Nitrocefin Applications:

Nitrocefin is a chromogenic β-lactamase substrate that undergoes colour change from yellow to red as the amide bond in the β-Lactam ring is hydrolyzed by β-lactamase. Nitrocefin undergoes colour changes induced by lactamases produced by Gram-positive and Gram-negative bacteria. Several studies have utilized the colour changing properties of Nitrocefin for the detection of β-lactamase activity from bacterial cell extracts by isoelectric focusing and spectroscopy. Nitrocefin has also been used in studies involving β-lactamase resistant antibiotics.

Chemical Properties

Nitrocefin Usage Method

1.Preparation of Cefnitrothiophene Storage Solution (10mg/ml) a) Before use, remove the product from the refrigerator and place it at room temperature for at least 20 minutes before centrifugation at low speed; b) Suck 100 μ Add 1mg of Cefnitrothiophene to high-quality DMSO, fully dissolve and mix well. It can be packaged according to a single dosage and stored in dark at -20 ° C.

2. The typical working concentration for preparing cephalosporin with a working solution of 1mg/ml is 0.5-1.0mg/ml, and this preparation is generally yellow in color. Take 20 μ Add 180 to the storage solution of Cefnitrothiophene (10mg/ml) μ After mixing with PBS buffer solution (0.1M, neutral pH), cephalosporin thiophene (1mg/ml) working solution is obtained. Excess working fluid can be stored in a dark place at -20 ° C and must be used up within 2 weeks.

3. Quick detection and analysis Once in contact β- Lactam producing bacteria, the samples to be tested can generally produce positive reaction quickly. It is usually recommended to have a reaction time of 20-30 minutes and set a negative control.

–3.1 Glass slide surface analysis method

a) Take a drop of Cefnitrothiophene working solution (1mg/ml) onto a clean glass slide surface;

b) Use a sterile inoculum ring to select a solid culture clone and mix it with liquid Chemicalbook drops evenly;

c) If red color can be observed within 20-30 minutes, it indicates that β- Lactam enzyme activity. If necessary, add more drops of Cefnitrothiophene working solution to prevent dryness

–3.2 Direct contact analysis method

a) Directly suck a drop of Cefnitrothiophene working solution (1mg/ml) onto the surface of the isolated colony;

b) If red color can be observed within 20-30 minutes, it indicates that β- Lactam enzyme activity.

–3.3 Broth culture medium analysis method

a) Take 1ml of broth culture medium and add 3-5 drops of Cefnitrothiophene working solution (1mg/ml);

b) If red color can be observed within 20-30 minutes, it indicates that β- Lactam enzyme activity.

–3.4 Cell lysate analysis method

a) Ultrasound lysis of 1ml cell suspension;

b) Add 3-5 drops of Cefnitrothiophene working solution (1mg/ml) to the lysis suspension; c) If red color can be observed within 20-30 minutes, it indicates that β- Lactam enzyme activity.

–3.5 Filter paper analysis method

a) Saturate a small piece of filter paper (~3x3cm) to absorb Cefnitrothiophene working solution (1mg/ml), and then place it in a clean culture dish;

b) Jump a separated clone and apply it to the impregnated filter paper;

c) If red color can be observed within 20-30 minutes, it indicates that β- Lactam enzyme activity.