Prelims

Microbiology

- Microbiology is the study of microbes or micro-organisms.

• Organisms that Make up the Microbial World

• Prokaryotes and Eukaryotes

• Because of their characteristics, microorganisms join all other living

organisms in two major groups of organisms: prokaryotes and eukaryotes
(Edelson, 2002).

• 1. Bacteria are prokaryotes (simple organisms having no nucleus or

organelles) because of their cellular properties.

• 2. Fungi, protozoa, and unicellular algae are eukaryotes (more complex

organisms whose cells have a nucleus and organelles).

• 3. Viruses are neither prokaryotes nor eukaryotes because of their simplicity

and unique characteristics.
• Five-Kingdom Classification

• Robert Whittaker of Cornell University devised the generally-accepted

classification of living things in 1969 (Gardner, 1991). He proposed a
Five Kingdom classification system:

1. Monera - includes Prokaryotes, such as bacteria and cyanobacteria.

2. Protista includes protozoa, unicellular algae, and slime molds, all of

which are eukaryotes and single-celled.

• 3. Fungi includes the molds, mushrooms, and yeasts (organisms that

are eukaryotes that absorb simple nutrients from the soil).
• 4. Plantae includes plants.
• 5. Animalia-includes animals.

• The Development of Microbiology

• Early Beginnings

Robert Hooke made key observations (Barbaree, 1993). He is reputed to have

observed strands of fungi among the specimens of cells he viewed.

In the 1670s and the decades thereafter, a Dutch merchant named Anton van
Leeuwenhoek made careful observations of microscopic organisms, which he
called animalcules. One of the first to provide accurate descriptions of
protozoa, fungi, and bacteria (Wachsmuth, Blake, & Olsvik, 1994).
• Louis Pasteur postulated the germ theory of disease, which states that
microorganisms are the causes of infectious disease.

• German scientist Robert Koch provided the proof by cultivating

anthrax bacteria apart from any other type of organism. Koch then
injected pure/ cultures of the bacilli into mice and showed that the bacilli
invariably caused anthrax.

• Golden Age of Microbiology – during this stage, many agents of

different infectious diseases were identified. Many of the etiologic
agents of microbial disease were discovered during the period,
leading to the ability to halt epidemics by interrupting the spread of
microorganisms.
• After World War II, antibiotics were discovered and introduced as
primary therapy for microbial infections. As such, the incidence of
pneumonia, tuberculosis, meningitis, syphilis, and many other diseases
declined with the use of antibiotics.

• Work with viruses could not be effectively performed until instruments

were developed to help scientists see these disease agents. In the
1940s, the electron microscope introduced, and the knowledge of
viruses developed rapidly. With the development of vaccines in the
1950s and 1960s, such viral diseases as polio, measles, mumps, and
rubella came under control.
• Microorganisms and Divisions of Microbiology

• Organisms of the microbial world are very diverse and include the
bacteria, cyanobacteria, rickettsiae, chlamydiae, fungi, unicellular
(single-celled) algae, protozoa, and viruses.

• Microorganisms are a collection of organisms that share the

characteristic of being visible only with a microscope.

• The majority of microorganisms contribute to the quality of human life

by doing such things as maintaining the balance of chemical elements
in the natural environment by breaking down the remains of all that dies
and by recycling carbon, nitrogen, sulfur, phosphorus, and other
elements.
• Some species of microorganisms cause infectious disease. They
overwhelm body systems by sheer force of numbers, or they produce
powerful toxins that interfere with body physiology.

• Viruses inflict damage by replicating within tissue cells, thereby causing

tissue degeneration.

• Bacteria

• Bacteria are relatively simple, prokaryotic organisms whose cells lack a

nucleus or nuclear membrane. The bacteria may appear as rods
(bacilli), spheres (cocci), or spirals (spirilla or spirochetes).
• Viruses

• Viruses are ultramicroscopic bits of genetic material (DNA or RNA) enclosed

in a protein shell and, sometimes, a membranous envelope.

• Viruses have no metabolism; therefore, it is difficult to use drugs to interfere

with their structures of activities. Viruses multiply in living cells and use the
chemical machinery of the cells for their own purpose.

• Protozoa

• Protozoa are eukaryotic, unicellular organisms. Motion is a characteristic

associated with many species and the protozoa can be classified according to
how they move. Some protozoa use flagella, others use cilia, and others use
pseudopodia. Certain species are nonmotile. Protozoa exist in an infinite
variety of shapes because they have no cell walls. Many species cause such
human diseases as malaria, sleeping sickness, dysentery, and toxoplasmosis.
• Fungi

• Fungi are eukaryotic microorganisms that include multicellular molds

and unicellular (single-celled) yeasts. The yeasts are slightly larger than
bacteria and are used in alcoholic fermentations and bread making.

• Certain yeasts such as Candida albicans are pathogenic. Molds are

filamentous, branched fungi that use spores for reproduction.

• The fungi prefer acidic environments, and most live at room

temperature under oxygen-rich conditions. The common mushroom is a
fungus.
• Algae

• Algae implies a variety of plantlike organisms. In microbiology, several

types of single-celled algae are important. Examples are the diatoms
and dinoflagellates that inhabit the oceans and are found at the bases
of marine food chains.

• Most algae capture sunlight and transform it to the chemical energy of

carbohydrates in the process of photosynthesis.
• Divisions of Microbiology

• According to Ackersvick (1992), there are currently five major divisions

of microbiology corresponding to the five major groups of
microorganisms.

• These include the following:

• 1. Bacteriology - study of bacteria;
• 2. Virology - study of viruses;
• 3. Mycology - study of fungi;
• 4. Phycology - study of algae; and
• 5. Protozoology study of protozoa (some scientists prefer to use
parasitology .
• Significance and Practical Applications of Microbiology

• Microbes have a profound impact on every facet of human life and

everything around us.

• Pathogens harm us, yet other microbes protect us. Some microbes
are pivotal in the growth of food crops, but others can kill the plants or
spoil the produce.

• Bacteria and fungi eliminate the wastes produced in the

environment, but also degrade things we would rather preserve. Clearly
they effect many things we find important as humans.
• Agriculture

• Aside from being important in biogeochemical cycling of nutrients, microbes

play vital role in maintenance of soil fertility and in crop protection.

• 1. Soil fertility microbes are being exploited in two important ways - bio-
fertilizers, and creating new nitrogen-fixing organisms.

• 2. Nitrogen-fixers through recombinant DNA technology efforts have been

made to introduce nitrogen-fixing genes (nif genes) into wheat, corn, and rice,
among others.

• 3. Biopesticides - several microbes (viruses bacteria, and fungi) are being

developed as suitable biopesticides for management of insect and nematodal
pests.
• 4. Bionematicides some fungi have good potential of their use as
bionematicides to control nematodal pests of vegetables, fruit and
cereal crops.

• Some bacterial and fungal products are also in use to control diseases
of roots and shoots of plants.

• 5. Bioweedicides several fungi have been found very useful in the

control of troublesome weeds of crop fields.
• Industrial Applications

• Microorganisms are used for commercial production of alcohols, acids,

fermented foods, vitamins, medicines, and enzymes, among others.

• 1. Development of pharmaceutical products.

• 2. Use of quality-control methods in food and dairy product production.
• 3. Production of vitamins, amino acids, enzymes, and growth
supplements.
• 4. Manufacture of many foods, including fermented dairy products (sour
cream, yogurt, and buttermilk), as well as other fermented foods such as
pickles, sauerkraut, breads, and alcoholic beverages.
• Environmental Conservation and Protection

• Several microbes could be shown very helpful in maintenance of

environmental quality through biodegradation of wastes into useful
products and also in biodegradation of harmful pesticides used in crop
protection and public health.

• . 1. Bioremediation - inexpensive and increasingly effective way of cleaning

up pollution such as those environments contaminated with crude oil,
polychlorinated biphenyls, and many other industrial wastes.

• 2. Treatment and recycling of large-volume sewage and waste-water in

metropolitan cities.

• 3. Valuable source of alternative energy from methane-producing bacteria.

• 4. Necessary for the fermentation of biomass into biofuels.

• 5. Environmental monitoring and biomonitoring environmental

pollutants can be detected by use of appropriate strains of microbes as
biosensors. Biosensor is a biophysical device used to detect the
presence and quantify the specific substances (sugars, proteins,
hormones, pollutants) in the specific environments.

