Control of

Microorganisms
Various Physical
and Chemical
Methods
INTRODUCTION

MICROORGANISMS
Some microorganisms are beneficial, some are
harmful.

MICROBIAL ACTIVITIES- food spoilage and

disease

CONCEPT OF MICROBIAL CONTROL:

Ignaz Semmel Weis (Hungarian Physician)
Joseph Lister (English Physician)

MICROBIAL GROWTH- controlled by physical and

chemical methods.

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TERMINOLOGY

STERILIZA
A treatment that
TION
kills or removes all
living cells,
including viruses
and spores, from a
substance or object

DISINFECT
A treatment that
ION
reduces the total
number of microbes
on an object or
surface, but does not
necessarily remove
or kill all of the
(chlorinatio
microbes
n) 3
TERMINOLOGY

SANITATIO
Reduction of the
N
microbial
population to levels
considered safe by
public health
standards

ANTISEPTI
C
A mild disinfectant
agent suitable for
use on skin surfaces

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TERMINOLOGY

GERMICID
“the agent kills”
E For
microbes.
example, a
bactericide agent
kills bacteria,
fungicide, virucide,
sporocide.

BACTERIOS
TATIC
The agent inhibits
growth.” For
example, a fungi
static agent inhibits
the growth of fungi,
but doesn’t
necessarily kill it.
(Refrigeration)
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TERMINOLOGY

DEGERMIN
G of
Removal
microbes from skin

ANTIMICRO
BIAL
AGENT
Agent kills
microorganisms or
inhibit their growth

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 CONDITIONS
INFLUENCING
DISCUSSION 1. Temperature
2. Type of Microbe
3. Physical State

ACTIONS
1. Kill
2. Inhibit Microbes
3. Damage plasma
membrane

GRAM POSITIVE BACTERIA-

Disinfectants and antiseptics
greater effect
GRAM NEGATIVE
BACTERIA- Pseudomonads
grow in some disinfectants
and antiseptics
ENDOSPORES-resistant to
chemical agents and physical
methods
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PHYSICAL Enzymes,
METHODS DNA,
Cytoplasmic
membranes
disrupt

Damaging cell
Used in food
components industry
Preparation of
culture media lab
ware, sterilization
of instrument

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1. HEAT

Kills microbes by denaturing THERMAL DEATH POINT (TDP)

enzymes. Heat resistance • Lowest temperature to kill all
vaies among different the bacteria in 10 mins.
microbes. • Useful in purifying water
RATE OF MICROBIAL DEATH • BOILING kills many Vegetative
• Bacteria usually die at a Cells
Constant rate • Inactivates viruses within 10
• Plotted logarithmically, this mins.
will give a straight line • No effectDEATH
THERMAL on spores
TIME (TDT)
• Time required to kill all the
bacteria at a given
temperature
• Developed for food canning
• Applications in cosmetics and
pharmaceuticals
DECIMAL REDUCTION TIME
(DRT)
• Length of time
• 90% of a bacterial population
killed at a given temperature
• Used in Commercial
Sterilization
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AUTOCLAVING

Autoclaving is a sterilization
method that uses high-pressure
steam. The autoclaving process
works by the concept that the
boiling point of water (or steam)
increases when it is under
pressure.

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 2.
PASTEURIZATI
ON

• Processused in preserving HIGH TEMPERATURE SHORT

heat sensitive foods like TIME
milk and beer • HTST
• 63°C for 30 mins. • Process
• Reduction of microorganism • 72°C for 15s (Quick heating
in milk and cooling)
ULTRA HIGH TEMPERATURE
• NOT a method of • UHT
sterilization • Sterilization
• Heating at 140°C for 3s
• Milk kept at room
temperature for 2 months
• Minimal changes in flavor
• EQUIVALENT
TREATMENTS- Different
methods produce same
effect
• MYCOBACTERIUM-
Tuberculosis

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 LOW
TEMPERATURES

• Decreasing temperature decreases

chemical activity
• Low temperature are NOT
BACTERICIDAL
• Restrict enzyme activity
• Ordinary refrigerator temperature 0°-
7°C
• Do NOT REPRODUCE
• Survive, restrict rate of growth
• USES:
-Food Preservation
-Drug
-Culture preservation

