Controlling of

Microbial Growth
Introductory Chemistry
Mr. Sanchez
Importance of Controlling Microbial Growth

• To prevent the transmission of diseases and

infection
• To stop decomposition and spoilage
• To prevent unwanted microbial
contamination
• Early civilizations practiced salting, smoking, pickling, drying,
and exposure of food and clothing to sunlight to control
microbial growth.
• Use of spices in cooking was to mask taste of spoiled food.
Some spices prevented spoilage.
• In mid 1800s Semmelweiss and Lister helped developed
aseptic techniques to prevent contamination of surgical
wounds.
Before then:
• Nosocomial infections caused death in 10% of surgeries.
• Up to 25% mothers delivering in hospitals died due to
infection
Factors Affecting
Microbial Growth
 Temperature

• Psychrophiles (cold loving microbes )

range 0 C – 20 C
• Mesophiles (moderate temp. loving microbes)
range 20 C - 40 C
• Thermophiles (heat loving microbes)
range 40 C - 100 C
 pH
• Most bacteria grow between pH 6.5 - pH 7.5
• Very few can grow at below pH 4.0
• Pathogenic bacteria even more narrow in tolerance range of
near neutral
• Yeast and moulds have much greater tolerance to acidic
(lower) pH
Microorganisms are classified based on their pH requirements
• Acidophilic microbes: pH range of 0.1–5.4,
• Neutrophilic microbes: pH range of 5.4–8.0
• Alkalophilic microbes: pH range of 7.0–11.5
 Moisture

• Microbes must have a supply of of water available

• Bacteria most restricted, then yeasts, then moulds
• Organisms tolerant of low water levels:
–halophilic bacteria osmophilic yeasts
–xerophilic molds
 Nutrient Availability

• Many nutrients are energy sources

• Nutrients serve as sources of C, O, N, P, & Sulfur
• Source for trace elements as Iron, Iodine, Zinc
 Oxygen Availability

• It affects microbial growth, and microorganisms can be

classified based on their oxygen requirements, such as
obligate aerobes, facultative anaerobes, and obligate
anaerobes.
• Obligate aerobes, • Facultative anaerobes, • Obligate anaerobes,
e.g. Mycobacterium, e.g. Saccharomyces e.g. Clostridium will
(yeast) will grow in the grow only in absence
will grow only in absence of oxygen, but
presence of free of oxygen which is
more slowly than if toxic to them
oxygen oxygen were present.
 Osmotic Pressure

• Microbes obtain almost all their nutrients in solution from

surrounding water

Tonicity
• isotonic
• hypertonic
• hypotonic
Bacterial Growth
 Bacterial Growth

• Increase in number of cells, not cell size

• One cell becomes colony of millions of cells

GENERATION TIME
• Time required for cell to divide/for population to double.
• Average for bacteria is 1-3 hours
• E. coli generation time = 20 min
– 20 generations (7 hours), 1 cell becomes 1 million cells!
Standard Growth Curve
 Standard Growth Curve
1. Lag phase – making new enzymes in response to new medium.
The length of lag phase depend upon
a.Type of bacteria.
b.Better the medium, shorter the lag phase.
c.The phase of culture from which inoculation is taken.
d.Size or volume of inoculum.
e.Environmental factors like temperature.
2. Log phase – Logarithmic (Exponential) phase: In logarithmic phase the
bacterial cell start dividing and their number increase by geometric
progression with time.
During this period…
a.Bacteria have high rate of metabolism
b.Bacteria are more sensitive to antibiotics and radiation during this period.
 Standard Growth Curve
3.Stationary phase – nutrients becoming limiting or waste products
becoming toxic.
death rate = division rate
In stationary phase after some time a stage comes when rate of multiplication and
death becomes almost equal. It may be due to:
a.Depletion of nutrients.
b.Accumulation of toxic products and sporulation may occur during this stage.
4.Death or Decline phase – In decline (death) phase, death exceeds
division. During this phase population decreases due to death of cells. The
factors responsible are:
a.Nutritional exhaustion
b.Toxic accumulation
c.Autolysinenzymes
Inhibiting microbial growth
 Sterilization
• Sterilization: Killing or removing all forms of microbial
life (including endospores) in a material or an object.
• Heating is the most commonly used method of
sterilization.
• Commercial Sterilization:Heat treatment that kills
endospores of Clostridium botulinum the causative
agent of botulism, in canned food.
• Does not kill endospores of thermophiles, which are
not pathogens and may grow at temperatures above
45oC.
 Disinfection
• Disinfection: Reducing the number pathogenic microorganisms to
the point where they no longer cause diseases.Usually involves the
removal of vegetative or non-endospore forming pathogens.
• May use physical or chemical methods.
Disinfectant: Applied to inanimate objects.
Antiseptic:Applied to living tissue (antisepsis).
Degerming: Mechanical removal of most microbes in a limited
area. Example:Alcohol swab on skin.
Sanitization: Use of chemical agent on food-handling equipment to
meet public health standards and minimize chances of disease
transmission. E.g: Hot soap & water
⚫Sepsis-refers to the presence of pathogens in blood or tissues

