BIOL 2042 Final Exam
Advantage and disadvantage of dilution
Advantage: A specific amount of bacteria are reduced with every dilution Disadvantage: Overnight incubation is required.
Who is the father of antibiotics? Why?
Alexander Fleming
What is human microbiome? Are the bacteria always harmful to us?
All bacteria in our body, not all of them are harmful to us and we need someneedsome of them
What is the use of TBE (Tris/Borate/EDTA) and/or TAE (Tris/Acetate/EDTA buffer)
Allows electricity to move throughout the system and helps the DNA to stay in the well
What is the purpose of PCR?
Amplification of DNA and the use of DNA sequences
Use of cycloheximide, why?
Antifungal agent, keeps any soil samples from potentially growing
Give an example of proper PPE while working with Tiny Earth project.
Gloves, mask, protective eyewear, labcoat
Glycerol stock:
Glycerol is mixed with bacterial culture so that when the cells are frozen, there will be no icicle formed and the cells survive. Glycerol stock is made to store the bacteria at -80°C freezer
Explain the difference between horizontal gene transfer and vertical gene transfer.
Horizontal gene transfer: Acquiring DNA from the environment (transformation), other bacteria (conjugation), or from viral bacteriophages (transduction). Vertical gene transfer: Parent cell passing DNA to offspring
Vertical versus horizontal transfer
Horizontal gene transfer: Acquiring DNA from the environment (transformation), other bacteria (conjugation), or from viral bacteriophages (transduction). Vertical gene transfer: Parent cell passing DNA to offspring
Why was it important to incubate at 30°C?
Human pathogens grow near temperature ranges of 37C. So its important to incubate at 30C to avoid growing these pathogens
What is the function of β lactams?
Inhibits bacterial cell wall synthesis
What is the purpose of Gel electrophoresis?
It allows for the separation of fragments of DNA- DNA migration to opposite side from cathode to anode
Why is fixing important?
It causes the bacteria to stick to the slide.
Purpose of Nutrient agar and supplement in this experiment (bacteriophage)
It is used to cultivate and maintain non-fastidious microorganisms - tryptone agar plate: general-purpose agar which will support the growth of a wide range of micro-organisms
Why use fewer rich media?
Less rich media doesn't allow contamination microbes to outgrow the soil microbes on the plate
Lytic and lysogenic cycle (clear vs. cloudy)
Lysis- death of host cell (clear) Lysogeny- prophage/integration of phage into host genes(cloudy)
Why pH of soil is important for shaping microbial life?
Microbes can only survive in a narrow range of pH. More acidic soils have more microbial life than basic soils do.
Microbe
Organisms that cannot be seen with the naked eye
Cyanobacteria characteristics
Oxygenic photosynthetic (in thylakoids) bacteria, - unicellular - filamentous - prokaryotic
Which is the RECIPIENT in conjugation?
P. putida 503CA
Which of the organisms used in this experiment (conjugation) had the plasmid:
PAW15
auxotroph vs prototroph in conjugation experiment
PAW15 is an auxotroph- requires Toluene to grow 503CA is a heterotroph- doesn't require Toluene
What are the phenotypes of each? (conjugation)
PAW15- Leu-,Tol+ 503CA- Leu+,Tol-
SLIDES: Anabaena, Spirulina, Gleocapsa
PICTURES
SLIDES: Trypanosoma spp, Trichomonas vaginalis, Euglena
PICTURES
SLIDES: Volvox, Spirogyra, Diatoms, Desmids
PICTURES
horizon
Parallel layer of the soil surface
oxygenic photosynthesis
Photosynthesis that oxidizes water to form oxygen; the form of photosynthesis is a characteristic of plants, protists, and cyanobacteria
acid fast staining procedure/organisms/appearance
Primary Stain: Carbolfuchin Decolorizer: Acid Alcohol Counterstain: Methlyene Blue M. smegmatis (used to detect mycobacterium) Mycobacterium appear red, non acid-fast bacteria appear blue
endospore staining procedure/organisms/appearance
Primary Stain: Malachite Green Counterstain: Safranin B. megaterum & C. sporogenes Vegetative cells appear pink, endospores appear green
Which organism (Genus, species, strain) is the DONOR? (conjugation)
Pseudomonas putida PAW15
Differentiate between pure and mixed culture. Which one is preferable?
