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    Practical 6 Identification of Gram Ve Bacteria

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    Mona Mostafa
    The document describes a laboratory experiment to identify gram positive bacteria through biochemical tests. The objectives are to familiarize students with major microbiology tests, understand positive and negative results, and use the tests to identify gram positive bacteria. The methods section outlines tests like catalase and coagulase to differentiate bacterial species. It provides background on each test, materials needed, procedures, and expected results to classify isolates.

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    Practical 6 Identification of Gram Ve Bacteria

    Uploaded by

    Mona Mostafa
    99 views8 pages
    The document describes a laboratory experiment to identify gram positive bacteria through biochemical tests. The objectives are to familiarize students with major microbiology tests, understand positive and negative results, and use the tests to identify gram positive bacteria. The methods section outlines tests like catalase and coagulase to differentiate bacterial species. It provides background on each test, materials needed, procedures, and expected results to classify isolates.

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    The document describes a laboratory experiment to identify gram positive bacteria through biochemical tests. The objectives are to familiarize students with major microbiology tests, understand positive and negative results, and use the tests to identify gram positive bacteria. The methods section outlines tests like catalase and coagulase to differentiate bacterial species. It provides background on each test, materials needed, procedures, and expected results to classify isolates.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    99 views8 pages

    Practical 6 Identification of Gram Ve Bacteria

    Uploaded by

    Mona Mostafa
    The document describes a laboratory experiment to identify gram positive bacteria through biochemical tests. The objectives are to familiarize students with major microbiology tests, understand positive and negative results, and use the tests to identify gram positive bacteria. The methods section outlines tests like catalase and coagulase to differentiate bacterial species. It provides background on each test, materials needed, procedures, and expected results to classify isolates.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 8

    Ms. Banan A.

    Atwah

    Practical 6

    1431 - 1432 H

    IDENTIFICATION OF GRAM POSITIVE BACTERIA


    1. Objectives:
    To know the major biochemical tests used in microbiology. To be familiar with the basic principles of those biochemical tests. To be able to read the positive and negative tests results. To know the application of those tests in identification of gram positive bacteria.

    2. Background:
    A variety of biochemical tests can be performed directly using single colonies seen on primary isolation plates. Depending on the nature of biochemical reactions, some reactions become positive within a minute or two, other take less than an hour, and most require incubation for 4-18 hours. Such tests are help in placing an isolate of known morphology into a further subdivision, thus directing the additional procedures needed for identification. Frequently, an isolate can be identified to a level that is clinically useful based on these assessments.

    3. Materials:
    1. 2. 3. 4. 5. Set of Gram stain Glass slides Wire loop & wooden sticks Normal saline Cultures of (Staphylococcus aureus, S. epidermidis, S. saprophyticus) on blood agars. 6. Cultures of [Streptococcus pyogenes "group A", S. agalactiae "group B"] on blood agars. 7. Culture of Enterococcus faecalis (group D) on blood agar 8. Cultures of (Streptococcus viridians, S. pneumoniae) on blood agars 9. 3% H2O2 (hydrogen peroxide) reagent 10. Human plasma and Agglutination paper
    1

    2nd Year Nutrition

    Ms. Banan A. Atwah

    Practical 6
    4. Methods:

    1431 - 1432 H

    Describe the gross colonial morphology of all given plates. Perform Gram staining of the available organisms and then comment on the results. Then biochemical tests will be demonstrated, you should know the test name, understand the principle and be able to differentiate between positive and negative results. The tests demonstrated as follows:

    1. Catalase test: Fig.1


    This test is used to differentiate those bacteria that produce the enzyme catalase, such as staphylococci, from non-catalase producing bacteria such as streptococci. Method of the test: 1. With a wooden stick take a part of single colony and put it on a slide. 2. Add one small drop of H2O2 to the bacteria. 3. Look for immediate bubbling by the production of gas

    2 H2O2 Results:

    Catalase Enz.

