University of the Philippines

Los Banos, Laguna

GARCIA, Kalel Liam M. Prof. John Daniel Ong

B.S. Biology MCB 11 ST

Microbiology - study of organisms and agents too small to be seen by the naked eye (<1mm)

Typical Bacterium : 2 μm with 10-12 g

Typical Viruses : 0.02 μm to 0.4 μm

Longest known bacterium - Epulopiscium (100 μm) [Epulopiscium fishelsoni]

- Thiomargarita namibensis (200 μm) [some reaches 3 times the size]

New Definition of Microbiology :

- The study of organisms that can exist as single cells, contain a nucleic acid genome for at
least some part of their life cycle, and are capable of replicating that genome

- Also include viruses, which microbiology texts traditionally discuss along with living
organisms

Importance of Microorganisms :

1. First living organisms on planet

2. Live everywhere life is possible

3. More numerous than any other kind of organisms

4. Global ecosystem depends on their activities

5. Influence human society in many ways

Focuses on :

1. Archaea

- Ancient or primitive (archaios)

- described in extreme environments (extremophile) *filia = love

- cell wall : Pseudopeptidoglycan

2. Algae (alga) [Phycology]

 - aquatic photosynthetic organisms

- kingdom : protista

- oxygen producers

- source of crude oil

3. Fungi (fungus) [Mycology]

- yeasts, rusts, smuts, mildews, molds, and mushrooms

- lack chlorophyll

4. Protozoan

- uses organic carbon as source of energy

- all are eukaryotes

- nonfilamentous

- dinoflagellates, amoebas, paramecia, malaria-causing Plasmodium

5. Viruses [Virology]

- infectious agent

- “slimy liquid” or “poison”

- not free-living (needs host cells)

- contain nucleic acid

6. Bacteria [Bacteriology]

Applied Microbiology :

1. Medical Microbiology

2. Immunology

3. Public Health Microbiology

4. Food and Dairy Microbiology

5. Industrial Microbiology

6. Agricultural Microbiology

Some challenges :
1. Infectious Diseases

2. New and Improved Industrial Process

3. Microbial Ecology ad Microbial Diversity

4. Biofilms

5. Microbes as Model Ecosystems

6. Genome Analysis

7. Assessment

Discovery of Microorganisms :

1. Zacharias Janssen and Hans Janssen (1597)

- First compound microscope (10x)

2. Robert Hooke (1665)

- First usable compound microscope (30x)

- “Mucor” - fungi (elongated stalks)

- Discovered cells (using cork)

3. Antony van Leeuwenhoek (1670) [Father of Microbiology]

- Single lens microscope (100x)

- “Wee Animalcules”

Spontaneous Generation Theory

- Life emerges from non-living matter

- Requires “vital force”

Biogenesis Theory

- Life begets life

Scientists who contribute into the debate between SG theory and Biogenesis Theory :

1. Francesco Redi

- maggots arise from the eggs of flies rather than directly from rotten meat

SG Supporters : life force doesn’t enter

*The discovery of microorganisms (1665) arose some scientist to point that microorganisms come
from boiled substances
2. John Needham - pro SG

- boiled mutton broth experiments (did not boil enough to kill microbes)

- concludes that organic matter contained a vital force that could confer the properties of life
on non-living matter

3. Lazzaro Spallanzani

- repeats and questioned Needham’s work

- boiled in 3/4 hours

- proposed that air carries germ to the culture medium

- sealing the flask destroyed its ability to support life

SG Supporters - air was required for the vital force to work

4-5. Franz Schulze/Theodor Schwann

- passed one through strong acids (Schulze)

- passed air through red-hot tubes (Schwann)

- Result : No microorganisms were formed

SG Supporters - strong acids and heat altered the air so it cannot support microbial grown

6-7. Georg Friedrich Schroder and Theodor von Dusch

- Filtered air through sterile cotton wool

- No microbial growth

8. Louis Pasteur - Father of Modern Microbiology

- uses swan-necked flasks and used nutrient rich broth called infusion

- each experimental variable was boiled

a. The broth provides a nutrient medium for the growth of unseen organisms in the air; life
comes from other life.

- A sterilized broth gives rise to life

b. The heat was killed the microorganisms in the air

- Sealing the flask prevents entry of the “life force”

c. The heat has killed the microorganisms in the air

 - Sterilizing the air kills life force

d. No living things will appear in the flask because microorganisms will not be able to reach
the flask

- If the ‘life force’ has free access to the flask, life will appear, given enough time.