• 6. Replenishment of the world's oxygen supply and control of global

warming through the removal of greenhouse gases by cyanobacteria.
• Biotechnology Applications

• Advances in biotechnology where microorganisms are used as living

factories to produce pharmaceuticals that otherwise could not be
manufactured.

• 1. Human hormone insulin

• 2. Human growth factor
• 3. Antiviral substance interferon
• 4. Numerous blood-clotting factors and clot-dissolving enzymes
• 5. Vaccines
• Evolution of Microbiology

• Invention of the Microscope

• Robert Hooke

• In 1664, Robert Hooke devised a compound microscope and used it to

observe fleas, sponges, bird feathers, plants and molds, among other items.

• Anton van Leeuwenhoek

• Anton van Leeuwenhoek developed a microscope that, although considered

crude by modern standards, was able to magnify samples greater than 200-
fold.
• Ferdinand Julius Cohn

• Ferdinand Julius Cohn was the first to carefully examine the world of
the microbe and made many observations of eukaryotic
microorganisms and bacteria.

• The following table lists some of the early advances that helped to
develop the practice of microbiology.

1964 Robert Hooke is the first to use a microscope to describe the

fruiting structures of molds

1673 Anton van Leeuwenhoek, a Dutch tradesman and skilled lens

maker, is the first to describe microbes in detail.
• 1872 - Ferdinand Julius Cohn publishes landmark paper on bacteria
and the cycling of elements. In it is an early 1872 classification scheme
that uses the name Bacillus.

• 1872 - Oscar Brefeld reports the growth of fungal colonies from single
spores on gelatin and the German botanist Joseph Schroeter grows
pigmented bacterial colonies on slices of potato.

• 1877 - Robert Koch develops methods for staining bacteria,

photographing, and preparing permanent visual records on slides.

• 1881 Koch develops solid culture media and the methods for obtaining
pure cultures of bacteria.
• 1882 - Angelina Fannie and Walther Hesse in Koch's laboratory
develop the use of agar as a support medium for solid culture.

• 1884 - Hans Christian Gram develops a dye system for identifying

bacteria [the Gram stain)

• 1887 First report of the petri plate by Julius R. Petri.

• 1915 M. H. McGrady establishes a quantitative approach for analyzing

water samples using the most probable number, multiple-tube
fermentation test.
• Ignaz Semmelweis

• Semmelweis made the first breakthrough in the true nature of disease.

He realized that a sepsis in obstetrical wards could prevent the
transmission of childbirth fever from patient to patient. He instituted a
policy for all attending physicians to wash their hands with chloride of
lime, calcium hypochlorite, and calcium chloride, between patients.
This innovation dropped the mortality rate or mortality from 18% to
2.4% (Burnbaker, 2000).

• Louis Pasteur Pasteur is better known for "pasteurization". He

observed that the process of converting sugar to alcohol is actually
performed by various yeast strains.
• Joseph Lister
• Lister was the first to greatly reduce the number of microorganisms on
surgical wounds and incisions by using bandages treated with phenic
acid, a compound that killed microorganisms.

• Identification of Viruses that Parasitize other Microorganisms

• In the latter half of the 19th century, the causative agents for anthrax,
tuberculosis, gonorrhea, diphtheria and many more were discovered.
• Vaccination as Effective Means of Protection

• As far back at the 11th century in India and China it was realized that liquid
from the pustules of a smallpox victim, when scratched on the skin of a
healthy patient, would most often cause mild disease. This intentional
infection, termed variolation, would also give life-long protection against the
virus.

• Edward Jenner

• In 1796, Edward Jenner, went in search of a more predictable and safer

method of protection against smallpox. Jenner hypothesized that cowpox
was related to smallpox and contraction of the former would protect against
the latter. Jenner inoculated a young patient with cowpox and later
challenged him with smallpox. The boy did not become ill and Jenner was
responsible for the creation of a safer method of protection against smallpox.
• Louis Pasteur

• Pasteur developed a method for creating cultures that would confer

immunity, but not cause disease. This involved multiple passes through
a susceptible host where the microorganism will spontaneously loose
their virulence. Pasteur's technique of weakening a strain by a
damaging treatment or passing it through a susceptible host was
termed attenuation and resulted in the creation of vaccines against
anthrax, plague, yellow fever, rabies and many other diseases.
• Development of Antimicrobials

• Paul Ehrlich - He developed salvarsan, the first effective

chemotherapeutic agent against Treponema pallidum, the causative agent
of syphilis.

• Alexander Fleming

• In September of 1928 Fleming cultured a fungus, a Penicillium mold, and

eventually isolated a soluble extract that could kill bacteria and treat
localized infection. He called the new compound penicillin after the mold
from which it came.
 • Chapter 2

• Basic Microbiology Equipment and Procedures in the Study of Bacteria

• Basic Microbiology Equipment

• Microscope
- Essentially the single most important equipment in microbiology, the
microscope allows for the visualization of microorganisms through
magnification. There are several types of microscopes. These can
include the light microscope, the electron microscope, and the scanning
probe microscope.
• Test Tube and Holder

• These are finger-like length of glass or clear plastic tubing which is

open in one end and rounded with a U-shaped bottom. Test tubes in
microbiology are used for handling and culturing all kinds of
microorganisms.
• Microscope Glass Slides

• A microscope slide is a thin flat piece of glass used to hold objects for
examination under the microscope.

• Inoculation Loop
• The inoculation loop is a simple tool that is used to retireve an inoculum
from a culture of microorganisms. It is also known as a smear loop, an
inoculation wand, or a microstreaker. The inoculation loop is used in
the cultivation of microbes on Petri dishes by transferring the inoculum
for streaking.
• Pipette
• A pipette is an instrument that is used to transport a measured volume
of liquid.

• Bunsen Burner

• Named after Robert Bunsen, the Bunsen burner is a laboratory

equipment that produces a single open gas flame for heating,
sterilization, and combustion.
• Petri Dish

• The Petri dish, named after Julius R. Petri, is a shallow cylindrical

covered container or dish that is often made of glass or plastic
(Gardner, 1991). It is used to culture cells or small moss plants.

• Medical Autoclave

• A medical autoclave is the simplest form of autoclve. It uses steam to

sterilize equipment and other objects in order to inactivate
microorganisms.
• Basic Microbiology Media

• Since the focus of microbiology is in the study of microorganisms, it is

essential that scientists are able to grow or culture them under
controlled conditions. Culture medium is a liquid or gel designed to
support the growth of microorganisms or cells or small plants.

• Types of Culture Media

• There are two fundamental types of culture media. These are cell
culture and microbiological culture. For the culture of microorganisms,
the most common include the following
• 1. Nutrient broths - contains amino acids and nitrogen although its
source may contain other compounds with unknown composition.
•
• a. These contain all the elements that most bacteria need to grow.
• b. These are non-selective so it is used for general cultivation and
maintenance of bacteria kept in laboratory culture collections.

• 2. Agar plates - a Petri plate that contains a growth medium in order to

culture or grow microorganisms or even small plants such as moss. In
some instances, selective growth compounds can be added to the
media, such as antibiotics.
• General Types of Media according to Purpose
• 1. Minimal media - contain minimum nutrients possible for colony
growth usually in the absence of amino acids.

• 2. Selective media - used for the culture of only select microorganisms.

3. Differential media also known as indicator media; used to

differentiate one microorganism from another grown on the same media
using the biochemical characteristics of microorganisms.

• 4. Transport media used as a temporary storage of specimens being

transported to the laboratory for cultivation; contains only buffers and
salt.
• 5. Enriched media - contain nutrients required to support the growth of
a wide variety of microorganisms, including fastidious species.

• Types of Blood Agar Plate

• 1. Blood agar plate - contains mammalian blood used to isolate

fastidious organisms and detect hemolytic activity such as
Streptococcal species.
• 2. Chocolate agar - a type of blood agar plate where the blood cells
have been lysed by heating the cells to 56°C and is used to culture
fastidious respiratory bacteria such as Haemophilus influenzae.
 3. Thayer-Martin agar - chocolate agar used to isolate Neisseria
gonorrheae.

• 4. Thiosulfate citrate bile salts sucrose-enriched agar a type of blood

agar plate the enhances the growth of Vibrio species including V
cholerae.

• Types of General Bacterial Media

• 1. Bile esculin agar - used to isolate Enterococci and group D

Streptococci.

• 2. Cetrimide agar - used to isolate Pseudomonas aeruginosa.