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3&4
DRY HEAT
• DIRECTSTERILIZATION
FLAMING- Burning
contaminants
• INCINERATION- Burns and
physically destroys organisms
-USED for needles, inoculating
wires, glassware, body parts
• HOT AIR STERILIZATION-
oxidation
-160°C for 2 hrs or 170°C for 1
hr
-USED for objects that won’t
melt, glassware,metal
FILTRATION
• The passage of a liquid or gas
through a filer with pores small
enough to retain microbes
• Separate bacteria from
suspending liquid
• FILTER- nitrocellulose, acetate
DEPTH FILTERS
• Fibrous or granular material
• Thick layer filled with twisting
channels
• Microorganisms sucked
through thick layer
• Microbes removed by physical
screening
MEMBRANE FILTERS
• Circular filters
• Porous membrane- 0.1mm
thick
• Made of cellulose acetate,
cellulose nitrate,
polycarbonate
HEPA FILTERS
HIGH-EFFICIENCY
PARTICULATE AIR FILTERS
• Filtration of small particles
• Capture minimum of 99.97% of
0.3 microns contaminants
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 6&
5 7
DESICCATION OSMOTIC PRESSURE
• Plasmolysis
• Removing water from
• High zone of salt and sugar
microbes • Salt- preservation of fish,
• Viruses and endospores can
meat, food
resist • HIGH OSMOTIC PRESSURE
• Disruption of metabolism
-low availability of sugar solution
• For food preservation
to prevent microbial growth
• Stops growth/ microbes are still
-HONEY; high sugar content
viable
preserved
• Freeze-drying
-Loss of H2O
(Lyophilization)- remove water
NON-IONIZING
from specimen
• Damage to DNA by UV Light
• Gonorrhea bacterium-
• Effective germicide wave
withstand for only 1 hours
length- 260nm
• Tuberculosis bacterium-
• Poor penetration
viable for months (mycolic
• UV RADIATION is only useful for
acid)
disinfecting outer surfaces
• Powdered milk- 85% water is
removed

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METHODS

CHEMICAL CONTROL
METHODS
• Phenols and Phenolics
• Halogens
• Alcohols
• Heavy metals and their compounds
• Surface-active agents
• Quaternary Ammonium Compounds
• Chemical food preservatives
• Aldehydes
• Antibiotics

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 What are
the types
of
disinfectan
ts?
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DISINFECT
ANTS

PHENOLS & PHENOLICS CHLORHEXIDINE

• Damages plasma membranes
• Another name for Carbolic
• USES: skin degerming, surgical
Acid/ Lysol/ Pine-Sol
scrubs
• Joseph Lister
• Only operates in narrow pH
• Exert influence by:
range (5-7)
1. Injuring plasma membranes
2. Inactivating enzymes IODINE
3. Denaturing proteins • Least toxic of the disinfectants
• Uses: skin surface, • Combines with amino acids
environmental surface, • Inactivates enzymes
instruments, mucus • Tincture/ Alcohol
membranes. • Iodophor
• Common: Cresols, • EXAMPLE: Betadine for wound
Hexachlorophene treatment
• PHENOLICS are long lasting
• No effect on spores
• Effective antibacterial agents,
fungi and many viruses

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DISINFECT
ANTS

HALOGENS
• Can be used alone or in ALCOHOL
solution • Denature proteins and dissolve
• Inactivated by sunlight lipids
• Alter cellular components • Evaporates
• Inactive enzymes • Fast acting
CHLORINE • Wet disinfectants
• Purifies drinking water 1. Aqueous Ethanol (60%-95%)
• 2-4 drops of chlorine per 2. Isopropyl Alcohol
liter/30 mins • Not effective against
• Forms an acid- endospore
HYPOCHLOROUS ACID- • USES: thermometer,
bactericidal instruments, before injection
• Gaseous form or in solution as
calcium hypochlorite
• Good disinfectants on clean
surfaces
• Inexpensive- Chlorox
• Never mix with other cleaning
agents
• Kills legionella species
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HEAVY METALS SILVER, MERCURY
Germicidal or antiseptic