*Some days later, the flask is still free of any living things. Disproving the SG Theory.

9. John Tyndall

- demonstrated that dust carries microorganisms

- also provided evidence for the existence of exceptionally heat-resistant forms of bacteria

*Tyndallization - Heating up to boiling point for 15 minutes, then cool, then boil.

Standard sterilization = Decontamination

10. Ferdinand Cohn - discovered the existence of heat-resistant bacterial endospores

*Endospores - a resistant sexual spore that develops inside some bacteria cells.

a. Vegetative - continuously replicating up to certain point

b. Dormant - resistant to different stress

*Endospore Staining :

a. Primary - Malachite Green Dye (7 Minutes)

- Cloth Method

b. Secondary - Safranin

Germ Theory of Disease

- Diseases are caused by specific agents called germs

1. Girolamo Fracastoro

- disease was caused by invisible living creatures

2. Agostino Bassi

- showed that a disease of silkworms was caused by a fungus

3. M.J. Berkeley

- demonstrated that the Great Potato Blight (caused by Phytophthera infestans) of Ireland was
caused by a fungus

4. Heinrich de Bay
 - showed that smut (Puceinia graminis) and rust fungi (Ustilago cynodotis) caused cereal crop
diseases

5. Ignaz Semmelweis

- asepsis in obstetrical wards to prevent the transmission of chilbirth fever from patient

- policy for all attending physicians to wash their hands with chloride of lime between
patients

6. Joseph Lister - “Father of Antiseptic Surgery”

- provided indirect evidence that microorganisms where the causal agent

- used phenol or carbolic acid in surgical dressings, heat-sterilized surgical instruments

7. Louis Pasteur

- showed that the pebrine disease of silkworms was caused by a protozoans (later was
discovered to be closely related to fungi)

8. Robert Koch

- established he relationship between Bacillus anthracis and anthrax

- used criteria developed by his teacher Jacob Henle

- derived the Koch’s Postulate

Koch’s Postulate :

1. The microorganism must be present in every case of the disease but absent from healthy

organisms

2. The suspected microorganism must be isolated and grown in a pure culture

3. The same disease must result when the isolated microorganism is inoculated into a healthy host

4. The same microorganism must be isolated again from the diseased host

Koch’s Postulate Limitations :

1. Causative agents of several human diseases do not cause disease in any known experimental
animals.

2. Some microbes are obligate intracellular parasites (like chlamydia or viruses) and are very
challenging, or even impossible to grow on artificial media.

3. Some diseases such as tetanus (Costridium tetani) have variable signs and symptoms between
patients.

4. Some diseases such as pneumonia and nephritis may be caused by a variety of microbes.

5. Some pathogens such as S. pyogenes, cause several different diseases.

6. Certain pathogens, such as HIV, cause disease in humans only --- it is unethical to purposefully
infect a human.

Variolation - method first used to immunize an individual against smallpox (Variola) with material
taken from a patient or a recently variolated individual in the hope that a mild, but protective
infection would result.

Edward Jenner - used a vaccination procedure to protect individuals from smallpox

*Last smallpox case in Somalia in 1977

*WHO declared smallpox as officially eradicated in 1979

Pasteur and his co-workers

- developed vaccines for chicken cholera, anthrax, and rabies

They discovered that :

- growing the pathogen in an abnormal host also weakens it

- incubation of cultures for long intervals between transfers caused pathogens to lose their
ability to cause disease

Vaccines - inoculation for hydrophobia (rabies)

1. Charles Chamberland - developed porcelain bacterial filter

- used to isolate first viruses studied

2. Paul Ehrlich - developed 606th compound SALVARSAN (inorganic arsenical to treat syphilis)

3. Alexander Fleming - discovered the “miracle drug” penicillin from penicillium which inhibits the
growth of Staphylococcus aureus.

Fermentation and Pasteurization:

Pasteurization - a process in which packaged and non-packaged foods are treated with mild heat to
eliminate pathogens and extend shelf-life.

1. Louis Pasteur - fermentations were the result of microbial activity

 - developed the process of pasteurization to preserve wine

2. Edward Buchner - cell-free fermentation

- extract from yeast cells

3. Sergei Winogradsky - microbial ecology

- chemosynthesis

- Winogradsky column

- biogeochemical cycles

- bacterial sulfate reduction (Beggiated sp.)

