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Klebsiella pneumonia

  • Gram-negative
  • Non-motile
  • Lactose-fermenting
  • Facultative anaerobic
  • Rod-shaped bacterium.
  • Mucoid Lactose fermenter on MacConkey agar.
  • Found in the normal flora of the mouth, skin, and intestines
  • Destructive changes to human and animal lungs if aspirated (inhaled), specifically to the alveoli (in the lungs) resulting in bloody sputum.

1- Gram Stain

2- MacConkey's Medium

3- Urease production test

4- IMViC

5- Triple Sugar Iron Agar (TSI)

6- Oxidase test

7- Motility agar

8- Gelatin hydrolysis

9- Catalase test

Klebsiella pneumonia is a Gram negative organism

MacConkey's Medium

  • Test the ability of the organism to ferment lactose and produce acid that covert the color of (Neutral red) into pink.

  • Klebsiella pneumonia is lactose fermenter so it turn the color of the colonies into Pink Mucoid color

Urease production test

• To test the ability of the organism to produce urease enzyme which split urea in urea media to form ammonia and CO2.

• The accumulation of the ammonia will produce alkaline PH which turns the color of the indicator (phenol red) into pink.

  • Klebsiella pneumonia is positive urease test.

IMViC

IMViC is abbreviation to the following tests

  • Indole
  • Methyl red (MR)
  • Voges-Proskauer (VP)
  • Citrate

A) Indole test

Test for the ability of the microorganism to break down tryptophan into indole.

Procedure:-

  • Incubate tryptone broth media with the tested organism.
  • The presence of indole can be detected through the use of Kovac’s reagent = (yellow in color)

(para dimethy aminobenzaldehyde in amyl alcohol).

  • Kovac's reagent, which is yellow, reacts with indole and produces a red color on the surface of the test tube.
  • E. coli is (+) with indole test.
  • K. pneumonia is (-) with Indole test.

B) Methyl red test

In methyl red test the test organism can ferment the glucose and produce acid which will change the color of the Methyl red indicator into red color

E. coli is one of the bacteria that produce acids, causing the pH to drop < 4.4.

The pH indicator methyl red will turn red

(a positive MR test)

Klebsiella and Enterobacter produce neutral products from glucose (e.g. Acetoin) causing the pH to rise < 6.2.

At this pH, methyl red indicator is a yellow

(a negative MR test).

C) Vogues-Proskaure’s test

The reagents used for the VP test are:-

  • Barritt's A (alpha-napthol)
  • Barritt's B (potassium hydroxide)

When these reagents are added to a broth in which acetoin (neutral product) is present, they produce a brick red color (a positive VP test).

This color may take 15 to 30 minutes to develop.

  • E. coli does not produce acetoin
  • Enterobacter and Klebsiella do

Methyl Red-Voges Proskauer Tests

  • E. coli is (MR+/VP-).
  • Enterobacter and Klebsiella are (MR-/VP+).

D) Citrate utilization test

The citrate test is used to determine the ability of a bacterium to utilize citrate as the only source of carbon.

  • Bacteria can break citrate into organic acids and carbon dioxide.
  • The carbon dioxide can form a basic compound, sodium carbonate.

A pH indicator Bromothymol Blue in the medium detects the presence of this compound by turning blue (a positive test).

Acidic (yellow), Green (neutral), Blue (basic)

  • E. coli is negative (emerald green) with citrate test.
  • Klebsiella pneumonia is positive (blue) with citrate test.

Whenever you see the name of this test i.e. Triple Sugar Iron Agar ,you have to remember that it’s a test which has three sugar (Lactose, Sucrose, and Glucose) and also iron; and it contains Agar Agar as solidifying agent

(TSI is a semi solid media having slant and butt).

Triple Sugar Iron Agar (TSI)

• 0.1% Glucose: If only glucose is fermented, only enough acid is produced to turn the butt yellow.

The slant will remain red

• 1.0 % lactose + 1.0% sucrose:

Large amount of acid turns both butt and slant yellow, thus indicating the ability of the culture to ferment either lactose or sucrose.

• Iron:

Ferrous sulfate: Indicator of H2S formation

• Phenol red:

Indicator of acidification

(It is yellow in acidic condition and red under alkaline conditions).

• It also contains Peptone which acts as source of nitrogen.

(Remember that when ever peptone is utilized under aerobic condition ammonia is produced)

Interpretation of Triple Sugar Iron Agar Test

1. If lactose (or sucrose) is fermented, a large amount of acid is produced, which turns the phenol red indicator --> yellow both in butt and in the slant.

Some organisms generate gases, which produces bubbles/cracks on the medium

2. If Lactose is not fermented but the small amount of glucose is, the oxygen deficient butt will be yellow

(Remember that butt comparatively have more glucose compared to slant i.e. more media more glucose)

But on the slant, less acid is produced by the organism and the slant will be red

(alkaline or neutral pH).

