MODULE 3: CONTROL OF BACTERIAL
GROWTH killed within 15 minutes.
2 METHODS OF STERILIZATION
1. PHYSICAL STERILIZATION
1.1 Application of heat
 Heat is the most commonly used
method for removal of microorganisms.
 Coagulates protein.
A. Moist Heat
o It destroys microorganisms through
coagulation of enzymes and structural
proteins and degradation of nucleic
acids.
o Water aids in the disruption of covalent
bonds
o Present in bacterial cell membranes.
A1. Boiling
 Destroys vegetative bacteria (Klebsiella
pneumonia)
 Temperature and time of exposure:
100oC for 10 to 15 minutes.
A2. Fractional Sterilization
i Tyndallization
- Steam for 30 minutes in 3
consecutive days.
- Temperature and time for
exposure: 100 C for 30 minutes
incubator 35 - 37 oC overnight.
ii Inspissation
- Temperature and time for
exposure: 70 – 80 C for 2 hours in
3 consecutive days.
- For protein media sterilization.
A3. Autoclaving
 Fastest and simplest method of
sterilization through which all organisms,
including those that contain spores, are  Bacteria which remain active in a dry
environment:
 Used to sterilize heat-stable objects. I. Neisseria gonorrhoeae
 Principle: Hot steam under pressure - viable for 1 hour
 121oC psi for 15 minutes for media, II. Mycobacterium tuberculosis
liquids, utensils, glass, pipettes, and - viable for several months
instrument assays III. Bacillus and Clostridium
 Decontamination of medial waste 132oC, - viable for 10 years
15psi for 30 – 60 minutes.
 Biological indicator: Bacillus 1.3 Radiation
stearothermophilus  Principle: Radiation passes through the
cells; free hydroxyl radicals and some
A4. Pasteurization peroxides are then created. These free
 Sterilization for milk radicals, in turn, cause different
 Eliminates food-borne pathogen, and intracellular damage.
organism responsible for food spoilage.
 Does not eliminate bacterial endospore. a) UV light: Nonionizing rad
 Temperature and time of exposure: - Causes damage to cellular
62oC for 30 minutes followed by rapid DNA by producing thymine
cooling (Flash Pasteurization) dimers.
- Forms thymine and dimers
B. Dry Heat ‘once it passes UV light’
o Denatures protein and kills by oxidation. (join)
o Does not require water.
b) X-rays: Ionizing rad/cold
o Dehydration via direct sunlight.
- With higher energy and
higher penetrating power
B1. Hot air oven
than UV light.
 Temperature and exposure: 160oC -
- More powerful than UV
170oC for 1.5 to 2 hours. light.
 Time depends on the penetration of heat
- It destroys vegetative cells
on objects to be sterilized. and endospores of both
 For metals and glassware. prokaryotes and
eukaryotes.
B2. Direct flame / Incineration
- Produces hydroxyl radicals
 Usually use for inoculating loops and by hydrolysis water and
needles. breaks down covalent
 Most common method for treating bonds on the DNA.
infectious waste and infected laboratory - Spores are resistant due to
animals. its low water content.
 Principle: Burning of materials into
ashes at 300 oC to 400 oC. c) Gamma rays: Ionizing rad/cold
- Breaks down chemical
 Hazardous materials: 870 oC to 980 oC.
bonds of DNA, inhibiting
bacterial growth and
1.2 Desiccation
integrity.
 Earliest method of sterilization. - Example: Filters –
Millipore & asbestos
1.4 Filtration
 Physical separation of microbes from
liquid.
 It may be used with both liquid and air
substances.
 Method of choice of sterilization for vax
 Example: asbestos, millipore,
nitrocellulose
1.5 Osmotic Pressure
 Plasmolysis
 Use high concentration of salts and
sugar in food to create a hypertonic
environment.
