MC 3: MIDTERM Notes
include percutaneous injury, ingestion, and mucous membrane
TOPIC 1: LABORATORY SAFETY
• Laboratories have certain standard safety equipment. These typically
include a:
A. General purpose fire extinguisher
B. Eyewash
C. Safety shower
D. cut-off switches for electrical and gas outlets.
• The microbiology lab has some additional safety considerations. Since
individuals work with potentially pathogenic organisms care must be taken
to prevent possible infection or transmission of the organisms from the
laboratory.
• Students must wear protective clothing (lab coats) while working the
laboratory. Lab coats may not be worn outside the laboratory. Intact skin
is an adequate barrier against microorganisms so gloves are not necessary
in lab. Gloves should be worn and students may wear gloves when
handling cultures if they so desire. Table tops must be disinfected before
and after lab using the disinfectant provided. Instruction in aseptic
technique will be provided. Aseptic technique must be followed while
working with microorganisms.
• Handwashing is a simple and effective way to prevent the transmission of
Sterilization, Disinfection and Antisepsis:
disease. While antibacterial soap may provide some additional protection
the major effect of handwashing is the mechanical removal of microbes
from the skin. Friction when washing hands is important to mechanically
remove organisms from the surface of the skin. Using a paper towel to turn
off the water prevents recontamination of the hands with microorganisms.
Hands must be washed whenever the student leaves the lab.
Microbiological Laboratory Equipment’s:
• There are basic equipment seen in microbiological laboratory:
1. Incubator- for bacterial and fungal culture.
2. Autoclave -used for sterilization of glass ware and media.
3. Biosafety cabinets- a place where we conduct experiments when
dealing with dangerous microorganisms. These biosafety cabinets
contain HEPA (high-efficiency particulate air filter). This type of air filter
can theoretically remove at least 99.97% of dust, pollen, mold, bacteria,
and any airborne particles with a size of 0.3 microns (µm)
a. Biosafety Level 1: Bacillus subtilis, Mycobacterium godornae
- Agents include those that have no known potential for infecting
healthy people.
b. Biosafety Level 2: HIV, HBV, Bacillus anthracis, Yersinia pestis
- Agents associated with the human disease; routes of transmission
exposure.
c. Biosafety Level 3: Mycobacterium tuberculosis, Mold stages of
systemic fungi, Francisella tularencis, Brucella species
- Indigenous/exotic agents that may cause serious or potentially
lethal disease through the inhalation route of exposure.
d. Biosafety Level 4: Arbovirus, Arenavirus, Filovirus, Smallpox virus
- Dangerous/exotic agents which post high individual risk of
aerosol-transmitted laboratory infections that are frequently fatal,
no tx/vaccine
4. Bunsen burner- used to work aseptic on the bench.
5. Candle jar - historically used for anaerobiosis; a lit candle was placed in
as air-tight jar such that when it went out it would be because it used up
all the available oxygen. The candle flame will consume all of the
oxygen that is inside the jar. It is used to grow bacteria requiring an
increased CO2 concentration (capnophiles).
6. Microscope and clean slides -to observe microscopic specimens that
cannot be seen by the naked eye.
7. Inoculation loops- used to inoculate test samples into culture media for
bacterial or fungal cultures, antibiograms, etc. Sterilized by passing
through a blue flame. They are usually made in platinum.
8. Petri-dish/agar plate- usually sterile before use, to act as a supporting
container to hold the culture medium in.
• Sterilization is the removal of all microbes, including endospores, and can
be achieved by mechanical means, heat, chemicals, or radiation. When
using heat, it may be either dry heat or moist heat. Traditionally, moist heat
under pressure is provided by autoclaving and dry heat by ovens.
• Disinfection is the process that eliminates most or all microorganisms, with
the exception of endospores. Disinfectants can be further subcategorized
as:
1. High-level disinfectants: which kill all microorganisms with the exception
of large numbers of endospores with an exposure time of less than 45
min
2. Intermediate-level disinfectants: which kill most microorganisms and
viruses but not endospores.
3. Low-level disinfectants: which kill most vegetative bacteria, some fungi,
and some viruses with exposure times of less than 10 min.
• Aseptic technique is a method that prevents the introduction of unwanted
organisms into an environment. When changing wound dressings aseptic
technique is used to prevent possible infection. When working with
microbial cultures aseptic technique is used to prevent introducing
additional organisms into the culture.
Disinfection vs Antiseptic Techniques:
• DISINFECTION: Disinfectants are used on inanimate objects and can be
sporostatic but are not usually sporocidal.
• ANTISEPTIC TECHNIQUES: Antiseptics destroy or inhibit the growth of
microorganisms in or on living tissues and can also be referred to as
biocides.
• The general aseptic procedure in the microbiological laboratory can be
summarized as follows:
1. Work Area Disinfection: The work area is first treated with a disinfectant
to kill any microorganisms that may be present. This step destroys
vegetative cells and viruses; endospores, however, are not destroyed in
this brief application of disinfectant.
2. Loops and Needles: The transport of organisms will be performed with
an inoculating loop or needle. To sterilize the loop or needle prior to
picking up the organisms, heat must be applied with a Bunsen burner
flame, rendering them glowing red-hot.
3. Culture Tube Flaming: Before inserting the cooled loop or needle into a
tube of culture, the tube cap is removed and the mouth of the culture
tube flamed. Once the organisms have been removed from the tube,
the tube mouth must be flamed again before returning the cap to the
tube.
4. Liquid Medium Inoculation: If a tube of liquid medium is to be
inoculated, the tube mouth must be flamed before inserting the loop
into the tube. To disperse the organisms on the loop, the loop should be
twisted back and forth in the medium. If an inoculating needle is used
for stabbing a solid medium, the needle is inserted deep into the
medium.
5. Final Flaming: Once the inoculation is completed, the loop or needle is
removed from the tube, flamed as before, and returned to a
receptacle. These tools should never be placed on the table top. The
inoculated tube is also flamed before placing the cap on the tube.
6. Petri Plate Inoculation: To inoculate a Petri plate, no heat is applied to
the plate and a loop is used for the transfer. When streaking the surface
of the medium, the cover should be held diagonally over the plate
bottom to prevent air contamination of the medium.
7. Final Disinfection: When all work is finished, the work area is treated with
disinfectant to ensure that any microorganisms deposited during any of
the procedures are eliminated.
TOPIC 2: INTRODUCTION TO MICROBIOLOGY AND PARASITOLOGY
YEAR MILESTONE
1530 Although microorganisms had been mentioned as a
Girolamo Fracastoro possible cause of disease by the Roman scholar Marcus
Varro in the 1st century B.C, Fracastoro's was the first
scientific statement of the true nature of contagion,
infection, disease germs, and modes of disease
 transmission. He proposed that epidemic diseases are
caused by invisible organisms, transferable pores.
1665 He published his discovery on cells in his book called
Robert Hooke Micrographia , and sees the first microorganisms on the
microscope.
1676 Anton van Leewenhoek observed the first bacteria.
Anton Von He was observing the lake water and found these
Leewenhoek organisms. This sparked the birth of Microbiology.
1768 Spallazani found that boiling broth would sterilise it and
Lazarro Spallazani kill any microorganisms in it. He also found that new
microorganisms could settle only in a broth if the broth
was exposed to the air.
1796 He was known to be the “Father of Immunology”
Edward Jenner After observing that cowpox infection seemed to protect
humans against smallpox, Jenner inoculated an eight-
year-old boy with cowpox matter from a blister on the
hand of an English milkmaid. He then repeatedly
attempted to “challenge” the cowpox inoculation by
exposing the boy to smallpox material—but the boy
never fell ill. Jenner had demonstrated smallpox
immunization.
1810 Appert is often regarded as the Father of Food
Nicholas Appert Microbiology. He developed a heating process in which
canned foods (for example, milk, meat, drinks, and
vegetables) could be preserved over a long period of
time and prevented from spoilage by microbial
fermentative action.
1835 Agostino Bassi made the important generalization that
Agostino Bassi many diseases of plants, animals and humans are
caused by animal or vegetable parasites.
Bassi demonstrated experimentally that a type of
silkworm disease was due to a ‘parasitic fungus.’ He
successfully isolated the parasite and used it to infect a
healthy animal. He was also responsible for intuitions on
the concepts of immunity, healthy carriers, predisposal
to contracting infections and the dynamics of
epidemics.
1838-1839 Proposed the Cell Theory.
Mathias They proposed that all organisms consist of cells.
Schleiden
Theodor
Schwann
1840 He embraced the unpopular microorganism theory of
Friedrich Gustav contagion put forth by the Renaissance forerunner of
Henle modern epidemiology, Girolamo Fracastoro, stating,
“The material of contagions is not only an organic but a
living one and is indeed endowed with a life of its own,
which is, in relation to the diseased body, a parasitic
organism.” He proposed that the germs cause disease.
John Snow Known as the “Father of Epidemiology”
Snow conducted studies of cholera outbreaks both to
discover the cause of disease and to prevent its
recurrence.
1864 Louis Pasteur develops pasteurization to destroy
Louis Pasteur organisms in wine. And also conducted an experiment
on Spontaneous Generation Theory:
Expanded upon Spallanzani’s findings by exposing
boiled broths to the air in vessels that contained a filter
to prevent all particles from passing through to the
growth medium. He also did this in vessels with no filter
at all, with air being admitted via a curved tube that
prevented dust particles from coming in contact with
the broth. By boiling the broth beforehand, Pasteur
ensured that no microorganisms survived within the
broths at the beginning of his experiment. Nothing grew
in the broths in the course of Pasteur’s experiment. This
meant that the living organisms that grew in such broths
came from outside, as spores on dust, rather than
spontaneously generated within the broth. Thus, Pasteur
dealt the death blow to the theory of spontaneous
generation and
supported germ theory instead.
1876 Established that a microbe can cause disease. He found
Robert Koch that the blood of cattle who were infected with anthrax
always had large numbers of Bacillus anthracis. Koch
found that he could transmit anthrax from one animal
to another by taking a small sample of blood from the
infected animal and injecting it into a healthy one, and
this caused the healthy animal to become sick. He also
found that he could grow the bacteria in a nutrient
broth, then inject it into a healthy animal, and cause
illness. Based on these experiments, he devised criteria
for
establishing a causal link between a microbe and a
disease and these are now known as Koch’s postulates.
1860 Joseph Lister found a way to prevent infection in
