Micro Lab Exam 2

What is a nutritive (general-purpose) medium?

A medium that supports growth of a broad range of nonfastidious microorganisms.

What is selective medium?

A medium containing ingredients that inhibit some organisms while permitting others to grow.

What is differential medium?

A medium containing substrates and indicators that reveal biochemical differences among organisms.

Can a medium be both selective and differential?

Yes. For example, MSA selects for salt-tolerant bacteria and differentiates mannitol fermenters.

What does a pH indicator reveal in culture media?

A change in acidity or alkalinity caused by microbial metabolism.

How does carbohydrate fermentation commonly change medium pH?

Acidic fermentation products lower pH, potentially changing an indicator's color.

Why does growth versus no growth matter on selective agar?

Growth suggests tolerance of the selective ingredients; lack of growth suggests inhibition.

Does a color change always mean the organism grew?

No. Interpret color together with visible growth and the medium's indicator.

Why is mannitol salt agar (MSA) selective?

MSA contains approximately 7.5% NaCl. This high osmotic pressure inhibits many salt-sensitive bacteria, especially many streptococci, while allowing salt-tolerant organisms such as staphylococci to grow. Growth indicates salt tolerance, not species identity.

Why is MSA differential?

MSA contains mannitol (fermentable carbohydrate) and phenol red (pH indicator). Organisms fermenting mannitol produce acidic products, lowering the pH and turning the medium yellow around colonies. Nonfermenters may grow but leave it red/pink.

What does yellow MSA around bacterial growth indicate?

Mannitol fermentation produces acid, turning phenol red yellow. [ADD IMAGE: yellow MSA plate]

What does growth on MSA without yellowing indicate?

The organism tolerates salt but did not detectably ferment mannitol. [ADD IMAGE: red/pink MSA plate]

What does no growth on MSA indicate?

The organism was inhibited by the selective conditions; mannitol fermentation cannot be evaluated.

What does the catalase test detect?

Catalase enzyme, which decomposes hydrogen peroxide into water and oxygen.

What is a positive catalase test?

Visible oxygen bubbles after hydrogen peroxide is added. [ADD IMAGE: catalase positive versus negative]

How do Staphylococcus and Streptococcus usually differ in catalase testing?

Staphylococcus is catalase-positive; Streptococcus is catalase-negative.

What is a dichotomous key?

A stepwise identification tool using paired alternative characteristics or test results. [ADD IMAGE: lab identification key]

What is hemolysis?

Damage or lysis of red blood cells, visible as changes around colonies on blood agar.

What is beta hemolysis?

Complete red blood cell lysis, producing a clear zone around colonies. [ADD IMAGE: blood agar]

What is alpha hemolysis?

Partial hemolysis with a greenish or brownish zone around colonies. [ADD IMAGE: blood agar]

What is gamma hemolysis?

No visible hemolysis or change in surrounding blood agar. [ADD IMAGE: blood agar]

What are hemolysins?

Microbial toxins or proteins that damage red blood cells; some contribute to virulence.

What is a virulence factor?

A microbial feature that contributes to causing disease or damaging a host.

How would catalase-positive cocci growing on MSA be approached with a key?

Follow the catalase-positive branch, then use MSA fermentation and any additional tests in the provided key.

What is an indicator organism?

An organism monitored as evidence of contamination or sanitary quality rather than necessarily being the pathogen itself.

Why are indicator organisms useful?

They provide a practical way to assess contamination risk when testing every pathogen is impractical.

What makes MacConkey agar selective?

Bile salts and crystal violet inhibit many Gram-positive bacteria.

What makes MacConkey agar differential?

Lactose and neutral red distinguish lactose fermenters from nonfermenters.

What does a pink/red colony on MacConkey agar suggest?

Lactose fermentation and acid production. [ADD IMAGE: MacConkey plate]

What does a colorless colony on MacConkey agar suggest?

Growth without detectable lactose fermentation. [ADD IMAGE: MacConkey plate]

What makes eosin methylene blue (EMB) agar selective?

Eosin Y and methylene blue dyes inhibit many Gram-positive bacteria.

What makes EMB agar differential?

Lactose fermentation alters colony color through acid effects on the dyes.

What does a metallic green sheen on EMB commonly indicate?

Strong lactose fermentation, classically seen with E. coli. [ADD IMAGE: EMB plate]

How do EMB and MacConkey agar compare?

Both select mainly for Gram-negative enterics and distinguish lactose fermentation; they use different dyes and appearance criteria.

What does Simmons citrate agar test?

Whether an organism can use citrate as its sole carbon source under the test conditions.