    2 H2O + O2 (gas)

    Gas production (bubbles) Catalase positive (all Staphylococcus species) No Gas production (No bubbles) Catalase Negative (all Streptococcus species)

    Figure 1: Catalase test 2

    2nd Year Nutrition

    Ms. Banan A. Atwah

    Practical 6
    2. Coagulase test:

    1431 - 1432 H

    This test is used to differentiate Staphylococcus aureus which produces the enzyme coagulase, from S. epidermidis and S. saprophyticus which do not produce coagulase. Principle: Coagulase causes plasma to coagulate (clot) by converting the plasma fibrinogen to fibrin.
    Bacterial coagulase

    Fibrinogen

    fibrin (clot) coagulation

    Two types of coagulase are produced by most strains of S. aureus: Free coagulase: converts fibrinogen to fibrin by activating a coagualse-reacting factor present in plasma. It is detected by clotting in the tube test (Fig. 2) Bound coagulase (clumping factor): converts fibrinogen directly to fibrin without requiring a coagulase-reacting factor. It can be detected by clumping of bacterial cells in the rapid slide test. Method for slide test (bound coaglulase): Fig.3 1. Add one drop of either human plasma or latex reagent on to paper slide 2. Using a plastic or wooden stick, take part of test colony to the slide 3. Mix well and look for clumping within 10 seconds. Results: Clumping or clots formed (plasma coagulated) within 10 seconds Coagulase produced (coagulase positive) the organism is S. aureus. No clumpimg within 10 seconds No coagulase produced (coagulase negative) the organism may be (other staphylococci S. epidermidis or S. saprophyticus) See chart on page 8

    2nd Year Nutrition

    Ms. Banan A. Atwah

    Practical 6

    1431 - 1432 H

    + v _
    Figure 2: Tube coagulase test

    + v
    Figure 3: Slide coagulase test

    2nd Year Nutrition

    Ms. Banan A. Atwah

    Practical 6
    Unknown culture identification (1)

    1431 - 1432 H

    1-Cultural characteristics (Colony gross morphology): table1


    Colony morphology Reaction on the plate

    Table 1: Gross Colony Characteristics No. Colony characteristics


    1 2 3 4 5 6 7 8 9 10 11 Size Form Elevation Margin Color Haemolysis Pigment production Odor Opacity Surface Consistency

    Observations
    Very small, Small, Medium, large, very large Punctiform, circular, filamentous, irregular, rhizoid, spindle Flat, raised, convex, pulvinate, umbonate Entire, undulate, lobate, erose, filamentous, curled White, grey, yellow, black, orange, pink, red, etc haemolysis , or Color of the pigment production Fruity, freshly cut apple, fishy, fecal or putrid, bleach, pungent Transparent, Opaque, Translucent Smooth, Glistening, Rough, dull Buttery, viscid, Brittle, mucoid

    2nd Year Nutrition

    Ms. Banan A. Atwah

    Practical 6

    1431 - 1432 H

    2-Gram Staining: Fig.4

    Gram reaction

    Gram positive

    Gram negative

    Shape

    Cocci

    Bacilli

    Spiral

    Arrangement

    Single

    Pairs Chain (diplo-) (strepto-)

    Cluster (staphylo-)

    irregular

    Figure 4: Main bacterial shapes & arrangements

    2nd Year Nutrition

    Ms. Banan A. Atwah

    Practical 6
    3-Biochemiacal test:

    1431 - 1432 H

    (write name of organism) Differential test


    Reaction results Positive (or sensitive) Catalase Coagulase Mannitol salt agar DNAse Novobiocin sensitivity test Bacitracin disk CAMP test optochin sensitivity disk Bile esculin hydrolysis Negative (or resistant)

    2nd Year Nutrition

    Ms. Banan A. Atwah

    Practical 6

    1431 - 1432 H

    Cocci G +ve Bacteria Bacilli

    Catalase

    +
    Staph.

    Strept. Nonsporing

    Endo Spores

    Sporing

    Coagulase

    Type of Haemolysis

    Coryne. diphtheria

    +
    S.Aureus

    (nonAureus)

    Optochin disk

    Bile Esculin hydrolysis

    Lactobacilli Presence of air

    DNase

    +
    S.Epi

    Novobiocin disk

    Bacitracin disk

    S+

    RS.sapro.

    S+
    St. gp A

    RB,C,G

    S+
    St.pneu.

    RSt.Viri

    Other St.gp D

    Aerobic

    +
    Enterococcus Faecalis

    Anaerobic

    CAMP test

    +
    St.gp B

    St.gpC,G

    Bacillus Spp.

    Clostridium

    e.g. Bacillus cereus Catalase +ve Streptococcus pyogenes Streptococcus agalactiae Streptococcus pneumoniae Streptococcus viridans 8

    2nd Year Nutrition

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