Controlling Microbial Growth

http://jameslunsford.com/microunknown2_exp_explain2_files/image002.jpg
Reminder: make sure you can define the following
Key Terms:

• Sterile
• Sterilization
• Commercial sterilization
• Disinfection
• Disinfectant Definitions are examinable!
• Antisepsis
• Antiseptic
• Sanitization
• Degerming

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 Learning objectives
• Discuss the basic principles of disinfection and use of
antiseptics.
• Explain bacterial growth control by altering growth
environment
• Explain bacterial growth control by interference with key
structures or metaboilsm
1. Inhibition of DNA replication
2. Inhibition of protein synthesis
3. Disrupting of cell structures
• Disinfectants
• Antiseptics
4. Blocking key enzymes or processes
• Discuss using a targeted control strategy to control bacterial
growth.
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 Control methods & type of surface

Type of surface Physical Chemical Therapeutic

Antimicrobials
Inert (e.g., table, floor) Non-ionizing Disinfectants -
Radiation
Microbiology Equipment Heat Certain -
UV disinfectants
Ethylene oxide
Laboratory media Autoclave - -
Filtration
Skin - Antiseptics Topical
antimicrobials
Mucous membranes - Antiseptics Antimicrobials

Tissues/Organs - - Antimicrobials

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 Control by Interfering with Growth Environment

• TEMPERATURE Above Max

1. Temporarily Inhibit Replication Below Min.

Optimum

Growth Minimum
rate Maximum

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Temperature
 Determining how effective a compound has been:
Microbial death

Log plot of data: can look at decrease as a measurement of the

percentage of the population affected:

This rate of death is usually described as a “Log” decrease:

• One Log decrease (106 cfu  105 )
= 90% of original population killed
• Two Log decrease (106  104)
= 99% of original population killed
• Three Log decrease (106  103)
= 99.9% of original population killed.
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 a) Moist heat (autoclaving)

• Lab equipment and media, instruments for surgery,

etc., need to be sterile.

• Achieved via use of an AUTOCLAVE

– Other options for sterilization include:
• ethylene oxide gas
• radiation.
Principle:
• Proteins are coagulated as tertiary & secondary
structures are destroyed

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 Moist Heat cont.
Steam is put under pressure  higher
temperature
• Steam @ 1 atm (normal pressure) = 100oC
• Steam @ 2 atm (+15 psi) = 121oC
– This temperature for 15 minutes kills most
microorganisms and their spores.

• Widespread uses for autoclaves:

microbiological culture media, surgical
instruments, glassware, solutions that are not
heat sensitive, etc. 8
 b) Dry heat

Temp oC Time (mins) for sterilization

Moist heat 121 15
Dry heat 121 600

• Action = OXIDATION
• Penetrates more slowly than dry heat.
• Objects are exposed to 121o C – 171oC for 1‐6 hours.

• Very high temperatures = incineration (burning).

– Used for destruction of contaminated materials

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c) Boiling
• Uses moist heat for reduction of microbial
numbers
• NOT effective in killing all ENDOSPORES
• Will destroy vegetative cells of most
bacteria and fungi and will inactivate some
viruses.

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 Type Temperature & Time Used for
Regular pasteurization 63oC for 30 mins milk, beer, fruit juices,
wine, yogurt, etc
High Temperature 72oC for 15 seconds milk
Short Time (HTST)
Ultra High Temperature 140oC for 3 seconds Milk, some juices
(“flash method”)

Product doesn’t require refrigeration

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 Comparison of Ionizing vs. Non‐ionizing
radiation

Ionizing Non‐ionizing

Gamma & X‐rays: 0.1‐40nm wavelength UV range: 50‐400 nm wavelength

Dislodges electrons from atoms Absorbed by purine & pyramidine

Damages DNA and produces peroxides bases: forms pyramidine dimers

Penetrates well Doesn’t penetrate well: limited to

Uses: sterilize heat or chemical‐
sensitive objects
E.g., some plastics, tissue grafts,
fruit & vegetables
surface sterilization
Uses: Sterilize some foods
E.g., nuts and spices.
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 CHEMICAL CONTROL
Basic principles
• Certain chemicals can inhibit or kill
microorganisms.
• Included as chemical control agents:
– Disinfectants
– Antibiotics
• Some have very general mechanism of action
– E.g., damage cell membranes
• Might also be toxic to human cells
• Some are very specific
– E.g., target unique structures or processes
• Less likely to be toxic to human cells

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Not in handout
 Inhibition of protein synthesis

Possible due to differences in ribosome structure:

– Prokaryotes: 70s (30s & 50s subunits)
– Eukaryotes: 80s (40s & 60s subunits)

• “selective toxicity”

• Inhibition achieved by:

– Halting chain elongation
– Blocking peptide bond formation

• Antibiotics include: Chloramphenicol, Streptomycin,

Tetracyline, Macrolides….
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 Description of compounds according to their effect:

Addition of compound

“STATIC”
Microbial
numbers

“CIDAL” or “LYTIC”

Time

May be concentration dependent

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Not in handout
 Disinfectants
NON‐LIVING SURFACES
Features:
• Inhibit or kill microorganisms present on INERT surfaces
• Non‐specific mechanisms:
– damage cell membranes
– denature proteins
– inactivate enzymes
• NOT suitable for use on living tissues (too
toxic/damaging).
• Can be combined with e.g., a surfactant (for better
cleaning).

