Microbial Growth Control

 Definition of Terms
Sterilization
- process of destroying all forms of microbial
life
Disinfection
- elimination of microorganisms from
inanimate objects and surfaces
Antimicrobial agents
- agents that inhibit the growth or completely
destroy the life of microorganisms

Anti-microbial agents could be:
1. Physical agents
2. Chemical agents
3. Chemotherapeutic agents
Disinfectant
- chemical agents that kills the growing forms but
not necessarily the resistant spore forming
bacteria

Antiseptic/Germicide
- chemical agents that kills or inhibits the growth and
metabolism of the microorganisms
- used in the body without damaging the tissues
Sanitizer
- agent that reduces the microbial population to safe
level

Bactericidal/Bactericide
- total killing of the microorganisms
Bacteriostatic/ Bacteriostasis
- condition in which bacterial growth are prevented
Decontamination
- treatment of an object or surfaces to make it safe
to handle

 Uses of Sterilization
- Culture media, reagents, containers, loops and
needles, glasswares, etc.
- Medical and surgical equipment or materials
used in penetrating normally sterile portions of
the human body
 Uses of Disinfection
- in cases in which sterility is unnecessary
- if steriliztion is impractical

 A. Physical Antimicrobial Control
1. Heat sterilization
- most reliable and universally applicable
- divided into Moist heat and Dry heat
a. Moist heat
1. Autoclaving
- steam under pressure
- 121 °C, 15 psi, 10 – 15 minutes

2. Boiling
- commonly employed but incompletely effective
3. Free flowing steam (Arnold sterilizer)
- also known as Intermittent sterilization or
Tyndallisation or fractionation
- consist of heating 80 to 100 ° C for 30 mins. For
3 consecutive days
- first heating: vegetative cells and some spores
will be killed
subsequent heating: more resistant spore

4. Pasteurization
- process used in food industries for milk and
and other dairy products, fruit juices, wine
beer and other heat sensitive substances
- exposing the product to 62 ° C for 3o mins.
- 71 °C for 15 seconds (flash pasteurization)

b. Dry heat (protein denaturation)
1. Flaming
- used in bacteriological loop and needles
2. Hot air oven
- for glasswares, petridishes, pipettes, flasks
- 180 °C for 1 – 2 hours
3. Incineration (Burning)

2. Radiation sterilization
- electromagnetic radiation (U.V., X-rays)
- causing DNA breaks
a. Ionizing radiation
- generates electrons, hydroxyl radicals, etc.
- alters DNA, cell death

3 . Filter sterilization
- for heat-sensitive substances
- consist of pores too small for passage of
microorganisms but large enough to allow
the passage of gas o liquid
e.g.
Membrane Filter
- composed of polymers with high tensile
strength
Nucleation tuck filter (Nucleopore)

- other filters
- Seitz filter (disks of asbestos-cellulose mixture)
- Chamberland filter (unglazed porcelain)
- Barkefeld filter (diatomaceous earth)
c. Low temperature (Freezing)
d. Desiccation
- Lyophilization (extreme dehydration)
e. Ultrasonic and Sonic Vibration (20 – 1000 kc)

 B. Chemical Antimicrobial Agents
Mode of action
1. Damage to cell wall or inhibition of cell wall
synthesis
2. Alteration of the cytoplasmic membrane
permeability
3. Alteration of the physical and chemical state
of proteins and nucleic acid
4. Inhibition of enzyme function
5. Inhibition of protein and nucleic acid synthesis

 Factors affecting the antimicrobial activity
1. Concentration of the agent
2. Temperature (increase Temp., increase reaction)
3. Contact time
4. pH
5. Nature of microorganism (species)
6. Growth phase of microorganisms.
7. Presence of special structure (spore, capsule)
8. Number of microorganisms
9. Presence of extraneous materials(blood,pus,etc)

 Qualities of good disinfectant
1. High co-effecient of disinfection
2. stable
3. water soluble
4. non-toxic, non-corrosive
5. easy to use
6. cheap