- nitrogen-fixing bacteria

4. Marthus Beijennck

- studied microorganisms in and around plants and in soul

- enrichment culture and selective media

Vaccination - originated from the Latin word vacca which means cow. (Cowpox cirus was used in
the first preparation for active immunization against smallpox)

- exposes a person to a specially prepared microbial (antigenic) stimulus which then triggers
the immune system to produce antibodies and limphocytes for future exposure to microbe.

* Originated in China through variolation (direct transfer of disease)

* First successful vaccination was done by Edward Jenner.

Principles of Vaccine Preparation :

1. Killed whole cells or inactivated viruses

2. Live, attenuated cells or viruses

3. Antigenic molecules derived from bacterial cells or viruses

4. Genetically engineered microbes or microbial agents

Creating Vaccines :

1. Killed or inactivated vaccines - prepared by cultivating the desired strain or strains of a

bacterium or virus and treating them with heat, formalin, radiation or some other agent that does
not destroy antigenicity.

- requires larger dose and more booster to be effective

2. Attenuated vaccines - any process that substantially lessens or negates the virulence of viruses or
bacteria.

- usually achieved by modifying the growth conditions or manipulating microbial genes in a

way that eliminates virulence factors

- includes long term cultivation, selection of mutant strains that grow at colder temperature,
passage of the microbe through unnatural hosts or tissue culture, and removal of virulence genes.

Advantages of Attenuated Vaccines :

1. Viable microorganisms can multiply and produce infection (but not disease) like the natural
organism.

2. They confer long-lasting protection.

3. They usually require fewer doses and boosters than other types of vaccines.

4. They are particularly effective at inducing cell-mediated immunity.

Disadvantages of Attenuated Vaccines :

1. They require special storage facilities

2. Can be transmitted to other people

3. Can Conceivably mutate back to a virulent strain

3. Acellular or subcellular vaccines - if the exact epitopes (the part of an antigen in which the
antibody attaches itself) that stimulate immunity are known, it is possible to produce a vaccine
based on a selected component of a microorganism.

4. Subunit vaccines - the antigen in these vaccines may be taken from culture of the microbes,
produced by genetic engineering or synthesized chemically.

Microscopes - used to view objects and areas of objects that cannot be seen with the naked eye.

Development in Microscopy :

1. Zaccharias Janssen and Hans Janssen (10x) - first compound microscope

2. Robert Hooke (40x) - first usable compound microscope

3. Anton van Leeuwenhoek (100x) - simle micsocrope (smaller lens)

4. Joseph Jackson Lister - developed better microscopes

5. Max Knoll and Ernst Ruska - electron microscope (1940)

Types of Microscopes :

1. Light Microscopes - light waves and mirrors

a. Simple

- Short Focal Length

- Only 1 lens

- Magnification - 300x

b. Compound or Complex

- 2 sets of lenses

- Magnification - 1000x

2. Electron Microscopes - electron beams and magnetic fields; in vacuum

- for examining objects smaller than 0.2μm in diameter

Features of a Good Microscope

1. Adequate Magnifying Power

2. Provide Good Contrast

3. Possess High Resolving Power

4. Serves Your Purpose

Types of Light Microscopes :

1. Bright Field

- Microscopic field brightly lit; objects under study are darker

 - Gross morphology

2. Dark Field

- Background is black; object bright or luminous

- For specimens that are :

a. Invisible in the ordinary light microscope

b. Cannot be stained by standard methods

c. Distorted by staining

3. UV

- Make use of shorter wavelength of light (180nm-400nm)

- Images are made visible by recording on a photographic emulsion or by displaying on a TV

screen

- Detection of substances (e.g. DNA)

4. Fluorescence

- modification of UV microscope

- makes use of fluorochromes (coating) (fluorescent dyes)

- detection of immunological reactions (antigen-antibody reaction)

5. Phase Contrast

- detailed examination of internal structure

- not necessary to fix or stain cells

- principle is based on variations in the refractive index

6. Differential Interference Contrast (surface)

- principle is based on variations in the refractive indices

- advantage: no diffraction halo associated with phase contrast

- disadvantage : the three-dimensional appearance may not represent reality

Types of Electron Microscopes :

1. Transmission Electron Microscope (TEM)

 - Examine viruses

- Internal ultrastructure in thin sections of cells

- uses Fluorescent Screen

2. Scanning Electron Microscope (SEM)

- Surface features of cells and viruses

- Lacks resolution of TEM but reveals a three-dimensional image

- uses Electron Detector

Special EM Stains : osmic acid, permanganate, lead, uranium, lanthanum, gold and silver

Examination of Microorganisms

1. Living or Natural State

Advantages :

