UNIT 5.

CONTROL OF MICROBIAL GROWTH

Definition of Terms:
Sterile
Free of life of every kind
Sterilization
Complete destruction or removal of all forms of microbial life including
endospore.
Usually done by steam under pressure, sterilizing gas, or ethylene oxide.
Disinfection
Destruction of vegetative pathogens on inanimate objects
Partial destruction through physical and chemical methods
Antisepsis
Destruction of vegetative pathogens on living tissue
Treatment is almost always by chemical antimicrobials
Sanitization
Treatment intended to lower microbial counts on eating and drinking
utensils to safe public health levels.
May be done with high temperature washing or by dipping into a chemical
disinfectant.
Degermining
Removal of microbes from a limited area, such as the skin around an
injection site.
Mostly mechanical removal by an alcohol-soaked swab
Microbicidal
Property of destroying microbes
Bactericidal, fungicidal, etc.
Microbiostatic
Property of inhibiting microbial growth and multiplication
Bacteriostatic, fungistatic, etc.
Disinfectant
Chemical agents applied on inanimate surface, too toxic to be applied
on living tissues
Antiseptic
Chemical agents which could have a cidal or static effect, applied
topically on living tissues.
Septic
Condition characterized by presence of pathogens (particularly on
living tissues)
Aseptic
Condition characterized by the absence of pathogens
Thermal death time
Minimum time required to kill suspension of microbes in a given
temperature in a specified environment
Thermal death point
Maximum temperature in a given time to destroy all microbes present

Several Factors that Influence the Rate at which Antimicrobial Agents

Work:
Exposure time of the agent
Numbers of microbes present
Relative resistance of microbes (for example, endospores versus vegetative
forms)
Activity of the agent (microbicidal versus microbiostatic)
2 General Methods of Microbial Growth
A. Physical methods - control is achieved by modifying environmental condtions
a. Scrubbing with soap and water
Washing based on standard operating procedure before and after
an activity
b. Filtration
Used to remove microbes for liquids or gases, with use of filters
0.2 to 0.45m pore.
Filters most bacteria, while viruses and small bacteria pass through
them.
For antibiotic solutions, CHO solutions, vaccines, culture media
which cannot be heated
Liquid: pulling solution through cellulose acetate or cellulose
nitrate medium with vacuum
Air: HEPA filters (removes organisms > 0.3m)
c. Sedimentation
Allowing solid or solutes or particulate matter to settle at bottom of
liquid
d. High temperature
i. Pasteurization
Use brief exposures to moderately high temp to reduce &
eliminate pathogens, w/o eliminating viable beneficial
microbes and w/o altering chemical nature of food.
Kills non-sporeforming organisms in heat sensitive materials
(milk products, wine, etc.)
Batch/LTH 63C for 30 mins
Flash/HTST 72C for 15 secs
ii. Moist Heat
Denatures bacterial proteins; water hastens breaking of Hbonds which hold proteins in their 3 dimensional
configurations; more penetrating
Boiling
100C for 15 mins or 30 mins
Kills vegetative cells but not spores
For sterilization of apparatus that are heat resistant
Suitable in situations from which endospores and
hepatitis virus are known to be absent
Autoclaving
Steam under pressure

 Most effective method

121C at 15lbs psi for 15mins
Destroys spores
For sterilization of culture media and surgical
instruments
Biologic indicator: Bacillus stearothermophilus
Tyndallization
Also Fractional/Discontinuous/ Intermittent Sterilization
For heat labile, spore-containing material
100C for 30 mins for 3 consecutive days; 60C for 1hr
for 5-6 days
Inspissation
75C - 80C for 2 hrs for 3 consecutive days
Principle: Thickening through evaporation
For high protein content media
e. Dry Heat
Denatures proteins; kills organisms by oxidation; requires higher
temperature and long exposure to ensure complete sterilization
Passing through a flame
For loops needles, mouth of tubes as well as plates
Hot Oven
For sterilization of glasswares
160C - 170C for 1.5 to 2 hrs
Incineration
For infectious wastes; burning wastes into ashes
870C - 980C (800C to 6500C)
Banned in the Philippines
Cremation
Burning dead bodies (with communicable disease) to
ashes
f. Low Temperature
Limits rate of microbial reproduction
Microbiostatic
Commonly used to preserve food, media, and cultures
5C for refrigeration temperature; 0C or subzero; freeze drying
through sublimation
g. Radiation
Ionizing Radiation
Short wavelength, high energy gamma rays and x-rays
For plastic syringes, catheter or gloves
Has deep penetrating power and works by causing
breaks in the DNA of target organisms
Non-ionzing Radiation
Long wavelength low E UV light
Very little penetrating power

