LEC 1

MICROBIOLOGY – The study of very small living organisms

Micro = very small
Bios = living organisms
Logy = the study of

Various categories of microbes:

 Viruses
 Bacteria
 Archaea
 Protozoa
 Certain types of algae
 Fungi

GERMS - It is a non-scientific term for microbes that causes disease

 Only about 3% of known microbes are capable of causing disease (pathogenic)
 Some nonpathogens are beneficial to us, whereas others have no effect at all

WHY STUDY MICROBIOLOGY?

 We have, living on and in our bodies, approximately 10x as many microbes as the
total number of cells.
 Some of the microbes that colonize our bodies are known as opportunistic pathogens.
 Microbes are essential for life.
 Many microbes are involved in the decomposition of dead organisms
(saprobes/saprophytic) and the waste products of living organisms.
 Some microbes are capable of decomposing industrial wastes.
 Many microbes are involved in elemental cycles.

Algae and bacteria - serve as food for tiny animals.

 Some microbes live in the intestinal tracts of animals, where they aid in the digestion of
food and, in some cases, produce substances that are of value to the host animal.
 Many microbes are essential in various food and beverage industries.

 Microbes are essential in the field of genetic engineering.

 Microbes have been used as “cell models”
 Microbes cause two categories of diseases: Infectious diseases and Microbial
intoxications.
EARLIEST KNOWN INFECTIOUS DISEASES

1. EGYPT, 3180 BCE

 Pestilence and Plague

2. GREECE, 1900 BCE

 Bubonic Plague (Near the end of Trojan War)

3. THEBES, EGYPT 1500 BCE

 Epidemic Fevers (Through writings in Ebers Papyrus found in tombs)

4. CHINA, 1122 BCE

 Smallpox

5. Epidemics of plague in the following places and time:

 ROME 790, 710, and 640 BCE
 GREECE 430 BCE

PIONEER MICROBIOLOGISTS & THEIR CONTRIBUTIONS

1. ANTON VAN LEEUWENHOEK

 First person to discover microscope and the presence of bacteria and spirochetes in the
mouth
 Saw a minute moving objects and called them “Little Animalcules”

2. JOHN TURBEVILLE NEEDHAM

 Irish priest
 Observed the appearance of microorganisms in putrefying meat
 Basis is the discovery of Anton van Leeuwenhoek
 Postulates the Theory of ABIOGENESIS
 Tried to establish that tiny microscopic animals, or “anguilles” in his own words, can be
developed spontaneously by natural forces, yet in a sealed container

3. LAZZARO SPALLANZANI
 Disproves the Theory of Abiogenesis by series of experimentations and became the
basis for Theory of Biogenesis
 Boiled beef broth for an hour, sealed the flasks and observed no appearance of
microorganisms and disproved the theory of spontaneous generation or abiotic origin of
life
 Said that every form of life takes its origin from their parents, germ cells, or seeds

4. RUDOLF VIRCHOW
 Challenged the case of spontaneous generation with the concept of BIOGENESIS
 Living cells can only arise from preexisting living cells

5. LOUIS PASTEUR
 Developed certain sterilization technique that kills bacteria at 40 – 60oC
(Pasteurization)
 Coined as the “Father of Microbiology”
 Postulates “Germ Theory of Disease”
o Diseases are caused by microorganisms

6. ROBERT KOCH
 Discovered the tubercle bacilli
 Coined as the “Father of Practical Bacteriology”
 Koch’s Postulates
 To link specific bacterial species with particular disease

*Koch’s Postulates
 The microorganism should be found in ALL cases of the disease in question, and its
distribution in the body should be in accordance with the lesions observed.
 The microorganism should be grown in pure culture in vitro for several generation
 When such a pure culture is inoculated into susceptible animal species, the typical
disease must result.
 The microorganism must again be isolated from the lesions of such experimentally
produced disease.

