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Microbiology

Week 4
presented by Amanda Arceo

sterilization
disinfectant
antiseptic
modes of action
physical methods
chemical methods
antibiotics
antibiotic resistance

Keywords

define disinfectant, antiseptic, sterilization and


sanitization
describe methods for evaluating antimicrobial agents
describe chemical and physical methods for controlling
growth
define modes of actions of common antibiotics
describe antibiotic resistance and how it occurs
describe antifungal and antiviral agents
compare physical, chemical and antibiotic methods of
controlling bacteria growth
explain how chemical agents operate at a cellular level
prepare a sterilization/sanitation plan
explain how to prevent antibiotic resistance

learning outcomes

Based on your knowledge from last week, and


what weve learned so far about microbes,
what factors can we control that will impact the
growth rate?

Welcome back!...

Difference?

sterilization

Difference?

killing all microbes

disinfectants

disinfection

killing enough
microbes that
infection is not
possible with the
number present

approved for use on


surfaces

antiseptics

approved for use on


living tissue

Microbial Control
Disinfectant=Dead; Antiseptic=Alive

Remember?

A bacteria has a 50% death rate per minute, what


percentage of population is going to die in the first
minute? What about the second minute?
If the culture began with 100 cells, how many cells
will be left after 5 minutes? What about a culture
that began with 200 cells? How will this affect
sterilization?
Do all bacteria have the same generation times
during the log phase? Do you suppose they will all
have the same death times during death phase?

Scenarios

a definite proportion of bacteria population


will die in a given time interval
the more organisms present, the longer it takes
to sterilize
different microbes have different
susceptibilities to antimicrobial agents

Things to consider

How can you tell if an


antimicrobial agent is
effective?

a means of comparing antimicrobial agents


compare its effectiveness to the original
disinfectant introduced by Lister: Phenol
if it is less efficient than Phenol, has a PC lower
than 1; if it is more efficient than Phenol, has a
PC higher than one
Salmonella typhi and Staphylococcus aureus are
the species we use to check (typically)

First, you need to know:


Phenol Coefficient

allows for evaluation of many different


disinfectants efficiencies on a single species of
bacteria
small circles of paper are soaked in antibiotic
and put on to an agar plate covered with a
single species of bacteria
if the bacteria are susceptible to the antibiotic,
there will be a clear ring around the filter
paper; if they are not susceptible, then the
bacteria will grow up to the filter paper

Filter Paper Method

Use-Dilution Test

be fast acting, even when organic


contaminants, such as body fluid, are present
be effective against all types of pathogens
without destroying tissue
easily penetrate material without destroying it
be easy to prepare and store
be inexpensive and easy to use
not have an unpleasant

6 qualities of a perfect disinfectant

chemical methods of control

reactions that impact proteins


reactions that impact membranes
reactions that impact cell components
reactions that impact viruses

chemical reactions that damage the cell


in the presence of a microbial agent

disrupt functional state by breaking hydrogen


and disulfide bonds
may be temporary or permanent (depending
on treatment)

denaturing proteins

membrane is primarily composed of lipids


substances which dissolve lipids can effectively
put holes in a cell membrane, breaking the
barrier between the cell and the environment
surfactants are soluble compounds that reduce
surface tension and/or dissolve lipids
wetting agents, such as detergent, increase the
effectiveness of a treatment but are not
antimicrobial themselves

impacting cellular membrane

structures of nucleic acids can be altered by


certain chemicals
other chemicals can interfere with cell wall
formation
some block metabolic pathways, preventing the
microorganism from growing

other ways to inhibit microbes

viruses are composed of a protein shell and


nucleic acids
anything that will control viruses must
somehow, then, impact the protein enough to
keep it from attaching to the host cell or
must impact/destroy the nucleic acids within
the virus
not all methods of microbial control that work
for bacteria will work for viruses

viruses

groups of 4 people (if everyone is present, there will


be some groups of five)
Tell me the mechanism of action, uses and
limitations for your assigned category of
antimicrobial agents.

soaps and detergents


heavy metals
halogens
alcohols
phenols
oxidizing agents
alkylating agents

Lets get into 7 groups

physical methods of control

heat
refrigeration
dessication
irradiation
filtration

used even before


we knew of microbes

preferred method for all materials that are not


damaged by the process

heat

thermal death point: temperature at which all


bacteria will be killed in 10 minutes
thermal death point: temperature at which all
bacteria will be killed in 10 minutes
Decimal reduction time (DRT, D value): the
length of time needed to kill 90% of the
population at a specified temperature.

dry

molecules are
oxidized
takes longer and can
only be used on items
that will not melt
under the
temperatures
open flame may be
used (ex: loops in lab)

moist

presence of water in the form


of steam disrupts protein
bonding and lipid structure
water transfers heat more
efficiently than air (would
you rather stick your hand in
an oven or boiling water?)

autoclave

increase atmospheric
pressure to increase boiling
point

pasteurization

most of the microbes are


killed to prevent disease
outbreaks

either 71.6 C for 15 seconds


or 62.9 C for 30 minutes

Types of heat sterilization

ultra high temperature processing spreads the


layer of milk (for example) so that its one
molecule thick and then raises the temperature
from 74C to 140C and back to 74C in less than 5
seconds
preserves flavor (for boxed, shelved milk)

alternative to
pasteurization (UHT)

If you work for Del Monte vegetable company


and they put you in charge of sterilization,
what factors would you consider when creating
a sterilization plan What information would
you need to collect? Do you want to use the
hottest temperature possible? Would you use a
long time?

