Lab Practical 2

UV is used to kill bacteria in

food processing, hospitals, sewage treatment, labs

____ range is effective for killing bacteria, but ____ is the opitmal wavelength for killing bacteria by damaging the bacterial DNA

240-300 nm, 260 nm

Two mutanagenic effects of UV light on DNA

deamination of cytosine, formation of thymine dimers

Deamination of cytosine

reaction converts cytosine to uracil and induces a mutation, structural change destabilizes the DNA molecule

Formation of thymine dimers

Formation of dimes between adjacent thymine on the same strand (covalent bond), DNA cannot be replicated or transcribed (DNA polymerase does not recognize)

Can DNA be repaired from UV damage?

yes

2 Methods of repairing thymine dimer repair

photoreactivation, biochemical excision repair

Biochemical Excision Repair

excision of damaged bases; reconstruction of a functional DNA from undamaged fragments; hydrolysis of ATP for energy

Photoreactivation is only for

thymine dimers

Photoreactivation

photolyase breaks apart thymine dimers, visible light powers photolyase

Spore forming bacteria can survive adverse conditions by

making spores

Spores have a thicker

outer core wall

Spores are resistant to

heat, chemicals, radiation

Spores can germinate and

develop into functional vegetative cells

Under conditions of nutrient starvation, a metabolically active vegetative cell starts developing an endospore through a process called

sporogenesis

Endospore structure

central core, PG cortex, two protein layers

Central core contains

outer core wall, cell membrane, nuclear region, and other cell components

Two protein layers endospore

spore coat and exosporium

An easy way to select for a sample containing endospore is to heat at ____ for 15-20 minute

70-80 degrees C

UV light wavelengths are between

100-400 nm

Germicidal

having the ability to kill germs or other microorganisms

The enzyme ____ can bypass the stalled DNA polymerase caused by thymine dimers, but this results in ______

translesion polymerase, high frequency of mutations

Antimicrobial drugs are used to treat infectious diseases by

killing microorganisms, inhibiting growth

Bacteriocidal

kills bacteria

Bacteriostatic

inhibits bacterial growth

Antimicrobial drugs include

antibacterials, antiviral, and antifungal drugs

Another name for disc diffusion test

Kirby Bauer

Disc diffusion test does not determine

if agent is bacteriostatic or bacteriocidal

Efficacy of Kirby Bauer is indicated by

diameter zone of inhibition

Factors affecting size of inhibition zone

sensitivity of organism, molecular size and concentration of agent, density of culture, diffusion rate of agent, size of inoculum, time and temperature of incubation, interactions between antibiotic and medium

Using disc diffusion, we don't get an ________ of an antibiotic that is most effective

accurate measure of concentration

Minimum inhibitory concentration (MIC)

the smallest concentration (highest dilution) of drug that visibly inhibits growth

Standard for MIC

.5 McFarland standard

First step of MIC lab

dilute bacterial culture to same turbidity as mcfarland standard

How do you incubate antibiotic plate

right side up

What agar is used for antibiotic disc diffusion

Mueller Hinton agar

What organism forms spores in the UV lab

Bacillus

Depending on the zone of inhibition, an organism is classified as ___, ____ or _____

resistant, intermediate, sensitive to the antimicrobial

Therapeutic concentration

high enough to inhibit bacterial growth but low enough to avoid tissue damage

Standard incubation time and temp for antibiotic disc diffusion

16-18 h, 33-37

MBC stands for

minimum bactericidal concentration

MBC is defined as

the minimum concentration that inhibits growth and induces cell death

If MBC is close to MIC

the agent is bactericidal

If MBC is much greater than MIC

the agent is considered bacteriostatic

Antiseptic

a chemical agent that can be safely used externally on living tissues to destroy microorganisms or inhibit growth

Antibiotics have to be used at a _____

therapeutic concentration

Disinfectant

a chemical agent used on inanimate objects to destroy microbes or inhibit their growth