7. JOSEPH LISTER
 “Father of Antiseptic Surgery”
 Deeply interested in the prevention of post- operative sepsis
 Introduced CARBOLIC ACID (phenol) to sterilize surgical instruments and to clean
wounds

8. ERNST HEINRICH PHILIPP AUGUST HAECKEL

  Discovered, described, and named thousands of new species
 Mapped a genealogical tree relating all life forms
 Classified microorganisms under the Kingdom Protista

14 CAREER PATHS FOR MICROBIOLOGISTS

1. Biotechnologists
- Biotechnologists work in the agriculture, environment, food, and clinical industries.
- They manipulate the genes of a microorganism.
- An environmental biotechnologist might develop microorganisms that clean polluted water.
- A medical biotechnologist could produce medicines using techniques such as cell culture.

2. Clinical Laboratory Scientists (Medical Technologists)

- They work in many areas including clinical, veterinary, and state and national health
laboratories.
- They analyze blood, urine, tissue and other body specimens in order to determine the cause
of an infection in a patient and what antibiotics are effective in treating the infection.
- This field is evolving as genetic and mass spectrophotometry techniques become common.
- Several employees at Microbiologists have backgrounds in clinical science.

3. Food Scientist and Technologists

- Food microbiologists test food and beverage products for pathogens such as Salmonella and
Listeria monocytogenes and for spoilage microorganisms such as lactic acid bacteria.
- Food microbiologists help their company meet standards for product safety and food quality.

4. Immunologists
- Investigates how a body defends itself against disease.
- Research areas include biodefense, biofilms, genetics, HIV/AIDS, immunologic mechanisms,
respiratory pathogens (including influenza) and vaccine development.

5. Mycologists
- Mycologists study disease-causing fungus and fungus that produce antibiotics.
- They often work in clinical, pharmaceutical, and research laboratories.
- They also work in environmental laboratories that analyze indoor air for mold spores.

6. Parasitologists
- They investigate how parasitic microorganisms infect living hosts, reproduce and cause
disease.

7. Personal Care Product and Cosmetic Scientists and Technologists

- Are responsible for ensuring the safety of products like shampoo, eye shadow, and baby
wipes.
- They test products for disease-causing microorganisms such as Pseudomonas aeruginosa.

8. Pharmaceutical Scientists and Technologists

- Are responsible for ensuring the safety of pharmaceutical products.
- Technologists test raw materials and finished products for disease-causing microorganisms
such as Staphylococcus aureus and Burkholderia cepacia.
- Scientists research and develop new drugs and therapies.

9. Sales
- Some microbiologists find sales to be a rewarding career.
- Because of their strong background in science, they are able to help customers choose the
best microbiology product for their situation.

10. Science Writers

- Readers appreciate well-written articles on current developments in microbiology.
- Strong communication skills combined with a background in microbiology can be the
foundation of a successful career as a science journalist or blogger.

11. Teachers and Professors

- Teachers and professors share their passion for microbiology by educating high school,
university, and post graduate students.
- They are responsible for creating and executing lesson plans that teach students the
characteristics of microorganisms and the latest developments in the field of microbiology.

12. Technical Support Specialists

- They provide technical assistance to customers using a manufacturer’s products.
- The Technical Support Department helps customer choose microorganism strains, instructs
customers on the use of products, and helps with hands-on customer and distributor trainings.

13. Virologists
- Virologists study viruses that affect humans, animals, insects, bacteria, fungi, and plants in
community, clinical, agricultural, and natural environments.
- They develop vaccines for influenza and other diseases.

14. Water Quality Laboratory Technicians

- Municipalities, water treatment plants, and state and local agencies need technicians to test
drinking water, treated water, and recreational water.
- Often the laboratories are testing for E. coli, an indicator of fecal contamination and a warning
sign that water-borne pathogens such as Salmonella and Shigella may be present.

LEC 2
CELL STRUCTURE AND TAXONOMY

Eukaryotes
 eu (true) and caryo (nucleus) ; they have a true nucleus because their DNA is enclosed
in a nuclear membrane.
Prokaryotes
 organisms with a primitive type of nucleus because it does not contain nucleoplasm
nor a nuclear membrane.

