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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
IMViC is abbreviation to the following tests
Test for the ability of the microorganism to break down tryptophan into indole.
Procedure:-
(para dimethy aminobenzaldehyde in amyl alcohol).
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:-
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.
Methyl Red-Voges Proskauer Tests
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.
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)
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).
• 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)
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
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 -).
• 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 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.
K. pneumonia is non motile.
Objective:
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:-
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.
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