SILVER NITRATE
Prevent genococcal eye
infections

COPPER SULFATE
ALGICIDE
MERCUROCHROME
Disinfects skin and
mucus membrane
Used for burn
MERCURIC
treatment and
CHLORIDE
denature proteins
COPPER SULFATE
Bacteriostatic
Destroy green algae
ZINC CHLORIDE
Ingredient in mouth wash
ZINC OXIDE
Anti fungal in paints

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 SURFACE ACTIVE
• Include soaps and detergents
AGENTS ORGANIC ACIDS
• SOAPS- anionic detergents CHEMICAL FOOD PRESERVATIVES:
• Skin degerming • Sorbic acid- inhibit fungus
• TRICLOCARBON- inhibit gram • Benzoic acid- inhibit fungus
positive bacteria • Propionic acid- inhibit fungus
• Decrease molecular surface • Nitrate and nitrite salts- for
tension meat
• Limited Germicidal Action
• Removal of organisms by • To prevent germination of
scrubbing clostridium botulinum
CATIONIC DETERGENTS endospores
• QUATERNARY AMMONIUM • CALCIUM PROPIONATE- for
COMPOUNDS (QUATS) bread
• Disrupt plasma membranes
• Most effective on gram-positive
bacteria
• Enzyme inhibition, preotein
denaturation
• EXAMPLE: Zephiran and
Cepacol
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 ALDEHYDES ANTIBIOTICS

• Antimicrobial • Used to preserve cheese

• Inactivate proteins • Used in feeds for animals
• Covalent crosslink formation
• FORMALDEHYDE- preserve
biological specimens
• GLUTARALDEHYDE- sterilize
hospital instrument
• Most effective of all chemical
disinfectants
• Carcinogenic
• Oxidize molecules inside cells

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2
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ANTIMICROBIA
L AGENT &
MECHANISM
OF
RESISTANCE
ANTIMICROBIALS
AGENTS

ANTIBIOTICS
A chemical substance produced by microorganism
which has the capacity to inhibit the growth of bacteria,
fungi, viruses, or protozoa. It has a high
chemotherapeutical index to reduce the active process
in organism in a diluted solution

ANTIBIOTICS = ANTI MICROORGANISMS = ANTI

MICROBES

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CLASSIFICATION OF
ANITBIOTICS
1. Based on chemical structures
2. Based on the sources
3. Based on mechanism of action
4. Based on spectrum of
action/activity
5. Based on modes of action

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 BASED ON CHEMICAL
STRUCTURES
1. Groups of sulfonamides ➜ sulfamethoxazole, sulfadiazine
2. Groups of Penicillin ➜ Penicillin G (Benzylpenicillin), Penicillin V, Ampicillin,
amoxicillin, nafcillin
3. Groups of cephalosporin ➜ cefalotin, cefazolin, cefamandole, cefuroxime,
cefotaxime, ceftriaxone
4. Groups of aminoglycoside ➜ streptomycin, neomycin, kanamycin, gentamycin,
tobramycin
5. Groups of chloramphenicol ➜ chloramphenicol, tiamphenicol
6. Groups of tetracyclines ➜ chlortetracycline, oxytetracycline, doxycycline,
minocycline HCl.
7. Groups of macrolides ➜ erythromycin, roxithromycin, spiramycin, azithromycin.
8. Groups of polyenes ➜ amphotericin B, nystatin
9. Groups of Lincomycins ➜ lincomycin, clindamycin
10. Groups of polymixins ➜ Polymyxin B, Polymyxin
11. Groups of sulfon ➜ dafsone
12. Other groups ➜ vancomycin, cycloxerine, bacitracin, metronidazole. 26
13. Groups of quinolones ➜ nalidixic acid, norfloxacin, ciprofloxacin, offloxacin
BASED ON THE
SOURCES

ANTIBIOTIC FROM ANTIBIOTIC FROM

MICROBES ALGAE
USNAT ACID
• A.B. from fungi- Penicillin
from P. notatum
ANTIBIOTIC FROM
• A.B. from bacteria-
1. A.B. from eubacteria 
HIGHER PLANTS
polymyxin from bacillus GARLISINA FROM ALLIUM
polymyxa SATIVUM
2. A.B. from
micromonosporaceae 
gentamyicin from ANTIBIOTIC FROM
micromonospora purpurea ANIMALS
ERITRINA FROM
HEMOGLOBIN OF COW

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BASED ON THE MECHANISM
OF ACTION

