Microbiology Lab Practical #2

Define plaques

Plaques are clear zones that represent where a phage is present and has killed the bacterium.

TSA/NA what was it used for? - Which organism(s) grow in this media? (Transformation lab)

Used as the control plate; ADP-1 and ADP-6

What are the PCR components?

Taq polymerase

dNTP's

Polymerase buffer

primers

di water

What are dNTP's?

mix of nucleotides building blocks of new DNA strand

What is the Phylogenetic Tree of Life?

Shows the evolutionary relationship between taxonomic groups

How are algae classified?

pigments, food reserves, cell walls

What is the reagent used in the oxidase lab?

TMPD

From the two organisms in the oxidase lab, E.coli and Pseudomonas putida, which tested positive?

Pseudomonas putida

Define coliphage

A type of bacteriophage that infects E. coli

Define bacteriophage

A virus that only infects bacteria

Define obligate intracellular parasite

Only replicate inside a living cell.

What was the coliphage name and the bacteria strain used in the bacteriophage experiment?

T-4; E. coli B

What is the lytic cycle?

phage causes lysis and death of host cell

What is the lysogenic cycle?

A viral replication cycle that does not kill the host immediately; phage incorporates its DNA into bacterial genome, forming a prophage

In the lytic cycle, do plaques appear clear or cloudy?

clear

In the lysogenic cycle, do plaques appear clear or cloudy?

cloudy

What was the purpose of nutrient agar and supplement in the bacteriophage experiment?

Tryptone agar plates: have less agar concentration than normal liquefied agar. It allows better diffusion of the viruses and better contact with the bacteria.

What is titer (PFU/ml)?

Titer=# of plaques X (1/d.f.) (PFU/mL)

What is the purpose of the bacteriophage experiment?

To determine the effect of the T-4 coliphage on E. coli B.

What is the purpose of incubating the phage/E. coli mixture at 37°C for 20 minutes or at Room temperature for 25 minutes in the bacteriophage experiment?

To ensure that the viruses can infect E.coli .

What is the conversion of 1 uL to mL?

1uL= 0.001 mL

What is the conversion of 1 mL to uL?

1mL=1000uL

How do you use a micropipette?

1) Press the plunger down to first stop

2) Immerse the tip of the pipette into the liquid

3) Lift the plunger slowly to get the liquid into your pipette.

4) Position the pipette over the receiving container

5) Press plunger to the second stop to release the liquid

6) Dispose of the pipette tip with the tip ejector

Advantage and disadvantage of dilution

Advantage: Isolation of organisms in soil (CFU's)

Disadvantage: High amount of saline can kill some organisms and there may be less variety of organisms

Why use fewer rich media in the Tiny Earth lab?

To limit growth/prevent overgrowth of organisms

Why was it important to incubate at 30°C?

To prevent the growth of pathogens

Precautions while starting the Tiny Earth project

Wear PPE

Treat every substance like it is pathogenic

Follow aseptic technique

Culture organisms below 35° C

Dispose of contaminated supplies in specified containers

Temperature for incubation of soil dilution plates after inoculation and why?

Soil dilutions were incubated at 30°C because it decreases the risk of growing pathogens.

What is the name of the antifungal agent used in the media for the tiny earth project? What does it do?

Cyclohexamide; breaks down fungal proteins and inhibits fungal growth

Define rhizosphere

soil that has direct contact with living plant roots

Define bulk soil

rest of the soil that isn't in direct contact with plant roots

Define horizon

Distinct layers in the soil

Define secondary metabolite

Organic compounds produced by bacteria that enhance their chances of survival, while being nonessential for fundamental growth processes (unlike structural molecules and DNA, for example).

What is an example of a secondary metabolite?

Antibiotics

What are the three main types of particles in soil?

sand, silt and clay

The importance of the collection of soil

There is a large variety of microorganisms living in soil

Why is the pH of soil important for shaping microbial life?

Organisms can only survive within a specific pH range.

Actinomycetes produce ______ percent of important antibiotics.

60%

Why choose so many isolates for the master plate?

To have a diverse group of organisms

Purpose of preparing a master plate?

Serves as a catalogue of the visibly distinct bacteria that were isolated from a sample

On what basis did we pick the soil isolates for making the master plates?

Unique colony morphology- pigment, shape, size

Process of making master plate:

Divide plate into 16 sections

Label the sections

Use sterile toothpick

Transfer colonies to both master plates, using NEW toothpicks per new colony

Incubate at 30 C

What is colony morphology?

The set of characteristics of a colony on a solid medium Ex: Shape, margin, or elevation

Why do we have to do dilutions?

To isolate colonies of bacteria so that we can see colonies instead of a lawn of bacteria.

What is VCN?

Viable Cell Number

What is CFU?

colony forming unit

How do you calculate viable cell number?

VCN=# colonies X 1/dilution factor (CFU/mL)

Why ESKAPE organisms are not used in the tiny earth project?

They are antibiotic resistant, making them unsafe to work with

Define ESKAPE "safe-relative" organism

ESKAPE safe-relatives are organisms that are similar/related to the ESKAPE pathogens, but are safer to use.

