Microbiology Bacteriological Techniques
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Questions and Answers

What is the correct order, after collecting a microbial sample, according to the text?

  • Incubate, then gram stain, then inoculate culture media.
  • Inoculate culture media, then gram stain, then incubate.
  • Gram stain, then inoculate culture media, then incubate. (correct)
  • Incubate, then inoculate culture media, then gram stain.

    • At what temperature are microbial cultures typically incubated, according to the text?

  • 37°C (correct)
  • 30°C
  • 25°C
  • 42°C

    • Which of the following is a liquid medium, according to the text?

  • Agar
  • Slant
  • Plate
  • Broth (correct)

    • What is a slant, according to the text?

    <p>A tube of agar that has solidified on an angle. (C)</p> Signup and view all the answers

    What is the purpose of selective media, according to the text?

    <p>To inhibit the growth of certain types of organisms, allowing specific bacteria to grow. (B)</p> Signup and view all the answers

    What is the primary purpose of differential media, according to the text?

    <p>To distinguish between certain bacteria based on biochemical reactions. (C)</p> Signup and view all the answers

    Which of the following is NOT an example of a selective media according to the text?

    <p>Salmonella-Shigella agar (SS) (A)</p> Signup and view all the answers

    What differentiates between lactose fermenters and non-lactose fermenters on Eosin Methylene Blue agar?

    <p>A pH indicator that stains lactose fermenters purple (C)</p> Signup and view all the answers

    What does the presence of 7.5% sodium chloride do on Mannitol Salt Agar?

    <p>It selects for halotolerant organisms. (A)</p> Signup and view all the answers

    What effect do colistin and nalidixic acid have on Columbia colistin-nalidixic acid agar (CNA), according to the text?

    <p>They inhibit the growth of gram-negative bacteria (D)</p> Signup and view all the answers

    What color change indicates a positive reaction on Bile Esculin agar?

    <p>Dark Brown to Black (A)</p> Signup and view all the answers

    What is the purpose of using sodium chloride 0.85% in API 20E testing?

    <p>To dilute Gram-negative bacteria (B)</p> Signup and view all the answers

    Which of the following best describes how the catalase test is performed?

    <p>Smear bacteria on a slide, add hydrogen peroxide, and look for bubbling (C)</p> Signup and view all the answers

    A positive coagulase test is indicated by what observation?

    <p>Plasma Gel (B)</p> Signup and view all the answers

    Which of the following is a critical requirement for storing antibiotic discs used in the Kirby-Bauer test?

    <p>They must be stored at 4°C (B)</p> Signup and view all the answers

    What is the primary purpose of control organisms in laboratory testing?

    <p>To ensure the accuracy and reliability of lab tests (A)</p> Signup and view all the answers

    The optochin sensitivity test is primarily used to identify which bacterial genus?

    <p>Streptococcus (A)</p> Signup and view all the answers

    In a standard oxidase test, which color indicates that bacteria produce cytochrome oxidase?

    <p>Blue (C)</p> Signup and view all the answers

    Which medium is designed to provide standardized results during antibiotic sensitivity testing?

    <p>Mueller-Hinton agar (MH) (C)</p> Signup and view all the answers

    The CAMP test is used to identify Streptococcus ______, a species causing mastitis.

    <p>agalactiae (B)</p> Signup and view all the answers

    In the context of bacterial identification, what does a black pigment within a colony on a selective agar typically indicate?

    <p>Hydrogen sulfide production (D)</p> Signup and view all the answers

    Triple sugar agar is used to determine if bacteria can ferment which of the following sugars?

    <p>glucose, lactose, and sucrose (C)</p> Signup and view all the answers

    What is the primary function of phenol red in mannitol-containing media?

    <p>Indicating pH changes (D)</p> Signup and view all the answers

    What is the purpose of adding blood to Mueller-Hinton agar?

    <p>To enrich the media for fastidious organisms (B)</p> Signup and view all the answers

    What is the purpose of ferric citrate in the selective agar mentioned?

    <p>To react with hydrogen sulfide, causing black pigment formation (B)</p> Signup and view all the answers

    Which enzyme does Urea agar slant test for, in bacterial cultures?

    <p>Urease (C)</p> Signup and view all the answers

    What is a primary characteristic of Escherichia coli based on the content provided?

    <p>Lactose fermenting (B)</p> Signup and view all the answers

    Besides Trypticase Soy Broth, which of the following is also a general-purpose broth?

    <p>Brain-heart infusion (BHI) broth (D)</p> Signup and view all the answers

    Which of the following is NOT mentioned as a culture medium?

    <p>Sodium chloride 0.85% (NaCl 0.85%) (D)</p> Signup and view all the answers

    Signup and view all the answers

    Study Notes

    Microbial Sample Sequence

    • Collect the sample.
    • Perform Gram stain.
    • Inoculate culture media.
    • Incubate for 18-24 hours at 37°C.
    • Check for growth.

