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    Biochemical Test of Bacteria

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    azizah

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    Biochemical Test of Bacteria

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    30 views26 pages

    Biochemical Test of Bacteria

    Uploaded by

    azizah

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    © All Rights Reserved
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    of 26

    Biochemical Tests of Bacteria

    Part one
    Assistant Prof. Dr. Fitua Al-Saedi
    Department of Clinical Laboratory Science
    College of Pharmacy
    Lab training
    Biochemical tests are important in the identification of bacterial
    species.

    These tests depend on the presence of certain enzymes. Different


    bacteria produce different enzymes such as catalase, oxidase,
    urease, gelatinase, etc., produced by the bacteria.
     Catalase Test
     Coagulase Test
     Optochin sensitivity testing
     Bacitracin sensitivity testing
     CAMP Test
     Bile Esculin Test
     Starch hydrolysis test
     Gelatinase Test
    Catalase Test
    To determine the ability of bacteria that produces Catalase enzyme which
    degrades the hydrogen peroxide.

    .
    In aerobic organisms, during aerobic respiration, oxygen serves as hydrogen
    acceptor and hydrogen peroxide is formed in the cell. High concentration of
    H2O2 is formed which is toxic to cell. Bacteria posses the catalase enzyme
    converts hydrogen peroxide into oxygen and water.
    Procedure:
    Catalase production can be determined by addition of the
    substrate H2O2 on bacterial culture.

    1. Slide Method:
    - Pure growth of the organisms will transfer to the clean slide by using
    inoculation loop or glass rod.
    - Immediately add a drop of 3% hydrogen peroxide on bacterial culture.
    - Observe the bubble formation.

    2. Tube Test:
    - Take one ml of 3 % hydrogen peroxide in test tube.
    - Small amount of bacterial culture introduce into the solution
    - Immediately observe the effervescence.
    Result Interpretation of Catalase Test and Examples

    Positive: Copious bubbles produced, active bubbling


    Examples: Staphylococcus spp. and the Micrococcus spp.

    Negative: No or very few bubbles produced.


    Examples: Streptococcus and Enterococcus spp
    Coagulase Test
    Staphylococcus aureus is known to produce coagulase, which can clot plasma into
    gel in tube or agglutinate cocci in slide. This test is useful in differentiating
    S.aureus from other coagulase-negative staphylococci.

    Most strains of S.aureus produce two types of coagulase, free coagulase and
    bound coagulase. While free coagulase is an enzyme that is secreted
    extracellularly, bound coagulase is a cell wall associated protein.

    Free coagulase is detected in tube coagulase test and bound coagulase is detected
    in slide coagulase test. Slide coagulase test may be used to screen isolates of S.
    aureus and tube coagulase may be used for confirmation..
    SLIDE COAGULASE TEST:

    Principle: The bound coagulase is also known as clumping factor.


    It cross-links the α and β chain of fibrinogen in plasma to form fibrin clot that
    deposits on the bacterial cell wall. As a result, individual coccus stick to each
    other and clumping is observed

    Procedure
    Interpretation result
    TUBE COAGULASE TEST:

    Principle: The free coagulase secreted by S. aureus reacts with coagulase


    reacting factor (CRF) in plasma to form a coagulase-CRF complex. The complex
    then reacts with fibrinogen to form the fibrin clot in a test tube

    Procedure
    5 ml of the diluted plasma (Add 0.2 ml plasma in 1.8 ml is added to
    saline)added to a test tube. About 5 drops of the test organism culture are
    added to the test tube.

    The test tube is mixed and incubated at 37°C for an hour.


    The tube is finally observed for the clot formation. If no clotting is
    observed, the tube should be examined at 30 minutes interval of up to 6
    hours.
    Interpretation result
    Optochin sensitivity testing
    This is a differential test used to distinguish between organisms sensitive to the
    antibiotic optochin and those not. Optochin is an antibiotic that interferes with

    the ATPase and production of adenosine triphosphate (ATP) in microorganisms.

    This test is used to distinguish Streptococcus pneumoniae (optochin sensitive


    (pictured on the right below)) from other a-hemolytic streptococci (optochin
    resistant (Streptococcus mitis is pictured on the left below).
    Bacitracin sensitivity testing

    Principle
    This is a differential test used to distinguish between organisms sensitive to
    the antibiotic bacitracin and those not. Bacitracin is a peptide antibiotic
    produced by Bacillus subtilis
    The antibiotic bacitracin inhibits the synthesis of bacterial cell walls by
    interfering the peptidoglycan synthesis of bacteria.