Joseph Lister wounds during and after surgery. He was the first to
apply the science of Germ Theory to surgery. Lister's
Antisepsis System is the basis of modern infection
control. His principles made surgery safe and continue
to save countless lives. He developed aseptic surgical
techniques,
and used carbolic acid(phenol) to treat dressings.
1881 Robert Koch introduces the Pure Culture Technique in the
laboratory.
1882 Walter and Franny Hesse introduce the agar plate as a
Angeline Fanny and solidifying gel for the culturing medium.
Walter Hesse Hesse and her husband Walther were part of Robert
Koch’s lab in Germany. While a student, Walther was
struggling with growing microorganisms from air due to
the inherent issues of working with gelatin in
1884 He invented the Gram staining technique and was a
Hans Christian Gram pioneering biologist who devised the system of
classification which led to as many as 30,000 formally
named species of bacteria being investigated. Gram
first dripped reagents, a substance designed to cause a
chemical reaction, onto lung tissue samples. He found
differences in the colouring of bacteria that are now
known to be Streptococcus pneumoniae and Klebsiella
pneumoniae.
Gram Stain Technique: The differences Gram observed
are a result of the composition of the bacterial cell wall.
Some bacteria have a cell wall composed of
peptidoglycan, a polymer of sugar and amino acids.
These “gram- positive” bacterial cells retain the colour
of a stain – usually a complex of crystal violet and
iodine, or methylene blue – and appear purple or
brown under the microscope. Others, that do not
contain peptidoglycan, are not
stained and are referred to as gram-negative, and
appear red.
1884 He discovered phagocytic cells which engulf bacteria.
Elle Metchnikoff According to Metchnikoff, the phagocyte is the first line
of defense against acute infection in most animals,
including humans, whose phagocytes are one type of
leukocyte, or white blood cell. This work formed the
basis of Metchnikoff’s cellular (phagocytic) theory of
immunity (1892), a hypothesis that engendered much
opposition, particularly from scientists who claimed that
only body fluids and soluble substances in the blood
(antibodies)—and not cells—destroyed invading
microorganisms (the humoral theory of immunity).
1928 British bacteriologist whose 1928 experiment with
Frederick Griffith bacterium was the first to reveal the “transforming
principle,” which led to the discovery that DNA acts as
the carrier of genetic information.
1929 He discovered Penicillin, the first form of antibiotic. He
Alexander Flemming grew cultures of bacteria on petri dishes in the hospital
where he worked. A fungal spore happened to
 contaminate one of the bacteria cultures and grew into
a fungal colony. The bacteria round the fungal colony
failed to grow as well as the bacteria on the other areas
of the petri dish. From this Fleming deduced that a
substance produced by the fungus must be slowing
down the
bacteria’s growth. He called this substance Penicillin
after the fungus in the petri dish, Penicillum notatum.
1944 Oswald Avery, Proposed that DNA is the cause of bacterial
Colin McLeod, transformation.
Maclin Carty
1953 The three-dimensional structure of DNA, first proposed by
Watson, Crick, James D. Watson and Francis H. C. Crick in 1953, consists
Franklin of two long helical strands that are coiled around a
common axis to form a double helix.
1971 Theodor Diener Theodor Diener demonstrates fundamental differences
between viroids and viruses.
1973 Herbert Boyer The first scientists to clone DNA and developed
and Stanley Cohen recombinant DNA technology, showing that genetically
engineered DNA molecules may be cloned in foreign
cells. Genetic engineering using living organisms was first
accomplished soon after it became feasible in the early
1970s.
1977 Carl Woese He classified organisms into three domains: the three
domains of cellular life — Bacteria, Archaea, and
Eukarya — remain objectively distinct.
1995 The first complete nucleotide sequence of a bacterial
chromosome is reported.
Concepts that honed Microbiology:
Theory of Spontaneous Generation:
Spontaneous generation is an obsolete body of thought on the ordinary
formation of living organisms without descent from similar organisms. Before the
discovery of microbes, it was widely thought that life, as in the case of rotting
food, arose from nothing. This idea was referred to as spontaneous generation.
Typically, the idea was that certain forms such as fleas could arise from inanimate
matter such as dust or that maggots could arise from dead flesh. A variant idea
was that of equivocal generation, in which species such as tapeworms arose from
unrelated living organisms, now understood to be their hosts.
Louis Pasteur’s 1859 experiment is widely seen as having settled the question. In
summary, Pasteur boiled a meat broth in a flask that had a long neck that curved
downward, like a goose. The idea was that the bend in the neck prevented falling
particles from reaching the broth, while still allowing the free flow of air. The flask
remained free of growth for an extended period. When the flask was turned so
that particles could fall down the bends, the broth quickly became clouded.
In detail, Pasteur exposed boiled broths to air in vessels that contained a filter to
prevent all particles from passing through to the growth medium, and even in
vessels with no filter at all, with air being admitted via a long tortuous tube that
would not allow dust particles to pass. Nothing grew in the broths unless the flasks
were broken open, showing that the living organisms that grew in such broths
came from outside, as spores on dust, rather than spontaneously generated
within the broth. This was one of the last and most important experiments
disproving the theory of spontaneous generation.
There were scientists that disprove the Spontaneous Generation Theory:
✔ 1670 - Francisco Redi - demonstrated larva present on meat was from eggs.
✔ 1765 - Spallanzani and others used sealed flasks and treated air to
prevent microbial growth however, others such as Needham
claimed a vitalistic force in air was necessary for spontaneous
generation to occur.
✔ 1860 - Louis Pasteur - “Father of Microbiology”
swan necked flasks. to disprove spontaneous generation. Also,
observed that heat could kill these microbes. This led to the development
of Pasteurization.
Germ Theory of Disease:
The germ theory of disease, also called the Pathogenic theory of medicine, is a
theory that proposes that microorganisms are the cause of many diseases.
Although highly controversial when first proposed, germ theory was validated in
the late 19th century and is now a fundamental part of modern medicine and
clinical microbiology, leading to such important innovations as antibiotics and
hygienic practices.
Prior to the discovery of microbes during the seventeenth century, other theories
circulated about the origins of disease. For example, the ancient Greeks
proposed the miasma theory, which held that disease originated from particles
emanating from decomposing matter, such as that in sewage or cesspits. Such
particles infected humans in close proximity to the rotting material. Diseases
including the Black Death, which ravaged Europe’s population during the Middle
Ages, were thought to have originated in this way.
In 1546, Italian physician Girolamo Fracastoro proposed, in his essay De
Contagione et Contagiosis Morbis, that seed-like spores may be transferred
between individuals through direct contact, exposure to contaminated clothing,
or through the air. We now recognize Fracastoro as an early proponent of the
germ theory of disease, which states that diseases may result from microbial
infection. However, in the sixteenth century, Fracastoro’s ideas were not widely
accepted and would be largely forgotten until the nineteenth century.
Koch’s Postulates:
Probably as important as his work on tuberculosis, for which he was awarded a
Nobel Prize in 1905, are Koch’s postulates. These postulates stated that to establish
that an organism is the cause of a disease, it must be found in all cases of the
disease examined. Additionally, it must be absent in healthy organisms prepared
and maintained in a pure culture capable of producing the original infection,
even after several generations in culture retrievable from an inoculated animal
and cultured again. By using his methods, Koch’s pupils found the organisms
responsible for diphtheria, typhoid, pneumonia, gonorrhoea, cerebrospinal
meningitis, leprosy, bubonic plague, tetanus, and syphilis.
Pure Culture Concept:
Perhaps the key method Koch developed was the ability to isolate pure cultures,
explained in brief here. Pure cultures of multicellular organisms are often more
easily isolated by simply picking out a single individual to initiate a culture. This is
a useful technique for pure culture of fungi, multicellular algae, and small
metazoa. Developing pure culture techniques is crucial to the observation of the
specimen in question. The most common method to isolate individual microbes
and produce a pure culture is to prepare a streak plate. The streak plate method
is a way to physically separate the microbial population and is done by spreading
the inoculate back and forth with an inoculating loop over the solid agar plate.
used
Upon incubation, colonies will arise and single cells will have been isolated from
the biomass.
Koch’s Postulates:
Probably as important as Robert Koch’s work on tuberculosis, for which he was
awarded a Nobel Prize in 1905, are Koch’s postulates. These postulates stated that
to establish that an organism is the cause of a disease, it must be found in all cases
of the disease examined. Additionally, it must be absent from healthy organisms
prepared and maintained in a pure culture capable of producing the original
infection, even after several generations in culture retrievable from an inoculated
animal and cultured again. By using his methods, Koch’s pupils found the
organisms responsible for diphtheria, typhoid, pneumonia, gonorrhoea,
cerebrospinal meningitis, leprosy, bubonic plague, tetanus, and syphilis.
Cell Theory:
While some scientists were arguing over the theory of spontaneous generation,
other scientists were making discoveries leading to a better understanding of
what we now call the cell theory. Modern cell theory has two basic tenets:
✔ All cells only come from other cells (the principle of biogenesis).
✔ Cells are the fundamental units of organisms.
Today, these tenets are fundamental to our understanding of life on earth.
However, modern cell theory grew out of the collective work of many scientists.
The Origins of Cell Theory:
The English scientist Robert Hooke first used the term “cells” in 1665 to describe the
small chambers within cork that he observed under a microscope of his own
design. To Hooke, thin sections of cork resembled “Honey-comb,” or “small Boxes
or Bladders of Air.” He noted that each “Cavern, Bubble, or Cell” was distinct from
the others (Figure 1). At the time, Hooke was not aware that the cork cells were
long dead and, therefore, lacked the internal structures found within living cells.
Despite Hooke’s early description of cells, their significance as the fundamental
 unit of life was not yet recognized. Nearly Cell wall Simple; present in plants and fungi Com
200 years later, in 1838, Matthias Schleiden (1804–1881), a German botanist who
Types of Microorganisms: Plasma membrane and Present Prese
made extensive microscopic observations of plant tissues, described them as
Cytoplasm
being composed of cells. Visualizing plant cells was relatively easy because plant Prokaryotes