What does a positive Simmons citrate result look like?

Visible growth is the key positive finding; the bromothymol blue indicator often changes from green to blue as alkaline products accumulate. Growth without a blue color change can still be citrate-positive. [ADD IMAGE: positive versus negative citrate slants]

What does a negative Simmons citrate result look like?

No growth and the medium remains green. [ADD IMAGE: citrate slant]

What does urease do?

Hydrolyzes urea, generating ammonia and raising pH.

What does a positive urease broth test look like?

Urease hydrolyzes urea into ammonia and carbon dioxide. Ammonia increases the pH; phenol red changes to a bright pink/fuchsia color. The rate and intensity depend on the organism and incubation. [ADD IMAGE: urease broth tubes]

What does a negative urease broth test look like?

No pink alkaline color change. [ADD IMAGE: urea broth]

What does TSIA stand for?

Triple Sugar Iron Agar.

Which three sugars are present in TSIA?

Glucose, lactose, and sucrose.

Why does TSIA contain less glucose than lactose or sucrose?

Low glucose (0.1%) and higher lactose/sucrose (1% each) help distinguish glucose-only fermentation from additional sugar fermentation.

What do A and K mean in TSIA?

A = acid (yellow); K = alkaline (red). [ADD IMAGE: TSIA A/K interpretation chart]

What is the correct order for writing a TSIA result?

Slant first, then butt: for example K/A means red slant and yellow butt.

What does A/A mean in TSIA?

Acid slant/acid butt: both yellow. Glucose is fermented, and sufficient lactose and/or sucrose fermentation maintains an acidic slant even in oxygen. Gas and H2S are reported separately. [ADD IMAGE: A/A tube]

What does K/A mean in TSIA?

Alkaline slant/acid butt: red slant, yellow butt. Glucose is fermented but lactose and sucrose are not detectably fermented. Once the small glucose supply is exhausted, aerobic peptone utilization re-alkalinizes the slant; the butt stays acidic. [ADD IMAGE: K/A tube]

What does K/K mean in TSIA?

Alkaline slant/alkaline butt: red/red, indicating no detectable fermentation of the three sugars, with alkaline reactions. Some nonfermenters instead produce K/NC (red slant and unchanged butt); distinguish these observations. [ADD IMAGE: TSIA chart]

Why can the TSIA slant become red while the butt stays yellow?

After limited glucose is depleted, aerobic metabolism of peptones on the slant raises pH, while the butt remains acidic.

How does oxygen availability differ between TSIA slant and butt?

The slant is relatively aerobic; the deep butt is relatively anaerobic.

What indicates gas production in TSIA?

Cracks, bubbles, or lifting/displacement of agar. [ADD IMAGE: gas-positive tube]

What indicates H2S production in TSIA?

Sulfur-containing compounds can be reduced to H2S, which reacts with iron salts to form a black ferrous sulfide precipitate. A blackened butt is interpreted as acid even when yellow is hidden. [ADD IMAGE: H2S-positive TSIA]

If TSIA butt is black, what should you assume about the butt's acid reaction?

A blackened butt is typically interpreted as acidic (A), even if yellow is obscured.

Can an A/A tube also be gas-positive and H2S-positive?

Yes. Sugar fermentation, gas production, and H2S production are separate observations.

A tube has red slant, black butt, and cracks. How is it interpreted?

K/A with H2S positive and gas positive (the black butt masks the acidic yellow). [ADD IMAGE: combined tube]

What is a phenol red indicator's general acid/alkaline color behavior?

Yellow under acidic conditions and red/pink under more alkaline conditions.

What does MRS stand for?

De Man, Rogosa and Sharpe medium.

What is the purpose of MRS agar?

To support and favor the growth of lactobacilli and other lactic acid bacteria.

How does MRS agar favor lactobacilli?

Its nutrient composition and relatively low pH favor acid-tolerant lactic acid bacteria over many competitors.

What metabolic process makes yogurt acidic?

Lactic acid fermentation of milk sugars.

How does lactic acid fermentation change yogurt taste?

Accumulating lactic acid gives yogurt its tart or sour taste.

Why does milk thicken during yogurt fermentation?

Falling pH destabilizes milk casein proteins, causing coagulation and gel formation.

What happens to yogurt pH during fermentation?

It decreases as lactic acid accumulates.

What is Sabouraud dextrose agar (SDA) used for?

Cultivating fungi, especially yeasts and molds.

How does SDA favor fungi over many bacteria?

Its acidic pH and nutrient formulation favor fungi and suppress many bacterial species.