 Works by creating dimers between adjacent

pyrimidines, which interferes with replication
B. Chemical Methods
a. Halogens
Chemicals based on elements from group VII of the periodic
table
i. Chlorine
Kills microbe by disrupting the plasma membrane
Chlorine gas, hypochlorites, chloramines all work by
disrupting disulfide bonds
Na hypochlorite or bleach (1:10)
ii. Iodine
Reactive by precipitating proteins and oxidizes essential
enzymes
I2 + detergent = iodophor
I2 in alcohol = I2 tincture
iii. Fluorides
In toothpaste, H2O supply
b. Phenolic Compounds
Act by disrupting lipid containing membranes, resulting in
leakage of cellular contents
Carbolic acid, Lysol, cresol-o-polyphenol
Standard disinfectant in the lab
c. Detergents
i. Anionic Detergents (Soaps)
Removing grease and soil that contains microbes
ii. Quaternary Ammonium Compounds (QUATS)
Cationic detergents
Act as surfactants that alter the membrane permeability of
some bacteria and fungi
Benzalkonium chloride (Zephiran)
For digestion and decontamination of sputum
Inactivated by ORGANIC SUBSTS
Disadvantage: Nonsporicidal; Nontuberculoidal
d. Alcohols
Most effective and most used
Act as surfactants, dissolving membrane lipids and
coagulating proteins of vegetative bacterial cells and fungi
Non-sporicidal
Used on skin (as antiseptic) and on thermometers and
injection vial rubber septum (as disinfectant)
Evaporates easily
i. Ethanol
70% is more effective than 95% EtOH
70% EtOH kills nearly 90% of cutaneous microbiota w/in 2
mins
ii. Isopropanol

 Highest bactericidal activity at 70-80%

Less activity against endospores, fungi and viruses
e. Aldehydes
Denature proteins and DNA by alkylation
Used for disinfecting surgical instruments
Formaldehyde and Glutaraldehyde (pH 7.5 kills Staphylococci
in 5 mins, tubercle bacilli in 10 mins and endospore for 12
mins
f. Acids destroy or inhibit microbial cells
i. Organic Acids
widely used in food preservation because they prevent spore
germination and bacterial and fungal growth and because
they are generally regarded as safe to eat
Acetic acid, Lactic acid, Benzoic acid, Sorbic acid
Vinegar (household disinfectant)
g. Alkalis inactivates proteins
i. Ammonium hydroxide
`reliably destroys prions
h. Dyes
antimicrobial effects are apparently due to the way they insert
into nucleic acids and cause mutations
interfere with cell wall synthesis
i. Hydrogen peroxide
produces highly reactive hydroxyl free radicals that damage
protein and DNA while also decomposing to O2 gas, which is
toxic to anaerobes
3 to 6% solutions antiseptic
6 to 25% solutions for sterilization mixtures, disinfects
implants, prostheses and contact lenses
Strong solutions are sporicidal
j. Ethylene oxide
Colorless gas, soluble in water and organic solvents used for
heat sensitive items
Used for sterilizing aircrafts for space explorations
Can be used for sterilizing disposable utensils, instruments and
prostheses
k. Ozone
Strong oxidizing agent
Oxidizes cellular biochemical
Disinfect drinking water
l. Heavy Metals
Acts with sulfhydryl groups of proteins, thus inactivates them
Inactivated by organic materials (e.g., blood)
Mercury-containing compound is no longer recommended toxic
to the environment
Silver Nitrate eyedrop used to prevent N. gonorrhoeae
infection in newborns

m. Chlorhexidine
surfactant and protein denaturant with broad microbicidal
properties, although it is not sporicidal
Solutions of chlorhexidine are used as skin degerming agents for
preoperative scrubs, skin cleaning, and burns
n. Antibiotics
Antimicrobial substances produced by microbes, used for
treating humans and animals
Modes of action: inhibits CW synthesis, inhibits CM function,
inhibits protein synthesis, inhibits nucleic acid synthesis