cells are clearly separated by their thick cell walls. Schleiden believed that cells - The primary distinguishing characteristics of the prokaryotes are their
formed through crystallization, rather than cell division. Bacteria
relatively small size, usually on the order of

Theodor Schwann (1810–1882), a noted German physiologist, made similar o 1 μm in diameter, and the absence of a nuclear membrane.
microscopic observations of animal tissue. In 1839, after a conversation with - The DNA of almost all bacteria is a circle with a length of about 1 mm;
Schleiden, Schwann realized that similarities existed between plant and animal Most prokaryotes have only a single chromosome.
tissues. This laid the foundation for the idea that cells are the fundamental - The chromosomal DNA must be folded more than 1000-fold just to fit within
components of plants and animals. the prokaryotic cell membrane.
Eukaryotes
In the 1850s, two Polish scientists living in Germany pushed this idea further,
culminating in what we recognize today as the modern cell theory. In 1852, - The “true nucleus” of eukaryotes is only one of their distinguishing
Robert Remak (1815–1865), a prominent neurologist and embryologist, published features. The membrane bound organelles, the microtubules, and the
convincing evidence that cells are derived from other cells as a result of cell microfilaments of eukaryotes form a complex intracellular structure unlike
division. However, this idea was questioned by many in the scientific community. that found in prokaryotes.
Three years later, Rudolf Virchow (1821–1902), a well-respected pathologist, - The agents of motility for eukaryotic cells are flagella or cilia—complex
published an editorial essay entitled “Cellular Pathology,” which popularized the multistranded structures that do not resemble the flagella of prokaryotes.
concept of cell theory using the Latin phrase omnis cellula a cellula (“all cells arise - Gene expression in eukaryotes takes place through a series of events
from cells”), which is essentially the second tenet of modern cell theory.[1] Given achieving physiologic integration of the nucleus with the endoplasmic
the similarity of Virchow’s work to Remak’s, there is some controversy as to which reticulum, a structure that has no counterpart in prokaryotes. Eukaryotes
scientist should receive credit for articulating cell theory. See the following Eye on are set apart by the organization of their cellular DNA in chromosomes
Ethics feature for more about this controversy. separated.
What is Microbiology? Prokaryotes versus Eukaryotes:
Characteristics: Eukaryotic Cells
It is a study of microorganisms, a large and diverse group of microscopic organisms
Definition Any cell that contains clearly defined
that exist as a single cells or cell clusters; it also includes which are microscopic but
nucleus and membrane bound
not cellular.
organelles.
Microbiology is a broad term which includes virology, mycology, parasitology,
Examples Animal, plant, protist cells
bacteriology, immunology, and other branches. A microbiologist is a specialist in
microbiology and these related topics. Microbiological procedures usually must Nucleus Present (membrane-bound)
be aseptic and use a variety of tools such as light microscopes with a Cell size Large (10-100 um)
combination of stains and dyes. As microbes are absolutely required for most
DNA Replication Highly regulated with selective origins
facets of human life (including the air we breathe and the food we eat) and are
and sequences.
potential causes of many human diseases, microbiology is paramount for human
Characteristics: Eukaryotic Cells
society.
Organism type Multicellular
- Microorganisms are divided into seven types: bacteria, archaea,
protozoa, algae, fungi, viruses, and multicellular animal parasites ( Examples Animal, plant, protist cells
helminths ).
Chromosomes More than one
- Each type has a characteristic cellular composition, morphology, mean of
locomotion, and reproduction. Ribosomes Large
- Microorganisms are beneficial in producing oxygen, decomposing
Growth rate/Generation Time Slower
organic material, providing nutrients for plants, and maintaining human
health, but some can be pathogenic and cause diseases in plants and Organelles Present
humans. Ability to store heriditary Yes
- Bacteria are unicellular organisms. The cells are described as prokaryotic
because they lack a nucleus. They exist in four major shapes: bacillus (rod
shape), coccus (spherical shape), spirilla (spiral shape), and vibrio
(curved shape). Most bacteria have a peptidoglycan cell wall; they
divide by binary fission; and they may possess flagella for motility. The
difference in their cell wall structure is a major feature used in classifying
these organisms.
- According to the way their cell wall structure stains, bacteria can be
classified as either Gram-positive or Gram- negative when using the
Gram staining. Bacteria can be further divided based on their response
to gaseous oxygen into the following groups: aerobic (living in the
presence of oxygen), anaerobic (living without oxygen), and facultative
anaerobes (can live in both environments).
- According to the way they obtain energy, bacteria are classified as
heterotrophs or autotrophs. Autotrophs make their own food by using the
energy of sunlight or chemical reactions, in which case they are called
chemoautotrophs. Heterotrophs obtain their energy by consuming other
organisms. Bacteria that use decaying life forms as a source of energy
are called saprophytes.
Prokaryotic Cells
Fungi
Any unicellular organism that does
not contain a- membrane
Fungi (mushroom, molds, and yeasts) are eukaryotic cells (with a true
bound nucleus
nucleus). Most fungi are multicellular and their cell wall is composed of
or organelles .
chitin.
Bacteria and archaea
- They obtain nutrients by absorbing organic material from their
Absent
environment (decomposers), through symbiotic relationships with plants
Small (<5 um) (symbionts), or harmful relationships with a host (parasites).
- They form characteristic filamentous tubes called hyphae that help
Replicates the entire genome at
absorb material. The collection of hyphae is called mycelium. Fungi
once.
reproduce by releasing spores.
Prokaryotic Cells
Archaea
Unicellular
- Archaea or Archaebacteria differ from true bacteria in their cell wall
structure and lack peptidoglycans. They are prokaryotic cells with avidity
Bacteria and archaea
to extreme environmental conditions.
One long single loop of DNA and plasmids
- Based on their habitat, all Archaeans can be divided into the following
Small groups: methanogens (methane-producing organisms), halophiles
(archaeans that live in salty environments), thermophiles (archaeans that
Faster
live at extremely hot temperatures), and psychrophiles (cold-temperature
Absent Archaeans).
Yes - Archaeans use different energy sources like hydrogen gas, carbon