• 3. Cysteine-lactose electrolyte deficient (CLED) agar used to isolate
and differentiate urinary tract bacteria through inhibition of Proetus
species from swarming; can also differentiate lactose-fermenters and
non-lactose fermenting bacteria.

• 4. Hektoen enteric agar (HEA) - used to isolate and recover fecal

bacteria of the family Enterobacteriaceae especially Salmonella and
Shigella species.

• 5. Mannitol salt agar (MSA) used to isolate organisms, especially

halophiles, that ferment mannitol which produce lactic acid.

• 6. McConkey agar (MAC) used to differentiate between Gm- and Gm+

bacteria through inhibition of Gm+ bacterial growth.
• 7. Mueller-Hinton agar (MHA) - used to test antibiotic susceptibility.

• 8. Nutrient agar used to culture non-fastidious organisms and observe

pigment production; one of the safest culture media since it does not
selectively grow pathogenic bacteria.

• 9. Onoz agar used for rapid diagnosis of Salmonella and Shigella

species as differentiated from other members of the
Enterobacteriaceae family.

• 10. Phenylethyl alcohol agar (PEA) used to isolate Staphylococcus

species through inhibition of Gram- bacilli such as E coli, Shigella, and
Proteus, among others.

• 11. R2A agar used for water analysis.

• 12. Tinsdale agar - used to isolate Corynebacterium diphtheriae.

• 13. Tryptic soy agar (TSA) - used as a general purpose media serving
as a base media for other agar types; it is used to support the growth of
Brucella, Corynebacterium. Listeria, Neisseria, and Vibrio species.

• 14. Xylose-lysine-deoxycholate agar (XLD) - used to culture stool

samples especially to allow Gm- bacterial growth and inhibiting Gm+
growth.
• Types of Fungal Media

• 1. Hay infusion agar - used to culture slime molds.

• 2. Malt extract agar - used to isolate fungal microorganisms.

• 3. Potato dextrose agar - used to culture some types of fungi.

• 4. Sabouraud agar used to culture fungi; has a low pH that inhibits

bacterial growth especially Gm- bacteria.
• Basic Microbiology Procedures

• Bright Field Light Microscopy

• The success of a student in a microbiology laboratory is directly related to

the competent use of the compound bright-field light microscope. Bright
field microscopy is the simplest of all types of optical microscopy
techniques.
• Procedure No. 1. Basic Microscopy
• 1. Make sure the x10 eyepiece is in place at the top of the body tube.

• 2. Raise the body tube to a few inches above the stage by looking from
the side and turning the coarse adjustment knob.

• 3. Rotate the revolving nosepiece and click the lowest power objective
into place above the stage (usually this is the x10).

• 4 Adjust the illumination if using a mirror by turning the flat side of the
mirror towards the light source so that light is reflected up towards the
condenser.

• 5. Rack the condenser up to within 2 mm below the stage and adjust the
iris diaphragm until it is half open.
• 6 Place the specimen on the stage making sure that the cover glass is
uppermost and secure it with either the stage clips or the mechanical stage
arms.

• 7. Adjust the angle of the mirror so that a spot of light appears on the slide
directly below the objective lens.

• 8. Look from the side and use the coarse adjustment knob to lower the
objective until it is just above the slide.

• 9. Look through the eyepiece and adjust the mirror to give an even amount of
illumination.

• 10. Use the coarse adjustment knob to slowly rack the objective upwards while
looking through the eyepiece until the specimen is in focus. It is often
sometimes easier to focus on the edge of the cover slip to start with as this
gives a nice clean edge when in focus whereas mucus can sometimes be
difficult to "find".
• 11. Use the fine adjustment knob to obtain the sharpest possible image.

• 12. If the light is too bright, either use a bulb of lower wattage or adjust
the iris diaphragm to reduce glare.

• 13. Focus the light source onto the slide by slowly racking down the
condenser. Be careful not to affect the angle of the mirror. Adjust the
condenser and iris diaphragm to give optimum illumination. Ideally,
once the condenser is set in the optimum position, there should not be
any need to keep altering it.
• Handwashing

• Proper handwashing technique performed by clinical personnel is the

most effective method of controlling infections, especially nosocomial
infections. A layer of oil and the structure of the skin prevent the
removal of microorganisms by simple handwashing (Weaver, 2000).

• Procedure No. 2. Handwashing

• 1. Wet your hands with clean running water (warm or cold) and apply
soap.

• 2. Rub your hands together to make a lather and scrub them well; be
sure to scrub the backs of your hands, between your fingers, and under
your nails
• 3. Continue rubbing your hands for at least 20 seconds. Need a timer?
Hum the "Happy Birthday" song from beginning to end twice.

• 4. Rinse your hands well under running water.

• 5. Dry your hands using a clean towel or air dry.

• Gram Staining Gram staining is a method of differentiating species of

bacteria into two large groups Gram positive or Gram negative based
on the chemical and physical properties of their cell walls. The Gram
stain is almost always the first step in the identification of a bacterial
organism (Barbaree, 1993). Although Gram staining is a valuable
diagnostic tool in both clinical and research settings, not all bacteria can
be definitively classified by this technique.
• Procedure No. 6. Gram Staining
• 1. Prepare heat-fixed smear of a bacteria
• 2. Place the slides on the staining rack
• 3. Flood smear with crystal violet and let stand for 30 seconds.
• 4. Rinse with water for 5 seconds.
• 5. Cover with Gram's iodine mordant and let stand for 1 minute.
• 6. Rinse with water for 5 seconds.
• 7. Decolorize with 95% ethanol for 15-30 seconds. Do not decolorize
too long. Add the decolorizer drop by drop until the crystal violet fails to
wash from the slide. Alternatively, the smear may be decolorized for 30-
60 seconds with a mixture of isopropanol-acetone, 3:1.
• 8. Rinse with water for 5 seconds.

• 9. Counterstain with safranin for about 60-80 seconds. Safranin

preparations vary considerably in strength, and different staining times
may be required for each batch of stain.

• 10. Rinse with water for 5 seconds.

• 11. Blot dry with bibulous paper and examine under oil immersion.

• 12. Gram positive organisms stain blue to purple, gram negative stain
pink to red.
• Acid-Fast Staining

• Acid-fast staining is a special bacteriological stain used to identify acid-

fast microorganisms such as Mycobacteria species. It can also be used
to stain few other bacteria like Nocardia. Acid-fast staining is also
known as Ziehl-Neelsen staining and it uses carbolfuschin, acid alcohol,
and methylene blue as its reagents. Acid-fast bacilli will be bright red
after staining (Beaman & Greenough, 1992).
• Procedure No. 7.
• Acid-fast Staining
• 1. Prepare a smear consisting of a mixture of microorganisms.
• 2. Allow the smear to air dry and then heat-fix.

• 3. Place the slide on a hot plate that is within a chemical hood (with the
exhaust fan on) and cover the smear with a piece of paper toweling that as
been cut to the same size as the microscope slide.

• 4. Saturate the paper with Ziehl's carbolfuschin and heat for 3-5 minutes. Do
not allow the slide to dry out and avoid excess flooding. Also prevent boiling
by adjusting the hot plate to a proper temperature A boiling water bath with a
staining rack or loop held 1-2 inches above the water surface also works well.

• 5. Remove the slide, let it cool and rinse with water for 30 seconds.
• 6. Decolorize by adding acid-alcohol drop by drop until the slide
remains only slightly pink. This requires 10-30 seconds and must be
done carefully.

• 7. Rinse with water for 5 seconds.

• 8. Counterstain with alkaline methylene blue for about 2 minutes.

• 9 Rinse with water for 30 seconds.

• 10. Blot dry with bibulous paper. 11. Examine the slide under oil
immersion. 12. Acid-fast organisms stain red; the background and other
organisms stain blue or brown.
• Preparation and Sterilization of Culture Media

• The survival and growth of microorganisms depend on available

nutrients and a favorable growth environment. In the laboratory, the
nutrient preparations that are used for culturing microorganism are
called media. The major difference among these media is that solid and
semisolid media contain a solidifying agent (agar) whereas a liquid
medium does not.

• Sterilization is the process of rendering a medium or material free of all

forms of life Autoclaving is sterilization of items through exposure to
steam at 121°C and 15 lbs of pressure for 15 minutes or longer. Dry-
heat sterilization and bacteriological filter are two other methods of
sterilization. Other methods include ultraviolet irradiation and
sterilization with ethylene oxide gas.
• Procedure No. 13. Autoclaving

• 1. Load the autoclave with the freshly prepared culture media.