EUKARYOTE PROKARYOTE
SIZE 10-30m in diameter (most Around 2-10m in diameter; 10
plants & animals); 10 times times smaller
larger
CELL WALL If present, it may contain Present; contains peptidoglygan
cellulose (algae & plants) and
chitin (fungi)
GLYCOCALYX absent Present (slime layers and
capsules)
CELL MEMBRANE present present
NUCLEUS Present; enclosed in nuclear Without nuclear membrane
membrane
DNA Linear DNA molecules and Single, long, supercoiled, circular
proteins DNA molecule;
No proteins
MICROTUBULES & Present Absent
ORGANELLES
 FLAGELLA / CILIA Has a complex structure if Flagella, when present, have a
present simple, twisted protein structure;
no cilia
RIBOSOMES Larger and bound to Smaller; free swimming in the
membranes (80S) cytoplasm (70S)
PHOTOSYNTHESIS Present (in plants) Present in cyanobacteria
REPRODUCTION Mitosis or meiosis Binary fission

EUKARYOTIC CELL STRUCTURE

CELL MEMBRANE
- “skin” of the cell
- Regulates the passage of nutrients, waste products, and secretions into
and out of the cell.
-The cell membrane has the property of “selective permeability”

NUCLEUS
- “True nucleus”
- Controls the functions of the entire cell and can be thought of as the “command center” of
the cell
- Has three components:

Nucleoplasm – the gelatinous matrix or base material of the nucleus

Chromosomes – contains the DNA molecules and proteins
Nuclear membrane - the “skin” of the nucleus

CYTOPLASM
- A semi-fluid, gelatinous, nutrient matrix
- Contains various organelles:

 Endoplasmic reticulum – highly convoluted transport network

 Ribosomes – consists mainly of rRNA and protein; play a role in protein
synthesis
 Consists of two subunits: 60S subunit and 40S subunit
  Golgi complexes – completes the transformation of newly synthesized proteins
to mature vesicles
 Mitochondria – referred to as the “powerhouse”, “power plants”, or “energy
factories” of the cells; energy is released from glucose molecules and other
nutrients to drive other cellular functions
 Centrioles
 Cytoskeleton – consists of microtubules, microfilaments, and intermediate
filaments; gives strength, support, and shape of the cell
 Lysosomes & Peroxisomes – aid in breaking down worn out parts of the cell and
is responsible for cell autolysis

CELL WALL
- Not all eukaryotic cells possess cell wall
- An external structure that provide rigidity, shape, and protection of the cell
- May contain cellulose, pectin, lignin, chitin, and some mineral salts

FLAGELLA AND CILIA

- Flagella
 The whipping motion of the flagella enables
cells to “swim” through liquid environment
 Flagella are whip-like
- Cilia
 Are also organelles of locomotion
 Tend to be shorter, thinner, and more numerous than flagella

STRUCTURAL PARTS OF THE PROKARYOTIC CELL

GLYCOCALYX
- slimy, gelatinous material produced by the cell membrane and secreted outside of the cell
wall.

2 types of Glycocalyx:
SLIME LAYER
- not highly organized; not firmly attached to the cell
wall;
- easily detaches from the cell wall and drifts away.
- enables bacteria to glide or slide along solid surfaces
CAPSULE
- highly organized and firmly attached to the cell wall
- usually consist of polysaccharide
- serve an antiphagocytic function, protecting the encapsulated bacteria from being
phagocytized by phagocytic WBCs
- virulence factor

OPSONIZATION
- The process by which antibodies (directed against bacterial capsules) bind with the capsule
to facilitate phagocytosis of the encapsulated bacteria by macrophages
- The macrophages or phagocytes bind with the Fc portion of the antibodies allowing them to
engulf the bacteria.

CELL WALL
- provides rigidity, strength and protection
- imparts shape of the organism (cocci or bacilli)
- primarily composed of peptidoglycan or murein layer

The peptidoglycan is composed of alternating subunits of 2 carbohydrate-containing amino

acids joined together by peptide links.