- Observation of unaltered/undistorted characteristics of the organism

- Cellular processes can be studied

- Motility can be observed

- Simple to prepare

Disadvantage :

- The refractive index of the cell is almost similar to that of water

Two Methods Used :

a. Wet Mount Technique

b. Hanging Drop Technique

2. Stained Preparations

Advantages :

- Provides contrast

- Slides can be preserved

- Specimens are killed

Disadvantages :

- More complicated and tedious to prepare

- More expensive

3 Basic Steps in Staining Microorganisms :

1. Smear Preparation

Smear - a thin dry film of microorganisms

2. Fixation

Heat Fixation : Direct Flame

Steam Fixation

Chemical Fixation : Alcohols

Purpose of Fixation :

a. Kills the cells

b. Makes the cells sticky so they adhere to the slide

c. Increases apparent diameter of cells

3. Staining - application of biological dyes

Dyes (Stains) - organic compound carrying chromophoric ions

Types of Stains : Basic or positively charged dye

Acidic or negatively charged dye

 Neutral

Mechanisms of Staining

Physical : Absorption (Dissolve), Adsorption (Adhere), Osmosis (High to Low), Capillary Action

Chemical : Ion-exchange

Staining Procedures

1. Simple Staining - only one dye

a. Positive/direct - cells same color as dye

Methylene Blue

Crystal Violet

b. Negative/indirect - cells colorless or luminous

India Ink

Nigrosin

2. Differential Staining - 2 or more dyes and/or reagents

Gram Staining - Gram Positive (blue/violet) (Peptidoglycan is thicker)

Gram Negative (red/pink) (Peptidocglycan is thinner)

*Gram’s Iodine - mordant (makes distinct and adhesion)

*Safranin (Secondary Stain)

Acid Fast Staining - Lycolic acids

- 95% ethanol (not heat)

- heat to let carbolfuschia absorb

- for Mycobacterium tuberculosis = Malachite Green

- Diagnosis of Tuberculosis
3. Structural Staining - 2 or more dyes and/or reagents

- coal method = 30 minutes

Endospore (Malachite Green for Primary Stain); Flagella; Capsule; Storage granules

*Lactophenol staining and slide preparation, has 3 components. The phenol kills any residual living
cells. The lactic acid preserves any fungal material, and cotton blue stain, stains fungal chitin. Chitin
is a major component of fungal cell walls. The staining mixture is simple to make, and easy to use.

Isolation and Cultivation of Microogranisms

Pure Culture (Axenic Culture)

- a culture which contains a single species of microorganism

- a population of cells arising from a single cell

Cultivation

- increasing the population of microorganisms by providing their nutritional and physical

requirements

Nutrients

- extracellular substances which provide the cell with materials for building protoplasm and
for energy generation

Culture Medium

- any nutrient material for growth and cultivation of microorganism in the laboratory

Uses of Culture Media :

a. For growth and maintenance of microbial cultures

b. To favor the production of particular compounds

c. To study microbial action on some constituents of the medium

Types of Culture Media :

1. According to Physical State

a. Liquid (Broth) - with no solidifying agent

b. Semi-solid - with 0.1% to 0.5% solidifying agent

c. Solid - with 1.5% to 2.0% solidifying agent (ex. Agar or Gelatin)

2. According to Chemical Composition

a. Synthetic - all components are chemically defined

b. Complex - not all components are chemically defined

e.g. Potato Infusion ( of plant origin )

Beef Extract ( of animal origin )

Yeast Extract ( of microbial origin )

3. According to Principal Function, Purpose or Application

a. General Purpose - can support most or almost all types of species

e.g. Nutrient Agar (NA)

b. Differential - distinguishes one type of bacteria from another

- with special reagents like pH indicators or dyes

e.g. Eosin Methylene Blue Agar (EMBA)

c. Selective - allows the growth of a specific type of microorganism only

- with selective agents (ex. Salts, Dyes, Antibiotics, Etc.)

e.g. Bacillus Cereus Agar (BCA)

d. Enrichment - used to increase the number of microorganism with unusual physiological

characteristics

- with special nutrients (ex. Blood, Serum)

e.g. Blood Agar

e. Assay - of prescribe composition used for assay of vitamins, amino acids and antibiotics
 - used to determine qualitative/quantitative production of such a compound by an
microorganism

Techniques :

1. Plating

- streak, spread or pour

*Colony - a macroscopically visible (sutface or subsurface) growth or cluster of microorganism

on a solid medium

2. Enrichment Culture

- isolation of specific types of microorganisms by a combination of nutrient and physical

conditions

- used for the isolation of unusual physiological types of microorganisms which are present in
small numbers and which grow slowly