Chemical Agents Mode of action Use
Alcohol (Ethanol,
Isopropanol)
Protein denaturant - topical antiseptic
- Disinfectant for
medical instrument
Phenol-containing
compounds
Disrupt cell membrane
Protein denaturant
-topical disinfectant
-Laboratory surfaces
Cat-ionic detergents
(Benzalkonium
chlroide)
Interact with phospholipid
of cell membrane
-topical disinfectant
-Disinfectant for medical
instrument, food
and dairy
Hydrogen peroxide Oxidizing agent Topical antiseptics
Iodine-containing
compounds
Oxidizing agent Topical antiseptics
Chlorine
gas/compound
Oxidizing agent Disinfectant for food, dairy
and water purification
Copper sulfate Oxidizing agent Algicide disinfectant in
swimming pools/water

Ethylene oxide Alkylating agent Steilant for heat-sensitive
materials
Formaldehde (3 – 8 %) Alkylating agent Surface disinfectant
Glutaraldehyde (2%) Alkylating agent
Mercuric chloride Combines with -SH
group
Disinfectant (surfaces)
Ozone Strong oxidizing agent Disinfectant for drinking
water
Orthoparaldehyde
(OPA)
High-level disinfectant
for medical instrument
Alkylating agent

C. Chemotherapeutic Agents
- chemical substances used
in treating diseases
Antibiotics
- special type of chemotherapeutic
agents used in treating infectious diseases
- usually obtained from living microorganisms

 Historical Highlight
1630 - European use natural quinine (from bark of
Cinchona tree) for malaria
1910 - Use of Salvarsan for syphilis by Paul Erlich
1935 - Use of Sulfonamides for large variety of
pathogens

1945 - use of Sulfadiazine and Sulfamerazine
(derivative of sulfonamides) for its anti-
bacterial effect
1929 - significant discovery of the “miracle” drug,
Penicillin by Alexander Fleming
- the drug was first out in the market in 1950’s

 Mechanisms of Action of Antibiotics
1. Inhibitionn of cell wall synthesis
2. Protein synthesis inhibition
-the beta-lactams: penicillin, cephalosphorins,
carbapenems and monobactams)
- binding to 50S ribosomal subunit (exhibited
by macrolides, chloramphenicol and
clindamycin) or to the 30S subunit (the
aminoglycosides, and tetracycline

3. Interference with nucleic acid synthesis
4. Inhibition of metabolic pathway
- sulphonamides
5. Disruption of bacterial membrane structure
-RNA, by rifampin, DNA, by fluoroquinolones
-by polymyxins, daptomycin

 Properties of a good Antibiotic
1. Selective toxicity
- harmful to microorganism but without being
harmful to the host
- relative rather than absolute
2. Broad spectrum
- inhibits many different species of pathogenic
microorganisms
3. Prevents development of genetically and
phenotypically resistant strains

4. Bactericidal rather than Bacteriostatic
5. Non-allergenic and no adverse reaction
6. Should remain active in the presence of plasma
and other body fluids.
7. Stable and water soluble

Different Groups of Antibiotics
1. Pinicillins
2. Cephalosphorins
3. Quinolons
4. Sulfonamides
5. Aminoglycosides
6. Macrolides (e.g.. Erythromycin)
7. Tetracyclin
8. Chloramphenicol
9. Anti-fungal
10. Anti-viral

 Why is drug resistance a concern?
1. Proliferation of resistant strains in Health care
institution
2. Treatment failure which causes serious consequences
especially critically ill person
3. Broader infection control problems for both in
Healthcare Institutions and communities as well
 Antibiotic resistance
- the ability of bacteria or other microbes to resist the
effects of antibiotic.

 Reasons why antibiotic resistance occurs
1. Over-prescription of antibiotics
2. Non-completion of prescribed antibiotics
3. Use of antibiotics in animals as growth
enhancers
4. Poor hospital hygiene
5. Indiscriminate use of antibiotics

 Mechanisms of Resistance to Antibiotics
1. Up-regulating the production of enzymes that
inactivate the antimicrobial agent.
2. Up-regulating pumps that expel the drugs from
the cell.
.