information dioxide, and sulphur. Some of them use sunlight to make energy, but not
 the same way plants do. They absorb sunlight using their membrane
pigment, bacteriorhodopsin. This reacts with light, leading to the
formation of the energy molecule adenosine triphosphate (ATP).
Protozoa
- Protozoa are unicellular aerobic eukaryotes. They have a nucleus,
complex organelles, and obtain nourishment by absorption or ingestion
through specialized structures.
- They make up the largest group of organisms in the world in terms of
numbers, biomass, and diversity. Their cell walls are made up of cellulose.
Protozoa have been traditionally divided based on their mode of
locomotion: flagellates produce their own food and use their whip-like
structure to propel forward, ciliates have tiny hair that beat to produce
Immunization, Antiseptics, and Antibiotics:
movement, amoeboids have false feet or pseudopodia used for feeding
and locomotion, and sporozoans are non-motile.
- They also have different means of nutrition, which groups them as
autotrophs or heterotrophs.
Algae
- Algae, also called cyanobacteria or blue-green algae, are unicellular or
multicellular eukaryotes that obtain nourishment by photosynthesis.
- They live in water, damp soil, and rocks and produce oxygen and
carbohydrates used by other organisms. It is believed that cyanobacteria
are the origins of green land plants.
Viruses
- Viruses are noncellular entities that consist of a nucleic acid core (DNA or
RNA) surrounded by a protein coat. Although viruses are classified as
microorganisms, they are not considered living organisms. Viruses cannot
reproduce outside a host cell and cannot metabolize on their own.
Viruses often infest prokaryotic and eukaryotic cells causing diseases.
- Viruses lack many of the attributes of cells, including the ability to
replicate. Only when it infects a cell does a virus acquire the key attribute
of a living system— reproduction. Viruses are known to infect all cells,
including microbial cells.
- Viral particles are generally small (eg, adenovirus is 90 nm) and consist of
a nucleic acid molecule, either DNA or RNA, enclosed in a protein coat,
or capsid (sometimes itself enclosed by an envelope of lipids, proteins,
and carbohydrates).
- Host–virus interactions tend to be highly specific, and the biologic range
of viruses mirrors the diversity of potential host cells. Further diversity of
viruses is exhibited by their broad array of strategies for replication and
survival.
Prions
- Prions are misfolded proteins with the ability to transmit their misfolded
shape onto normal variants of the same protein. They characterize
several fatal and transmissible neurodegenerative diseases in humans
and many other animals.
- Prions attack nerve cells producing neurodegenerative brain disease.
- Prion related diseases include Creutzfeldt-Jakob disease (CJD),
Gerstmann-Sträussler-Scheinker disease, and fatal familial insomnia affect
humans.
Multicellular Animal Parasites
- A group of eukaryotic organisms consisting of flatworms and roundworms,
which are collectively referred to as the helminths.
- Although they are not microorganisms by definition, since they are large
enough to be easily seen with the naked eye, they live a part of their life
cycle in microscopic form.
- Since parasitic helminths are of clinical importance, they are often
discussed along with the other groups of microbes.
- Understanding microbes gives us the ability to fight pathogens using
immunization, antiseptics, and antibiotics.Surprisingly, most microbes are
not harmful to humans. In fact, they are all around us and even a part of
us. However, some microbes are human pathogens; to combat these, we
use immunization, antiseptics, and antibiotics.
- Immunization is the process by which an individual’s immune system
becomes fortified against an agent (known as the immunogen).
- When the immune system is exposed to molecules that are foreign to the
body, it will orchestrate an immune response. It will also develop the ability
to respond quickly to subsequent encounters with the same substance, a
phenomenon known as immunological memory. Therefore, by exposing a
person to an immunogen in a controlled way, the body can learn to
protect itself: this is called active immunization.
- Vaccines against microorganisms that cause diseases can prepare the
body’s immune system, thus helping it fight or prevent an infection. The
most important elements of the immune system that are improved by
immunization are the T cells, the B cells, and the antibodies B cells
produce. Memory B cells and memory T cells are responsible for the swift
response to a second encounter with a foreign molecule. Through the use
of immunizations, some infections and diseases have been almost
completely eradicated throughout the United States and the world. For
example, polio was eliminated in the U.S. in 1979. Active immunization and
vaccination has been named one of the “Ten Great Public Health
Achievements in the 20th Century. ”
- By contrast, in passive immunization, pre-synthesized elements of the
immune system are transferred to a human body so it does not need to
produce these elements itself. Currently, antibodies can be used for
passive immunization. This method of immunization starts to work very
quickly; however, it is short-lasting because the antibodies are naturally
broken down and will disappear altogether if there are no B cells to
produce more of them. Passive immunization occurs physiologically, when
antibodies are transferred from mother to fetus during pregnancy, to
protect the fetus before and shortly after birth. The antibodies can be
produced in animals, called” serum therapy,” although there is a high
chance of anaphylactic shock because of immunity against animal serum
itself. Thus, humanized antibodies produced in vitro by cell culture are
used instead if available.
- In early inquiries before there was an understanding of microbes, much
emphasis was given to the prevention of putrefaction. Procedures were
carried out to determine the amount of agent that needed to be added
to a given solution in order to prevent the development of pus and
putrefaction. However, due to a lack of understanding of germ theory, this
method was inaccurate. Today, an antiseptic is judged by its effect on
pure cultures of a defined microbe or on their vegetative and spore forms.
- Antiseptics are antimicrobial substances that are applied to living tissue to
reduce the possibility of infection, sepsis, or putrefaction. Their earliest
known systematic use was in the ancient practice of embalming the
dead. Antiseptics are generally distinguished from antibiotics by the
latter’s ability to be transported through the lymphatic system to destroy
bacteria within the body, and from disinfectants, which destroy
microorganisms found on non-living objects. Some antiseptics are true
germicides, capable of destroying microbes (bacteriocidal), while others
are bacteriostatic and only prevent or inhibit bacterial growth.
Microbicides that destroy virus particles are called viricides or antivirals.
- An antibacterial is a compound or substance that kills or slows down the
growth of bacteria. The term is often used synonymously with the term
antibiotic; today, however, with increased knowledge of the causative
agents of various infectious diseases, the term “antibiotic” has come to
denote a broader range of antimicrobial compounds, including anti-
fungal and other compounds.
- Antibiotic Testing: Discs soaked with various compounds are put onto a
lawn of bacteria. If the compound on the disc kills or slows bacteria growth,
a “halo” of clear media is seen.
Branches of Microbiology:
Bacteriology Study of Bacteria
Mycology Study Fungi
Protozoology Study of Protozoans
Phycology Study of Algae
Parasitology Study of Parasites
Virology Study of Viruses
Immunology Study of immune mechanisms
Nematology Study of Nematodes
TOPIC 3: BACTERIAL CELL ANATOMY, MORPHOLOGY AND REPRODUCTION
Eukaryotic Cells VS Prokaryotic Cells:
Eukaryotic Cells:
- notable characteristics of Eukaryotes are the presence of the membrane
enclosed cell organelles that specific cellular function. Such as:
▪ Nucleus-provide membrane closure for chromosomes
▪ Lysosomes- provide environment for controlled enzymatic degradation
of intracellular substances.
▪ Mitochondria- generate energy (ATP)
▪ Golgi Bodies-processes substances for transport outside the cell
▪ Endoplasmic Reticulum – process and transport proteins
Prokaryotic Cells:
▪ They do not have organelles. All functions take place in the cytoplasmic
membrane of the cell.
▪ Cell walls of most prokaryotic cells are made up of peptidoglycan layer.
Bacterial Morphology
▪ most clinically relevant species range in a size of 0.25 to 1 um in width and 1
to 3 um in height.
▪ differences in the cell wall provide the basis for the Gram Stain, which is the
most fundamental test used in bacterial identification schemes.
▪ Gram Stain – the staining procedure separates almost all bacteria that are
medically important bacteria into two different types:
✔ Gram-positive: deep blue to purple color
✔ Gram-negative: pink to red color
Bacterial Cell Components:
CELL ENVELOPE
▪ ✔ Cell motility
🡪 comprises of:
I. Outer Membrane
🡪 mostly found in Gram-negative bacteria
🡪 function: cell’s initial barrier to the environment; serve as permeability
barriers to hydrophilic and hydrophobic compounds.
🡪 it is a membrane bi-layered structure composed of lipopolysaccharide –
gives a the surface of a Gram negative bacteria a net negative charge
🡪 The lipopolysaccharide plays a significant role in a certain ability of the
bacteria to cause a disease.
🡪 Porins are water-filled protein structures that are scattered throughout
the lipopolysaccharide that control the passage of nutrients and other
solutes including antibiotics through the outer membrane.
▪ CELL WALL
🡪 Also referred to the Peptidoglycan layer or Murein layer.
