Exercise 28 - Gram Negative Bacteria

What is a characteristic feature of the cell wall structure in gram-negative bacteria?

Gram-negative bacteria possess a thin layer of peptidoglycan in their cell wall.

What additional structural feature is found in the cell wall of gram-negative bacteria?

Gram-negative bacteria have an outer phospholipid bilayer membrane containing lipopolysaccharides (LPS).

What is the role of Lipid A (Endotoxin A) in gram-negative bacteria?

Lipid A, a component of lipopolysaccharides (LPS), accounts for symptoms of endotoxic shock.

Where are gram-negative bacilli commonly found?

Gram-negative bacilli are found in soil, fresh water, salt water, and occur in abundance in the intestinal tracts of mammals.

Which are the three tests for hydrogen sulfide?

1. TSI

2. SIM

3. LIA

Any black color on any of these tests is positive for H2S, unlike the bile esculin slant, which needs to be at least ½ black to be positive.

MacConkey

True or false? MacConkey agar is both selective and differential.

True

MacConkey

What does MacConkey agar contain and what does it select for?

Bile salts and crystal violet

Selects for Gram-negative bacteria

MacConkey

What is MacConkey agar’s carbohydrate?

Lactose

MacConkey

What is MacConkey agar’s pH indicator?

Neutral red

MacConkey

Name one strong lactose fermenting Gram negative bacilli on MacConkey agar.

Escherichia coli

MacConkey

Name one weak lactose fermenting Gram negative bacilli on MacConkey agar.

Enterobacter aerogenes

MacConkey

Name one non-lactose fermenting gram negative bacilli on MacConkey agar.

• Morganella morganii

• Salmonella

MacConkey

Pink growth on the MacConkey agar means that the organism is able to ferment ______________.

Lactose

MacConkey

Growth on MacConkey agar means the organism is Gram -_____.

Gram-negative

MacConkey

Colorless growth on MacConkey agar means the organism is…?

Non-lactose fermenting

Urease Test

In the urease test, what is the main enzyme?

Urease

Urease Test

In the urease test, what is the substrate and the pH indicator?

Substrate - Urea

pH indicator - Phenol red

Urease Test

In the urease test, what is produced if the test is positive?

Ammonia, CO2, and water

Ammonia raises the pH and produces a hot pink color

Urease Test

In the urease test, is ammonia considered acidic or alkaline? What would be the approximate pH?

Alkaline, with a pH above 7.

Urease Test

Name three bacteria that produce urease.

• Proteus vulgaris

• Proteus mirabilis

• Morganella morganii

Urease Test

The enzyme urease breaks down urea into which three components?

Ammonia, CO2, and H2O

Citrate Utilization

The citrate agar slant contains what enzyme?

Citrase

Citrate Utilization

What is the pH indicator in the citrate test?

Bromthymol blue

Citrate Utilization

Name two citrate positive bacteria.

• Enterobacter aerogenes

• Klebsiella pneumoniae

Citrate Utilization

What color is produced if the citrate utilization test is positive?

Prussian blue

Nitrate Reduction

In the nitrate reduction test, which enzyme allows bacteria to anaerobically reduce nitrate to nitrite?

Nitrate reductase

Some bacteria further reduce the nitrite to ammonia or completely to molecular nitrogen.

Nitrate Reduction

In the nitrate broth, do the enzymes require oxygen to function?

No, the nitrate reduction process is anaerobic.

Nitrate Reduction

What are the steps in nitrate reduction?

Nitrate → Nitrite → Ammonia → Molecular Nitrogen

NO₃ ——> NH₂ ——> NH₃ ————> N₂

Nitrate Reduction

What reagents are added to a nitrate broth?

• Nitrate Reagent A

• Nitrate Reagent B

Nitrate Reduction

What are the results for each test? Positive or negative?

What is added to each test?

• Test 1 (Positive)

  • Reagent A and B added

  • Nitrate has been reduced to nitrite

• Test 2 (Negative)

  • Reagent A and B added

  • Bacteria did not reduce nitrate to nitrite

  • Next step is to add zinc (2a & 2b)

• Test 2a (Negative)

  • Zinc is added

  • Zinc reduced the nitrate, not the bacteria

• Test 2b (Positive)

  • Zinc is added but there is no color change

  • Bacteria is positive for nitrate reduction

  • Bacteria reduced nitrates beyond nitrites to ammonia or molecular nitrogen

SIM Test

How is the Sulfide Indole Motility (SIM) test done?