The 2 CHO-containing amino acids are: N- acetylglucosamine and N-acetylmuramic acid.

The functions of the cell membrane includes:

CELL MEMBRANE (lipid bilayer)

 transport of solutes into and out of the cell

 houses enzymes involved in outer membrane synthesis, cell wall synthesis, and in
the assembly and secretion of extracytoplasmic and extracellular substances
 generation of ATP and cell motility
 mediates chromosomal segregation during replication
 houses molecular sensors that monitor chemical and physical changes in the
environment

CHROMOSOME
- consists of a single, long, supercoiled, circular DNA molecule that serves as the control center
of the cell
- no nucleoplasm nor nuclear membrane; the chromosome is suspended or embedded in the
cytoplasm

NUCLEOID – the DNA-occupied space within a bacterial cell

PLASMID – extrachromosomal DNA; small, circular molecules of double- stranded DNA that are
not part of the chromosome

CYTOPLASM
- consists of a complex mixture of all the materials required by the cell for its metabolic
functions (water, enzymes, dissolved oxygen, waste products, essential nutrients,etc.)

CYTOPLASMIC PARTICLES
- includes ribosomes (polyribosomes) and granules (metachromatic )
- Metachromatic granules are large inclusion which stores inorganic phosphate that can be
used in the synthesis of ATP. They are also known as volutin.

PILI (pilus) or FIMBRIAE (fimbria)

- hair like structures most often observed on gram negative bacteria
- composed of polymerized CHON molecules called PILIN
- thinner than flagella; rigid structure; not associated with motility
- 2 types: COMMON PILI and SEX PILI
- virulence factor

CONJUGATION
- The passing of genetic information between from one bacteria to another, often via often
- A bacterial cell possessing a sex pilus (called a DONOR CELL) is able to attach to another
bacterial cell (called a RECIPIENT CELL) by means of the sex pilus
- Genetic material (usually in the form of plasmid) is then transferred through the hollow sex
pilus from the donor cell to the recipient cell

SPORES
- referred to as endospores; for perpetuation only
- means of survival when bacterial moisture and nutrient supply is low
- spores are formed through the process called SPORULATION
- virulence factor formed by Bacillus and Clostridium
SPORULATION
 - A copy of the bacterial chromosome will be made.
- The plasma membrane begins to invaginate enclosing the chromosome duplicate with
some cytosol.
- A multilayered protective coat (consisting of membranes, peptidoglycan mesh, a
keratin-like CHON and an outer layer called exosporium) will be formed around the
enclosed chromosome.
- The bacterium will release the endospore through cell lysis.
- When the spore lands on a nutrient-rich environment, it germinates, and a new
vegetative bacterium emerges.

AXIAL FILAMENTS
- organ of locomotion for spirals
- alternate contraction and expansion causes the movement of the bacterium

FLAGELLA (flagellum)
- protein filaments that enable bacteria to move (virulence factor)
- 10-20 nm thick so it is not visible under the compound microscope
- it is attached to the bacteria by a basal body. The basal body spins around and spins the
flagellum causing it to undulate in a coordinated manner.

FLAGELLA
Monotrichous Amphitrichous

Lophotrichous Peritrichous
MICROBIAL TAXONOMY

The science of classification...

 From Ancient Greek words: Taxis meaning “arrangement” and Nomia meaning
“method”
 Taxonomy is the branch of science dealing with naming, grouping of organisms on
the basis of the degree of similarity and arranging them in an order on the basis of
their evolutionary relationship
 Taxonomy is related to nomenclature, classification, and phylogeny of organisms

AIMS OF TAXONOMY
- Classification of organisms
- Show relationships among organisms
- Way to provide universal identification of an organism

BACTERIAL TAXONOMY
- the science of classification of living organisms
- was established based on the binomial system developed Carolus Linnaeus

It consists of 3 separate but interrelated areas:

- Classification - arrangement of organisms into taxonomic groups based on similar
morphologic, physiologic, and genetic traits
- Nomenclature - assignment of names to the various taxa according to international rules, set
by International Code of Nomenclature of Bacteria (ICBN) or Bacterial Code (BC)
- Identification - process of determining whether an isolate belongs to one of the established,
named taxa or represents a previously unidentified species