🡪 Gives the bacteria cell shape and strength to withstand changes in the
environmental osmotic pressure that would otherwise result in cell lysis
and also protects the cell against mechanical disruption.
🡪 This feature has been the primary target for the development and
design of antibiotics.
🡪 The structure of the cell is composed of disaccharide pentapeptide
subunits.
🡪 The notable difference between the cell walls of gram-positive versus
gram negative cell wall is that peptidoglycan layer of the gram-positive
bacteria is thicker.
🡪 Gram positive cell wall also contain Techoic acids.
🡪 some Gram positive bacteria like the Mycobacterium is rich in mycolic
acid that make their cells refractory to toxic acids.
▪ PERIPLASMIC SPACE
🡪 Only found in Gram negative bacteria
🡪 It is bounded by the internal surface of the outer membrane and the
external surface of the cellular membrane.
🡪 It consists of gel-like substances that help secure nutrients from the
environment and also contain enzymes that degrade macromolecules
and detoxify environmental solutes including antibiotics that enter
through the outer membrane.
▪ CYTOPLASMIC INNER MEMBRANE
🡪 Present in both Gram-positive and Gram-negative bacteria and is the
deepest layer of the cell envelope. And the structure of the cell
membrane for both are similar.
🡪 It is functionally similar to that of a Eukaryotic cell’s organelles.
🡪 Functions include:
✔ Transport solutes into and out of the cell.
✔ Housing enzymes involved in the outer membrane synthesis, cell wall
synthesis, and the assembly and secretion of extracytoplasmic and
CELL INTERIOR
extracellular substances
- They are the structures that are bounded internally by the cytoplasmic
✔ Generation of chemical energy (like the ATP).
✔ Mediation of chromosomal segregation during the replication.
✔ Housing molecular sensors that monitor chemical and physical
changes in the environment.
CELLULAR APPENDAGES
▪ play a role in causing infections and in laboratory identification, varies
among bacterial species and even among strains of the same species.
● Capsule
🡪 Immediately exterior to the peptidoglycan/murein layer of gram-
positive bacteria and outer membrane of the gram-negative
bacteria.
🡪 Often referred to as the slime layer
🡪 Composed of high molecular weight polysaccharides whose
production may depend on the environment and growth
conditions surrounding the bacterial cell.
🡪 It does not function as an effective permeability membrane
barrier or add strength to the cell envelope but only protects the
bacterial from attack by cells of the human defense system.
(Immune system)
● Fimbriae or Pili:
🡪 It is a hair-like, proteinaceous structures that extend from the cell
membrane into the external environment .
🡪 some may be up to 2 um in length.
🡪 There are two general types:
a. Common Pili- are adhesins that help bacteria attach to
animal host cell surfaces., often as the first step in establishing
infection.
b. Sex Pili- serves as the conduit for the passage of DNA from
donor to recipient during conjugation.
🡪 *Bacterial conjugation: this process occurs between two living
cells, involves cell-to-cell contact and requires mobilization of the
donor bacterium’s chromosome.
● Flagella:
🡪 They are complex structures, mostly composed of the protein
flagellin, intricately embedded in the cell envelope.
🡪 These structures are responsible for bacterial motility.
🡪 Although not all bacteria are motile, motility plays an important
role in survival and the ability of certain bacteria to cause
disease.
🡪 Depending on the bacterial species, a flagella may be:
🡪 Monotrichous flagella –located at one end of the cell
🡪 Lophotrichous flagella- located at both ends of the cell
🡪 Peritrichous flagella- entire cell is covered with flagella
membrane.
▪ Cytosol:
🡪 It is where nearly all the other functions not conducted by the cell
membrane occur.
🡪 It contains thousands of enzymes and is the site of protein synthesis.
🡪 It has granular appearance caused by the presence of many
polysomes (messenger RNA complexed with several ribosomes
during translation and protein synthesis. )
▪ Inclusions:
🡪 It includes storage reserve granules. Two common types of granules:
a. Glycogen- storage form of glucose.
b. Polyphosphate granules- a storage form of inorganic phosphates
that are microscopically visible in certain bacteria stained with
specific dyes.
🡪 Unlike eukaryotic chromosomes, bacterial chromosomes exist as a
nucleoid- highly coiled DNA intermixed with RNA, polyamines, and
various protein that lend structural support
🡪 Depending on the stage of cell division, there may be more than
one chromosome per bacterial cell.
▪ Plasmids:
 🡪 are the other genetic elements that exist independently in the In cell division: - It starts out as a small, spherical cell approximately 1 to 2 µm in diameter.
cytosol and their numbers vary from none to several per bacterial This cell is referred to as a baeocyte (which literally means "small cell").
- Most bacteria divide by binary fission into two equal progeny cells. In a
cell. - The baeocyte begins to grow, eventually forming a vegetative cell up to 30
growing culture of a rod-shaped bacterium such as E coli, cells elongate
▪ Endospore: µm in diameter. As it grows, the cellular DNA is replicated over and over,
and then form a partition that eventually separates the cell into two
🡪 Under adverse physical and chemical conditions, or when nutrients and the cell produces a thick extracellular matrix.
daughter cells.
are scarce some bacterial genera are able to form spores - The vegetative cell eventually transitions into a reproductive phase where it
- The partition is referred to as a septum and is a result of the inward growth
(sporulate). undergoes a rapid succession of cytoplasmic fissions to produce dozens or
of the cytoplasmic membrane and cell wall from opposing directions until
🡪 Sporulation involves substantial metabolic and structural changes in even hundreds of baeocytes. The extracellular matrix eventually tears
the two daughter cells are pinched off.
the bacterial cell. open, releasing the baeocytes.
- The chromosomes, which have doubled in number preceding the division,
🡪 The spore state is maintained until favorable conditions for growth - Observed in cyanobacterium Staneria
are distributed equally to the two daughter cells.
are again encountered.
- Although bacteria lack a mitotic spindle, the septum is formed in such a Budding:
🡪 This survival tactic is demonstrated by a number of clinically relevant
way as to separate the two sister chromosomes formed by chromosomal
bacteria and frequently challenges our ability to sterilize materials - Budding has been observed in some members of the Planctomycetes,
replication.
and food for human use. Cyanobacteria, Firmicutes (a.k.a. the Low G+C Gram-Positive Bacteria)
- This is accomplished by the attachment of the chromosome to the cell
and the prosthecate Proteobacteria.
BACTERIAL REPRODUCTION membrane.
- Although budding has been extensively studied in the eukaryotic yeast
- According to one model, completion of a cycle of DNA replication triggers
Binary Fission Saccharomyces cerevisiae, the molecular mechanisms of bud formation in
active membrane synthesis between the sites of attachment of the two
bacteria are not known.
- Most bacteria rely on binary fission for propagation. sister chromosomes. The chromosomes are then pushed apart by the
- A schematic representation of budding in a Planctomyces species is
- Conceptually this is a simple process; a cell just needs to grow to twice its inward growth of the septum, one copy going to each daughter cell.
shown:
starting size and then split in two. But, to remain viable and competitive, a
In cell groupings:
bacterium must divide at the right time, in the right place, and must Intracellular offspring production:
provide each offspring with a complete copy of its essential genetic - If the cells remain temporarily attached after division, certain characteristic
- Epulopiscium spp., Metabacterium polyspora and the Segmented
material. groupings result.
Filamentous Bacteria (SFB) form multiple intracellular offspring
- Understanding the mechanics of this process is of great interest because it - Depending on the plane of division and the number of divisions through
- - or some of these bacteria, this process appears to be the only way to
may allow for the design of new chemicals or novel antibiotics that which the cells remain attached, the following may occur in the coccal
reproduce. Intracellular offspring development in these bacteria shares
specifically target and interfere with cell division in bacteria. forms: chains (streptococci), pairs (diplococci), cubical bundles (sarcinae),
characteristics with endospore formation in Bacillus subtilis.
or flat plates. Rods may form pairs or chains.
- - Instead of placing the FtsZ ring at the center of the cell, as in binary fission,
- After fission of some bacteria, characteristic post-division movements
(A) Z rings are placed near both cell poles in Epulopiscium. (B) Division forms
The cell must copy its genetic occur. For example, a “whipping” motion can bring the cells into parallel
material (DNA) and segregate a large mother cell and two small offspring cells. (C) The smaller cells
these copies to opposite ends of positions; repeated division and whipping result in the “palisade”
the cell. contain DNA and become fully engulfed by the larger mother cell. (D) The
arrangement characteristic of diphtheria bacilli.
internal offspring grow within the cytoplasm of the mother cell. (E) Once
Corynebacterium diptheriae and its characteristic "palisade arrangement seen offspring development is complete the mother cell dies and releases the
using Gram Stain offspring.
As division occurs, the cytoplasm is Then the many types of proteins
cleaved in two, and in many that comprise the cell division
bacteria, new cell wall is machinery assemble at the future
synthesized. division site.

