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    Detection of Beta Lactam

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    Rajkishor Yadav
    The document describes four methods for detecting beta-lactamase enzymes: the nitrocef disk test, rapid acidometric method, iodometric method, and modified hodge test. It provides details on the reagents, procedures, and interpretations for each method.

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    Detection of Beta Lactam

    Uploaded by

    Rajkishor Yadav
    17 views18 pages
    The document describes four methods for detecting beta-lactamase enzymes: the nitrocef disk test, rapid acidometric method, iodometric method, and modified hodge test. It provides details on the reagents, procedures, and interpretations for each method.

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    The document describes four methods for detecting beta-lactamase enzymes: the nitrocef disk test, rapid acidometric method, iodometric method, and modified hodge test. It provides details on the reagents, procedures, and interpretations for each method.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    17 views18 pages

    Detection of Beta Lactam

    Uploaded by

    Rajkishor Yadav
    The document describes four methods for detecting beta-lactamase enzymes: the nitrocef disk test, rapid acidometric method, iodometric method, and modified hodge test. It provides details on the reagents, procedures, and interpretations for each method.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    of 18

    Detection method for

    beta-lactamase
    Pratima Pandey
    BSC.MLT 3rd year
    METHODS:

    Beta-lactamase enzymes activity can be detected by ;

    1) Nitrocef disks test.


    2) Rapid acidometric method.
    3) Iodometric method.
    4) Modified hodge test.
    1. Nitrocef disk test
    Nitrocef Disks™ are impregnated with nitrocefin, a
    chromogenic cephalosporin.

    As the amide bond in a beta-lactam ring is hydrolyzed by a


    beta-lactamase, nitrocefin changes color from yellow to red.

     Bacteria which produce beta-lactamase in significant amounts


    produce this yellow to red color change on the Nitrocef
    Disk™.
    These beta-lactamases are capable of inactivating
    "penicillinase-labile-penicillins", such as amoxicillin,
    ampicillin, penicillin, carbenicillin, mezlocillin, and
    piperacillin.

     Most positive bacterial strains will produce a color change


    within 5 minutes. Some staphylococci, however, may take up
    to 60 minutes for a positive result.
    Showing positive (left disk) and negative (right disk) reactions for
    Nitrocef Disks™ (Cat. no. Z7301). 
    Each disk was moistened with deionized water and growth from cultures
    of Haemophilus influenzae(ATCC ® 33533) and Moraxella (Branhamella)
    catarrhalis (ATCC ® 25240) was applied to the left and right disks,
    respectively. The orange/red color development was indicative as beta-
    lactamase positive. The yellow color development was indicative as beta-
    lactamase negative. 
    2. Acidimetric method
    Reagents: 2 ml of the 0.5% phenol red solution was added to 16.6 ml sterile
    distilled water. After mixing, this solution was added to a vial of crystalline
    benzyl penicillin G which contained 20 million units. This solution was then
    placed in sterile container.

    Since this solution was at an acidic pH due to citrate buffer in the penicillin, 1 N
    sodium hydroxide was added drop-wise to the solution till development of reddish
    violet colour (pH 8.5). This solution should be stable for several months only if it
    doesn’t turn yellow.

    Procedure: 100 μl of penicillin phenol red solution was placed in the well of
    microtitre plate. Several colonies were suspended in the solution to get dense
    suspension. The solution turned yellow within 10-15 minutes if β Lactamase
    enzyme was produced.
    Note: Colour changes after 15 minutes is not of significance
    since it represents decomposition of substrate. Overnight broth
    culture should not be used as the organism may have produced
    an acid in growth medium.
    3. Iodometric method
    Reagents- Sterile potassium or sodium penicillin G powder (6 lac units) was
    dissolved in potassium phosphate buffer PH 6.0, 0.05 M at a concentration of 6000
    μg / ml (to be prepared fresh when required) Starch solution was prepared by adding
    1 gm of soluble starch to 100 ml distilled water and slowly bringing it to boil.

    The solution should become clear opalescent in appearance and can be stored at 4 ºC
    for several days.

    Iodine solution: This was prepared by dissolving 2.03 g of iodine and 53.2 g of
    potassium iodide in 100 ml distilled water. It should be stored in ground glass,
    stopper brown bottle.

    β lactamase producing strain of S. aureus ATCC 29213was used as positive control.


    Procedure
    100 μl of the penicillin solution was dispensed into a well of
    microtitre plate. Several colonies of the organism to be tested
    were emulsified into the solution to get dense suspension.

     Two drops of starch were added and then the plate was kept at
    room temperature for 30-60 minutes.

    One drop of iodine was then added which turn the solution
    blue. If the blue colour disappeared in 10 minutes, the organism
    was considered as β lactamase positive. Negative control with
    penicillin alone was kept without any culture suspension.
    4. Modified hodge test for carbepenemase
    The Modified Hodge Test (MHT) detects carbapenemase
    production in isolates of Enterobacteriaceae.

    Other carbapenemase, like the metallo β lactamase (MBL) and the


    SME-1 in Serratia marcescens, can also produce a positive MHT.

    Carbapenemase production is detected by the MHT when the test


    isolate produces the enzyme and allows growth of a carbapenem
    susceptible strain (E.coli ATCC 25922) towards a carbapenem disk.

    The result is a characteristic cloverleaf-like inde


    Prepare a 0.5 McFarland dilution of the E.coli ATCC 25922 in
    Procedure
    5 ml of broth or saline.

     Dilute 1:10 by adding 0.5 ml of the 0.5 McFarland to 4.5 ml of
    MHB or saline.

    Streak a lawn of the 1:10 dilution of E.coli ATCC 25922 to a
    Mueller Hinton agar plate and allow to dry 3–5 minutes.

    Place a 10 µg meropenem or ertapenem susceptibility disk in
    the center of the test area.
    In a straight line, streak test organism from the edge of the disk
    to the edge of the plate. Up to four organisms can be tested on
    the same plate with one drug.

    Incubate overnight at 35OC ± 2OC in ambient air for 16–24
    hours
    Interpretation of result
    After 16–24 hours of incubation, examine the plate for a clover
    leaf-type indentation at the intersection of the test organism and
    the E. coli 25922, within the zone of inhibition of the
    carbapenem susceptibility disk.

    MHT Positive test has a clover leaf-like indentation of the


    E.coli 25922 growing along the test organism growth streak
    within the disk diffusion zone.

    MHT Negative test has no growth of the E.coli 25922 along


    the test organism growth streak within the disc diffusion.
    Thank you………

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