If neither lactose/sucrose nor glucose is fermented,

both the butt and the slant will be red.

The slant can become a deeper red-purple (more alkaline) as a result of production of ammonia from the oxidative deamination of amino acids

(Remember peoptone is a major constitutents of TSI Agar) .

If H2S is produced, the black color of ferrous sulfide is seen.

H2S + Fe ---> FeS ppt + H2

Expected results

1- Alkaline slant/no change in butt (K/NC)

i.e Red/Red =

glucose, lactose and sucrose non-fermenter

2-Alkaline slant/Alkaline butt (K/K) i.e Red/Red =

glucose, lactose and sucrose non-fermenter

3-Alkaline slant/acidic butt (K/A);

i.e. Red/Yellow =

glucose fermentation only,

gas (+ or -), H2s (+ or -)

4-Acidic slant/acidic butt (A/A);

i.e.Yellow/Yellow =

glucose, lactose and/or sucrose fermenter

gas (+ or -), H2s (+ or -).

Oxidase production

• It indicates the presence of cytochrome C in the respiratory chain.

• Aerobic microorganisms with cytochrome c can oxidize certain colorless amines to form colored products

MATERIALS:

• Filter paper 1% N,N,N'N' tetramethyl-p-henylenediamine

METHOD:

• Pre-wet filter paper with oxidase reagent

(N,N,N',N'-p-phenylenediamine) and allow to dry.

• Pick a bacterial colony with a sterile toothpick.

• Gently scratch the colony onto the filter paper.

Result: A purple color is produced.

Klebsiella pneumonia is oxidase negative

Motility agar

Motility agar is a differential medium used to determine whether an organism is equipped with flagella and thus capable of swimming away from a stab mark.

The results of motility agar are often difficult to interpret.

  • If the entire tube is turbid, this indicates that the bacteria have moved away from the stab mark (are motile). The organisms in the two tubes pictured on the right are motile.
  • If, however, the stab mark is clearly visible and the rest of the tube is not turbid, the organism is likely nonmotile (tube pictured on the left).

K. pneumonia is non motile.

Gelatin hydrolysis

Objective:

  • To test the ability of the microorganism to produce exoenzyme gelatinsae which liquefy gelatin
  • The gelatin hydrolysis is indicated by loss in ability to solidify even after refrigeration
  • K. pneumonia is negative

Catalase test

The catalase test is used to detect the presence of catalase enzymes by the decomposition of hydrogen peroxide to release oxygen and water with production of air bubbles.

Upon addition of H2O2 on the bacterial growth, the presence of air bubbles means that the organism produces catalase enzyme.

K. pneumonia is positive catalase test

Methods

1- Tube or bottle method

2- Agar slant method

3- Cover-slip method

Enterobacter aerogenes

Enterobacter aerogenes:-

  • Gram-negative rod-shaped bacterium.
  • Nosocomial and pathogenic bacterium that causes opportunistic infections including most types of infections.
  • Some of the infections result from specific antibiotic treatments, venous catheter insertions, and/or surgical procedures.
  • Found in the human gastrointestinal tract and does not generally cause disease in healthy individuals.
  • Live in various wastes, hygienic chemicals, and soil.
  • Has some commercial significance – the hydrogen gas produced during fermentation has been experimented with using molasses as the substrate.

Klebsiella pneumonia

Laboratory identification

Muhammad El-adawy

Demonstrator of Microbiology & Immunology

Gram Stain

+VP

-VP

1-Gram Stain

Enterobacter aerogenes is a Gram negative organism

2-McConkey's Medium

Enterobacter aerogenes is lactose fermenter so it turn the color of the colonies into pink color.

3-Urease production test:

Enterobacter aerogenes is negative urease test.

Composition of Triple Sugar Iron Agar (TSI)

4-IMViC

A-Indole test:

Enterobacter aerogenes is negative with Indole test.

B- Methyl red test:

​​ Enterobacter produce neutral products from glucose (e.g. Acetoin) causing the pH to rise above 6.2. At this pH, methyl red indicator is a yellow (a negative MR test).

C- Vogues-Proskaure’s test

​​ Enterobacter produce acetoin.

Methyl Red-Voges Proskauer Tests

​​ Enterobacter is (MR-/VP+).

D-Citrate utilization test:

• Enterobacter aerogenes is positive with citrate test.

5- Triple Sugar Iron Agar (TSI)

Name of the organisms Slant Butt Gas H2S

Enterobacter Acid (A) Acid (A) Pos (+) Neg (-)

6- Oxidase test

Enterobacter aerogenes is oxidase negative

Enterobacter aerogenes

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