SIM agar deep with a needle stabbing the butt a single time

SIM Test

What three things does the SIM agar deep tube test for?

1. Indole production

2. Hydrogen sulfide production

3. Motility

SIM Test

What amino acid does the SIM agar tube contain?

Tryptophan

SIM Test

If an organism produces the enzyme tryptophanase in the SIM test, it will degrade tryptophan into what?

Indole, pyruvate, and ammonia

SIM Test

What is added to the SIM agar tube after incubation?

Kovac’s Reagent

SIM Test

Which test tubes are considered motile?

• Tubes 1,3,4,5

SIM Test

Which tube is positive and which is negative?

SIM Test

If the SIM agar test tube produces thiosulfate reductase, sulfur is reduced to what gas? What color will be produced?

Hydrogen sulfide gas is produced with a black precipitate.

SIM Test

In this SIM test, indicate if there is a presence of:

• Indole

• Hydrogen sulfide

• Motility

SIM Test

Diffuse growth throughout without seeing a stab line also shows motility.

LIA Test

In the LIA slant test, is lysine deamination an aerobic or anaerobic process? Where does it occur?

Aerobic process that occurs in the slant

LIA Test

In the LIA slant test, is lysine decarboxylation an aerobic or anaerobic process? Where does it occur?

Anaerobic process that occurs in the butt of the media

LIA Test

How is the lysine irone agar (LIA) test performed?

A sterile needle is used to stab the butt and streak the surface of the slant of the LIA medium with the organism.

LIA Test

What is the pH indicator of the LIA media?

Bromcresol purple

LIA Test

What are the colors of both positive deamination and decarboxylation?

Dark Red / Purple

LIA Test

What are the colors of both negative deamination and decarboxylation?

Purple / Yellow

LIA Test

In this tube, what is the result for deamination and decarboxylation?

Deamination - Positive (Dark red)

Decarboxylation - Negative (Yellow)

LIA Test

In this tube, what is the result for deamination and decarboxylation?

Deamination - Negative (Purple)

Decarboxylation - Positive (Purple)

LIA Test

In this tube, what is the result for deamination and decarboxylation?

Deamination - Negative (Purple)

Decarboxylation - ???

LIA Test

In this tube, what is the result for deamination and decarboxylation?

Deamination - Positive

Decarboxylation - Negative

LIA Test

What color is produced if the organism positively decarboxylates (butt) lysine?

Purple color of the butt

LIA Test

What color is produced if the organism does NOT decarboxylate (butt) lysine?

Yellow color of the butt

LIA Test

What color is produced if the organism positively deaminates (slant) lysine?

Dark red color of the slant

LIA Test

What color is produced if the organism does NOT deaminate (slant) lysine?

Purple color of the slant

LIA Test

Hydrogen sulfide gas will produce what precipitate in the LIA test?

Black precipitate

LIA Test

What components of the LIA media test for H₂S production?

• Sodium thiosulfate

• Ferric ammonium citrate?

MRVP

What are the two components of the MRVP test?

1. MR-Methyl Red Test

2. VP-Voges Proskauer Test

MRVP

What is the purpose of the MRVP test?

To determine if the organism is able to ferment glucose and what types of fermentation acids are produced.

MRVP

The MRVP test was initially designed to distinguish between which two organisms?

• Escherichia coli

• Enterobacter aerogenes

MRVP

What would the results be for the MR and VP test for the following bacteria?

• Escherichia coli

• Enterobacter aerogenes

• Escherichia coli

  • MR Positive (Red broth)

  • VP Negative

• Enterobacter aerogenes

  • MR Negative

  • VP Positive (Red diffusing band)

MRVP

What reagent is added to the MR test?

Methyl red

MRVP

In the methyl red test, a positive E. coli test produces which three acids?

1. Lactic acid

2. Acetic acid

3. Formic acid

MRVP

In the methyl red test, Enterobacter aerogenes only produces which acid in the methyl red test?

Acetic acid

MRVP

In the methyl red test, the acids produced by E coli are strong enough to overcome the buffering capacity of the phosphate buffer. Does this increase or decrease the pH?

Decreases the pH

MRVP

MRVP broth contains which three components?

• Peptones

• Phosphate buffer

• Glucose

MRVP

In this methyl red test, which is positive and which is negative?

MRVP

In the methyl red test, a cherry red positive color indicates what pH?

pH of 4.4 or lower

MRVP

In the VP test, what is the pH of acetylmethylcarbinol?

pH 6.2

MRVP

In the methyl red test, an orange color indicates that the test is positive or negative?

