Enterobacteriaceae عملي

THE Enterobacteriaceae

جراثيم / د.انسام / طب عام ثالثعملي 7/3/2017 *

Common charactestics

They are Gram-negative bacilliFacultative anaerobes.Fermenting sugars (glucose).Reduce nitrate to nitrite. Lack cytochrome oxidase (oxidase negative). Non-spore formingCatalase-positive. Grow well on MacConkey’s agar at 37°C.Most have many flagella (motile), few are non-motile. *

Principles of identification

Gram’s stain: gram - bacilliMotiliy: all motile /Shigella, Klebsiella Oxidase: -veColonial morphology Biochemical testsSerological typing *

E. Coli on MacConkey’s agarSmall round convex entire edge colonies smooth surface pink due to lactose fermentation *

Klebsiella species

K. pneumoniae subspecies aerogenes is the most frequently isolated species. All grow readily on ordinary media, are non-motile and are capsulated. Colonies are large, high convex, mucoid tend to coalese, pink due to lactose fermentation.
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Mucoid colonies

Mucoid large colonies tend to coalese LF
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IMViC REACTIONS
IMViC reactions are a set of four reactions (tests) that are commonly employed in the identification of members of family enterobacteriaceae. The four reactions are: Indole test, Methyl Red test, Voges Proskauer test and Citrate utilization test.
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INDOLE TEST

Medium: peptone water, which contains amino acid tryptophan and the MO inoculated and incubated overnight at 37oC. Following incubation few drops of Kovac’s reagent are added. Kovac’s reagent consists of para-dimethyl aminobenzaldehyde, isoamyl alcohol and conc. HCl.Formation of a red or cherry coloured ring at the top is taken as positive.Example: Escherichia coli: Positive; Klebsiella pneumoniae: Negative *

Positive indole

Negative indole
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METHYL RED (MR) TEST

Principle: Some bacteria produce large amounts of acids from glucose fermentation. Methyl Red is a pH indicator, which remains red in color at a pH of 4.4 or less. Medium: glucose phosphate peptone water broth, inoculated and incubated at 37C for 48 hours. Reagents: 5 drops of MR reagent. Development of red color is taken as positive. MR negative organism produce yellow color. Example: Eschericihia coli: Positive; Klebsiella pneumoniae: Negative
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Methyl Red (MR)

Positive red color (e.g E.coli) Negative yellow color (e.g klebsiella)
positive
negative
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VOGES PROSKAUER (VP) TEST
Principle: VP test detects butylene glycol producers. Acetyl-methyl carbinol (acetoin) Medium: glucose phosphate broth and incubated for at least 48 hours. Reagent: 0.6 ml of alpha-naphthol is added to the test broth and shaken. 0.2 ml of 40% KOH is added to the broth and shaken. The tube is allowed to stand for 15 minutes. Appearance of red color is taken as a positive test. Examples: Escherichia coli: Negative Klebsiella pneumoniae: Positive
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Vogus proskour (VP)

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CITRATE UTILIZATION TEST

Principle: detects the ability of an organism to utilize citrate as the sole source of carbon and energy.Procedure: Bacteria are inoculated on a medium containing sodium citrate and a pH indicator bromothymol blue. Production of NH3 from utilization of sodium citrate and ammonium salt respectively results in alkaline pH. This results in change of medium’s color from green to blue.Examples: Escherichia coli: Negative; Klebsiella pneumoniae: Positive *

Citrate Utilization

Positive blue color Negative green color
Klebsiella positive
E. Coli negative
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Citrate utilization

VP
MR
Indole
Bacterium
-
-
+
+
E. coli
+
+
-
-
K.pneumoniae
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Carbohydrate utilization test
Fermentation is an oxidation reduction reaction that takes place in an anaerobic condition and is detected by observing color change of PH indicator as acid products are formed. Gas is best detected by using a broth CHO-fermentation medium into which small inverted durham tubes have been placed. Even trace amounts of gas which collect as bubbles under the Durham tube can be detected. The resulting gas is usually a mixture of H2 and CO2.
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API 20E

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Genus Proteus

Microscopical appearance: gram negative bacilli, motile, non spore formingCulture MacConkey’s agar: NLFSmall round colonies entire edge convexSmooth surface colorless because nonLactose fermentor *

Proteus

Blood and nutrient agar: swarming (concentric zone of growth cover the whole surface of the medium)
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Proteus

Urease +ve Urease medium Pink color= +ve
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Proteus
Biochemical tests IMViC: + + - v P.vulgaris - + - v P. Mirabilis
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Salmonella

MacConkey’s agar :colorless colonies non lactose fermentor *

Salm. SS agar

Colorless colonies With black center Due to H2S production
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Salmonella TSI agar

Slant: alkaline Butt: Acid Gas: +- Black color due to H2S production
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Widal testslide agglutination test

Agglutination is a positive test result and if the positive reaction is observed with 20 ul of test sample, it indicates presence of clinically significant levels of the corresponding antibody in the patient serum No agglutination is a negative test result and indicates absence of clinically significant levels of the corresponding antibody in the patient serum.
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Tube method

Serial dilutions of patients serum
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Shigella

Gram negative non motile bacilliCulture:MacConkey’s agar: Colorless colonies non lactore fermenter *

Shigella

SS agar: colorless colonies no black center
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Shigella

TSI: Slant: alkaline Butt: acid No black color
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