ESKAPE pathogens/Safe relatives

Enterococcus spp = Enterococcus raffinosus

Staphylococcus aureus - Staphylococcus epidermidis

Klebsiella pneumoniae - Escherichia coli

Acinetobacter baumanni - Acinetobacter bayly

Pseudomonas aeruginosa - Pseudomonas putida

Enterobacter species - Enterobacter aerogenes

John Snow

Mapped the occurence of cholera in London

Louis Pasteur

Proved microorganisms are present in the air; Created pasteurization

Robert Koch

Discovered the bacteria that causes anthrax; Came up with Koch's postulates

Define pasteurization

Application of high heat for a short time to kill harmful bacteria in beverages

Define broad spectrum antibiotics

They are effective against a broad range of gram-negative and gram-positive bacteria

Define narrow spectrum antibiotics

Affect a narrow range of bacteria

What is the human microbiome? Are the bacteria always harmful to us?

Human microbiome describes the bacteria that live in/on us and do not harm us most of the time.

What is antagonism? How did we test for the production of antibiotics by our isolates?

Interaction between organisms where one benefits at the expense of the other; By checking for zones of inhibition

Alexander Fleming

Discovered penicillin; "Father of antibiotics"

Paul Ehrlich

Came up with the "magic bullet theory" which states that Chemicals can be made to kill pathogens without harming the host

Selman Waksman

discovered the antibiotic streptomycin

coined the term "antibiotic"

Alma Whiffen

discovered the antifungal agent cycloheximide

How and why is antibiotic resistance a concern?

The effect of the over use of antibiotics and antibiotic resistance in the environment

How does antibiotic resistance arise?

spontaneous mutations, horizontal gene transfer (via conjugation)

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 antibiotics

Targets of antibiotics

cell wall, protein synthesis, nucleic acid synthesis, metabolism

Define antibiotic

a chemical that inhibits the growth of or kills bacteria

What is the function of β lactams?

Prevents cell wall formation by preventing the cross-linking of peptidoglycan (especially with gram-positives)

60% of the antibiotics are produced by which bacteria?

Streptomyces

Why do antibiotics kill bacteria and not us?

They only affect bacterial components Ex: bacterial cell wall (not present in humans)

Give five modes of actions of antibiotics.

1. Inhibit cell wall synthesis

2. Protein synthesis

3. Injury to plasma membrane

4. Inhibit nucleic acid synthesis

5. Inhibits folic acid synthesis

Give 5 mechanisms of antibiotic resistance within a cell

Enzymatic destruction or inactivation of the drug

Prevention of penetration to the target site

Alteration of the drug's target site

Rapid efflux

Variations of mechanisms of resistance

What is horizontal gene transfer?

Transfer of genes between cells of the same generation

What is vertical gene transfer?

transfer of genes from an organism to its offspring

Definition transformation

uptake of naked DNA by a cell

What organisms were involved in the transformation lab?

Acinetobacter spp. ADP1 and Acinetobacter spp. ADP6

What did we use to lyse the ADP 1 cells in the transformation lab?

TENS solution

What does the tris-HCl buffer do? (TENS solution)

Maintains pH

What does EDTA do? (TENS solution)

chelating agent

What does NaOH do? (TENS solution)

breaks the cell wall

What does SDS do? (TENS solution)

Detergent that solubilizes the cell membrane

Which organism was lysed in the transformation lab?

ADP-1

Which organism was transformed in the transformation lab?

ADP-6

What kinds of media were used for the transformation lab?

TSA/NA and POB

Which organism grew on the POB medium. Why?

ADP-1 grew on the POB plate because it contains the enzyme protocatechuate oxygenase that can break down the medium.

What is conjugation?

Unidirectional transfer of genetic material between bacteria through direct contact

What is required for conjugation?

cell to cell contact (pilus); conjugable plasmid; Opposite mating types

Name the organisms (STRAINS) used in the conjugation lab.

E.coli, Pseudomonas putida PAW-15, Pseudomonas putida 503cA

What is the role of the pilus?

Initiates contact from the donor to the recipient to exchange genes.

What is a plasmid?

small circular piece of DNA found in the cytoplasm of bacteria

Name the plasmid responsible for degrading toluic acid

TOL plasmid

Which of the organisms used in the conjugation lab had the plasmid?

Pseudomonas putida PAW-15

Which organism (Genus, species, strain) is the DONOR from the conjugation lab? What's the phenotype?

Pseudomonas putida PAW-15; Leu- Tol+

Which is the RECIPIENT from the conjugation lab? What's the phenotype?

Pseudomonas putida 503cA; Leu+ Tol-

Which media were used to conduct the conjugation experiment?

(GMM) Glucose minimal media (Control plate); M-toluate minimal + leucine plate (Control plate); and M-toluate plate (Test plate)

What's an auxotroph? Which organism was the auxotroph in the conjugation lab?

Organism that has a growth requirement; Pseudomonas putida PAW-15 (requires leucine)

What's a prototroph? Which organism was the prototroph in the conjugation lab?

Organism that doesn't have a growth requirement; E. coli is the prototroph

What is the phenotype of the daughter cell in the conjugation experiment? (in reference to presence of plasmid and leucine requirement)

Leu+ Tol +

What is PCR amplification?

A laboratory technique for rapidly amplifying millions to billions of copies of a specific segment of DNA.

What is thermal cycler?

Used to amplify DNA sequence by increasing and decreasing temperature