⚫Asepsis means the absence of pathogens

⚫Antisepsis-is the prevention of infection

⚫Antiseptic technique- developed by Joseph Lister in

1867,refers to the use of antiseptics.
⚫Sterile technique-is practice to exclude all microorganisms from a
particular area, so that the area will be sterile.
Using Physical Methods
Of Microbial Control
Physical Methods of Microbial Control:

1- HEAT: Kills microorganisms by denaturing their enzymes and other

proteins.

Thermal Death Point (TDP): Lowest temperature at which all of the

microbes in a liquid suspension will be killed in ten minutes.

Thermal Death Time (TDT): Minimal length of time in which all bacteria will
be killed at a given temperature.

Decimal Reduction Time (DRT): Time in minutes at which 90% of bacteria

at a given temperature will be killed. Used in canning industry.
 Physical Methods of Microbial Control:

Moist Heat: Kills microorganisms by coagulating their proteins.

(In general, moist heat is much more effective than dry)

Boiling: Heat to 100oC or more at sea level. Kills vegetative forms of bacterial
pathogens, almost all viruses, and fungi and their spores within 10 minutes or less.
Endospores and some viruses are not destroyed this quickly. However brief boiling
will kill most pathogens.

Hepatitis virus: Can survive up to 30 minutes of boiling.

Endospores: Can survive up to 20 hours or more of boiling.
- Reliable sterilization with moist heat requires temperatures
above that of boiling water.
 Physical Methods of Microbial Control:

Autoclave: Chamber which is filled with hot steam under pressure.

Preferred method of sterilization, unless materialis damaged by heat,
moisture, or high pressure.
-Temperature of steam reaches 121C at twice atmospheric pressure.
- Most effective when organisms contact steam directly or are contained
in a small volume of liquid.
- Allorganisms and endospores are killed within 15 minutes.
Pasteurization: Developed by Louis Pasteur to prevent the spoilage of
beverages. Used to reduce microbes responsible
for spoilage of beer, milk, wine, juices, etc.
 Physical Methods of Microbial Control:

Dry Heat: Kills by oxidation effects.

Direct Flaming: Used to sterilize inoculating loops and needles. Heat
metal until it has a red glow.
Incineration: Effective way to sterilize disposable items (paper cups,
dressings) and biological waste.

Hot Air Sterilization: Place objects in an oven. Require 2 hours at

170oC for sterilization.

Dry heat is transfers heat less effectively to a cool body, than moist heat
 Physical Methods of Microbial Control:

2- FILTRATION:
Removal of microbes by passage of a liquid or gas through a screen like material
with small pores.

Used to sterilize heat sensitive materials like vaccines, enzymes,

antibiotics, and some culture media.