4. Inhibition of protein
1. Inhibition of cell wall
synthesis:
synthesis leads to the
• This antibiotics inhibit one
death of the bacteria lysis
of the reactions in the
(bactericidal effect)
process of transcription
• penicillin, cycloserine,
a. Inhibition of translation
vancomycin, bacitracin,
process of microbes
cefottaxime, ceftriaxone.
b. Inhibit ribosome on the 30
2. Disruption of cell
S subunit:streptomycin,
membrane function 
tetracylines, netilmicin,
polymyxin (polymyxin B,
kanamycin
polymyxin E), polyenes,
c. Inhibit ribosome on the 50
nystatin
S subunit:
3. Inhibits spesific metabolic
chloramphenicol,
reaction
clyndamycin, lincomycin
• Inhibits the enzymatic
• Inhibits the transcription
reactions  sulfonamides,
process of microbes:
INH, PAS, trimethoprim
Rifampin, actinomycin

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BASED ON THE SPECTRUM
OF ACTION

Broad spectrum: Effective to Gram

+, Gram - bacteria, mycoplasmas,
chlamydiae, rickettsiae, sometimes
protozoa:

chloramphenicol, tetracyclines

Narrow spectrum: Effective to Gram

+ / Gram - bacteria only:

penicillins, cephalosporins,
erythromycins, polymyxins

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 BASED ON MODES OF
ACTION
• 1938 : N. gonorrhoeae are sensitive to
• 1948 : N. gonorrhoeae became resistant, sulfa was no
longer used
• N. gonorrhoeae that resistant to penicillin -----
penicillinase producer - strains.
• Staphylococcus that resistant to penicillin beta-
lactamase enzymes.
• Paul Ehrlich (1902 – 1909) mice infected with
trypanosoma and treated with azo dyes, organic
arsenyl and triphenyl methone trypanosoma became
30
resistant after contacted with the drugs.
MECHANIS
MS OF
RESISTANC
E

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Bacteria produce enzymes that destroy
the active drugs such as beta-lactamases
which will destroy beta-lactams
antibiotics.
• Natural resistance :
1. Genetic: chromosomal resistance and
extrachromosomal resistance
• Genetic resistance happen because of
genetic changes
2. Non genetic
• Non genetic resistance happen
because of antibiotics come into
contact with bacteria which have
active metabolism.

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• Example : M. tbc can persist in
the tissues for a long time. The
bacteria persist for years after
infection without replication, due
to the good immune system of
the patient.
• In this condition M .tuberculosis
can not be killed by antibiotics
• Acquired resistance: Sensitive
bacteria will get this resistance
properties through plasmid which

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 WEINSTEIN,
1984
1. Alteration of cell membrane permeability, such alteration inhibit penetration of
antibiotics to bacterial cell - Staphylococcus against tetracyclines
2. Alteration in bacterial cell, so that a big ammount of antibiotic destroy enzymes are
produced
- β- lactamase against penicillins and cephalosporins
- Acetyltransferase against chloramfenicol
- Phosphorilase, acetylase and adenylase against aminoglycosides
3. Alteration of receptors usually affects bacterial ribosomes. The mutation alters the
DNA that produces a ribosomal protein receptor so the a antibiotics cannot bind to
it- erythromycin receptor in staphylococcus
4. Alteration of a metabolic pathway in bacterial cell, to bypass a reaction inhibited 34
by
an antimicrobial agent - dehidrofolate by trimethoprim, sulfanomide, INH and PAS
SOME APPROACHES TO SOLVES
RESISTANCE PROBLEMS
1. Administration of
antibiotics prescription only
if the clinical signs and
tests indicated that certain
bacteria are the most
probable caused of
infection
2. Bacteriologic diagnosis
must be sought and
susceptibility tests must be
determined
3. Avoid the usage of
antibiotics which have
been known resistance in
one population
4. Reduce the usage of
topical antibiotics, use
antiseptics instead.
5. Limit the period of
consuming antibiotics
6. Reduce the usage of
prophylactic antibiotics.
7. Use narrow spectrum
antibiotics
8. Always follow directions
for use of antibiotics
9. Prescrible antibiotics
based on clinical situation
and not on patient’s will or
pharmaceutical
advertisements.
Rational drug: drugs given
after accurate diagnosis. It
will be effective with minimal
side effects.
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 FACTOS INVOLVED IN THE USAGE OF AB RATIONALLY,
EFFECTIVELY, AND SAFELY