Other components of the division

then assemble at the FtsZ ring. This
machinery is positioned so that
division splits the cytoplasm and
does not damage DNA in the
process

- A key component of this machinery is the protein FtsZ. Protein monomers of

Other forms of Bacterial Reproduction:
FtsZ assemble into a ring-like structure at the center of a cell.
Baeocyte Production: TOPIC 3: MICROBIAL TAXONOMY, PHYSIOLOGY, METABOLISM AND GENETICS
Morphologic changes during growth:
Taxonomy –the vocabulary of Medical Microbiology
What is Taxonomy? - the most basic taxonomic group defined as a collection of bacterial strains
- It comes from a Greek word, ‘Taxon’ meaning arrangements. that share many common physiologic and genetic features and as a group Example of Taxonomic ranks:
- It is defined as the classification of organisms in an ordered system that differ notable from other bacterial species. Formal rank example
indicates a natural relationship. Subspecies- taxonomic subgroups within a species. Kingdom Prokaryotae
- Allows all biologists to use a common label for every organism they study - biotype, serotype, or genotype are given to groups below the subspecies Division Gracilicutes
within their particular disciplines. levels that share specific but relatively minor, characteristics. These Class Scotobacteria
- In diagnostic microbiology, classification, nomenclature, and identification subgroups are still taxonomically important but their use in diagnostic Order Eubacteriales
of microorganisms play a central role in providing an accurate and timely microbiology is limited. Family Enterobacteriaceae
diagnosis of infectious diseases. Genus – is the next higher taxon and comprises different species that have several Genus Escherichia
Role of Taxonomy: important features in common but differ sufficiently to still maintain their status as Species coli
Clinically, taxonomy facilitates communication among technologists, physicians, individual species. Subtype Escherichia coli O157: H7
microbiologists and scientists by assigning universally useful names to clinically NOMENCLATURE – refers to the naming of an organism by international rules
relevant microorganisms. This is essential for: (established by a recognized group of medical professionals) according to its IDENTIFICATION – it is the process in which a microorganism’s key features are
● Recognizing new and emerging pathogenic microorganisms. characteristics. Genus and species are the groups of most concern to delineated. Once those features are established the profile is compared with
● Recognizing the changes in the types of infections or diseases caused by microbiologists. The discussion of rules governing microbial nomenclature is limited those other previously characterized microorganisms so that the organism in
familiar microorganisms to these two taxons designations. question can be classified within the most appropriate taxa (classification) and
● Understanding the mechanism of antimicrobial resistance and detecting - In this Binomial (two-name) system of nomenclature: can be assigned an appropriate genus and species (nomenclature).
new resistance mechanisms exhibited by a particular microorganism. ● Every organism is assigned a genus or species name of Latin or Greek It is practical use of a classification scheme to:
● There are three areas that make up taxonomy in relation to diagnostic derivation 1. isolate and distinguish desirable organisms from undesirable ones
microbiology: ● The scientific label consists of two parts: 2. verify the authenticity or special properties of a culture in a clinical setting,
a. Classification a. The Genus designation – which is always capitalized and
b. Nomenclature b. the species designation – which is never capitalized 3. isolate and identify the causative agent of a disease.
c. Identification ● Both components should be in Italics or underlined in script. For example: Identification Methods:
CLASSIFICATION – is the organization of microorganisms that share similar Streptococcus pneumoniae - A wide variety of methods and criteria are used to establish a
morphologic, physiologic and genetic traits into specific groups or taxa. Notes: microorganism’s identity. These methods can be separated into two
Classification Hierarchy: 1. The name may be abbreviated using the upper-case form of the first letter categories:
Kingdom of the genus designation followed by a period (.) and the full species name A. Genotypic Characteristics –relate to the organism’s genetic makeup, including
Division which is NEVER abbreviated. For example: Streptococcus pneumoniae = S. the nature of the organism’s genes and constituent nucleic acids.
Class pneumonia B. Phenotypic characteristics are based on the features beyond the genetic level
Order
2. Frequently an informal designation for example: streptococci, and include readily observable characteristics and those characteristics that may
staphylococci; may be used to label a particular group of organisms but require extensive analytic procedures to be detected.
Family
such designations are not capitalized or italicized.
Genus
● As more information is gained regarding organism classification and Identification Criteria and Characteristics for Microbial Classification:
Species
identification, a particular species may be moved to a different genus or Phenotypic Criteria Examples:
1. Kingdom-comprised of similar divisions
assigned to a new genus. Macroscopic Characteristics of microbial growth patterns on artificial media
2. Division- composed of similar classes
● The such changes are documented in the International Journal for Morphology as observed when inspected with the unaided eye. Examples:
3. Class- composed of similar order
Systematic Bacteriology. size, texture and pigmentation of bacterial colonies
4. Order- composed of similar families
● In diagnostic laboratory, changes in the nomenclature are phased in Microscopic Size, shape and intracellular inclusions, cellular appendages,
5. Family-composed of similar genera
gradually so that physicians and laboratorians have ample opportunity to Morphology and arrangement of cells when observed with the aid of the
6. Genus- composed of similar species
recognize the changes in the naming. microscope magnification.
7. Species
- This is usually accomplished by using the new genus designation while
Staining Ability of the organism to reproducibly stain with a particular
- Classification of bacteria requires experimental and observational
continuing to provide the previous designation in parenthesis:
Characteristics color with the application of specific dyes and reagents.
techniques; this is because biochemical, physiologic, genetic, and
Stenotrophomonas (Xanthomonas) maltophilia
Staining is usually used in conjunction with microscopic
morphologic properties are oft en necessary for an adequate description of
morphology.
a taxon.
Environmental Ability of the organism to grow at various temperature, in the
requirements presence of oxygen and other gases , at various pH levels, or in
the presence of ions and salts.
Species
 Nutritional Ability of the organism to utilize various carbon and nitrogen c. The mechanisms by which genetic information is changed and exchanged The viruses integrate their DNA into the bacterial cell’s chromosome, where viral
requirements sources as nutritional substrates when grown under specific among bacteria. replication and expression is directed
environmental conditions. NUCLEIC ACID STRUCTURE AND ORGANIZATIONS: 1. When the production of viral products is completed, viral DNA is excised/ cut
Resistance Exhibition of a characteristic inherent resistance to specific - DNA is most common form macromolecule that encodes genetic from the bacterial chromosome and packaged within protein coats.
Profiles antibiotics, heavy metals, or toxins by certain microorganisms information in bacteria 2. The viruses are then released when the infected bacterial cell lyses.
Antigenic Establishment of profiles of microorganisms by various serologic - RNA is most common in viruses 3. In transduction, the virus not only packages its own DNA but may also
Properties and immunologic methods that are useful for determining the - DNA consists of deoxyribose sugars connected by phosphodiester bonds package a portion of a bacterium’s DNA. When the viruses infect another
relatedness among various microbial groups - the bases are covalently linked to each deoxyribose sugar are the key to bacterial cell, they release all their DNA contents.