    Bacteriological Media

    • Agar: A semisolid medium.
    • Broth: A liquid medium.
    • Plate: A flat, round container of agar.
    • Tube: A screw-top container, potentially containing agar or broth.
    • Slant: A tube of agar that has solidified on a slant.
    • Selective Media: Medium containing compounds that inhibit the growth of certain organisms, useful for isolating particular types of bacteria. Examples include MacConkey II Agar, Bismuth Sulfite Agar, Eosin Methylene Blue, Columbia Colistin-nalidixic acid agar (CNA), and Mannitol Salt Agar. Gram-positive or Gram-negative bacteria can be selected for based on the compound.

    Differential Media (Examples)

    • MacConkey II Agar (Mac): Differentiates lactose fermenters from non-lactose fermenters; neutral red indicator stains lactose fermenting colonies purple. This agar inhibits overgrowth of E. coli vs Klebsiella spp.
    • Salmonella-Shigella agar (SS): Selects for pathogenic enteric Gram-negative bacteria, differentiating colonies based on lactose fermentation.
    • Mannitol Salt Agar (MSA): Selects for halotolerant organisms (example: Staphylococcus) and differentiates organisms that can ferment mannitol from those that cannot. A lower pH causes phenol red to turn yellow.

    Hemolysis

    • Hemolysis is the destruction of red blood cells (RBCs), releasing their contents into the surrounding fluid.

    Eosin Methylene Blue (EMB) Agar

    • Dyes inhibit growth of Gram-positive bacteria.
    • Selects for Gram-negative bacteria.
    • Differentiates between lactose fermenters and non-lactose fermenters.

    Columbia Colistin-nalidixic acid agar (CNA)

    • Colistin and nalidixic acid inhibit Gram-negative organisms.

    Phenylethyl alcohol (PEA) Agar

    • Used for the growth of Gram-positive bacteria.

    Mannitol Salt Agar (MSA)

    • Contains 7.5% sodium chloride (a high salt concentration), used to select for organisms that are halotolerant (example: Staphylococcus spp.).
    • Mannitol and phenol red (a pH indicator) can differentiate organisms that ferment mannitol from those that cannot.

    Mueller-Hinton Agar (MH)

    • A general-use medium (commonly used in antibiotic sensitivity testing).
    • Used in the Kirby-Bauer test and can be enriched with blood for fastidious organisms.
    • Measuring zone diameters can help determine an organism's susceptibility.

    Salmonella-Shigella Agar (SS)

    • Selecting pathogenic enteric Gram-negative bacteria.
    • Differentiating colonies based on lactose fermentation (pink colonies)
    • Differentiating Hydrogen Sulfide (H2S)-producing bacteria with ferric citrate in the formula (black pigment).

    Gram-negative lactose fermenter

    • Escherichia coli

    Gram-negative, non-lactose fermenter, hydrogen sulfide producer

    • Salmonella

    Gram-negative, non-lactose fermenter, non-hydrogen sulfide producer

    • Shigella

    Broths for general use

    • Trypticase soy broth (TSB).
    • Brain heart infusion (BHI).

    BHI use in turbidity

    • Brain heart infusion (BHI) broth is used in preparing a bacterial solution at a specific turbidity level.

    Enrichment medium

    • Brain heart infusion broth, or BHI, can be used as an enrichment medium for growing Salmonella and Shigella when used during fecal culturing.

    Urea Broth/Agar

    • Determines if bacteria produce the enzyme urease, which degrades urea into ammonia, water, and carbon dioxide.

    Bile Esculin Agar (BE)

    • Identifying bacteria that hydrolyze esculin (enterococci).
    • Positive test produces a dark brown to black colour change.

    Simmons Citrate Agar

    • Identifies organisms that use citrate as sole carbon source.
    • A positive reaction turns the agar a deep blue color.

    Triple Sugar Iron Agar (TSIA)

    • Determines whether bacteria ferment three types of sugars: glucose, lactose, and sucrose.

    Sodium Chloride 0.85% (NaCl 0.85%)

    • A sterile solution used for diluting Gram-negative bacteria before testing (API 20E).

    Catalase Test

    • Detects the enzyme catalase.
    • Positive result is indicated by bubbling.
    • Examples of catalase-positive bacteria: Staphylococcus. Examples of catalase-negative bacteria: Streptococcus.

    Coagulase Test

    • Tests for the presence of coagulase enzyme production, differentiating Staphyloccocus species (which can produce the enzyme), from other bacteria. Positive test indicated by a clear gel formation of the sample mixture.

    Control Organisms

    • Organisms used in lab tests to verify accuracy and reliability.

    Optochin Sensitivity Test

    • Tests for Streptococcus pneumoniae (a bacterial species). Arrangement of Streptococcus pneumoniae: Diplocci.

    Oxidase Test

    • Detects cytochrome oxidase.
    • Positive result is indicated by a colour change (pink, blue, or yellow).

    CAMP Test

    • Tests for Streptococcus agalactiae.

    Mastitis

    • Inflammation of the mammary glands in animals.

    California Mastitis Test (CMT)

    • Cow-side test for mastitis. Detects somatic cells in the milk.

    Common Mastitis Pathogens

    • Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, and Escherichia coli.

    CMT Positive Next Steps

    • Milk culture, BAP, MAC (or specialized mastitis media) and subculturing.

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