What are hyphae?

Filamentous fungal structures that grow and collectively form mycelium.

What is mycelium?

A network or mass of fungal hyphae.

What is a filamentous bacterium?

A bacterium that grows as elongated cells or branching filaments, potentially resembling fungal hyphae.

How can filamentous bacteria and fungi be distinguished structurally?

Fungi are eukaryotic and have chitin-containing walls; filamentous bacteria are prokaryotic and have bacterial cell envelopes.

What ecological role do many soil fungi play?

Decomposition and nutrient cycling by breaking down organic matter.

What ecological role do many filamentous soil bacteria play?

Decomposition and nutrient cycling; some also produce antimicrobial compounds.

Why can colony appearance alone be insufficient to distinguish soil fungi from filamentous bacteria?

Both may form fuzzy or filamentous colonies, so microscopy and other tests may be needed. [ADD IMAGE: soil colony comparisons]

What is a plasmid?

A typically small, extrachromosomal DNA molecule capable of independent replication.

How are bacterial plasmids commonly structured?

Often circular, double-stranded DNA.

What is an R plasmid?

A plasmid carrying one or more antibiotic resistance genes.

How can plasmids contribute to antibiotic resistance spread?

They can carry resistance genes and, in some cases, move between bacteria through horizontal gene transfer.

What is horizontal gene transfer?

Movement of genetic material between organisms other than from parent to offspring.

What is bacterial conjugation?

DNA transfer between bacteria requiring direct cell-to-cell contact.

What is bacterial transformation?

Uptake of free extracellular DNA by a competent bacterium.

What does competent mean in bacterial transformation?

Able to take up extracellular DNA.

How can bacteria be made artificially competent?

Laboratory treatments such as calcium chloride plus heat shock or electroporation.

What is electroporation?

Application of a brief electrical pulse that transiently increases cell membrane permeability to DNA.

What is a transconjugant?

A recipient bacterium that acquired DNA through conjugation.

How can selective medium identify transconjugants?

Choose conditions requiring markers from both donor and recipient, so only recipients acquiring the transferred trait can grow.

Why include donor-only and recipient-only controls in a conjugation experiment?

Plate each parental strain alone on the same double-selective medium used for the mating mixture. Neither parent should grow if selection is specific. Growth in the mixture plus absence of growth in controls supports transfer rather than parental contamination or incomplete selection.

What result supports successful conjugation?

Growth of appropriately selected mixed-culture transconjugants with no corresponding growth in relevant parental controls.

How does conjugation differ from transformation?

Conjugation transfers DNA by direct contact between donor and recipient, often involving conjugative plasmid machinery. Transformation is uptake of extracellular free DNA by a competent recipient, without direct donor contact. Both are horizontal gene transfer.

Does acquisition of antibiotic resistance always mean a new mutation occurred?

No. Resistance genes may have been acquired by horizontal gene transfer.

What is a bacteriostatic antimicrobial?

A drug that inhibits bacterial growth without necessarily killing cells.

What is a bactericidal antimicrobial?

A drug that kills bacteria.

What is the purpose of the Kirby-Bauer disk diffusion test?

A standardized lawn of bacteria on Mueller-Hinton agar is exposed to antimicrobial-impregnated disks. After incubation, measure each zone diameter in mm and compare it with organism/drug-specific breakpoints to classify susceptibility; zone size alone is not an MIC or a direct measure of killing.

What is a zone of inhibition?

A visible area around an antimicrobial disk where bacterial growth is prevented. [ADD IMAGE: Kirby-Bauer plate]

What is measured in Kirby-Bauer testing?

The diameter of the inhibition zone, typically in millimeters.

Why must Kirby-Bauer zone size be interpreted using standards?

Zone size depends on drug diffusion and organism/drug-specific breakpoints; bigger is not automatically clinically susceptible.

Why is Mueller-Hinton agar used in disk diffusion testing?

It supports reproducible bacterial growth and standardized antimicrobial diffusion.

What properties of Mueller-Hinton agar support reliable testing?

Controlled composition, appropriate pH and cation levels, and good diffusion characteristics.

Can a Kirby-Bauer plate alone establish whether a drug is bactericidal?

No. Disk diffusion measures inhibition, not directly whether cells were killed.

What do susceptible, intermediate/increased exposure, and resistant categories represent?

Interpretive susceptibility categories based on validated antimicrobial-specific standards.

How do yeast and molds differ morphologically?

Yeasts are generally unicellular; molds grow as multicellular filamentous hyphae.

How do many yeasts reproduce asexually?

By budding or fission.