Subcellular Establishment of the molecular constituents of the cell that are the genetic code within the DNA molecule 4. Therefore, the newly infected cell is the recipient of donor DNA introduced

properties typical of a particular taxon, or organism group, by various - four bases: by infecting bacteriophage and recombination between DNA from two

analytic methods. 1. Two Purines: Adenine and Guanine different cells may occur.

Some examples include cell wall components, components of 2. Two Pyrimidines: Cytosine and Thymine CONJUGATION – this process occurs between two living cells, involves cell-to-cell

the cell membrane and enzymatic content of a microbial cell. - In RNA, Uracil replaces Thymine. contact, and requires mobilization of the donor bacterium’s chromosome
- When taken together, the sugar, the phosphate and a base form a single 1. The contact is mediated by the sex pilus. The sex pilus from the donor

Genotypic Criteria Examples unit referred to as nucleotide. bacterial cell established a conjugative bridge that serves as the conduit for

DNA base DNA comprises four bases (guanine, cytosine, adenine, - DNA and RNA nucleotide polymers and the order of bases along a DNA or DNA transfer from donor to recipient cell.

composition ratio thymine). The extent to which DNA from two organisms is RNA strand is known as the base sequence. This sequence provides the 2. With intracellular contact established, chromosomal mobilization is

made up of cytosine and guanine relative to their total information that specifies the proteins that will be synthesized by microbial undertaken and involves DNA synthesis. One DNA strand is produced by the

base content can be used as indicator of relatedness, or cells. The sequence is the genetic code. donor and is passed to the recipient. Where a strand complimentary to the

lack thereof DNA MOLECULAR STRUCTURE: donor strand is synthesized.

Nucleic acid The order of the bases along a strand of DNA or RNA are - is composed of two nucleotide polymers. Each strand has a 5’ and a 3’ 3. The amount of DNA transferred depends on how long the cells are able to

(DNA and RNA known as the base sequence and the extent to which the hydroxyl terminus. The two strands run anti-parallel, with the 5’ terminus of maintain contact, but usually the portions of the chromosome are

base sequence sequences are similar (homologous) between two one strand opposed to the 3’ terminal of the other transferred. In any case the new DNA is then available to recombine with the

analysis microorganisms can be determined directly or indirectly by - The strands are bound via two hydrogen bonds. recipient’s chromosome.

various molecular methods. The degree of similarity in the Adenine bound to Thymine (bound to two hydrogen bonds) - in addition to chromosomal DNA, genes encoded in non-chromosomal

sequences may be a measure of the degree of organism Guanine bound to Cytosine (bound to three hydrogen bonds) genetic elements, such as plasmids and transposons may be transferred by

relatedness. conjugation as well.

MECHANISMS OF GENE TRANSFER: Transposition – is the process in which these genetic elements excise from one

BACTERIAL GENETICS, METABOLISM AND STRUCTURE: Three mechanisms by which bacteria physically exchange DNA include genomic location and insert into another.

Knowledge regarding genetic, metabolic, and structural characteristics of Transformation, Transduction and Conjugation. - Transposons do not exist independently within the cell. They are referred to

microorganisms provides the basis for understanding almost every aspect of TRANSFORMATION – donor bacterial cell dies and undergoes lysis recipient as the “jumping genes”, because their ability to change location within and

diagnostic microbiology, including: bacterial cell receives the free DNA. even between the genomes of bacterial cells.

● The mechanism by which microorganisms cause disease - The free DNA are seen as fragments in the environment. Certain bacteria is - Transposons carry genes whose products help mediate transpositional

● Developing, implementing optimum techniques for microbial detection, able to uptake the free DNA and then undergoes the transformation. process as well as genes that encode for antimicrobial resistance.

cultivation, identification, and characterization - bacteria is usually competent on this manner. - Plasmids and transposons play a key role in genetic diversity and

● Understanding microbial action and resistance - examples: Neisseria, Haemophilus and Streptococcus genera. dissemination of genetic information among bacteria.

● Developing and implementing tests for antimicrobial resistance detection Process: (Please put picture about bacterial transport or video of each processes)

● Designing strategies for disease therapy and control. 1. Once the donor DNA, usually singular strand, gains access to the interior of BACTERIAL METABOLISM:

BACTERIAL GENETICS: the recipient cell, recombination with the recipient’s homologous DNA can Bacteria uses various strategies for obtaining essential nutrients from the

Genetics is the process of heredity and variation and is the starting point from all occur. environment, but the common goal is to transport these substances from the

other cellular pathways, functions and structures originate. The ability of the 2. The mixing of DNA between bacteria via transformation and recombination external environment to the cell’s interior.

microorganisms to maintain viability, adapt, multiply and cause disease is founded plays a major role in antibiotic resistance and in the dissemination of genes Microbial metabolism consists of the biochemical reaction’s bacteria use to break

by genetics. that encode factors essential to the organism’s ability to cause disease. down organic compounds and the reactions they use to synthesize new bacterial

Three major aspects of microbial genetics include: TRANSDUCTION – a mechanism wherein DNA from two bacteria may come parts from the resulting carbon skeletons. Energy for the new constructions is

a. Structure and organization of genetic material together in one cell, thus allowing for recombination. This process is mediated by generated during the metabolic breakdown of the substrate.