• 2 Close and lock the autoclave door.
• 3. Set the autoclave time for 15 minutes or longer and select a slow rate of
exhaust.

• 4. Make certain that the autoclave temperature is set to 121°C.

• 5. Start the autoclave by pushing the start button or twisting the knob to the
start position.

• 6. When the period of sterilization is completed and the pressure in the

chamber reads 0, carefully open the door and remove the containers, using
heat-proof gloves.
• Procedure No. 14. Preparing Agar Plates

• 1. Use some of the sterilized tryptic soy agar to prepare agar plates.

• 2. When the plates are cool, invert them to prevent condensing

moisture from accumulating on the agar surfaces.

• 3. All plates and tubes should be incubated for at least 24 hours to

ensure sterility before you use them.
• Isolation of Normal Flora

• The microorganisms that constitute the normal flora of the human body
are usually harmless, although some are potential pathogens or
opportunists. These latter microorganisms may cause disease under
certain circumstances.

• Procedure No. 27. Throat Culture

• 1. Swab the person's throat around the tonsillar area using a sterile
cotton swab.

• 2. Use a sterile tongue depressor to keep the tongue from interfering

with the swab.
• . 3. Roll the swab back and forth near one edge of a blood agar plate then
streak from this area with a sterile loop.

• 4. Label the plate with the body site and the type of medium used.
• 5. Incubate the plate, inverted, at 35°C for 24 to 72 hours.
• 6. Observe the blood agar plate for the various types of colonies.
• 7. Characterize the colonies and hemolytic types.

8. Select a number of well-isolated colonies that fit the descriptions of

common throat organisms.

9. Stain each colony you select with the Gram stain and perform a catalase
test on the same colonies you gram stained. Extreme care must be exercised if
a colony is taken from a blood agar plate. Erythrocytes contain catalase, and a
carryover of only a few blood cells can give a false-positive reaction.
• Procedure No. 30. Disinfection
• 1. Select one of the disinfectants and, if necessary, dilute it according to
product specifications.

• 2. Place 5 mL of disinfectant into two sterile tubes and add 0.05 mL. of P.
aeruginosa to one tube and 0.05ml of s. aureus to the other. (Other
microorganisms may be used)

• 3. Label the tubes accordingly.

• 4. Mix each tubes in order to obtain a homogenous suspension.

• 5. At intervals of 1, 2, 5, 10, and 15 minutes, transfer 0.1 mL. of the mixture

containing the bacteria and the disinfectant to separate tubes of tryptic soy
broth. Do this for both bacteria.
• 6. Inoculate two tubes of broth with 0.1 mL. of both bacteria and mark
these "controls".

• 7. Incubate all tubes for 48 hours at 35°C

• 8. Shake and observe each of the tubes for growth.

• 9. Record the presence of growth as and the absence of growth as –

• Procedure No. 31. Use of Antimicrobial Agents

• 1. Mark the lid of each Mueller-Hinton agar plate with the date and the
name of the bacterium to be inoculated. Use a separate sterile cotton
swab for each bacterium. The swab is immersed in the culture tube and
the excess culture is squeezed on the inner side of the test tube. If
there are sufficient supplies you may wish to analyze the antimicrobial
sensitivity of microorganisms from your throat

• 2. The swab is then taken and streaked on the surface of the Mueller-
Hinton plate three times, rotating the plate 60-degrees after each
streaking.
• 3. Run the swab around the edge of the agar to ensure that the whole
surface has been seeded.

• 4. Allow the culture to dry on the plate for 5-10 minutes at room
temperature with the top in place.
• 5. Dispense antibiotics onto the plate either with the multiple dispensers
or individually with the single unit dispenser. Make sure no contact is
made between the antibiotic disk and the culture by gently pressing the
disk with alcohol-flamed forceps. Do not press the disk into the agar
and do not move the disk once it is placed on the agar.
• 6. Incubate the plates for 16-18 hours at 35°C. Do not invert the plates.
• 7. Measure the zones of inhibition to the nearest mm for each of the
antibiotics tested. For each antibiotic, determine whether the bacteria re
resistant or susceptible.
• Procedure No. 35. Stool Specimen Collection

• 1. Collect the stool in a dry, clean, leakproof container. Make sure no

urine, water, soil or other material gets in the container.

• 2. Fresh stool should be examined, processed, or preserved

immediately. An exception is specimens kept under refrigeration when
preservatives are not available; these specimens are suitable for
antigen testing only.

• 3. Preserve the specimen as soon as possible. If using a commercial

collection kit, follow the kit's instructions. If kits are not available, the
specimen should be divided and stored in two different preservatives,
10% formalin and PVA (polyvinyl-alcohol), using suitable containers.
Add one volume of the stool specimen to three volumes of the
preservative.
• 4. Insure that the specimen is mixed well with the preservative. Formed stool
needs to be well broken up.

• 5 Insure that the specimen containers are sealed well. Reinforce with
parafilm or other suitable material. Insert the container in a plastic bag.

• 6. Certain drugs and compounds will render the stool specimens

unsatisfactory for examination. The specimens should be collected before
these substances are administered, or collection must be delayed until after
the effects have passed. Such substances include: antacids, kaolin, mineral
oil and other oily materials, non-absorbable antidiarrheal preparations,
barium or bismuth (7-10 days needed for clearance of effects), antimicrobial
agents (2-3 weeks), and gallbladder dyes (3 weeks).

• 7. Specimen collection may need to be repeated if the first examination is

negative. If possible, three specimens passed at intervals of 2-3 days should
be examined.
 • Chapter 3

• Microbial Control
• Techniques for Controlling Pathogenic Microorganisms

• The control of microbial growth means to inhibit or prevent growth of

microorganisms. This control is affected in two basic ways.

• 1. By killing microorganisms, and

2 By inhibiting the growth of microorganisms

The control of microbial growth usually involves the use of agents.

Agents which kill cells are called “cidal” agents while agents which inhibit
the growth of cells ( without killing them) are referred to as “static” agents.
Thus, the term bactericidal refers to killing bacteria and bacteriostatic
refers to inhibiting the growth of bacterial cells. There are three principles
types of agents used in the control of microbial growth (Alouf, 1991):
1. Physical agents are used exclusively on objects outside the body;

2 Chemical agents are used on inanimate objects as, well as on the body
surface; and

3. Chemotherapeutic agents, which are most often used inside the living
body.

Physical Control of Pathogenic Microorganisms

The chief agent used in this type of control is heat, applied in different
forms. Furthermore, there are some other methods in which different
types of physical agents are used.
• The aim of heating is sterilization which is the complete removal of all life
forms in an item. It can be done in a variety of ways, such as the following
(Spice, Cruz-Reyes & Ackers, 1992).

• 1. Direct flame - also known as incineration.

• a. Often used to sterilize the inoculation loop and bacteriological needle
before removing a certain sample from a culture tube and after preparing a
smear.
• b. The tip of the tube is also flamed in order to destroy the microbes which
may have come in contact with it.

• 2. Hot air oven.

• a. Uses dry heat to sterilize items which are "baked" at 160°C for a period of
two hours to kill bacterial spores as well as other microbial structures.
• b. This is often used to sterilize dry powders, oily substances, and glassware.
• 3. Boiling water - moist heat is used to sterilize items where heat penetrates
better because of the immersion of the whole item in boiling water.
• a. However, because water's boiling point is at 100°C, it will usually require at
least two hours of exposure for complete bacterial spore destruction.

• b. Moist heat kills microbes by coagulating and denaturing their proteins.

• 4. Autoclaving - uses the pressure of steam to increase the temperature and

reduce the length of time required to fully sterilize an item.

• a. The pressure is usually maintained at 15 lb/sq inch above normal

atmospheric pressure and the water molecules are superheated to at least
121.5°C. This allows the moisture to rapidly conduct heat into the items being
sterilized up to 15 meters.

• b. This is often used to sterilize culture media, glassware, and metal-ware

among others.
• 5. Fractional sterilization also called tyndallization or intermittent
sterilization because sterilization is performed in an on-off manner.

• a. Items are first sterilized using free-flowing steam at 100°C for 30

minutes allowed to cool overnight to let the bacterial spores germinate
into vegetative Cells and the exposed again to the bacterial spores
germinates on the second day.

• b. The item is then allowed to cool overnight and sterilized one last time
on the third day.