Pure Culture: Contain a single strain of bacteria; are the most preferable culture Mixed Culture: Contains any number of bacterial strains
Phylogenetic Tree of Life
Represents the relative relatedness of all life based on the assumption that all life comes from a common ancestor - bacteria, archaea, eukarya
why does sand limit the growth of microbes?
Sand is more permeable than finer soil, allowing for the passage of water and leaching of soluble molecules, which depletes the soil of necessary nutrients, limiting the amount of life that can be sustained by it.
What are the three main types of particles in soil?
Sand, slit, and clay
How do slit and clay restrict the growth of microbes?
Slit and clay are less aerated than coarse soils, restricting the growth of microbes that need oxygen gas below the surface.
What should you do if you have a small spill on the table? What should you do if you have a large one?
Small spills: Immediately flood the area with disinfectants, then wipe Large spills: Inform TA immediately
bulk soil
Soil outside the rhizosphere
Function of the following dyes used in electrophoresis: staining dye and loading/tracking buffer
Staining dye: Gel green stain- allows the visualization of DNA on a Gel Loading and Tracking buffer: bromophenol blue (for color, stain), glycerol (for weight)- stains the DNA, allows samples to be seen when loading onto gel
60% of the antibiotics are produced by which bacteria?
Streptomyces
How did we test for the production of antibiotics by our isolates?
Swabbing 6 bacterial organisms onto TSA plates and checking for a zone of inhibition
Coliphage and bacteria name used in lab for bacteriophage experiment
T4 and E.coli
What did we use to lyse the cells?
TENS solution
Name the plasmid responsible for degrading toluic acid
TOL plasmid degrades toluic acid
What kinds of media were used for this experiment?
TSA/NA - used as a control plate (ADP1 and ADP6 grows) POB (para-hydroxy benzoate) - one was used as a control plate and the other 2 were used for transformation (ADP1 grew)
PCR components and functions
Taq Polymerase: polymerization of dNTP into a DNA strand dNTP's: mix of nucleotides building blocks of new DNA strand. Polymerase Buffer -creates optimum activity of Taq polymerase Primers- small nucleotide sequence - locate target DNA fragments
Thermus aquaticus
Taq polymerase: polymerization of dNTP into a DNA strand
Oxidase test:
Tests for the presence of CYTOCHROME C OXIDAS neg = yellow pos. = purple indicator: an artificial electron donor TMPD
molecular phylogeny
The study of evolutionary relationships using comparative genomics
microbial ecology,
The study of microbes' interactions with each other and with their environments.
biodiversity and bioactivity
The types of organisms present in an ecosystem and what their activities entail, respectively
Paul Ehrlich
Theorized the "magic bullet" drug.
Why ESKAPE organisms are not used in the tiny earth project?
They are dangerous and pathogenic to humans
What is titer (units) Be able to calculate phage titer.
Titer - number of phages in a sample # of plaques x 1/dilution factor (PFU/mL)
Sequencing data. Why are samples sent for DNA sequencing?
To identify unknown organisms by nucleotides/proteins
What is the function of the Center for Disease Control and Prevention?
To promote health and quality of life by preventing and controlling disease, injury, and disability.
TENS components and purpose:
Tris buffer: provides a pH buffer to maintain the DNA EDTA: removes divalent cations that are required by DNA degrading enzymes NaOH: breaks the cell wall SDS: solubilizes cell membrane
Buffer compositions (Tris-HCl, EDTA, MgCl2, KCl, Triton)
Tris-HCl: Maintain pH EDTA: Keeps DNA intact MgCl2: Cofactor over Taq polymerase KCl: Neutralizes charges in DNA template Triton: Stabilizes DNA
Algae characteristics
Unicellular or multicellular - Photosynthetic - Some are motile - Sexual (via gametes) or asexual reproduction - Cell wall contains cellulose
How does antibiotic resistance arise?
Vertical and horizontal gene transfer Also due to the overuse and misuse of antibiotics.
VCN (units)
Viable Cell Number VCN = # colonies X 1/dilution factor ex) 251 x 10^-6 = 2.51 x 10^8 cfu/ml
How do we "read" the DNA migration?