3. Acquisition of genes for
antibiotic resistance
through horizontal gene
transfer
4. Down regulating or altering
an outer membrane protein
channel that the drug
requires

 Antimicrobial activity in vivo
A. Drug-Pathogen relationship
1. Environment
- influence the effect to microorganism
(microorganisms are located in different
tissues and parts of the body
a. State of metabolic activity
- diverse biosynthetic activity of microorganisms
may results to dormancy which often survive
upon exposure to drugs (causes clinical relapse

b. Distribution of drugs
- unequal distribution of drugs to the tissues
and body fluids
c. Location of organisms
- drugs enter the cells at different rates
d. Interfering substances present in the host
- drugs may react with blood, tissue proteins,
exudates, necrotic debris which is unfavourable to
drug action

2. Concentration
- organisms inside the body may not be exposed
to constant concentration of drugs
- factors:
a. Absorption
b. Distribution
c. Variability of concentration
d. Post-antibiotic effect
- delayed re-growth of bacteria after
exposure to antimicrobial agent

 B. Host-Pathogen Relationship
1. Alteration in Tissue response (Inflammatory)
2. Alteration of Immune response
3. Alteration of Microbial flora
C. Host – Drug Relationship
1. Absorption
2. Metabolism 4. Toxicity
3. Distribution 5. Excretion

 Dangers of indiscriminate use of Antibiotics
1. Widespread sensitization (Hypersensitivity rxn)
2. Changes in normal flora of the body
(super-infection due to drug-resistant strain)
3. Masking serious infection
4. Direct drug toxicity
(renal damage, auditory nerve damage)
5. Development of drug resistance in microbial
population

 Antimicrobial Chemoprophylaxis
- administration of antimicrobial drugs to prevent
infection
- use of specific drugs to specific microorganisms
- the risk of acquiring infection is weighed against
the toxicity, cost, inconvenience and enhanced
risk of super-infection

 Antimicrobial Activity in vitro
Antimicrobial Susceptibility Test
-used to select effective antimicrobial drugs for
treatment and control of infectious diseases
especially when caused by resistant pathogens
- predicts the in-vivo success or failure of antibiotic
therapy
- MIC and MBC determination

 Methods
A. Dilution Method
- cumbersome, limited to special circumstances
- provides quantitative information on MIC and
MBC
1. Two-fold dilution
2. Agar dilution

B. Diffusion Method
- most widely used is the disk diffusion test
- filter paper disk containing a measured quantity
of a drug is placed on the surface of a solid
medium that has been inoculated on the surface
with the test organism
- after incubation, the diameter of the clear zone of
inhibition surrounding the disk is taken as a
measure of the inhibitory power of the drug
against the particular test organism
- subject to many physical and chemical factors in
addition to the simple interaction of drug and
organisms

 Kirby-Bauer Technique
- use of commercially available antibiotic disk
- culture medium: MHA
- inoculum: turbidity = 0.5/1.0 McFarland
= 3.8 x 10 raised 8 cfu
- indicator: zone of inhibition (mm units)
- Interpretation: Susceptible/Sensitive
Intermediate
Resistant

Factors affecting the antimicrobial activity in vitro
1. pH of the environment
- some drugs are active at acidic condition some
at alkaline like aminoglycosides
2. component of the media
- some additives will inhibit the growth of other
microorganisms (e.g., PABA from tissue
extract: antigonize sulfonamides, NaCl:
enhance growth of MRSA)
3. stability of the antibiotic

4. Size of the inoculums
- larger bacterial inoculums the lower the
apparent susceptibility
5. Length of incubation
- the longer the incubation the greater the
chance for resistant mutant strain to immerge
6. metabolic activity of the microorganism
- in general, actively growing microorganisms
are more susceptible to those in resting phase

Microbial Growth Control

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