Micro Lab Practical 2

Know how pili are used to transfer plasmids from one cell to another

Transfer pilus makes contact w/ cell → outer membranes fuse Donor cell & recipient cell

What is normal flora? What is transient flora?

Transient - microbes present for short time w/o causing disease

What are Durham tubes and why are they used?

Traps CO2 produced indicating fermentation

List some ways to reduce the risk of antibiotic resistance

Use when necessary finish the prescription narrow-spectrum > broad-spectrum

Insertion-

addition of 1+ nucleotides into DNA sequence

Genetic selection -

choosing indv. w/ desirable genetic traits for breeding

What mutations are typically NOT reversible?

deletion

Genotype -

genetic makeup

Suppressors -

mask / reverse effects of a mutation

How can a second-site mutation result in reversion?

most mutations restore original phenotype by compensating for the effect of the original mutation

Phenotype -

observable traits

Deletion-

removal of 1+ nucleotides from DNA sequence

Reversion -

reversal of mutation

Codon -

sequence of 3 nucleotides forming a unit of genetic code in DNA/RNA

What does a positive fermentation results look like? What does a negative result look like?

(+) - Orange/yellow, (-) stays red

Describe the test and what a positive/negative result looks like

(+) - bubbles → staph

Urease Test Results

(+) - pink

What does a positive test look like in Cytochrome? Negative test?

(+) purple → it was oxidized

What is a nosocomial infection?

Acquired at the hospital

How do you finish the nitrate test after incubation?

Add nitrate (+) red(-) true negative or (+) w/ addition of zine (go clear)

Compare and contrast aerobic respiration and anaerobic

Aerobic- 60% effective, high ATP (36) Anaerobic - less effective, lower ATP (2)

What colonies are tested in a genetic screen? Is this a selection experiment or are all colonies allowed to grow prior to testing?

Allows cells to grow & develop into colonies something is sprayed allowing researcher to differentiate between mutants & wild type

Bactericidal and bacteriostatic? Cytocidal and cytostatic?

Bacteria or cell

What are anabolic and catabolic reactions?

Break down build up

What is starch?

CHO containing multiple glucose chains

What is an antiseptic? List some examples

Can be safely applied to skin (iodine, alcohol)

What are competent cells?

Can take up extracellular material

What are antibiotics?

Chemical sec. As secondary metabolites to inhibit/ kill other organisms

What is a plasmid and how do we use it in the laboratory setting?

Circular pieces of DNA that replicate inside cells separately from Chromosomal DNA

What is mutagenesis?

Creating DNA mutations

Define transformation

DNA into cell

Would bacteria with DNase be pathogenic or non-pathogenic? Why?

DNAse secretion inc. virulence of pathogen → inflicts damage on host cell

Explain the DNase test and how the DNA is complexed with a green dye

Degraded DNA released the bound dye (+) = zone of clearing

What type of bacteria would we use a SIM test for? Why?

Differentiate members of Enterobacteriaceae - monitoring sugar fermentation pathways

What bacteria does this test differentiate between?

Enterococcus from Staph

Why do we use gram's iodine in this experiment?

Flooded to show halo - (+) → zone of clearing = amylase presence

Know the different types of genetic mutations.

Frameshift - insertion/deletion of nucleotides Nonsense - introduces stop codon Missense - changes codon → protein change

What organisms are tested on this

Gram positive, differentiates enterococcus from staph

What kinds of bacteria are urease positive?

Helicobacter, Klebsiella, & campylobacter

What is the Ames test used for?

Identify mutagenic chemicals

Why could the test be a false negative in Nitrate Reduction Test?

Incomplete reduction, slow growth, interfering substances

What does each letter stand for in IMViC? Describe positive and negative results for each.

Indole - Tyr hydrolysis (+) black Methyl Red - glucose fermented into mixed acids (+) red Vogues Proskauer - acetoin presence (+) red/brown @ top Citrate - citrate as C source (+) blue

What mutation can cause a frameshift mutation?

Insertion / deletions of 1+ nucleotides

What is a disinfectant? List some examples

Kill or prevent growth of microorganisms (inanimate objects) Chlorine or ethanol

What is the difference between broad-spectrum and narrow-spectrum antibiotics?

Kill wide range v. select species

What test do we use to determine antibiotic sensitivity? Describe this test

Kirby-Bauer method - bacteria mixed w/ conc. Of antibiotics & measure zone of clearing

Fermentation, examples?

Metabolism of sugars for energy - Ex.) yeast/butyric/lactic acid fermentation

Why do you use a sterile wooden stick instead of metal in Cytochrome Oxidase Test?

Metal could give false positive due to surface oxidation

What is the difference between microbicidal and microbiostatic?

Microbicidal- against kill microorganism Microbiostatic- inhibit growth

What is a pathogen? What are opportunistic pathogens?

Microorganisms that cause disease v. dont normally but could

What is casein?

Milk protein too large to enter microbial cell

What is replica plating? How is it done?

Mutant analysis to look for a mutant unable to use CHO Plate cells → plate pressed onto velvet → plate w/o CHO pressed → colonies that don't grow on media can be found of 1st plate & chosen

How do the byproducts affect the pH in Urease test?

NH4+ inc. pH → used as N2 source to synthesize

What bacteria is used for the Ames test? Why?

Nonpathogenic strains of salmonella typhimurium- intentionally mutated

What is electroporation?

Not naturally competent bacteria made competent thorough chemicals or electrical field exposure

Why are we using selective media with antibiotics for this experiment?

Observe & record outcome

What is genetic exchange?

Occurs when DNA is transferred to 2nd cell (transformation, conjugation, & transduction)

What is denitrification?

Oxidized N2 reduced to Molecular Nitrogen

What was the first antibiotic discovered?

Penicillin

What is the purpose of histidine?

Presence Allows chemical metabolism to take place

Why can only certain bacteria use this as a nutrient source? How is it broken down?

Proteolytic enzyme presence - peptide bonds broken through hydrolysis

List some common mutagens

Radiation / chemicals

What are hydrogen peroxide and superoxide radicals? Why are they bad for the cell?

Radicals degrade other molecules → apoptosis

Define conjugation

Requires that the bacteria be in actual contact

What does each letter in SIM stand for? Describe positive and negative results for each.

S - H2S, (+) black I - Tyr hydrolysis, (+) red ring M - Motility (+) cloudy

What is metabolism

Scope of how cells live, function, & replicate in their environment & rxns that occur during these fxns

Why and when can mutations occur?

Spontaneously / DNA replication errors

What 3 things does a SIM test tell us about the bacteria?

Sulfur, Indole, Motility

What two enzymes are necessary to protect against superoxide radicals? Describe the biochemical reaction formula for each

Superoxide dismutase (SOD) - converts radicals → H2Catalase - converts H2 → H20 + O2

Describe the starch hydrolysis experiment

Tested ability to break down starch