We can then view the gel under UV light, asDNA dye is fluorescent The further it travels, the smaller the molecular weight
Define antibiotic
a medicine that inhibits the growth of or destroys microorganisms
coliphage
a phage that specifically attacks E. coli.
epidemiologist
a specialist in the study of outbreaks of disease within a population group
broad spectrum
ability of a drug to be effective against a wide range of microorganisms
Motility Test: Pos/neg, what it detects
ability of an organism to move from inoculation site neg = no growth Pos = black growth outward from stab
What is agarose? How does agarose work during gel electrophoresis?
agarose gel is used to slow the movement of DNA and separate by size
Define ESKAPE pathogen, "safe-relative" organism
an acronym comprising the scientific names of six highly virulent and antibiotic resistant bacterial pathogens safe-relative is an alternative organism similar to the ESKAPE pathogens, but is safe to use in the classroom
Pandemic
an epidemic that is worldwide/global
The Methyl (M) Red Test: Pos/neg, what it detects, indicator used
another test for glucose fermentation - Red = positive result - Yellow = Negative result indicator = methyl red
Antagonism and how did you detect it
antagonism- inhibition of action of one organism by another (one organism benefits while the other is harmed) detection- zones of inhibition
narrow spectrum
antimicrobials effective against a limited array of microbial types
What is % of identity?
how similar the query sequence is to the target sequence, The higher the percent the more significant the match
How do you know if the microbes are living?
if they form colonies on an agar plate
How you are going to label a petri dish? Why?
label back of the dish, Label with your initials, type of medium used, a date, and an identifying number.
Importance of algae in microbiology
makes agar, is important for aquatic food chain, and is in petroleum
Importance about collection of soil
many antibiotics come from the soil
obligate intracellular parasites
microparasite which is capable of growing and reproducing inside the cells of a host ex. bacteriophage
What is MMWR?
morbidity and mortality weekly report published by the CDC
The Voges (V) - Proskauer Test: Pos/neg, what it detects, reagent used
tests for neutral end product neg= no change pos = red ring on top reagent = Solution A (alpha-Napthol) + Solution B (KOH)
Epidemiology
the science that deals with why, when, and where diseases occur and how they are transmitted among individuals in a community
Rhizosphere
the soil layer that surrounds actively growing roots
Louis Pasteur
this man discovered that heat could kill bacteria, pasteurization
doubling time
time it takes for a bacterial population to double in size.
Urea test: Pos/neg, what it detects, indicator
to determine if organism has the enzyme Urease neg: orange pos: pink indicator: phenol red
What is the purpose of incubating the phage-E. coli mixture at 37 C 20 minutes at Room temperature in the experiment (bacteriophage)
to kill or lyse by the coliphage
What are the purpose of epidemiological studies?
to learn how disease is spread within a population
What is the purpose of this experiment? (bacteriophage)
to observe the infection of bacteria by bacteriophages and identify plaques
role of pilus in conjugation
transfer of DNA between bacteria
Pasteruization
treating a substance with heat to kill or slow the growth of pathogens
What is the DNA ladder? (1 Kb-1.5Kb ladder)
used as a reference, DNAs of known sizes on the gel, makes it easy to determine the sizes of unknown DNA
16sRNA gene
utilized as a template for sequencing
Name the organisms (STRAINS) used in the experiment conjugation
◦ Pseudomonas putida PAW-15 ◦ Pseudomonas putida 503CA ◦ E. coli
What percent of ethanol is used in the lab as a disinfectant?
70-85%
Actinomycetes produce ______ percent of important antibiotics.
90
What is Query coverage?
A Percent value of found nucleotide sequence percentage overlap with found nucleotide sequence from the database
Paramecium
A ciliated (it propels itself via cilia) protist that lives in fresh water and eats other tiny organisms for food.
sporadic
A disease that occurs infrequently and irregularly.
What is thermal cycler?
A machine programmed to adjust temperatures at timed intervals
media
A mixture of nutrients contained within a liquid or solid form.
outbreak
A sudden rise in the incidence of a disease
What is a heterocyst?
A type of cell in Cyanobacterium where nitrogen fixation occurs
Bacteriophage
A virus that infects bacteria
Which organism WAS lysed in transformation experiment?
ADP1
Which organism was transformed?