• 6. Pasteurization-used to reduce the bacterial population of a liquid

such as milk.
• a. It involves heating the liquid to about 62.9°C for 30 minutes to kill
both the Mycobacterium tuberculosis and Q-fever agents.
• 7. Flash pasteurization - pasteurization at 71.6°C for 15 seconds.
• 8. Ultra-pasteurization pasteurization at 82°C for 3 seconds.
• 9. Hot oil used by some physicians and dentists in the sterilization of
instruments at 160°C for one hour.

• Radiation

• 1. Ultraviolet Irradiation - bacteria is often destroyed by energy

wavelengths of 265 nm damaging the bacterial DNA. UV light has an
energy wavelength of 100-400 nm. This is often used to reduce air
contamination.
• 2. lonizing Radiation x-rays and gamma rays can also be used to
destroy bacteria through the ejection of electrons out of organic
molecules.
• Preservation Methods
- Preservation methods are often employed to control the growth of
microbes on food.

- 1. Drying - used on meats, cereals, fish, fruits, and other food items.
- 2. Salting used on syrups, jams, jellies. Based on the principle of
osmotic concentration and the presence of salt where water comes out
of cells because of exosmosis.
- 3. Low temperatures and freezing - retards spoilage by reducing the
metabolism of microorganisms.
• Chemical Control of Pathogenic Microorganisms
• Chemical methods of controlling pathogenic microorganisms involve
the removal of the microorganisms from an object or body part. Unlike
physical methods, these methods rarely achieve sterilization (Tateishi,
Bzolon & Kitamoto, 1995). Rather, chemical methods achieve a state of
disinfection.

• 1. Disinfectant a chemical agent that removes microorganisms from an

inanimate object.

• 2. Antiseptic - a chemical agent that removes microorganisms from a

living object such as a body tissue

• Phenol Phenol and phenolic compounds are the standard disinfectants

especially useful against Gram+ bacteria.
• Halogens

• Chlorine and iodine are the two halogens most commonly used as chemical
agents. Chlorine is often used in water treatment facilities to keep bacterial
populations at low levels. lodine is usually used as an antiseptic for wounds
as tincture of iodine.

• Alcohol
• Alcohol is an effective antiseptic applied on the skin. It may come as ethyl
alcohol, propyl, butyl, and pentyl alcohol. 70% ethyl alcohol is often used as
antiseptic.

• Alkylating Agents

• Alkylating agents include formaldehyde, ethylene oxide, beta propiolactone,

and glutaraldehyde. Formaldehyde inactivates viruses in vaccine
preparations and in the production of toxoids from toxins. Ethylene oxide
sterilizes plastic materials used in the laboratory.
• Hydrogen
• Peroxide Hydrogen peroxide is used in rinsing wounds, scrapes, and
abrasions.

• Soaps and Detergents

• The pH of most soaps is 8.0 which destroys some bacteria that are
susceptible to alkali solutions.

• Soap is also used for the mechanical washing of the skin surface. It wets,
emulsifies, and solubilize particles that cling to the skin surface.

• Detergents are synthetic chemicals developed for their ability to be

strong wetting agents and surface tension reducers.
• Acids

• The most common acids used as either disinfectants or antiseptics

include benzoic acid, salicylic acid, and undecylnic acid for tinea
infections in the skin. Lactic acid and acetic acid are used as food
preservatives.

• Surgical and Medical Asepsis

• Aseptic Technique Asepsis is the condition of being free from disease-

producing microorganisms. Implies all those procedures that reduce or
eliminate pathogens and their actions or minimize their areas of
existence
• Medical Asepsis

• Medical asepsis includes all of the procedures used to protect the

patient and his environment from the spread of infectious organisms.
Medical asepsis or the aseptic technique is based on maintaining
cleanliness to prevent the spread of pathogenic microorganisms and to
ensure that the environment is free from microbes as much as possible.

• Basic Procedures of Medical Asepsis

• 1. Perform patient care handwash.
• 2. Disinfect materials as required.
• 3. Maintain clean patient care environment.
• 4. Store and handle linen properly.
• Sterile Technique

• Sterile technique refers to the way sterile materials are handled in order
to keep them free of living microorganisms (germs). Sterile technique
prevents contamination.

• Principles of Surgical Asepsis

• Surgical asepsis is known as the sterile technique. It requires strict

compliance ordered and specific procedures which render an area free
from all microorganisms including bacterial spores. Surgical asepsis is
used in the operating room, delivery room during surgical
procedures catheterization, and during dressing changes.
• The following are the principles of surgical asepsis:
• 1. Only a sterile object can touch another sterile object.

• 2. Open sterile packages so that the first edge of the wrapper is

directed away from the worker in order to avoid the possibility of a
sterile wrapper touching unsterile clothing.

• 3. Avoid spilling any solution on a cloth or paper used as a field for a

sterile setup.

• 4. Hold sterile objects above the level of the waist.

• 5. Avoid talking, coughing, sneezing, or reaching over a sterile field or
object.
• 6. Never walk away from or turn your back on a sterile field.

• 7. All items brought into contact with broken skin or used to penetrate
the skin in order to inject substances into the body, or to enter normally
sterile body cavities, should be sterile.

• 8. Use dry sterile forceps when necessary.

• 9. Consider the edge (outer 1 inch) of a sterile field to be contaminated.

• 10. Consider an object contaminated if you have any doubt as to its

sterility.
• Antimicrobial Agents in Therapy

• Antimicrobial Agents Antimicrobial agents are chemicals that eliminate

pathogenic mi organisms in man. These agents can be chemical in origin
such as the sulfonamides or derived from other living microorganisms,
such as the penicillins.

• Antibacterial Agents

• Antibacterial agents are antimicrobials designed specifically against

bacteria. The following are the different drug classes of antibacterial
agents used in the management of bacterial infections.
• 1. Aminoglycosides - kill bacteria through the inhibition of bacterial
protein synthesis.

• a. Used against Gram- microorganisms that produce urinary tract

infections, meningitis, wound infections, and life-threatening
septicemias.

• b. Standard of treatment for nosocomial Gram-infections due to

Acinetobacter, Citrobacter, Enterobacter, Escherichia coli, Kleibsiella,
Providencia, Pseudomonas, Salmonella, and Shigella species.

• c. Agents amikacin, gentamicin, kanamycin, neomycin, netilmicin,

streptomycin, and tobramycin.
• . 2. Cephalosphorins - kill bacteria through the inhibition of bacterial cell wall
synthesis.

• a. First generation effective against Gram+ microorganisms such as

Staphylococcus and Streptococcus species.

• i. Mild antibacterial activity against Gram- microorganisms.

• ii. Agents include cefadroxil, cefazolin, cephalexin, cephalotin, cephradine, and
cephapirin.

• b. Second generation:

• i. Greater antibacterial activity against Gram- microorganisms such as Escherichia,

Kleibsiella, and Proteus species than teh first generation cephalosphorins.

• ii. Agents include cefaclor, cefamandole, cefmetazole, cefonicid, cefoxitin, cefprozil,

loracarbef, and cefuroxime.
• c. Third generation:

• i. Generally less active than the first generation cephalosphorins as

Pseudomonas against Gram+ microorganisms. Especially effective
against penicillinase-producing bacteria aeruginosa.
• ii. Agents include cefdinir, cefixime, cefoperazone, cefotaxime, cefotetan,
cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, and ceftriaxone.

• d. Fourth generation:

• i. Broad spectrum cephalosphorin effective against both Gram+ and

Gram- microorganisms.
• ii. Agents include cefepime. e. Management of some urinary tract
infections, respiratory tract infections, abdominal infections, septicemia,
meningitis, and osteomyelitis.
• 3. Macrolides - act by inhibiting bacterial protein synthesis.

• a. Used for respiratory infections, gastrointestinal tract infections, skin and

soft tissue infections, and sexually-transmitted infections especially if
penicillin, cephalosphorin, and/or tetracycline is contraindicated in the patient.

• b. Agents include azithromycin, clarithromycin, dirithromycin, erythromycin,

and troleandomycin.

• 4. Penicillins - Kills bacteria through the inhibition of bacterial cell wall

synthesis.

• a. Very effective against bacteria that rapidly multiply.

• b. Used to treat otitis media, pneumonia, meningitis, urinary tract infections,
syphilis, and gonorrhea.
• c. It is also used as a prophylactic antibiotic in surgical and dental
procedures especially in patients with history of rheumatic fever.

