Glucose phosphate broth

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Glucose phosphate broth - Wikipedia

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Glucose phosphate broth is used to perform methyl red (MR) test and Voges–Proskauer test (VP).

Glucose – 5 g/L
Dipotassium phosphate – 5 g/L
Proteose Peptone – 5 g/L
Distilled water – 1000 mL

pH – 6.9

Methyl red test

Principle

It is used to determine the ability of an organism to produce mixed acids by fermentation of glucose and to overcome the buffering capacity of the medium.

Procedure

Inoculate MacConkey's (Glucose phosphate broth) with pure culture of test organism. Incubate the broth at 35 °C for 48–72 hours.

After incubation add 5 drops of methyl red directly into the broth, through the sides of the tube.

Interpretation

The development of stable red color in the surface of the medium indicates sufficient acid production to lower the pH to 4.4 and constitute a positive test. Since other organism may produce lesser quantities of acid from the test substrate, an intermediate orange color between yellow and red may develop. This does not indicate positive test.

Controls

Positive and negative controls should be run after preparation of each lot of medium.

Positive control: Escherichia coli

Negative control: Klebsiella

Voges Proskauer test

Principle

It is used to determine the ability of some organisms to produce a neutral end product, acetyl methyl carbinol (acetoin) from glucose fermentation. The production of acetoin, a neutral reacting end product produced by members such as Klebsiella, Enterobacter etc., is the chief end product of glucose metabolism and form less quantities of mixed acids. In the presence of atmospheric oxygen and 40% KOH, acetoin is converted to diacetyl and α-naphthol serves as catalyst to bring out red color complex.

Media

Glucose Phosphate Broth

Reagents

A: α-naphthol – 5 g

Absolute ethyl alcohol – 100 mL – 0.6 mL – 3 parts
B: KOH – 40 g
Distilled water – 100 mL – 0.2 mL – 1 part

Procedure

Inoculate a tube of glucose phosphate broth with a pure inoculum of test organism and incubate at 35 °C for 24 hours.

To 1 mL of this broth add 0.6 mL of 5% α-Naphthol followed by 0.2 mL of 40% KOH. Shake the tube gently to expose the medium to atmospheric oxygen and allow the tube to remain undisturbed for 10–15 minutes.

Interpretation

A positive test is represented by the development of red color in 15 minutes or more after addition of the reagents, indicating the presence of diacetyl, the oxidation product of acetoin. The test should be red, after standing for 1 hour because negative VP cultures may produce copper-like colour potentially resulting in a false positive interpretation, also because due to action of the reagents when mixed.

Controls

Positive and negative controls should be run after preparation of each lot of medium.

Positive control: Klebsiella

Negative control: Escherichia coli^[1]

References

^ Myer's and Koshi's Manual of Diagnostic Procedures in Medical Microbiology and Immunology. Vellore, India: Christian Medical College & Hospital. 2001.

Growth media / agar plates

Selective media

Gram positive

Actinomycetota	Mycobacterium tuberculosis Löwenstein–Jensen medium Middlebrook 7H9 Broth Middlebrook 7H10 Agar Middlebrook 7H11 Agar Mycoplasma pneumoniae Eaton's agar
Bacillota	Corynebacterium diphtheriae Hoyle's agar Enterococcus Bile esculin agar Lactobacillus Lee's Agar MRS agar Lactococcus M17 agar Listeria Fraser broth PALCAM Staphylococcus Mannitol salt agar Baird-Parker agar Vogel–Johnson agar

Gram negative

Alphaproteobacteria	Brucella Brucella agar Farrell's medium
Betaproteobacteria	Neisseria Thayer–Martin agar New York City agar
Gammaproteobacteria	Aggregatibacter actinomycetemcomitans Tryptic soy-serum-bacitracin-vancomycin Bordetella Bordet–Gengou agar Enterobacteriaceae VRBD agar Haemophilus influenzae/Legionella pneumophila Buffered charcoal yeast extract agar Pseudomonas aeruginosa Cetrimide agar Salmonella XLT agar DCA agar Salmonella/Shigella XLD agar

Differential media

Fungal media

Nonselective media

Other/ungrouped media

Categories:

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Contents

Methyl red test

Principle

Procedure

Interpretation

Controls

Voges Proskauer test

Principle

Media

Reagents

Procedure

Interpretation

Controls

References