1. Accurate diagnosis
2. Accurate choices of antibiotics
3. Deliver accurate dose
4. Accurate dosing interval
5. Accurate examinations of patophysiologic conditions of the patient
6. Factors involve in choosing antibiotics
• Disease factors
• Drug factors
• Recipient factors

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 FACTOS INVOLVED IN THE USAGE OF AB RATIONALLY,
EFFECTIVELY, AND SAFELY
1. DISEASE FACTORS
• Selective for etiologic bacteria susceptibility test
• Types and doses depend on location of infection
• Enough penetrating potentials to cross :
- blood-brain barrier in
- abscess walls

2. Drugs factors -> Ideal antibiotics

• Have a narrow spectrum, affect only to etiologic bacteria
• Have a bactericidal effect, unless none is sensitive, bacteriostatic drugs can be delivered
• Effective even in the presence of body fluids exudate, protein or enzymes.
• Ability to reach the infected tissue, enough drug concentration during the span of a dosing
interval in blood / infected area.
• Do not cause resistance
• Have a minimal toxic effects for the patient
• Safe for pregnancy and pediatric patients
• Low costs
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 FACTOS INVOLVED IN THE USAGE OF AB RATIONALLY,
EFFECTIVELY, AND SAFELY

3. PATIENT FACTORS:
• Age
• Genetics
• Pregnancy
• Accompanying diseases

• Antibiotic prophylaxis: An antibiotic is used to anticipate infection from certain bacteria

which are sensitive to the drug.
• Goal : To minimize the surgical wound infection, by treating with antibiotic in lethal
concentration for microorganisms at the beginning of surgery until it finished (done).

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SENSITIVITY
TESTS
OR
RESISTANCE
TESTS

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QN AND QL
Strokes
method,
Ericcson
method, Kirby-
QUANTITA Bauer method,
TIVE Comparison
method

MIC, MIC PLATE

QUALITATI
VE

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• The Kirby-Bauer Method:
Commonly in microbiology use the
Kirby-Bauer Method . It use medium of
Mueller-Hinton Agar on the
susceptibility test
• Mueller-Hinton Agar
- Sensitive : clear area (zones of
inhibition)
- Resistance : No zones of inhibition

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SIDE EFFECTS OF
ANITIBIOTICS

1. ALLLERGIC 2. TOXIC REACTION

REACTION - Can happen in
- a respond in sensitive individual depend on
individual due to the the doses of drugs in
abnormality in his the body -> Hearing
immune system: disorder because of
Penicillin, gentamycin
Sulfonamides, - Manifestation can
Streptomycin occur :
- Mild symptoms are - Temporary and
skin rashes and permanently
itching. - After a prolonged
- Severe symptoms are use/ acute respond
anaphylactic shock.
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ANTIVIRAL AGENTS
1. Inhibit viruses coverage
2. Inhibit DNA and RNA synthesis in the viruses
3. Inhibit protein synthesis in the viruses
4. Inhibit specific enzymes activities in the
viruses
5. Inhibit the growth of viruses
6. Promote immunity system of the body
7. Prevent virus infection to the body

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The Drugs that use :

- To inhibit viruses coverage: Amantadine, Rimantidine

- To inhibit DNA and RNA synthesis: Acyclovir, Ganciclovir, Foscarnet, Ribavirin, Valacyclovir,
vidarabine, cidofovir
- To inhibit return transcription from nucleocid: Zidovudine, Didanosine, Zalcitabine
- To protease enzyme Other antiviral drugs: Idoxuridine, Trifluridine, Fluorouracil,
Interferons, Immunoglobulins
- Notes : Antiviral drugs only inhibit the early stage of replication

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 GROUPS OF
ANTIFUNGAL AZOLE
AGENTS IMIDAZOLES
Ketoconazole,
miconazole, clotrimazole,
GROUPS OF fluconazole, itraconazole
POLYENES
Amphotericin
B, Nystatin

GROUPS OF
ALILAMIN
Terbinafin, Nafitin

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Other fungal agents: griseofulvin, flucytosine 5