3. Serial Dilution

- used if the desired microorganism is present at a higher level than any other
microorganism

- uses a series of tubes with diluents

4. Single-Cell Isolation Technique

- uses a micropipette or a microphobe to physically pick a single cell and transfer it to an agar
medium

5. Membrane Filter Technique

- for samples with low population

- uses a sterile membrane filter having a pore size that retains microorganism

Basic Steps in Preparing Pure Cultures :

1. Isolation

2. Transfer desired colony to a slant or stab

3. Verify the purity

 a. Microscopic

b. Restreak on Agar Medium

c. Subject to Different Physiological/Biochemical Tests

4. If colony is pure, make stock cultures

a. Purity is maintained

b. Viability is retained

Culture Preservation Methods

Objective : To retain the viability of the stock culture for a long period of time while maintaining
its purity and trait of being “true-to-type”

1. Periodic Transfer to Fresh Media

Considerations :

a. Time Interval of Transfers

b. Proper Medium

c. Proper Storage Temperature

2. Overlaying Cultures with Mineral Oil

Aim : Limit the availability of O2 to reduce the metabolic rate

Advantages :

a. Simple

b. Enables one to remove some growth under the oil and inoculate it in a fresh medium
and still preserve the initial culture

Disadvantages :

a. Viability of microorganisms varies with species

3. Freeze-Drying (Lyophilization)

- employs rapid drying in frozen state

 - uses dry ice in alcohol ( -70 degrees Celsius)

Advantages :

a. Long-term survival

b. Less opportunity for changes in the characteristics of culture

c. Smallness of storage containers

4. Freezing with Liquid Nitrogen

- temperature at -196 degrees Celsius

- specimens are frozen along with a protective agent (Glycerol) in liquid-nitrogen refrigerators

5. Drying

- for spore- and cyst- formers

- drying temperature at 45 degrees Celsius

Banking Microbes

Culture Collections

- organization which maintain authentic pure cultures of microorganisms

- provide ‘type’ strains to microbiologists throughout the world

Examples :

1. American Type Culture Collection (ATCC) [Maryland]

2. National Collection of Type Cultures [London]

3. Japanese Type Culture Collection

4. Philippine Natural Collection of Microorganisms [BIOTECH-UPLB]

Sterilization (Latin sterilis = barren) – killing or removal of ALL viable organisms in an

object or habitat

Disinfection

– killing, inhibition or removal of microorganisms that may cause disease

Disinfectants : agents used to carry out disinfection and are normally used only on inanimate
object (does not sterilize an object)

Sanitization

– reduction of microbial population to levels considered safe by public health standards

Antisepsis – prevention of infection or sepsis in living tissues using chemicals

Antiseptics : generally not as toxic as disinfectants; can be used on living tissues

Microbial control can be achieved through:

1. Inhibition of microbial growth

2. Destruction of the microorganism i.e. sterilization

Reasons for controlling microbial growth:

1. To prevent/ limit spoilage or destruction of valuable substances/ commodities

2. To prevent infections

3. To prevent contamination of the cultures, the person and the environment

Agents Used In Microbial Control

Physical Agents :

A. Heat

1. Moist heat (MOA: denaturation/coagulation of proteins)

a. Boiling or flowing steam – kills vegetative cells and eukaryotic spores within 10 minutes

b. Pasteurization – process that uses relatively brief exposures to moderately high

temperature to reduce microbial population and to eliminate human pathogens

1. low temperature holding (LTH) – 62.8°C for 30 mins

2. high temperature short-time (HTST) – 72°C for 15 secs

3. ultra-high temperature (UHT) – 140-150°C for 1 -2 secs

c. Steam under pressure (autoclaving) – 121°C for 15 minutes

basis: spores of Bacillus stearothermophilus

d. Tyndallization/ fractional steam sterilization/ intermittent sterilization

- for materials destroyed at temperature > 100°C

- sterilization at 90-100°C for 30 mins for 3 consecutive days and incubated at 37°C in
between

2. Dry heat

a. Direct flame (incineration) – MOA: burning to ashes

b. Hot air (mechanical convection oven) – 170 – 180°C for 1 hour

MOA: oxidation of cellular components

B. Low Temperature (MOA: limits growth due to decreased rates of cell reactions and

changes to some proteins)

1. Refrigeration – 4°C

2. Freezing/ Deep freezing – -0° to -95°C

C. Filtration (MOA: exclusion of microorganisms)

Used for:

Heat sensitive materials – e.g. enzymes, toxins, vitamins

 - Membrane filters

Air – High efficiency particulate air (HEPA) filter removes 99.97% of 0.3 μm particles

D. Desiccation (MOA: microbiostasis)

1. Drying (sun, air, oven)

2. Freeze-drying/ lyophilization

E. Increased osmotic pressure (MOA: microbiostasis) – increase solute concentration

eg. by adding sugar, salt

F. Radiation

1. Ionizing radiation (MOA: free radicals formation)

- X-rays, gamma rays

- very short wavelength →cause atoms to lose electrons or ionize

- result in DNA destruction

- excellent sterilizing agent (penetrates deep into objects)

2. Non-ionizing radiation (MOA: formation of thymine dimers)

- UV rays

- most lethal at 260 nm →absorbed readily by DNA

- DNA damage

- does not penetrate objects – surface sterilization only

Chemical Agents :

Antimicrobial agents: chemicals that kill microorganisms or prevent their growth

-cide (Latin cida = kill) – substances that kill organisms

e.g. Germicide

Bactericide

Fungicide

-static (Greek statikos = causing to stand or stopping) – do not kill but prevents growth

eg. Bacteriostatic/ bacteriostat

Fungistatic/ fungistat
Examples of Antimicrobial Agents

1. Phenol and phenolics (MOA: disruption of plasma membrane, protein denaturation, and
inactivation of enzymes

phenol – used as standard for the effectiveness of other disinfectants (phenol coefficient)

phenolics – derivatives of phenol

eg. cresols, xylenols, and orthophenylphenol

2. Alcohols (MOA: denature proteins and dissolve membrane lipids)

- bactericidal and fungicidal but not sporicidal

eg. ethanol and isopropanol

3. Halogens (MOA: oxidation of cell constituents)

eg. Iodine – can also inhibit protein function

Chlorine

- chlorine gas

- sodium hypochlorite

- calcium hypochlorite

4. Heavy Metals (MOA: denatures enzymes and other essential proteins)

1% silver nitrate →prevents ophthalmic gonorrhea

silver sulfadiazine →used on burns

copper sulfate →algicide

merthiolate →disinfects skin mucous membrane

5. Surface-active Agents (surfactants)

a. Soaps and acid-anionic detergents

(MOA: mechanical removal of microorganisms)

eg. bath soaps

b. Cationic detergents/quaternary ammonium compounds

(MOA: disrupt plasma membrane and denature proteins)

6. Organic Acids (MOA: inhibits metabolism)

MOA not related to acidity/pH

widely used in foods/ cosmetics

eg. sorbic acid, benzoic acid, parabens, calcium propionate

7. Sterilizing Gases (MOA:protein denaturation)

used on heat-sensitive items like catheters, plastic Petri dishes and syringes

rapidly penetrate packing materials

microbicidal and sporicidal

eg. ethylene oxide

8. Aldehydes (MOA: protein inactivation)

chemical sterilants

eg. formalin (37% aqueous solution of formaldehyde) used in vaccines

glutaraldehyde used in medical equipment

9. Oxidizing Agents (MOA: oxidation of cell components)

eg. hydrogen peroxide (H2O2)

Conditions influencing the effectiveness of antimicrobial agent activity :

1. Population size – larger population, longer time for the microorganisms to die

2. Population composition – microorganisms differ markedly in their susceptibility

eg. bacterial endospore more resistant than vegetative cells

younger cells more readily destroyed than mature organisms

3. Concentration or intensity of antimicrobial agent

- often but not always, ↑concentration = more rapid killing of microorganisms

 - sometimes, ↓concentration is more effective

eg. 70% ethanol is more effective than 95% ethanol

4. Duration of exposure – Longer exposure →More organisms are killed

5. Temperature – increase in T° at which a chemical acts often enhances its activity

6. Local environment – environmental factors surrounding the microorganisms may

either protect or aid in their destruction

eg. microorganisms in acidic foods are readily killed by pasteurization than

microorganisms in neutral foods like milk

Points to Remember :

1. Few chemical agents achieve sterility; most merely reduce microbial populations to safe levels or
remove vegetative forms of pathogens from objects

2. Rough spectrum of susceptibility of microorganisms to disinfectants:

Most Susceptible:

vegetative bacteria

fungi

lipid-containing viruses

Less Susceptible:

mycobacteria

non-lipid containing viruses

Generally Resistant:

spores