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MCB 11 ST Q1
MCB 11 ST Q1
MCB 11 ST Q1
Microbiology - study of organisms and agents too small to be seen by the naked eye (<1mm)
- 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 :
Focuses on :
1. Archaea
- kingdom : protista
- oxygen producers
- lack chlorophyll
4. Protozoan
- nonfilamentous
5. Viruses [Virology]
- infectious agent
6. Bacteria [Bacteriology]
Applied Microbiology :
1. Medical Microbiology
2. Immunology
5. Industrial Microbiology
6. Agricultural Microbiology
Some challenges :
1. Infectious Diseases
4. Biofilms
6. Genome Analysis
7. Assessment
Discovery of Microorganisms :
- “Wee Animalcules”
Biogenesis Theory
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
*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
SG Supporters - strong acids and heat altered the air so it cannot support microbial grown
- No microbial growth
- uses swan-necked flasks and used nutrient rich broth called infusion
a. The broth provides a nutrient medium for the growth of unseen organisms in the air; life
comes from other life.
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
- 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.
*Endospores - a resistant sexual spore that develops inside some bacteria cells.
*Endospore Staining :
- Cloth Method
b. Secondary - Safranin
1. Girolamo Fracastoro
2. Agostino Bassi
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
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
Koch’s Postulate :
1. The microorganism must be present in every case of the disease but absent from healthy
organisms
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
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.
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.
- incubation of cultures for long intervals between transfers caused pathogens to lose their
ability to cause disease
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.
Pasteurization - a process in which packaged and non-packaged foods are treated with mild heat to
eliminate pathogens and extend shelf-life.
- chemosynthesis
- Winogradsky column
- biogeochemical cycles
- nitrogen-fixing bacteria
4. Marthus Beijennck
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.
Creating Vaccines :
- 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.
1. Viable microorganisms can multiply and produce infection (but not disease) like the natural
organism.
3. They usually require fewer doses and boosters than other types of vaccines.
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 :
Types of Microscopes :
a. Simple
- Only 1 lens
- Magnification - 300x
b. Compound or Complex
- 2 sets of lenses
- Magnification - 1000x
1. Bright Field
2. Dark Field
c. Distorted by staining
3. UV
4. Fluorescence
- modification of UV microscope
5. Phase Contrast
Special EM Stains : osmic acid, permanganate, lead, uranium, lanthanum, gold and silver
Examination of Microorganisms
Advantages :
- Simple to prepare
Disadvantage :
2. Stained Preparations
Advantages :
- Provides contrast
Disadvantages :
- More expensive
1. Smear Preparation
2. Fixation
Steam Fixation
Purpose of Fixation :
Mechanisms of Staining
Physical : Absorption (Dissolve), Adsorption (Adhere), Osmosis (High to Low), Capillary Action
Chemical : Ion-exchange
Staining Procedures
Methylene Blue
Crystal Violet
India Ink
Nigrosin
- Diagnosis of Tuberculosis
3. Structural Staining - 2 or more dyes and/or reagents
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.
Cultivation
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
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
2. Enrichment Culture
- 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 micropipette or a microphobe to physically pick a single cell and transfer it to an agar
medium
- uses a sterile membrane filter having a pore size that retains microorganism
1. Isolation
a. Purity is maintained
b. Viability is retained
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”
Considerations :
b. Proper Medium
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 :
3. Freeze-Drying (Lyophilization)
Advantages :
a. Long-term survival
- specimens are frozen along with a protective agent (Glycerol) in liquid-nitrogen refrigerators
5. Drying
Banking Microbes
Culture Collections
Examples :
Disinfection
Disinfectants : agents used to carry out disinfection and are normally used only on inanimate
object (does not sterilize an object)
Sanitization
2. To prevent infections
Physical Agents :
A. Heat
a. Boiling or flowing steam – kills vegetative cells and eukaryotic spores within 10 minutes
- sterilization at 90-100°C for 30 mins for 3 consecutive days and incubated at 37°C in
between
2. Dry heat
B. Low Temperature (MOA: limits growth due to decreased rates of cell reactions and
1. Refrigeration – 4°C
Used for:
Air – High efficiency particulate air (HEPA) filter removes 99.97% of 0.3 μm particles
2. Freeze-drying/ lyophilization
F. Radiation
- UV rays
- DNA damage
Chemical Agents :
e.g. Germicide
Bactericide
Fungicide
-static (Greek statikos = causing to stand or stopping) – do not kill but prevents growth
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)
Chlorine
- chlorine gas
- sodium hypochlorite
- calcium hypochlorite
used on heat-sensitive items like catheters, plastic Petri dishes and syringes
chemical sterilants
1. Population size – larger population, longer time for the microorganisms to die
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
Most Susceptible:
vegetative bacteria
fungi
lipid-containing viruses
Less Susceptible:
mycobacteria
Generally Resistant:
spores