BIOL 2042 Final Exam

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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


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