaline

red;
yellow (salmon);
Hot pink

Results:

Salmonella typhimurium - yellow (salmon) - Urea Hydrolysis
Proteus vulgaris - Hot pink + Urea Hydrolysis

Ornithine Decarboxylation test

A. Enterobacter cloacae - purple (+)
B. Original color of test
C. Klebsiella pneumoniae - yellow (-)

Is this medium selective, differential, or both?

Differential

How is it different? Based on....

Ornithine decarboxylation

What kind of molecule is ornithine?

amino acid

This test is helpful in differentiating between what 2 organisms? (give species name)

Enterobacter cloacae & Klebsiella pneumoniae

What 5 characteristics are shared by the 2 organisms listed above?

G (-)'s, bacillus, sucrose, lactose non-fermenters, Both have same IMViC results (- - + +), enteric normal flora

What is the name of the enzyme produces by bacteria undergoing ornithine decarboxylation?

decarboxylase

Purpose of the mineral oil: creates __________conditions that promote fermentation. Accumulation of acid end products from fermentation is necessary because decarboxylase enzymes work only in an _______ environment. Decarboxylase enzymes then catalyze chemical reactions that produce _________end products.

anaerobic;
acidic;
basic

What is name of the pH indicator in this medium?

bromcresol purple

This pH indicator is _________(color) when acidic (negative result) and __________ (color) when basic (positive result)

Yellow;
purple

Results

A. Enterobacter cloacae - purple (+)
B. Original color of test
C. Klebsiella pneumoniae - yellow (-)

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