b. Replication and expression of genetic information viruses that infect bacteria (bacteriophages)
Process:
The occurrence of all biochemical reactions in the cell depends on the presence
and activity of specific enzymes. Thus, metabolism can be regulated in the cell
either by:
● regulating the production of an enzyme itself (a genetic type of regulation,
in which production of the enzyme can be induced or suppressed by
molecules present in the cell) or
● by regulating the activity of the enzyme (via feedback inhibition, in which
the products of the enzymatic reaction or a succeeding enzymatic reaction
inhibit the activity of the enzyme).
Diagnostic schemes analyze each unknown microorganism for:
1. utilization of various substrates as a carbon source,
2. production of specific end products from various substrates, and
3. production of an acid or alkaline pH in the test medium.
Knowledge of the biochemistry and metabolism of bacteria is important in the
clinical laboratory.
For nutrients to be internalized they must cross the bacterial cell envelope, a
complex cell structure that helps protect the cells from environmental insults,
maintains intracellular equilibrium and transports substances into and out of the cell.
● Water, carbon and oxygen enter the cell through passive diffusion across the
envelope
● Other nutrients require energy and selectivity by the cell envelope:
Active Transport is among the most common methods used for uptake of amino
acids, organic and inorganic acids.
- The mechanism, which is driven by an energy-dependent pump, involves a
carrier molecule embedded in the membrane portion of the cell envelope.
These carriers combine with the nutrients, transports them across the
membrane and releases them within the cell.
FERMENTATION AND RESPIRATION:
Bacteria use biochemical pathways to catabolize (break down) carbohydrates
and produce energy by two mechanisms—fermentation and respiration
(commonly referred to as oxidation).
Fermentation is an anaerobic process carried out by both obligate and facultative
anaerobes. In fermentation, the electron acceptor is an organic compound.
- Fermentation is less efficient in energy generation than respiration (oxidation)
because the beginning substrate is not completely reduced; therefore, all
the energy in the substrate is not released.
- When fermentation occurs, a mixture of end products (e.g., lactate,
butyrate, ethanol, and acetoin) accumulates in the medium. Analysis of
these end products is particularly useful for the identification of anaerobic
bacteria.
- End-product determination is also used in the Voges-Proskauer (VP) and
methyl red tests, two important diagnostic tests used in the identification of
the Enterobacteriaceae.
- (The term fermentation is often used loosely in the diagnostic microbiology
laboratory to indicate any type of utilization—fermentative or oxidative—of
a carbohydrate—sugar—with the resulting production of an acid pH.)
Respiration (not an act of breathing) is an efficient energy generating process in
which molecular oxygen is the final electron acceptor.
- Obligate aerobes and facultative anaerobes carry out aerobic respiration,
in which oxygen is the final electron acceptor.
- Certain anaerobes can carry out anaerobic respiration,in which inorganic
forms of oxygen, such as nitrate and sulfate, act as the final electron
acceptors.
BIOCHEMICAL PATHWAYS FROM GLUCOSE TO PYRUVIC ACID
The starting carbohydrate for bacterial fermentations or oxidation is glucose. When
bacteria use other sugars as a carbon source, they first convert the sugar to
glucose, which is processed by one of three pathways. These pathways are
designed to generate pyruvic acid, a key three-carbon intermediate.
The three major biochemical pathways bacteria use to break down glucose to
pyruvic acid are:
1. Embden-Meyerhof-Parnas (EMP) glycolytic pathway
2. pentose phosphate pathway and
3. Entner-Doudoroff pathway
- Pyruvate can be further processed either fermentatively
or oxidatively.
EMP GLYCOLYTIC PATHWAY:
● Major pathway in conversion of glucose to pyruvate
● Generates reducing power in the form of NADH2
● Generates energy in the form of ATP
● Anaerobic; does not require oxygen
● Used by many bacteria, including all members of Enterobacteriaceae
PENTOSE PHOSPHATE (PHOSPHOGLUCONATE) PATHWAY
● Alternative to EMP pathway for carbohydrate metabolism
● Conversion of glucose to ribulose-5-phosphate, which is rearranged into
other 3-, 4-, 5-, 6-, and 7-carbon sugars
● Provides pentoses for nucleotide synthesis
● Produces glyceraldehyde-3-phosphate, which can be converted to
pyruvate
● Generates NADPH, which provides reducing power for biosynthetic
reactions
● May be used to generate ATP (yield is less than with EMP pathway)
● Used by heterolactic fermenting bacteria, such as lactobacilli, and by
Brucella abortus, which lacks some of the enzymes required in the EMP
pathway
ERTNER-DOUDOROFF PATHWAY:
● Converts glucose-6-phosphate (rather than glucose) to pyruvate and
glyceraldehyde phosphate, which can be funneled into other pathways
● Generates one NADPH per molecule of glucose but uses one ATP
● Aerobic process used by Pseudomonas, Alcaligenes, Enterococcus faecalis,
and other bacteria lacking certain glycolytic enzymes
Anaerobic Utilization of Pyruvic Acid (Fermentation)
- Pyruvic acid is a key metabolic intermediate. Bacteria process pyruvic acid
further using various fermentation pathways. Each pathway yields different
end products, which can be analyzed and used as phenotypic markers
Some fermentation pathways used by the microbes that inhabit the human
body are as follows:
● Alcoholic fermentation: The major end product is ethanol. This is the pathway
used by yeasts when they ferment glucose to produce ethanol.
● Homolactic fermentation: The end product is almost exclusively lactic acid.
All members of the Streptococcus genus and many members of the
Lactobacillus genus ferment pyruvate using this pathway.
● Heterolactic fermentation: Some lactobacilli use this mixed fermentation
pathway, of which, in addition to lactic acid, the end products include
carbon dioxide, alcohols, formic acid, and acetic acid.
● Propionic acid fermentation: Propionic acid is the major end product of
fermentations carried out by Propionibacterium acnes and some anaerobic
non–spore-forming, gram-positive bacilli.
● Mixed acid fermentation: Members of the genera Escherichia, Salmonella,
and Shigella within the Enterobacteriaceae use this pathway for sugar
fermentation and produce a number of acids as end products—lactic,
acetic, succinic, and formic acids. The strong acid produced is the basis for
the positive reaction on the methyl red test exhibited by these organisms.
● Butanediol fermentation: Members of the genera Klebsiella, Enterobacter,
and Serratia within the Enterobacteriaceae use this pathway for sugar
fermentation. The end products are acetoin (acetyl methyl carbinol) and
2,3-butanediol.
Detection of acetoin is the basis for the positive VP reaction characteristic of these
microorganisms. Little acid is produced by this pathway. Thus, organisms that have
a positive VP reaction usually have a negative reaction on the methyl red test, and
vice versa.
● Butyric acid fermentation: Certain obligate anaerobes, including many
Clostridium species, Fusobacterium, and Eubacterium, produce butyric acid
as their primary end product along with acetic acid, carbon dioxide, and
hydrogen.
Aerobic Utilization of Pyruvate (Oxidation)
- The most important pathway for the complete oxidation of a substrate under
aerobic conditions is the Krebs or tricarboxylic acid (TCA) cycle. In this cycle,
pyruvate is oxidized, carbon skeletons for biosynthetic reactions are created,
and the electrons donated by pyruvate are passed through an electron
transport chain and used to generate energy in the form of ATP. This cycle
results in the production of acid and the evolution of carbon dioxide
Carbohydrate Utilization and Lactose Fermentation
- The ability of microorganisms to use various sugars (carbohydrates)for growth
is an integral part of most diagnostic identification schemes.
- The fermentation of the sugar is usually detected by acid production and a
concomitant change of color resulting from a pH indicator present in the
culture medium.
- Bacteria generally ferment glucose preferentially over other sugars, so
glucose must not be present if the ability to ferment another sugar is being
tested.
- An important step in classifying members of the Enterobacteriaceae family
is the determination of the microorganism’s ability to ferment lactose.
- These bacteria are classified as either lactose fermenters or lactose
nonfermenters. Lactose is a disaccharide consisting of one molecule of
glucose and one molecule of galactose linked together by a galactoside
bond.
- Two steps are involved in the utilization of lactose by a bacterium.
1. The first step requires an enzyme, β-galactoside permease, for the
transport of lactose across the cell wall into the bacterial cytoplasm.
2. The second step occurs inside the cell and requires the enzymeβ-
galactosidase to break the galactoside bond, releasing glucose, which
can be fermented. Thus, all organisms that can ferment lactose can also
ferment glucose.