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Ideal characteristics for a disinfectant:
i. Be fast acting
ii. Be effective against all types of infectious agents
iii. Be inexpensive and easy to obtain and use
iv. Not smell unpleasant
v. Be easy to prepare and stable even when exposed
to the environment.

No single disinfectant is all of these thing!

• Therefore: most disinfectants are used in certain
circumstances.

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 Assaying disinfectant effectiveness
1. Use‐dilution test

• Two bacterial cultures:

– species of Salmonella and P. aeruginosa.

• Metal rings  dipped into cultures  briefly dried 

placed into various concentrations of disinfectant 
rinsed with water  transferred to nutrient broth 
number of bacteria determined.

• Measures effectiveness of disinfectant based on

number of resulting bacteria following treatment.
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2. Filter paper disc method
Sterile filter
Bacterial culture swabbed onto
paper disk
agar plate

Disinfectant
solution

Placed onto
surface
Incubate

Measure zones of inhibition

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 You should be familiar with use and how the following
work:

Chemical Compound When it might be used Mechanism of action

Quarternary Ammonium Surfaces & Equipment Lipid solvent &protein

Compounds denaturation

Halogens Surfaces & Equipment Oxidizing agent

(Chlorine, Iodine)

Peroxygens Surface & topical Oxidizing agent

(Hydrogen Peroxide) antiseptic

Alchohols Surfaces & Equipment Lipid solvent &protein

(Ethanol, Iso-propyl & topical antiseptic denaturation
alcohol)

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 Antiseptics
LIVING TISSUE

• How good/useful an antiseptic is depends on:

– Antimicrobial activity
– Toxicity to living tissues

Applications for Antiseptics:

1. Reduce numbers of microorganisms from mucous
membrane before surgery
 E.g., Iodine
2. Remove microorganisms from skin before an injection
 E.g., alcohol

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Murray, Table 10‐4, page 91.

Agents Bacteria Mycobacteria Bacterial Spores Fungi Viruses

Disinfectants

Alcohol + + - + +/-
Hydrogen peroxide + + +/- + +
Formaldehyde + + + + +
Phenolics + + - + +/-
Chlorine + + +/- + +
Iodophors + +/- - + +
Glutaraldehyde + + + + +
Quaternary ammonium +/- - - +/- +/-
compounds

Antiseptic Agents

Alcohol + + - + +
Iodophors + + - + +
Chlorhexidine + + - + +
Parachlorometaxylenol +/- +/- - + +/-
Triclosan + +/- - +/- +

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 Antibiotics
Blocking enzymes or key processes

SELECTIVE TOXICITY =
– Unique to the microorganism
– Highly specific
– Eukaryotic enzymes may be slightly different than those
from prokaryotes or viruses.
• Can be difficult to achieve!!
• Interference with host cells or processes  side‐effects.

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 Inhibitors of Peptidoglycan synthesis (most bacteria)

Functional cell wall is essential for strength, shape,

basic structure, etc. Blocking some component
Rationale associated with the cell wall formation (e.g., the
cross-linking of the peptidoglycan) will weaken the
bacteria and might prove fatal.
Why is this a good Peptidoglycan is UNIQUE to bacteria – not found
strategy? anywhere in Eukaryotic cells.
ENZYMATICALLY block cross-linking of the
Example of a peptidoglycan.
Mechanism o How? Antibiotic binds to the enzymes
(transpeptidases) responsible for the cross-linking.
Examples of
β-lactam antibiotics
compounds
o Penicillins (e.g., Methicillin, Amoxicillin)
with this
o Cephalosporins
mechanism
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 PROBLEMS with this general strategy of
inhibiting peptidoglycan synthesis:
• Changes to the pathway, e.g., use of a different or
modified enzyme, can mean the microorganisms aren’t
affected by the compound.
• Some microorganisms lack peptidoglycan (e.g.,
Mycoplasma sp.), meaning that they aren’t sensitive to
the compound.
• Some bacteria produce an enzyme that inactivates the
antibiotic and prevents it from working.
• Very slowly growing or metabolically inactive bacteria
aren’t synthesising peptidoglycan and are therefore
minimally unaffected/completely unaffected.

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 TAILORING THE CONTROL STRATEGY

• Major Considerations:
1. The object being treated: surfaces vs. living tissues
2. Number of microorganisms and their impact on
effectiveness
3. Which microorganisms are present – microbial
susceptibility

4. Achievable goals

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Kirby‐Bauer Sensitivity testing (Disk diffusion
technique)

Conc. of
antibiotic

Distance from
disc

Which antibiotics are the bacteria NOT

SENSITIVE/SUSCEPTIBLE to?

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http://phil.cdc.gov/phil/details.asp; Image # 3902
 Broth dilution method:
MIC = 1st tube WITHOUT visible growth
1.6 g/ml

Plate out onto agar

MBC = plate without

visible colonies

1.6 g/ml 3.2 g/ml 6.4g/ml

 Quiz
• I have placed a quiz on the BPM3 sakai site for
you to test yourself on methods to control
bacterial growth.