Negative

MRVP

What is the end product of a positive MR test?

• Lactic acid

• Acetic acid

• Formic acid

The bacteria produces a mixture of acids from the fermentation of glucose that is in the media.

MRVP

What is the end product of a positive VP test?

Only acetic acid

The bacteria is able to ferment the glucose but is only able to produce one acid end-product.

MRVP

In the VP test, acetic acid is converted to…?

Acetylmethylcarbinol

MRVP

In the VP test, what is added to the empty tube with the MRVP broth?

• Barritt’s reagent A(alpha naphthol)

• Barritt’s reagent B( 40% KOH)

MRVP

What characteristic is shown in a positive VP test after 20 minutes?

Dark red band at the top of the broth, which will diffuse over time. This means that the organism fermented glucose to produce acetic acid.

MRVP

Can an organism be positive for both the MR and VP test?

No

MRVP

Why can’t both tests be positive for a certain organism?

• Different metabolic pathways: The MR-positive pathway favors strong acid production, whereas the VP-positive pathway converts acids to neutral products.

• Bacteria typically specialize in one pathway, not both simultaneously, due to enzyme regulation and resource allocation.

• If the organism produces enough stable acids to turn MR positive, it generally lacks the capacity to convert those acids into neutral products needed for a VP-positive result, and vice versa.

TSI

What are the ingredients in the TSI agar slant?

• Glucose/Sucrose/Lactose

• Phenol red

• Peptones

• Sodium thiosulfate

• Ferrous sulfate

TSI

What are the carbohydrates and their concentrations in the TSI slant?

• Glucose 0.1%

• Sucrose 1.0%

• Lactose 1.0%

TSI

What is the pH indicator of the TSI slant?

Phenol red

TSI

How is the triple-sugar iron agar test performed?

A sterile needle is used to stab the butt and a zig zag streak is made on the surface of the TSI agar slant.

TSI

In this TSI agar test, indicate:

• Is the slant/butt acidic or alkaline

• Is there gas present

• Is there hydrogen sulfide present

TSI

What carbohydrates are being fermented?

Glucose

TSI

What carbohydrates are being fermented?

Glucose

TSI

What carbohydrates are being fermented?

1st glucose, then sucrose and/or lactose

TSI

What carbohydrates are being fermented?

None, no carbohydrates are being fermented

TSI

In an alkaline slant/acid butt, what has occurred?

Only glucose fermentation has occured

TSI

In an acid slant/acid butt, what has occurred?

Glucose fermentation plus lactose and/or sucrose fermentation has occurred

TSI

In an alkaline slant/alkaline butt, what has occurred?

No carbohydrate fermentation has occurred

TSI

Is hydrogen sulfide production in the TSI test, acidic or alkaline?

Acidic. The black color of hydrogen sulfide will mask the yellow color of the pH indicator.

TSI

Which components of the media test for H₂S production?

• Sodium thiosulfate

• Ferrous sulfate

TSI

TSI

What are the following results for this tube?

• Acidic or Alkaline?

• Presence of gas?

• Presence of hydrogen sulfide?

• Acid/Acid

• Gas +

• H2S +

Lactose and/or sucrose fermentation has occurred.

Hydrogen sulfide production is always acidic, even though black coloration masks yellow color of pH indicator.

TSI

What are the following results for this tube?

• Acidic or Alkaline?

• Presence of gas?

• Presence of hydrogen sulfide?

• Alkaline/Acid

• Gas (Negative)

• H2S +

Only glucose fermentation has occurred

TSI

What are the following results for this tube?

• Acidic or Alkaline?

• Presence of gas?

• Presence of hydrogen sulfide?

• Alkaline/Alkaline

• Gas (Negative)

• H2S (Negative)

No carbohydrate fermentation has occurred. Instead, peptones are catabolized resulting in an alkaline pH due to the production of ammonia.

TSI

Fermentation of glucose, sucrose, and lactose will result in what color of the slant and/or butt of a TSI agar?

Yellow, as the acids will react with the phenol red

TSI

Production of ammonia will result in what color of the slant and/or butt of a TSI agar?

Red, as the ammonia, produced by peptones, will react with the phenol red.

TSI

What is being catabolized when a TSI agar is all alkaline with an all over orange/red color?

• Peptones are being catabolized, not carbohydrates

  • Rise in pH due to ammonia production

  • Peptones react with phenol red

  • Color changes to a deeper red