SEQUENCE OF TAXONOMIC GROUPS (TAXA)

 Kingdom
 Division or Phylum
 Class Order
 Family Genus
 Species

MICROBIAL CLASSIFICATION (Binomial System)

 Only the first letter of the genus is written in CAPITAL letters.
 The rest of the letters of the genus and specific epithet is written in small letters.
  The genus may be abbreviated. (Ex. E. coli )
 “sp.” is used to designate a single species.
 “spp.” is used to designate more than one species.

EXAMPLE OF CLASSIFICATION

Family Microcococceae
Genus Staphylococcus
Species aureus
Accepted abbreviation S. aureus
Informal staphylococci

LAB 1
MICROBIOLOGY LABORATORY

Biohazards - are biological substances that may present a health risk to humans.

Clinical specimens are potential biohazards to laboratory personnel because the specimen may
contain pathogenic strains of microorganisms including parasites.

• Guidelines ensure safety in the laboratory have been compiled by several agencies, including :
a. the Occupational Safety and Health Administration (OSHA),
b. the Centers for Disease Control and Prevention (CDC), and
c. the Joint Commission (JC).

• Of particular significance is the publication of standards for bloodborne pathogens by OSHA,

which were published in the Federal Registry in 1991.

SAFETY PROCEDURES AND PRECAUTIONS

• The microbiology laboratory, whether in a classroom or a working diagnostic laboratory, is a

place where cultures of microorganisms are handled and examined.
• Even if the microorganisms you are studying are not usually considered pathogenic (disease
producing), any culture of any organism should be handled as if it were a potential pathogen.
• It is important to prevent contamination of your hands, hair, and clothing with culture
material and also to protect your neighbors from such contamination.
• The importance of asepsis and proper disinfection is stressed throughout this manual and
demonstrated by the experiments.

UNIVERSAL PRECAUTIONS AND STANDARD PRECAUTIONS

• Universal precautions are recommendations that describe the handling of clinical specimens
by health care personnel, first introduced by the CDC in 1987.
• According to Clinical Laboratory and Standards Institute (CLSI), universal precautions are a
set of preventive measures designed to reduce the risk of transferring HIV, hepatitis B virus, and
other bloodborne pathogens in the health care setting.
• Universal precautions apply to all human blood and all other body fluids that contain visible
blood.
• However, universal precautions do not apply to feces, nasal secretions, saliva except in the
dental setting, sputum, sweats, tears, urine, and vomitus unless they contain visible blood.

Universal Precautions
1. Blood and body fluids from all patients must be considered infectious. The medical student
must assume that all patients, and thus, specimens are infectious
2. All specimens must be placed in secure containers that should not leak during transport.
3. All persons processing blood and body fluid should wear gloves.
4. Students must discard gloves and wash their hands when they are done processing
specimens.
5. Needles should only be used when there is no alternative.
6. All spills and accidents should be reported to the laboratory instructor.
7. Laboratory work areas should be decontaminated with an appropriate chemical agent after a
spill.
8. Contaminated materials, including containers used in laboratory tests, should be
decontaminated and disposed of according to institutional policy.
9. Hand-washing is required after completion of laboratory duties.

PERSONAL PROTECTIVE EQUIPMENT

• Personal protective equipment (PPE) is a significant part of standard precautions;

• OSHA requires that employees must be protected from hazards encountered during
work.
• In the laboratory, PPE includes protective laboratory clothing, disposable gloves, eye
protection, and face masks.