• d. Agents include amoxicillin, ampicilin, carbenicillin, cloxacillin,

dicloxacillin, mezlocillin, nafcillin, oxacillin, penicillin G potassium or
sodium, penicillin V potassium, piperacillin, and ticarcillin.

• e. Combination drugs include amoxicillin-clavulanate, ticarcillin-

clavulanate, amipicillin-sulbactam, and piperacillin-tazobactam.

• 5. Quinolones - often effective against a wide variety of Gram- and

Gram+ bacteria.

• a. Kill bacteria through the inhibition of the bacterial DNA gyrase activity
to prevent the replication of bacterial DNA.
• b. It is often used in a variety of infections: .

• Ciprofloxacin - treatment of infections caused by organisms that cause

enteritis, gonococci, meningococci, Hemophilus influenzae, Methicilin-
resistant staphylococci and Pseudomonas aeruginosa.

• Levofloxacin broad spectrum activity against Gram+, Gram-,

anaerobic bacteria. Treatment of maxillary sinusitis, acute bacterial
• exacerbations of chronic bronchitis, community-acquired
pneumonia, skin and soft tissue infections, urinary tract infections,
and acute pyelonephritis.

Ofloxacin lesser activity against P aeruginosa but greater activity against

N gonorrheae, C trachomatis, and genital ureaplasma.
• 6. Sulfonamides act through the inhibition of bacterial synthesis of folic acid
leading to bacterial cell death.

• a. Used in the treatment of urinary tract infections and otitis media and as
prophylaxis against streptococcal infections.

• b. Agents include sulfadiazine, sulfamethizole, sulfasalazine, sulfisoxazole,

sulfamethoxazole, sulfamethoxazole-trimethoprim trimoxazole), and
erythromycin-sulfisoxazole.

• 7. Tetracyclines - act by inhibiting bacterial protein synthesis.

• a. Used in the treatment of some sexually transmitted infections, urinary

tract infections, upper respiratory tract infections, pneumonia, and
meningitis, especially if the patient is allergic to penicillin.
• c. Agents include demeclocycline, doxycycline, minocycline,
oxytetracycline, and tetracyline.

• 8. Other antibacterials:

• a. Chloramphenicol - inhibits bacterial protein synthesis. Used in

rickettsial infections, meningitis, and typhoid fever.

• b. Clindamycin inhibits bacterial protein synthesis. Used against Gram-

aerobes and Gram+ and Gram- anaerobes.
• c. Metronidazole has antibacterial, trichomonacidal, and protozoacidal
activity. Treatment of trichomoniasis, giardiasis, amebic dysentery,
amebic liver abscess, and anaerobic bacterial infections.

• d. Vancomycin - inhibits bacterial cell wall synthesis.

• i. Used only against Gram+ bacteria such as Streptococcus,

Staphylococcus, Clostridium difficile, and Corynebacterium.

• ii. Treatment of endocarditis, osteomyelitis, meningitis, pneumonia, and

septicemia.
• Antiviral Agents

• 1. Abacavir inhibits the activity of HIV-1 reverse transcriptase to

prevent viral DNA growth. Management of HIV-1 infection in
combination with zidovudine and lamivudine.

• 2. Acyclovir – inhibits viral cell replication. Management of genital

herpes and threatening mucocutaneous herpes simplex infections in
immunocompromised patients.

• 3. Amantadine - dopaminergic agent that has a specific activity against

Influenza A virus.
• 6. Efavirenz a non-nucleoside reverse transcriptase inhibitor that prevents
the replication of HIV-1. Management of HIV-1 infection in combination with a
protease inhibitor or a nucleoside analog reverse transcriptase inhibitor.

• 7. Famciclovir inhibits viral cell replication. Treatment of genital herpes and

acute herpes zoster.

• 8. Lamivudine a nucleoside reverse transcriptase inhibitor that prevents viral

cell replication. Management of HIV-1 infections and hepatitis B infections.
•
• 9. Oseltamivir - inhibits neuroaminidase to prevent viral cell reproduction
and spread. Management of uncomplicated illness due to influenza.

• 10. Ribavirin - effective against respiratory viruses. Management of viral

infections due to Adenoviridae, Herpesviridae, and Poxviridae viral families.
Also effective against RNA viruses such as influenza, parainfluenza, and
respiratory syncytial viruses.
• 11. Valacyclovir inhibits viral cell replication. Management of acute
herpes zoster shingles in immunocompromised patients.

• 12. Zanamivir inhibits neuraminidase important for viral cell particle

reproduction and spread. Management of uncomplicated acute
influenza infection.

• 13. Zidovudine inhibits viral cell replication. Management of HIV-1

infections especially in patients with confirmed absolute CD4 count of
less than 500/mm³.
• Anti-Fungal Agents
• 1. Topical antifungal agents alters fungal cell membrane to increase
permeability, induce leakage of amino acids and electrolytes, and impair
the uptake of essential nutrients necessary for fungal growth.

• a. Treatment of topical fungal infections due to Tinea pedis, Tinea

corporis, Candida albicans.

• b. Treatment of athlete's foot, jock itch, ringworm, oral thrush, diaper

rash.

• c. Agents include clotrimazole, ketoconazole, miconazole, nystatin.

• 2. Systemic antifungal agents:
• a. Amphoterecin B disrupts cell membrane of fungal cells leading to
loss of cell contents. Treatment of systemic fungal infections, meningitis,
and topical candidiasis.

• b. Fluconazole - inhibits certain fungal metabolic pathways to inhibit

cell wall synthesis. Treatment of cryptococcal meningitis and local and
systemic candidiasis.

• c. Flucytosine inhibits fungal RNA and protein synthesis. Treatment of

candidal septicemia, endocarditis, urinary tract infections, cryptococcal
meningitis, and pulmonary infections.

• d. Ketoconazole - similar to fluconazole and itraconazole.

• Anti-Parasitic Agents

• 1. Anti-protozoal Agents:

• a. Used to kill parasites that cause intestinal amoebiasis, malaria,

toxoplasmosis.

• b. Agents include chloroquine, hydroxychloroquine, metronidazole

• 2. Anti-trematodal Agents:

• a. Used in the treatment of parasitic infections caused by flukes such as

Schistosoma species, Fasciola.
• b. Agents include triclabendazole, ivermectin.

• 3. Anti-nematodal Agents:

• a. Treatment of roundworm infections such as those caused by Ascaris,

hookworms.

• b. Agents include albendazole, ivermectin, thiabendazole,

mebendazole, and praziquantel.
• 4. Anti-cestodal Agents:

• a. Treatment of infections caused by flatworms or tapeworms.

• b. Agents include benzimidazole, albendazole.

• Anti-Tuberculous Agents

• 1. Ethambutol - alters cellular RNA synthesis and phosphate

metabolism.

• 2. Isoniazid - disruption of cell wall and inhibition of replication.

• 3. Rifampin prevents RNA synthesis through inhibition of DNA-

dependent RNA polymerase to prevent key metabolic pathways
required for mycobacterial growth and replication.
 • Chapter 4

• Infection and Host Resistance

• Definition of Terms

• 1. Infection - the replication of organisms in the tissue

• 2. Carrier (colonized individual) - a person in whom organisms are

present and may be multiplying, but who shows no clinical response to
their presence.

• 3. Incubation period - the interval in the preclinical period between the

time at which the causative agent first infects the host and the onset of
clinical symptoms. During this time the agent is replicating.

• 4. Convalescent period - when the patient recovers and returns to

normal but may still continue to be a source of infection even if feeling
better.
• Chain of Infection

• The chain of infection includes the three factors that lead to infection: the
etiologic agent, the method of transmission, and the host (Remington & Klein,
1993).

• Mechanism of Bacterial Infectious Disease Processes

• We are constantly exposed to bacteria (including air, water, soil and food).
Normally due to our host defenses most of these bacteria are harmless.

• In compromised patients, whose defenses are weakened, these bacteria

often cause opportunistic infectious diseases when entering the bloodstream
(after surgery, catheterization or other treatment modalities).
• Transmission and Adhesion

• In transmission, specific bacterial species (or strains within a species) initiate

infection after being transmitted by different routes to specific sites in the
human body.

• Bacterial infections are usually initiated by adherence of the microbe to a

specific epithelial surface of the host. If not, the organism is removed by
peristalsis and defecation, ciliary action, coughing and sneezing, or urination.

