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