ADP6
What are the three types of record keeping?
databases, lab notebook and photographs
The Indole (I) Test: Pos/neg, what it detects, reagent used
detects the bacteria which has the tryptophanase enzyme. Neg = no color change pos = red ring on top Reagent used for this test: Kovac's reagent
Sulfide Test (SIM): Pos., what it detects, reagent used
determines the production of H2S production from the reduction of sulfur in the medium pos= forms black precipitate reagent: ferrous salts
Alexander Fleming
discovered penicillin - father of antibiotics
Selman Waksman
discovered streptomycin
Alma Whiffen
discovered the antifungal agent cycloheximide
epidemic
disease that spreads throughout a population of people in a short time period
(identify protozoa used in lab) - characteristics or protozoa
eukaryotic, unicellular, lack a cell wall
John Snow
father of epidemiology
why choose so many isolates for the master plate?
for diversity
Transformation
(genetics) modification of a cell or bacterium by the uptake and incorporation of exogenous DNA
Why are we focusing on bacteria for our research?
- Bacteria are everywhere - Bacteria make specialized chemical compounds which have human applications - Bacteria greatly impact our health
How do plaques relate to the Lytic and lysogenic cycles of phage infection? (sizes/clarity)
- Clear plaques = lytic cycle of phage infection - Cloudy plaque = Lysogenic cycle of phage infection - Size = The longer the incubation the larger the plaque will grow
importance of protozoa
- Play important roles in the fertility of soil - ecologically imp. in food webs and decomposing organic matter
What is the E value? (BLAST)
- The number of hits which are expected from the database - The smaller the e-value, the more likely the match is true
Precautions while starting the Tiny Earth project
- Treat all environmental samples as a source of potentially pathogenic microorganisms - Culture organisms bellow 35°C - Follow aseptic technique - Wear appropriate PPE - Dispose of contaminated supplies in designated containers
importance of algae
- basis of aquatic food chains - source of most atmospheric O2 - food for humans - source of chemicals (agar)
Organisms (strains) involved in transformation experiment:
Acinetobacter spp. ADP1 and Acinetobacter spp. ADP6
Process of making master plate
-Identify Colonies of Interest - Divide plates into 16 sections on both - Write numbers on back of the plates - Transfer the colonies to both plates using fresh toothpick each time. - Incubate at 30°C
Why is air-drying important?
-attempting to heat fix before a slide is completely dry will cause the bacteria to lyse
PCR applications
-cloning genes -Diagnosis of inherited diseases -Detection of viruses (HIV) -Studies of gene expression during development -Forensics (DNA fingerprinting) -Evolution (amplification of DNA of extinct species)
Conversion from μl to ml
1 μl = .001 mL ^^divide by 1000
Write 3 lab etiquettes you should follow while working in the lab.
1. Always wash your hands before and after lab work 2. Never place personal belongings on benchtops or surfaces used for lab work 3. Never remove lab equipment from lab
Which media were used to conduct this experiment?
1. Glucose minimal media 2. m-Toluate minimal and leucine plate 3. m-Toluate minimal plate
Give 4 mechanisms of antibiotic resistance within a cell.
1. Limiting uptake of a drug 2. Modifying a drug target 3. Inactivating a drug 4. Active drug efflux
Gram stain procedure, organisms used, gram +/-
1. Primary stain (Crystal violet- a basic stain) 2. Mordant ( iodine) (something that helps fix dye on or in a cell) 3. decolorizing agent (alcohol) 4. Counterstain (Safranin- a basic stain) - B. meg, E. coli, & P. putdia Gram + cells retain the crystal violet, staying purple Gram - cells do not retain the crystal violet; turn pink due to safranin
What are three things you should include during record keeping?
1. Record of all used materials 2. Record of all data and observations collected 3. Record of positive and negative results
Characteristics of antibiotic
1. They are therapeutic agents 2. They are selectively toxic towards microorganisms 3. They kill or inhibit the growth of bacteria 4. These are small secondary metabolites 5. The term "antibiotics" is used for either antibiotics produced by microbes or for synthetic antibiotic
Give 3 examples of aseptic techniques that need to be followed.
1. Work near an open flame 2. Do not place petri dish lids face down on any surface 3. Point tubes away from your face and body when working with them
What percent of bleach is used in the lab as a disinfectant?
10%
How can we use 16s RNA to detect bacteria and eliminate our eukaryotes from the isolates obtained as a lab?