• Penetration and Spread

• Some bacterial pathogens reside on Epithelial surfaces. Other species are

able to penetrate these barriers but remain locally. Others pass into the
bloodstream or from there onto other systemic sites. This often occurs in the
intestine, urinary tract and respiratory tract and much less through the skin.
• Mechanism of Viral Infectious Disease Processes

• Initiation of Infection

• To infect a cell, the virus must attach to the cell surface, penetrate into the cell, and
become sufficiently uncoated to make its genome accessible to viral or host
machinery for transcription or translation.

• Viral Pathogenesis

• Pathogenesis is the process by which an infection leads to disease. Pathogenic

mechanisms of viral disease include the following (Remington & Klein, 1993):

• 1. Implantation of virus at the portal of entry - virions implant onto living cells mainly
via the respiratory, gastrointestinal, skin-penetrating, and genital routes although
other routes can be used.
• Viral Pathogenesis

• Pathogenesis is the process by which an infection leads to disease.

Pathogenic mechanisms of viral disease include the following
(Remington & Klein, 1993):

• 1. Implantation of virus at the portal of entry - virions implant onto living

cells mainly via the respiratory, gastrointestinal, skin-penetrating, and
genital routes although other routes can be used.
• 2. Local replication - most virus types spread among cells extracellularly,
but some may also spread intracellularly.

• 3. Spread to target organs (disease sites) - viral spread can be

facilitated in one of two ways:

• a. Viremic the most common route of systemic spread from the portal of
entry through the circulation, which the virus reaches via the lymphatics.

• b. Neural dissemination via nerves usually occurs with rabies virus and
sometimes with herpesvirus and poliovirus infections.
• 4. Spread to sites of shedding of virus into the environment - although the
respiratory tract, alimentary tract, urogenital tract and blood are the most
frequent sites of shedding, diverse viruses may be shed at virtually every site.

• Mechanism of Fungal Infectious Disease Processes

• Entry

• Fungi infect the body through several portals of entry.

• The first exposure to fungi that most humans experience occurs during birth,
when they encounter the yeast C. albicans while passing through the vaginal
canal.

• Another fungus, Malassezia furfur, is common in areas of skin rich in

sebaceous glands.
• Fungi rarely cause disease in healthy, immunocompetent hosts, even
though we are constantly exposed to infectious propagules. It is only
when fungi accidentally penetrate barriers such as intact skin and
mucous membrane linings.

• Fungi gain access to host tissues by traumatic implantation or

inhalation.

• Fungal Factors
• Most of the fungi that infect humans and cause disease are classified
by tissue or organ levels that are primary sites of colonization.

• 1. Superficial fungal infections involve only the outermost layers of the

stratum corneum of the skin or the cuticle of the hair shaft
• 2. Dermatophyte infections are caused by fungi that colonize skin, hair,
and nails on the living host.

• 3. Subcutaneous mycoses involve fungi that are abundant in the

environment and have a low degree of infectivity.

• 4. Systemic mycoses are fungi that have the innate ability to cause
infection and disease in humans and other animals. The primary site of
infection is the respiratory tract.
• Mechanisms of Helminthic Infectious Processes

• Transmission of Infection Helminthes are transmitted to humans in

many different ways. These can include the following:

• 1. Accidental ingestion of infective eggs or larvae (some hookworms);

• 2. Presence of larvae that actively penetrate the skin (hookworms,
schistosomes, Strongyloides);
• 3. Larvae are contained in the tissues of the intermediate host and are
taken in when a human eats that host (Clonorchis in fish, tapeworms in
meat and fish, Trichinella in meat).

• The levels of infection in humans therefore depend on standards of

hygiene (as eggs and larvae are often passed in urine or feces).
• Host Factors Influencing Susceptibility

• Human behavior is a major factor influencing susceptibility to infection.

If the infective stages of helminthes are present in the environment,
then certain ways of behaving, particularly with regard to hygiene and
food, will result in greater exposure.
• Non-Specific Host Responses

• Antigen

• An antigen is any substance that elicits an immune response (Seeley,

2003). It can be a virus, a bacteria, or a simple foreign object in a certain
body part where it is not supposed to be found.

• Innate Immunity

• The innate immunity system is what a person is born with and it is

nonspecific. It is genetically based and is passed on to children.
• Physical Barriers or Mucosal Immunity

• An intact and continuous skin is considered to be the body's first line of

defense against microbial infections. Other non-specific mechanisms that act
to provide as physical barrier or flushing mechanism include the following:

• Ciliary action in the respiratory airways;

• Coughing and sneezing:
• Flushing action of tears, saliva, and urine;
• Sloughing off of skin;
• Sticky mucus in respiratory and gastrointestinal tracts;
• Acid pH (<7.0) of skin secretions;
• Sebum secreted by hair follicles that contains lactic acid and fatty acids;
• Slightly acidic vaginal secretions (after the onset of menses)
• Inflammation

• Inflammation is nonspecific response to any trauma occurring to tissues. It is

accompanied by signs and symptoms that include heat, swelling, redness,
and pain. Inflammation mobilizes components of the immune system area
sets into motion repair mechanisms and encourages phagocytes to come to
the area and destroy any microorganisms present.

• Fever

• Fever is considered a nonspecific defense mechanism because it develops in

response to numerous traumas. Fever is initiated by circulating substances
called pyrogens, which affect the brain's hypothalamus and cause the latter
to raise the temperature. Although excessive fever can be dangerous, fever is
believed to have a beneficial role because it retards the growth of
temperature-sensitive microorganisms and it increases the metabolism of
body cells while stimulating the immune reaction.
• Vaccines in the Elimination of Disease

• Vaccines

• Vaccination is the administration of an antigenic material - known as a

vaccine - to produce immune protection against a disease.

• Active Immunization

• In active immunization, the person is given an antigen usually a part of a

virus or bacteria - to stimulate the immune system to produce antibodies
against the pathogen.

• The person is only given enough antigens to stimulate the immune system
but not enough to elicit a full clinical course of the disease.
• Passive Immunization

• Passive immunization is similar to active immunization in that the

substance is given in commercially-prepared forms. However, actual
antibodies are administered instead of antigens that stimulate the
production of antibodies.

• Types of Vaccines

• The four main types of vaccines that are currently in clinical use
presenting a foreign antigen to the immune system in order to evoke an
immune response are as follows (Remington & Klein, 1993):
• 1. Inactivated - consists of virus particles which are grown in culture and
then killed using a method such as heat or formaldehyde.

• a. The virus particles are destroyed and cannot replicate.

• b. Viral capsid proteins are intact enough to be recognized and

remembered by the immune system and evoke a response.

• c. Since the properly produced vaccine does not reproduce, booster

shots are required periodically to reinforce the immune response.

• 2. Attenuated - live virus particles with very low virulence are

administered.
• a. The virus will reproduce, but very slowly.

• b. Since they reproduce and continue to present antigen beyond the

initial vaccination, boosters are required less often.

• 3. Virus-like particle - vaccines that consist of viral protein(s) derived

from the structural proteins of a virus.

• 4. Subunit vaccine - works by presenting an antigen to the immune

system without introducing viral particles, whole or otherwise.
 • Chapter 5

• Normal Microbial Flora and Pathogenic Bacteria

• Normal Human Microbial Flora
.
• Microorganisms of the normal flora may aid the host, may harm the host, or
may exist as commensals. Even though most elements of the normal
microbial flora inhabiting the human skin, nails, eyes, oropharynx, genitalia,
and gastrointestinal tract are harmless in healthy individuals, these
organisms frequently cause disease in compromised hosts.

• Normal Flora of Skin

• 1. Staphylococcus epidermidis - the major inhabitant of the skin, and in some

areas it makes up more than 90 percent of the resident aerobic flora.
• 2. Staphylococcus aureus - present in the nose and perineum.
• 3. Micrococci not as common as staphylococci and diphtheroids; however,
they are frequently present on normal skin.
• Respiratory Tract Flora

• The normal flora of the oral cavity and the upper respiratory tract
include the following:

• 1. General distribution - Bordetella parapertussis, Eikenella corrodens.

2. Nose - Cardiobacterium species and Staphylococcus aureus.
• 3. Nasopharynx - Acinetobacter species, Campylobacter sputorum,
Hemophilus species,.
• 4. Pharynx - Candida albicans, Hemophilus parainfluenzae, Hemophilus
paraphrophilus,.
• 5. Upper respiratory tract - Kingella species, Peptococcus species, and
Streptococcus pyogenes.
• Gastrointestinal Tract Flora

• The normal flora of the gastrointestinal tract includes the following:

• 1. General distribution - Bacteroides fragilis, Campylobacter coli.