SUMMARY OF BIOSAFETY LEVELS (BSLs) FOR INFECTIOUS AGENTS

• Biosafety Level 1 (BSL 1): No known pathogenic potential for immunocompetent individuals.
• Typical examples include Bacillus subtilis.
• Most undergraduate laboratory course operate under BSL 1 precautions.
• Precautions include adherence to standard laboratory techniques.
• Biosafety Level 2 (BSL 2): Level 1 practice plus laboratory coats, protective gloves, limited
access, decontamination of all infectious waste, and biohazard warning signs.
• Apparatus includes partial containment equipment (such as classes I and II biological
safety cabinets) when procedures may lead to the production of infectious aerosols.
• This category includes the most common microorganisms associated with laboratory-
acquired infections, including HBV, HIV, Staphylococcus, and enteric pathogens such as
Salmonella and Shigella.
• Biosafety Level 3 (BSL 3): Level 2 procedures plus special laboratory clothing and controlled
access are recommended for handling clinical material suspected of containing Mycobacterium
tuberculosis, Brucella, Coccidioides immitis, Rickettsia, and specific viruses such as arbovirus.
• The air movement must be carefully controlled to contain the infectious materials.
• Biosafety Level 4 (BSL 4): Level 3 practices plus entrance through a separate room in which
street clothing is changed and replaced with laboratory clothing.
• Maximum containment includes the use of a class II biological safety cabinet and the
decontamination of all personnel and material before leaving the area.
• This level is primarily used in research facilities and includes a limited number of exotic
viruses including filovirus and arenavirus.

ENGINEERING CONTROLS

• Engineering controls are needed to protect employees from the hazards that may occur
during the performance of laboratory procedures.
• All laboratories must adhere to a minimum of Biosafety Level 2 guidelines.
• Hazardous areas should be identified and labeled accordingly. The biohazard label should be
used to identify those areas of the laboratory where infectious specimens or cultures are stored
or present.
• Air in the microbiology laboratory should move from areas of low risk to high risk and should
not be recirculated after it passes through the microbiology laboratory.
• BSCs protect laboratory workers from aerosols through sterilization by either heat, ultraviolet
light, or passage of air through a HEPA filter that removes particles larger that 0.3mm.
• Cabinets are classified as class I, II, or III based on performance characteristics with
regard to biological containment.

• Class I and II BSCs - provide effective partial containment for procedures involving moderate-
and high-risk microorganisms or Biosafety Levels 2 and 3 agents.
• Class I cabinets - are open-fronted, negative-pressure, ventilated cabinets.
• Unsterilized room air enters and circulated within the cabinet, and the exhaust air
from the cabinet is filtered by a HEPA filter.
• Class II BSCs - sterilize both the air entering and circulating within the cabinet and the
exhaust air.
• Type II vertical laminar-flow biological cabinets have HEPA-filtered, recirculated
airflow within the workspace.
• The exhaust air from the cabinet also is filtered by a HEPA filter.
• Class III BSCs - provide the highest level of safety, and all air entering and leaving cabinet is
sterilized with a HEPA filter.
POST-EXPOSURE PLAN

• All laboratory accidents or safety incidents must be reported promptly.

• Appropriate treatment must be prompt.
• Appropriate serological and clinical follow-up of the employee must
be provided.
• There must be documentation of the accident with a report verifying the incident and follow-
up.

DISPOSAL OF HAZARDOUS WASTE

• Microbiological waste must be decontaminated before disposal.

• All contaminated materials should be placed into two leak-proof plastic bags; double bagging
protects against infectious materials from leaking or falling from a single bag.
• Contaminated needles, scalpels, and other implements that have a puncture risk should be
placed into sharps containers.
• The autoclave is often used to decontaminate these materials.
• Specimens or infectious materials shipped to reference laboratories must be packages
according to the requirements of the government.
CHEMICAL SAFETY

• In 1987, United States Occupational Safety and Health Administration (OSHA) published the
Hazard Communication Standard providing institutional educational practices to ensure all
laboratory personnel have a thorough working knowledge of the hazards of chemicals with
which they work.
• Called “Right-to-Know”, which mandates that all hazardous chemicals in the workplace be
identified and clearly marked with National Fire Protection Association (NFPA) label, stating
the health risks and the hazard class.