16s RNA is utilized as a template in the laboratory
Temperature for incubation of soil dilution plates after inoculation and why
30C, to prevent pathogenic organisms from growing
culturable bacteria.
Bacteria that we can culture in a lab
What is nucleotide BLAST?
Basic Local Alignment Search Tool-- search nucleotide and protein databases for possible matches of unknown DNA
Give five mode of actions of antibiotics.
Cell wall synthesis Plasma membrane Folic Acid Synthesis Nucleic acids Protein synthesis
Cysts (protozoa)
Cysts = trophozoite The two stages of protozoa
DNA migration
DNA migration to opposite side from cathode to anode
The first step in bacterial growth is __
DNA synthesis
What are the steps of PCR? What happens in each step?
Denaturation: Unwinding the double helix at 95C for 30 Annealing: Priming the DNA at 56C for 30s Extension: Adding on complementary nucleotides at 72C for 90s
IMVC tests:
Detects coliform bacteria, and differentiates E. coli from Enterobacter ssp. - E. coli indicates fecal contamination IMVC (++--) - Enterobacter ssp. Is indicative of no fecal contamination (--++)
Robert Koch
Developed the culture plate method to identify pathogens
Given a soil sample, how can you isolate microbes from soli into a medium?
Dilution, then transfer to growth medium.
What are "communicable diseases"?
Disease that can spread from person to person through direct or indirect contact
Why do we have to do dilutions
Done for Salt count - to reduce the number of bacteria for proper plating results Serial dilutions (1:10 or 1:100?) b. 1/10 1 ml into 9ml of sterile water c. 1/100 0.1ml into 9.9 ml of sterile water
How to cross out an error during documenting in lab notebook?
Draw a line through the error and new text should continue in the next available space
ESKAPE pathogens
Enterococcus faecium Staphylococcus aureus Klebsiella pneumoniae Acinetobacter baumanii Pseudomonas aeruginosa Enterobacter ssp.
ESKAPE safe relatives
Enterococcus raffinosus Staphylococcus epidermis Escherichia coli Acinetobacter baylyi Pseudomonas putida Enterobacter aerogenes
classification of algae
based on color, food reserves, cell walls
classification of protozoa
based on motility
cyanobacteria importance
bases of many food chains, consumed by filter feeders and small fish, used for water filters, some produce useful toxins
What are the two common mechanisms bacteria use to reproduce?
binary fission and budding
What is the purpose of performing the BLAST analysis?
can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families.
Importance of bacteria count
can help figure out the duration of life
Targets of antibiotics
cell wall, protein synthesis, nucleic acid synthesis, metabolism
Blepharisma
cilia, not photosynthetic, eat bacteria or each other if no food source
plaques
clear circular zone on an agar surface resulting from bacterial lysis by bacterial viruses
What is the plasmid?
closed, circular extrachromosomal DNA that can self replicate.
On what basis you picked the soil isolates for making the master plates?
colony morphology
Secondary metabolites in plants
compounds naturally produced by plants that are not involved in the primary growth and development of plants
Amoeba
one common sarcodine with an unusual adaptation for movement and getting nutrients
What does PCR stand for?
polymerase chain reaction
DNA molecules loaded into gel migrate towards the _____________electrode because DNA is __________charged molecule.
positive, a negative
nitrogen fixation
process of converting nitrogen gas into nitrogen compounds that plants can absorb and use
How and why is antibiotic resistance a concern?
resistance to antibiotics makes it harder to treat severe infections
colony morphology
shape, margin, elevation, texture, pigment production
DNA fragments separate based on what three features during gel electrophoresis?
size, charge, buffer solution
What dictates how far a fragment will travel?
size, the smallest fragments will travel the greater distance
What are primers? Which primers were used for the 16S rRNA?
small nucleotide sequence, locate target DNA fragments 27F and 1492R
Purpose of preparing a master plate?
so you can go back and compare and contrast
Phycology
study of algae
Why does antibiotic kills bacteria not us?
target components found exclusively in cell walls
Citrate test results: Pos/neg, what it detects, indicator
tests ability to use citrate as sole carbon source for growth neg: green pos: blue indicator: bromothymol blue
glucose/lactose biochemical test: Pos/neg, what it detects, indicator used
tests for either glucose/lactose fermentation neg: phenol red pos: yellow indicator = phenol red