    A disk saturated with a small amount of bacitracin (0.04 units) is placed on an


    agar plate, allowing the antibiotic to diffuse into the medium and inhibit the
    growth of susceptible organisms. After incubation, the inoculated plates are
    examined for zones of inhibition surrounding the disks.

    If the organism grows up to the edge of the disk, it is resistant to the antimicrobial
    compound infusing the disk. If there is a zone around the edge of the disk where
    the organism has not grown, the organism is susceptible to the antimicrobial in
    the disk.
    Bacitracin sensitivity test is commonly used to distinguish between the b-hemolytic

    streptococci: Streptococcus agalactiae (bacitracin resistant)

    and Streptococcus pyogenes (bacitracin sensitive).


    Starch hydrolysis test ( amylase production test)

    This test is used to identify bacteria that can hydrolyze starch (amylose and

    amylopectin) using the enzymes α-amylase and oligo-1,6-glucosidase.

    Because of the large size of amylose and amylopectin molecules, these can not

    pass through the bacterial cell wall. In order to use these starches as a carbon

    source, bacteria must secrete α-amylase and oligo-1,6-glucosidase into the

    extracellular space. These enzymes break the starch molecules into smaller

    glucose subunits which can then enter directly into the glycolytic pathway.
    Procedure
    On Starch agar (a simple nutritive medium with starch added). Inoculate of organism
    to be tested on to labeled plate and incubate it for 48 hours at 37°C. Following
    incubation, flood the surface of the plates with iodine solution with a dropper for 30
    seconds. Pour off the excess iodine.
    Examine for the clear zone around the line of bacterial growth.
    Result Interpretation of Test and Examples

    Positive test: A clear zone around the line


    of growth after addition of iodine solution
    indicates that the organism has
    hydrolyzed starch.

    Negative test: A blue, purple, or black


    coloration of the medium

    Bacillus subtilis is positive for starch hydrolysis


    (pictured below on the left).

    The organism shown on the right is negative


    for starch hydrolysis (E. coli).
    Bile Esculin Test
    Principle :

    Bile-esculin test is based on the ability of the group D streptococci


    and Enterococcus species, to hydrolyze esculin in the presence of bile.

    Bile esculin agar contains bile salts to inhibit the growth of gram positive
    organisms other than enterococci and group D streptococci.
    It also contains nutrients, esculin, and ferric citrate.

    When an organism hydrolyzes the glycoside esculin to form esculetin


    and dextrose, the esculetin reacts with the ferric citrate to produce a dark
    brown or black phenolic iron complex.
    Results
    CAMP Test

    CAMP factor is a diffusible, heat-stable protein produced by group B streptococci

    This is a synergistic test between Staphylococcus aureus and Streptococcus

    agalactiae. S. agalactiae produces CAMP factor

    The two bacteria are streaked at 90o angles of one another.


    As a result, an arrow of beta-hemolysis is produced between the two streaks
    Gelatinase Test
    Principle of Gelatin Hydrolysis Test

    This test is used to determine the ability of an organism to produce extracellular

    proteolytic enzymes, gelatinases that hydrolyze gelatin. The reaction occurs in two

    sequential steps: in first reaction gelatinases hydrolyze gelatin into polypeptides

    and then polypeptides are further converted into amino acids

    The amino acids are taken up by the cell and used for metabolic purposes.
    It distinguishes the gelatinase-positive, pathogenic Staphylococcus aureus from

    the gelatinase-negative, non-pathogenic S. epidermidis

    The test can also be used to differentiate genera of gelatinase-producing bacteria

    such Serratia and Proteus from other members of the family Enterobacteriaceae.
    Procedure of Gelatin Hydrolysis
    Test
    There are several methods for
    determining gelatin hydrolysis test.
    The most commonly used method
    is the nutrient gelatin stab method

    -Stab a nutrient
    gelatin tube -
    with bacteria and incubate for
    24h at 37ºC
    Expected results

    Positive: liquefaction of the inoculated tube even after exposure to cold


    temperature of ice bath or refrigerator (4°C)

    Negative: Complete solidification of the inoculated tube


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