Biofilms are made of _____ secreted by bacterial cells

exopolysaccharides

Exopolysaccharides act as a protective mechanism against

nutrient/water loss

Biofilms are commonly foun

on teeth, on catheters, on drains, on contacts

Some organisms form biofilms at _____ interface when inoculated in a test tube

air-liquid

What is in the tube in the biofilm experiment

glucose and Lb

What organisms do we use for the biofilm former

P. aeruginosa or S. aureus

Four stages of biofilm formation

reversible attachment, non-reversible attachment, maturation, active dispersal

What happens during reversible attachment

motile bacteria bump into a surface and then swim away

Nonreversible attachment is due to an increase in production of

cell strucutres (pili, glycocalyx_ and surface proteins

What happens during maturation stage

actively growing cells produce polysaccharide and typically lose flagella

Active dispersal occurs when

the surrounding environment becomes. more favorable and flagella are produced so motile cells can escape the biofilm

Cells in the biofilm community may differentiate to express or silence genes, producing an antibiotic tolerant subpopulation of

persister cells

What are used as disinfectants

aldehydes, chlorine, and derivatives

Disinfectants typically work by

destroying the cell membrane of microorganisms or interfering with their metabolism

bacterial replication is ____, meaning that a single organism will divide into two, maintaining _____ gene transfer

binary, vertical

bacteria can aquire new genetic information from ______

horizontal gene transfer

conjugation

direct cell to cell contact that mediates transfer DNA

transduction

genetic information carried by bacterial virus from one bacterium to another

transformation

acquire DNA from surrounding environments

Ways for bacteria to aquire new genetic information

conjugation, transduction, transformation

plasmid

small circular DNA molecule that can replicate independently of the chromosome

extra genetic info from plasmid could be responsible for

antibiotic resistance, virulence factors, nutrient utilization, among other benefits

The contact during conjugation is formed by a

sexual pilus (f pilus)

F pilus is produced by a plasmid called

fertility factor, or F factor

A cell that has F factor is called a ____ cell

F+ cell

During conjugation, a copy of F factor is _________

nicked, unwound, and a single strand is transferred to the recipient cell

What is the recepient cell called during conjugation

F- cell

once a recepient cell obtains F factor it replicates the _____ to make a _____ molecule

single stranded DNA, double stranded DNA

Transconjugate

recipient cell that has received a copy of F factor

The F factor is a type of ____ that contains specific genes within the _____

plasmid, tra-operon

Tra-operon contains genes responsible for

transferring the plasmid from donor to recipient cell

R plasmid

F plasmids that carry more than one gene for antibiotic resistance

Which F factor do we use

F'128 factor

F'128 factor also contains the ____ operon, and a gene ____

lac, cat that codes for resistance to chloramphenicol

The F factor is present in ____ in the donor cell

multiple copies

The chromosome of the donor cell does not have genes responsible for ______

lactose utilization

Only one cell among _____ donor or recpient cells is transconjugate

10^5 or 10^6

What gene does the recepient cell contains

kan, that codes for resistance to kanamycin

The recipient cell is naturally _____ negative

lac

Occasionally, the F factor transfer will be interrupted before the transfer is complete, meaning ____ will be transferred but not ____

cat, lacZ

The interrupted mating process can be used to determine the _____ between the cat gene and the lacZ gene on the F factor

genetic distance

MacConkey+chloramphenicol

F+: cells grow, lac+
F-: no growth
trans: majority donor cells, very few transconjugate, both lac +

MacConkey + kanamycin

F+: no growth
F-: cells grow, lac-
Trans: mixture of lac- recipient and lac+ trans (very few)

MacConkey+chloramphenicol+kanamycin

F+: no growth
F-: no growth
Trans: trans cells only, most lac+

Bacteriophage

virus that bacteria is susceptible to

In some estimates, there as many as ___ times as many phages as there are bacteria on earth

10

Phages contain their own ____

genetic material that is transcribed and translated within the host cell

PHages exist _____ in an infective but nonliving state called

extracellularly, virion

Viral reproduction occurs exclusively inside a host cell, and can occur in one of two cycles

lytic cycle, lysogenic cycle

Basic structure of a virion

capsid (protein shell) that encloses nucleic acid (either DNA or RNA)

Examples of virion shapes

helical, polyhedral, binal

With the binal phage, all of the viral nucleic acid is located in the ____

head, and the tail is used for attachment

Stages of lytic infection

attachment/absorption, penetration, biosynthesis, maturation, release