 Example:
a. Immersion of meat in salt solution
b. Fruits and vegetables in sugar
solution
1.6 Sonic Vibration, Trituration, Agitation
 Mechanical methods that disintegrate
bacteria
a. Sonic vibration – sound waves
b. Trituration – grinding
c. Agitation – shaking
1.7 Lyophilization or Freeze-dying
 Low temperature dehydration
 Preservation method
2. CHEMICAL METHODS
2.1 Antiseptic
 The process of reducing or inhibiting the
growth of microorganisms or pathogens
on living tissues to prevent infection.
2.2 Disinfectant
 strong chemicals used to disinfect
inanimate objects in which it may not be
used for living organisms.
 Aims to kill disease-causing microbes
but not spore formers.
2.3 Bactericidal o Both antiseptic and disinfectant.
 A drug or chemical agent that kills
bacteria. B. Modification of Protein
1. Halogens
Variables of Disinfection 1a. Chlorine
1) Concentration • Hypochlorite in restaurants (drinking water)
2) Time > survival and swimming pools
3) Temperature • CDC there is a limit because it is highly
4) ph toxic
• 4 milligrams per liter of chlorine (in purification
of water, beyond 4 milligrams is already toxic for the human body)
o Increased concentration Decreased • Example: hypochlorite in restaurants
o Increased time survivors
and swimming pools
2.4 Bacteriostatic
1b. Iodine
 A drug or chemical that inhibits the
• most effective skin antiseptic
growth and reproduction of bacteria
• for minor scrapes and cuts
• Example: Tincture of Iodine – 2%
solution of KI in ethanol Iodophors
CHEMICAL AGENTS
• Tincture is weak (2 -3% of iodine)
A. Disruption of Cell membrane • A.KA “Povidone iodine or betadine
1. Alcohol
2. Heavy Metals
o denatures protein and disorganizes lipid • inhibits enzymatic activities
structure in the cell membrane. • Denature protein (in the DNA)
• Example:
2. Detergents/ soaps – surfactants Hg – Merthiolate
o A.K.A surfactants Ag – AgNO3, Silver sulfadiazine
o surfactants interact with the lipid in the
cell membrane and with the surrounding 3. H2O2 – hydrogen peroxide
water • for cleansing wounds and contact lenses
o increases the surface tension • oxidizes microbes.
o Example: Quaternary ammonium • Mouthwash (but in small amounts only, dilute:1 is to
(Quats or Zephiran) 1, hindi pwedeng derecho or lunukin sa bunganga lang daw or else
mamamatay)
Friction is needed in washing clothes to increase surface tension with the detergent.
• topical antiseptic used in wound
cleaning which kills pathogens thru
3. Phenols
oxidation burst and local oxygen
o original disinfectant of Lister production.
o Denature protein
o Example: Carbolic acid/cresol (Lysol)
Topical: Surface of the skin
o JOSEPH LISTER – Father of antiseptic
surgery
4. Formaldehyde 2. Malachite green – BASIC DYE 2. Ampicillin
• 37% formalin in water (pure form of formalin) – • Used in parasitology. o semisynthetic drug
dilute to 5% in formaldehyde • Effective against fungi and gram + o for Gram + and Gram –
effective disinfectant. bacteria. o derived from penicillin
• 0.2%– 0.4% it can inactive bacteria and • Example: in L-J Medium for MTB o improved version of penicillin
viruses. kills other bacteria except MTB
• denatures protein 3. Bacitracin
• bactericide and fungicide ANTIMICROBIAL DRUGS o topical antibiotic ointment, intramuscular
Paul Ehrlich o for Staphylococcus, Streptococcus,
5. Glutaraldehyde - CARCINOGENIC Father of chemotherapy Neisseria, Haemophilus
• 10X more effective than formaldehyde Discovered salvarsan 606 for syphilis.