NFPA LABEL;
BLUE – Health Hazard
RED – Fire Hazard
YELLOW – Reactivity
WHITE – Specific Hazard

•Chemical hygiene plan – includes guidelines on proper labeling of chemical containers,

Manufacturer’s Material Safety Data Sheet (MSDS), and the written chemical safety training
and retraining programs

Employer’s Responsibility:
1. establish laboratory work methods and safety policy
2. provide supervision and guidance to employees
3. provide safety information, training, PPE, and medical surveillance to employees
4. provide and maintain equipment and laboratory facilities that are adequate for the tasks
required
5. know and comply with the established laboratory work methods
6. have a positive attitude toward supervisors, coworkers, facilities, and training
7. give prompt notification of unsafe conditions to the immediate supervisor
8. engage in the conduct of safe work practices and use of PPE

Material Safety Data Sheet includes:

1. Substance name
2. Name, address, and telephone number of manufacturer
3. Hazardous ingredients
4. Physical and chemical properties
5. Fire and explosion data
6. Toxicity
7. Health effects and first aid
8. Stability and reactivity
9. Shipping data
10. Spill, leak, and disposal procedures 11. PPE
12. Handling and storage

Safety Equipment:
1. safety showers deliver 30-50gal/min of water at 20- 50psi
2. eye-wash stations
3. fire extinguishers
4. fire blankets, spill kits, first aid supplies
5. mechanical pipetting must be used for manipulating all types of liquids
6. hoods
▪ fume hoods – required to expel noxious and hazardous fumes
▪ biosafety cabinet – remove particles that may be harmful to the employee who is working
with infective biologic specimens with biosafety levels

Chemical Spill (C-L-E-A-N)

•Contain the spill
•Leave the area
•Emergency: eye wash, shower and medical care •Access MSDS
•Notify supervisor

Actions in case of fire: (R-A-C-E)

1. Rescue any injured individuals
2. Activate the fire alarm
3. Contain the fire; close the doors
4. Extinguish the fire, if possible
TO OPERATE AN EXTINGUISHER:
Pull the pin
Aim nozzle at the base of the fire
Squeeze the handle
Sweep nozzle side to side
LAB 2
THE MICROSCOPE

What is a MICROSCOPE?
From the Ancient Greek words
- mikrós or “small”
- skopeîn or “to look or see”

Microscope - a tool that is used to view smaller objects that the human eye can see.

Microscopy - scientific field of study which is used to study minute structures and objects by a
microscope.
- The most basic microscopes used in various institutions today make use of a series of
lenses that collect, reflect, and focus light into the specimen, which is the object under
inspection.
- Without the presence of light, microscopes won’t work.
- The use of different microscope lenses promotes magnification without altering the
quality of the image produced.

TYPES OF MICROSCOPES

1. SIMPLE MICROSCOPE
- is simply a large magnifying glass with a shorter focal length that has a convex mirror with a
small focal area.
- most common examples of this type of device are the handheld lens and eyepiece lens.
- when a material is held close to the lens of the microscope, its focus is created, and the
original object becomes magnified and more erect.
- since it’s only a simple microscope, it only has one magnification level depending on what
lens is used.

Simple microscopes are only used for reading and magnifying non-complex items.

2. COMPOUND LIGHT MICROSCOPE

- the most common type of microscope used today, which mechanism is explained earlier.
- it is basically a microscope that has a lens or a camera on it that has a compound medium in
between.
- this compound medium allows for magnifications in a very fine scale.
 While the simple microscope only requires natural light to see the object, a compound light
microscope needs an illuminator to view the specimen.

MAGNIFICATION: This pertains to making the specimen look larger through the microscope
through zooming in the lenses. Magnification is a quantified property which ranges from 40x,
100x, 400x, and up to 1000x.
RESOLUTION: This refers to how good the image is captured by the compound microscope
lens. A higher resolution means that the image will be clearer and more detailed. Also, it has an
improved visual clarity as it has more layers of magnifications.
CONTRAST: Like in photography, the background’s darkness relative to the focus or specimen
is referred to as contrast. Excellent contrast is typically achieved through staining the specimen
so its colors would stand out when viewed in the microscope.