High Efficiency Particulate Air Filters (HEPA):

Used in operating rooms and burn units to remove bacteria from air.
Physical Methods of Microbial Control:

3-LOW TEMPERATURE:
Effect depends onmicrobe and treatment applied.
Refrigeration: Temperatures from 0 to 7oC.Bacteriostatic effect. Reduces
metabolic rate of most microbes so they cannot reproduce or produce toxins.
Freezing: Temperatures below 0oC.
Flash Freezing: Does not kill most microbes.
Slow Freezing: More harmful because ice crystals disrupt cell structure.
Over a third of vegetative bacteria may survive 1 year.
Most parasites are killed by a few days of freezing.
 Physical Methods of Microbial Control:

4- RADIATION: radiation kill microbes:

A.Ionizing Radiation:
• Gamma rays, X rays, electron beams, or higher energy rays.
• Have short wavelengths (less than 1 nanometer).
• Dislodge electrons from atoms and ions.
• Cause mutations in DNA and produce peroxides.
• Used to sterilize pharmaceuticals and disposable medical supplies. Food
industry is interested in using ionizing radiation.

Disadvantages: Penetrates human tissues.

May cause genetic mutations in humans.
 Physical Methods of Microbial Control:

B . Ultraviolet light (Nonionizing Radiation):

• Wavelengthis longer than 1 nanometer. Damages DNA by

producing thymine dimers, which cause mutations.
• Used to disinfect operating rooms, nurseries, cafeterias.

Disadvantages: Damages skin, eyes. Doesn’t penetrate paper,

glass, and cloth.
Using Chemical Methods
Of Microbial Control
 Chemical Methods of Microbial Control:

1 PHENOLS AND PHENOLICS:

Phenol (carbolic acid) was first used by Lister as a disinfectant.
- Rarely used today because it is a skin irritant and has strong odor.
- Used in some throat sprays and lozenges.
- Acts as local anesthetic.
Phenolics are chemical derivatives of phenol (carbolic acid).
Cresols: Derived from coal tar (production of coke and coal gas from coal)
(Lysol) (Disinfectant Spray, sanitizers, wipes etc).
Biphenols (pHisoHex): Effective against gram-positive staphylococci
and streptococci.
Used in nurseries. Excessive use in infants may cause neurological damage.
- Destroy plasma membranes and denature proteins.
-Advantages: Stable, persist for long times after applied, and remain active
in the presence of organic compounds.
 Chemical Methods of Microbial Control:

2- HALOGENS: Effective alone or in compounds. (Consisting of five chemically

related elements: fluorine (F), chlorine (Cl), bromine (Br), iodine (I), and astatine (At)
A.Iodine:
- Tincture of iodine (alcohol solution) was one of first antiseptics
used.
- Combines with (amino acid-tyrosine) in proteins and denatures proteins.
- Stains skin and clothes, somewhat irritating.
- Iodophors: Compounds with iodine that are slow releasing, take several minutes
to act. Used as skin antiseptic in surgery.
- Not effective against bacterial endospores.
- Betadine ((Povidone-iodine) is a topical antiseptic)
- Isodine (Antiseptic)
Chemical Methods of Microbial Control:

B. Chlorine:
- Whenmixed in water forms hypochlorous acid
• Used to disinfect drinking water, pools, and sewage.
• Chlorine is easily inactivated by organic materials.
• Sodium hypochlorite(NaOCl):Is active ingredient of bleach.
• Chloramines: Consist of chlorine and ammonia. Less
effective as germicides.
 Chemical Methods of Microbial Control:

3- ALCOHOLS:
- Kill bacteria, fungi, but not endospores or naked viruses.
- Act by denaturing proteins and disrupting cell membranes.
- Evaporate, leaving no residue.
- Used to mechanically wipe microbes off skin before injections or blood drawing.
- Not good for open wounds, because cause proteins tocoagulate.
- Ethanol: Drinking alcohol. Optimum concentration is 70%.
- Isopropanol: Rubbing alcohol. Better disinfectant than ethanol.
- Also cheaper and less volatile. (easily evaporated at normal temperatures)
 Chemical Methods of Microbial Control:

4- HEAVY METALS:
- Include copper, selenium, mercury, silver, and zinc.
- Oligodynamic action: Very tiny amounts are effective.
A.Silver:
- 1% silver nitrate used to protect infants against gonorrheal eye infections until recently.
B.Mercury:
- Organic mercury compounds like merthiolate and mercurochrome are used to
disinfect skin wounds.
C.Copper:
- Copper sulfate is used to kill algae in pools and fish tanks.
D. Selenium:
- Kills fungi and their spores. Used for fungal infections.
- Also used in dandruff shampoos.
E. Zinc:
- Zinc chloride is used in mouthwashes.
 Chemical Methods of Microbial Control:

5- QUATERNARY AMMONIUM COMPOUNDS (QUATS):

- Widely used surface active agents.
- Cationic (positively charge) detergents.
- Effective against gram positive bacteria, less effective
against gram-negative bacteria.
- Also destroy fungi, amoebas, and enveloped viruses.
- Zephiran, Cepacol, also found in our lab spray bottles.
- Pseudomonas strains that are resistant and can grow in presence of Quats are a big
concern in hospitals.

Advantages: Strong antimicrobial action, colorless, odorless, tasteless, stable, and

nontoxic.
Diasadvantages: Form foam. Organic matter interferes with
effectiveness. Neutralized by soaps and anionic detergents.
 Chemical Methods of Microbial Control:

6- ALDEHYDES:
- Include some of the most effective antimicrobials.
- Inactivate proteins by forming covalent crosslinks with several functional groups.

A. Formaldehyde gas:
- Excellent disinfectant.
- Commonly used as formalin, a 37% aqueous solution.
- Formalin was used extensively to preserve biological specimens and inactivate
viruses and bacteria in vaccines.
- Irritates mucous membranes, strong odor.
- Also used in mortuaries for embalming (preserve).
Chemical Methods of Microbial Control:

B. Glutaraldehyde:
- Less irritating and more effective than formaldehyde.
- One of the few chemical disinfectants that is a sterilizing agent.

- A 2% solution of glutaraldehyde (Cidex) is:

- Bactericidal, tuberculocidal, and viricidal in 10 minutes.
- Sporicidal in 3 to 10 hours.
- Commonly used to disinfect hospital instruments.
- Also used in mortuaries for embalming.
 Chemical Methods of Microbial Control:

7- GASEOUS STERILIZERS:
- Chemicals that sterilize in a chamber similar to an autoclave.
- Denature proteins, by replacing functional groups with alkyl groups.

A. Ethylene Oxide:
- Kills all microbes and endospores, but requires exposure of 4
to 18 hours.
- Toxic and explosive in pure form.
- Highly penetrating.
- Most hospitals have ethylene oxide chambers to sterilize mattresses and
large equipment.
 Chemical Methods of Microbial Control:

8. PEROXYGENS (OXIDIZING AGENTS):

- Oxidize cellular components of treated microbes.
- Disrupt membranes and proteins.

A. Ozone:
- Used along with chlorine to disinfect water.
- Helps neutralize unpleasant tastes and odors.
- More effective killing agent than chlorine, but less stable and more expensive.
- Highly reactive form of oxygen.
- Made by exposing oxygen to electricity or UV light.
 Chemical Methods of Microbial Control:

B. Hydrogen Peroxide:
- Used as an antiseptic.
- Not good for open wounds because quickly broken down by catalase present in
human cells.
- Effective in disinfection of inanimate objects.
- Sporicidal at higher temperatures.
- Used by food industry and to disinfect contact lenses.

C. Benzoyl Peroxide:
- Used in acne medications.
Chemical Methods of Microbial Control:

D. Peracetic Acid

• One of the most effective liquid sporicides available.


Sterilant :

• Kills bacteria and fungi in less than 5 minutes. Kills
endospores and viruses within 30 minutes
• Used widely in disinfection of food and medical instruments
because it does not leave toxic residues.