Question

cold can control microbial growth


bacteriostatic: slows growth but does not kill
microbes
freezing significantly slows metabolism

cold sterilization?

dessication is when you remove the water from


something (drying)
it significantly slows metabolism
many will die without moisture, though some
are capable of regenerating when water is readded

yeast
endospores

dessication

ultraviolet light

damages DNA and proteins and is highly effective


for inactivating viruses

ionizing radiation
X-rays and gamma rays (short wavelengths)

Low doses needed


lab tools and food preservation

microwave radiation

many microorganisms have DNA repair mechanisms


that allow them to withstand more UV radiation than
expected
can only penetrate air, limiting its resourcefulness
in labs for tools and in sewage treatment

longer wavelengths
requires rotation

strong visible light

mostly in violet-red wavelengths


may oxidize certain light-sensitive chemicals inside
bacteria

radiation

adding salt or sugar preserves by forcing


desiccation

example?

osmotic pressure change

antibiotic modes of action

Ehrlich first described chemotherapy as the use of


chemical substances to kill pathogens without
harming the human population. Today we consider
it the use of chemicals to treat disease.
antibiotic: a chemical substance produced by a
microorganism which has the capacity to inhibit the
growth of bacteria and even destroy bacteria and
other microorganisms in a dilute solution.

What was the first antibiotic discovered and by


whom?
In order for something to be considered antibiotic,
it must actually be produced by a microorganism
(the answer to the question above is produced by
fungus, for example)

antibiotic

selective toxicity

chemotherapeutic index is defined as the


maximum tolerable dose per kg of body weight
divided by the minimum dose per kg of body
weight that will cure the disease
antibiotics with a high chemotherapeutic index
will have a large gap between the dose required
to kill the bacteria and the dose that will kill its
host (presumably a human)

selective toxicity

Spectrum activity: range of bacteria species that the antibiotic can treat
Broad Spectrum Antibiotics? *
Narrow Spectrum Antibiotics?

Broad-spectrum antibiotics kill not only the


targeted bad bacteria, but the good microbes
too

this means the host is now susceptible to a superinfection

infestation of microbes that were not destroyed by


the original treatment

susceptibility to superinfection

similar to disinfectants and antiseptics,


antibiotics perform chemical reactions inside
the cell to disrupt its ability to grow
these modes of action are not always fully
understood for antibiotics before they are put
into use

if it works, were going to use it!

antibiotic function

How do antibiotics work?


5 main functions

takes advantage of the fact that microbes have


rigid cell walls and humans do not
peptidoglycan synthesis is often the site of
disruption and because of this, this method is
most effective against gram positive microbes

WHY?

Inhibition of cell wall synthesis

takes advantage of the fact that microbes, like


all living cells, need to synthesize protein

Inhibition of protein synthesis

all cells have cell membranes, but bacteria and


fungi have certain molecules that make them
distinguishable from animal cells.
Disrupting the cell membrane is most effective
against gram positives due to extra lipid layer,
as well as fungi

Cell membrane disruption

takes advantage of the fact that bacterial


ribosomes are smaller than that of eukaryotes

Inhibition of nucleic acid synthesis

substances that prevent a cell from carrying out


metabolism by either competitively inhibiting
enzymes or being erroneously incorporated into
important biomolecules, such as nucleic acids

sulfanilamide is chemically similar to the


precursory molecule needed to create folic acid and
can competitively inhibit the enzyme to prevent
folic acid creation
humans ingest folic acid and therefore are
unaffected by the drug
some chemicals are similar in composition to
specific nucleic acids and can replace these
nucleotides during synthesis and be used in
antivirals

antimetabolites

Antibiotic Resistance

they resist!

transduction
conjugation

both will be discussed further in unit 6

bacteria can trade genes via plasmids

5 mechanisms of
resistance

changing the site that the antibiotic binds and


acts against to no longer allow the antibiotic to
bind

alteration of targets

changes occur in the membrane transport


system so the antibiotic can no longer penetrate
the cell

alteration of membrane
permeability

new enzymes are created that destroy or


inactivate the antibiotic

development of enzymes

the active site that was previously inhibited is


changed so the antibiotic can no longer bind to
it

alteration of an enzyme

the pathway that was previously blocked by


the antibiotic is bypassed and/or replaced by a
different pathway

alteration of a metabolic
pathway

take the entire course of antibiotics prescribed


using 2 antibiotics simultaneously

this can result in an additive effect, synergism


MAY result in decreased effect, antagonism

the prescribing doctor should be up to date on this


topic when attempting to prescribe two or more
antibiotics simultaneously

only use antibiotics when absolutely necessary

3 ways to limit drug


resistance

solubility in body fluids


selective toxicity
toxicity is not easily altered
non-allergenic
stability: maintain a constant concentration in
blood and tissue
resistance not easily acquired
long shelf-life
reasonable cost

ideal antibiotics

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