and less toxic Discovered the first stain for gram B. Inhibition of Protein Synthesis (protein synthesis -ribosomes)

• Kill bacteria spores staining which is methylene blue 1. Chloramphenicol – bacteriostatic

• used for respiratory therapy equipment Before crystal violet, methylene blue o derived from Streptomyces venezuelae
Salvarsan 606: A.K.A magic bullet
was first utilized. o typhoid, cholera, conjunctivitis, otitis
6. Ethylene oxide media
• Colorless but flammable CHARACTERISTICS OF ANTIMICROBIAL o for Gram + and Gram –
• Used for sterilization of heat-sensitive DRUGS o high intake or dosage can cause anemia
equipment (aplastic anemia - bone marrow produces less
RBC)
• most effective cold sterilization 1. Selective toxicity (Kailangan organism lang ang namamatay)
technique 2. Wide spectrum of activity (Eg: Penicillin) 2. Erythromycin
3. Host should not become allergic to the o other than bacteria can treat parasitic
7. Acids and Alkalis antibiotic (pero madami allergic kaya kailangan samahan ng antihistamine
• denatures protein or epinephrine para counteract and allergic reaction)
infection.
• weak acids are used as food 4. The microorganism should not become readily o Used to treat pneumonia, acne, dental
preservatives (bacteriostatic) resistant to the antibiotic. (pag sinabing 1 week antibiotic 1 week abscesses, ear infection and STD
• Example: benzoic, propionic, citric acids dapat or else it would cause mutation to the bacteria tapos magiging resistant si chlamydia
patient)
o freezes the ribosome
C. Modification of Nucleic Acids (2 dyes)
4 FUNCTIONS
3. Tetracycline
A. Inhibition of Cell Wall Synthesis
1. Crystal Violet (Gentian Violet) o other than bacteria can treat parasitic
1. Penicillin – bacteriostatic
• Gram staining in bacteria is the most infection
o derived from fungi Penicillin notatum o broad spectrum
important staining method in
o discovered by Alexander Fleming o Used to treat UTI, STD, acne, cholera,
microbiology (because you will be able to group the 2 most
or biggest groups of bacteria in microbiology according to their cell o Susceptible: Gram + bacteria, malaria.
wall)
spirochetes
• 2 biggest, gram + (thick cell wall, they can retain crystal
violet), gram – (safranin) (thin cell wall, after washing 4. Streptomycin
Disadvantages:
out crystal violet they can retain the color safranin; gives the pink o derived from Streptomyces griseus
color)
- destroyed by acids o used to treat Neisseria gonorrhoeae and
• skin antiseptic
- destroyed by penicillinase or MTB (mycobacterium tuberculosis)
• binding of (+) charged dye molecule to
B – lactamase (enzymes) 5. Gentamycin, Lincomycin, Clindamycin and
the (–) charged PO4 groups of nucleic
- mostly for Gram +, Neisseria and Aminoglycosides
acid.
Treponema pallidum only
C. Injury to Cell Membrane LABORATORY EXPERIMENT 5:
1. Amphotericin B Antimicrobial Susceptibility Testing
- from Bacillus polymyxa
- for Gram – ANTIMICROBICS
- Example: Brucella abortus, Klebsiella 3 Categories of Antimicrobics
pneumonia
2. Tyrocidin 1. Antibiotics
3. Gramicidin S
 Derived from microorganisms
4. Imidazole and Triazole  Example: (Alexander Fleming)
- Penicillin – Penicillin notatum
D. Inhibition of Nucleic Acid Synthesis - Streptomycin – Streptomyces griseus
1. Rifampin Streptomycin: can be used for tuberculosis and tularemia
2. Quinolones
2. Semisynthetic
3. Sulfonamides
4. Trimethoprim  Chemically derived
 Example: Ampicillin – derived from
Drug resistant penicillin
- the ability of a microorganism to grow in
3. Synthetics
the presence of antibiotics that would
normally kill or inhibit its growth.  Antimicrobial drugs synthesized by
chemical procedures in the laboratory
 Example: Sulfonamides, Quinolones

Disk Diffusion Method (Kirby Bauer Method)