• 2. Mouth - Actinomyces species, Aggregatibacter
actinomycetemcomitans.

• 3. Gingiva - Bacterionema matruchotii.

• 4. Saliva- Lactobacillus species

5. Sputum - Lactobaciliter freundi, Gordonia bacterium, Mycobacterium

chelonae, and Neisseria sicca.
• Gram Positive Bacteria Pathogenic to Man

• Bacteria Gram positive bacteria stain blue or violet by Gram staining because
they are able to retain the crystal violet stain because of their peptidoglycan
cell wall.

• Actinomyces

• Actinomyces are characterized by contigious spread, suppurative and

granulomatous inflammation, and the formation of multiple abscesses and
sinus tracts that often discharge sulfur granules.

• Clostridium
• Clostridia are rod-shaped obligate anaerobes that produce endospores. They
are involved in a variety of human diseases, the most important of which are
gas gangrene tetanus, botulism, pseudomembranous colitis and food
poisoning.
• 2 Clostridial species that produce tetanus include C tetani.

• a. Tetanus is a severe disease caused by the toxin of C tetani.

• i. This organism grows in a wound and secretes a toxin that invades

systemically and causes muscle spasms.

• ii. Cramping and twitching of muscles around a wound symptom -

initial symptom

• iii. No fever but sweats profusely and begins to experience pain,

especially in the area of the wound and around the neck and jaw
muscles (trismus).
• iv. Portions of the body may become extremely rigid, and opisthotonus
(a spasm in which the head and heels are bent backward and the body
bowed forward) is common.

• v. Complications include fractures, bowel impaction, intramuscular

hematoma, muscle ruptures, and pulmonary, renal, and cardiac
problems.

• Prevention and control include the following:

• - Meticulous wound care using aseptic technique

• - Prophylactic injections of tetanus toxoid
• - Local debridement, after the patient's spasms are controlled by
benzodiazepines.
• - Penicillin or metronidazole is usually administered to kill the bacteria, but
may not be a necessary adjunct in therapy.

• - Human tetanus immunoglobulin (HTIG) injected intramuscularly: dosage

recommendations vary from 500 IU in a single intramuscular injection to
3000-6000 IU injected intramuscularly in several sites.

• - Supportive measures, such as respiratory assistance and intravenous fluids.

• Corynebacterium

• Corynebacteria are rod-shaped, aerobic or facultative anaerobic, non-spore-

forming and non-motile bacteria that are straight or slightly curved. Its most
important specie, C diphtheriae, causes diptheria. Diphtheria is most
commonly an infection of the upper respiratory tract and causes fever, sore
throat, and malaise.
• 3. Prevention and control include the following:

• a. Adequate immunization with diphtheria toxoid: formaldehyde-inactivated

diphtheria toxin that remains antigenically intact.

• b. Penicillin and erythromycin

• Enterococcus

• Enterococci are a common commensal of the intestines. These are

facultative anaerobic bacteria that occur in pairs (diplococci) or short chains.

• It is often the cause of urinary tract infections, bacteremia, bacterial

endocarditis, diverticulitis, and meningitis. Enterococci exhibit a high level of
antibiotic resistance even to Beta-lactam and aminoglycoside antibiotics.
• Staphylococcus

• Staphylococci are facultative anaerobic commensals of the skin and the

nares. They appear round and form in grape-like cluster. Most of the
species are harmless and reside normally on the skin and mucus
membranes. Pathogenic species produce toxins or penetrate and
invade tissues to cause disease in humans.

• 1. S. aureus - major cause of nosocomial and community-acquired

infections.

• a. Superficial skin infections - boils, furuncles, styes, impetigo

• b. Serious infections in immunocompromised or immunosuppressed

patients - pneumonia, deep abscesses, meningitis.
• Streptoccocus

• Streptococci are non-motile and non-spore-producing spherical bacteria

that causes scarlet fever, rheumatic heart disease, glomerulonephritis,
and pneumococcal pneumonia.

• 1. Group A streptococcus include S pyogenes which is a beta-hemolytic

streptococci, meaning it completely destroys blood cells.

• a. Leading cause of streptococcal pharyngitis or "strep throat", acute

rheumatic fever, scarlet fever, acute glomerulonephritis, and necrotizing
fasciitis.

• b. May also produce sinusitis, otitis, joint or bone infections, meningitis.

• Gram Negative Bacteria Pathogenic to Man

• Overview of Gram Negative Bacteria

• Gram negative bacteria do not retain the crystal violet stain such that
the final stain is characterized by a pink or pale red stain.

• Bordetella

• Bordetella pertussis belongs to the genus Bordetella in the family

Alcaligenaceae, which contains several species of closely related
bacteria with similar morphology. В pertussis and B parapertussis cause
whooping cough (pertussis) in humans.
• 1. Clinical manifestations:

• a. Incubation period of 1 to 2 weeks

• b. Catarrhal phase-1-2 weeks

• i. Whooping cough begins
• ii. Low-grade fever, rhinorrhea, and progressive cough; the patient is highly infectious

• Prevention and control include the following:

• a. Tetracycline, erythromycin, and chloramphenicol

• b. Human hyperimmune pertussis globulin is still used occasionally.

• c. Susceptible children (unimmunized children without a history of whooping cough) should

have no contact with pertussis patients during the first 4 weeks of illness, although such
isolation is often difficult.
• Chlamydia

• Chlamydia is a group of obligate intracellular parasitic bacteria that are the most
common cause of bacterial sexually transmitted infectious blindness. Chlamydial
species of medical interest include C trachomatis, C pneumoniae, and C psittaci.

• - Tetracycline and erythromycin are the antibiotics commonly used to treat

chlamydial infections in humans.

• Escherichia
• Escherichia are non-spore-forming, facultatively anaerobic and rod-shaped bacteria
which are normal inhabitants of the gastrointestinal tract. As long as these bacteria
do not acquire genetic elements encoding for virulence factors, they remain benign
commensals.

• 1. There are three groups of E coli are associated with diarrheal diseases.

• a. ETEC (enterotoxigenic) - E coli strains that produce enterotoxins.

• b. EIEC ( enteroinvasive) – E coli strains have invasion factors and cause tissue
destruction and inflammation resembling the effects of Shigella.

• c. EPEC (enteropathogenic) – E coli associated with outbreaks of diarrhea in

newborn nurseries, but produce no recognizable toxins or invasion factors.

• Helicobacter

• Helicobacter is a group of microaerophilic bacteria that lives in the lining

of the upper Gl tract and has been implicated in the development of
peptic ulcers, chronic gastritis duodenitis, and gastric cancer.
Helicobacter pylori is the type specie of the genus.
• Neisseria

• The Neisseria family of bacteria includes Neisseria, Moraxella, Kingella,

and Acinetobacter with Neisseria as the only significant contributor of
human pathogens. Neisseria gonorrhea and N meningitidis are the
agents of gonorrhea and acute bacterial meningitis, respectively.

• Most common symptom of uncomplicated gonorrhea:

• i. Discharge that may range from a scanty, clear, or cloudy fluid to one
that is copious and purulent

• ii. Very often with dysuria

Prevention and control include:

• a. Use of condoms in preventing N gonorrhea transmission

• b. N gonorrhea antibiotic therapy with penicillin, tetracycline,
erythromycin, cephalosphorins, and quinolones
• c. N meningitidis antibiotic therapy with sulfonamides, rifampin

• Pseudomonas

• Most pseudomonads known to cause disease in humans are associated

with opportunistic infections.
• 1. Species include the following:

• a. P. aeruginosa produces localized infection following surgery or burns

results in a generalized and frequently fatal bacteremia.

• i. Urinary tract infections following introduction of P aeruginosa on

• catheters or in irrigating solutions. .

• ii. Cystic fibrosis patients are chronically colonized with P aeruginosa.

•
• iii. Necrotizing pneumonia may occur in other patients following the use of
contaminated respirators.

• iv. Pseudomonas aeruginosa can cause severe corneal infections following

eye surgery or injury.
• 3. Prevention and control include the following:

• a. Proper isolation procedures, aseptic technique, and careful cleaning

and monitoring of respirators, catheters, and other instruments.

• b. Topical therapy of burn wounds with antibacterial agents such as

mafenide or silver sulfadiazine, coupled with surgical debridement.

• c. Gentamicin, tobramycin