3. DARK FIELD MICROSCOPE

- In a dark field microscope, the object appears bright against a dark background. This is made
possible by the use of a special dark-field condenser.
- It is used to identify the living, unstained cells and thin bacteria like spirochetes which cannot
be visualized by light microscopy.

4. PHASE CONTRAST MICROSCOPE

- It is used to visualize the living cells by creating a difference in contrast between the cells and
water.
- It converts slight differences in refractive index and cell density into easily detectable
variations in light intensity.
- It is useful for studying:
a. Microbial motility
b. Determining the shape of living cells
c. Detecting bacterial components such as endospores and inclusion bodies.

6. FLUORESCENCE MICROSCOPE
- When fluorescent dyes are exposed to ultraviolet rays (UV) rays, they become excited
and are said to fluoresce, i.e. they convert this invisible, short- wavelength rays into the
light of longer wavelengths (visible light).
7.
6. STEREO MICROSCOPE
- the stereo microscope, dissecting or stereoscopic microscope, is an optical microscopy
version designed specifically for low magnification imaging of a biological specimen.
- it works through reflecting light off the specimen’s surface rather than transmitted through
its medium.
- This type of microscope is often used in chemistry laboratories where more detailed, three-
dimensional images are required that would be possible with an electron microscope or other
high-powered microscope.

7. SCANNING ELECTRON MICROSCOPE (SEM)

- is a very popular type of scanning electron microscopes, which produces images of a material
by scanning the sample with a high-powered beam of electrons.
- the electrons interacting with atoms within the sample create different signals which contain
data about the structure and topography of the material.

5. TRANSMISSION ELECTRON MICROSCOPE (TEM)

- is an optical microscopy method in which an electrical beam of electrons is transmitted
through an unstained sample to create an optical image of the sample.
- Instead of sending electrons to scan and bounce off the specimen as what SEMs do, TEMs
allow the electrons to pass through the thin sample.

PARTS OF THE MICROSCOPE (COMPOUND)

Three Structural Parts:

Head- also known as the body, it carries the optical parts in the upper part of the microscope.
Base- acts as microscopes support. It also carries microscopic illuminators.
Arms - the part connecting the base and to the head and the eyepiece tube to the base of the
microscope. It gives support to the head of the microscope and it is also used when carrying
the microscope.

OPTICAL PARTS OF COMPOUND MICROSCOPE

EYEPIECE OR OCULAR LENS

- Also known as the ocular.
- This is the part used to look through the
microscope.
- It is found at the top of the microscope.
- Its standard magnification is 10x with an optional eyepiece having magnifications from 5X –
30X.

OBJECTIVE LENSES
- These are the major lenses used for specimen visualization.
- They have a magnification power of 40x-100X.
- There are about 1- 4 objective lenses placed on one microscope, in that some are rare facing
and others face forward.
- Each lens has its own magnification power.

MECHANICAL PARTS OF COMPOUND MICROSCOPE

NOSEPIECE
- Also known as the revolving turret. It holds the objective lenses.
- It is movable hence it can revolve the objective lenses depending on the magnification power
of the lens.

ADJUSTMENT KNOBS
- These are knobs that are used to focus the microscope.
- There are two types of adjustment knobs:
a. fine adjustment knobs
b. coarse adjustment knob

STAGE
- This is the section on which the specimen is placed for viewing.
- They have stage clips that hold the specimen slides in place.
- The most common stage is a mechanical stage, which allows the control of the slides by
moving the slides using the mechanical knobs on the stage instead of moving it manually.

APERTURE
- This is a hole on the microscope stage, through which the transmitted light from the source
reaches the stage.

ILLUMINATING PARTS OF COMPOUND MICROSCOPE

SUB STAGE CONDENSER

- These are lenses that are used to collect and focus light from the illuminator into the
specimen.
- They are found under the stage next to the diaphragm of the microscope.
- They play a major role in ensuring clear sharp images are produced with a high magnification
of 400X and above.
- The higher the magnification of the condenser, the more the image clarity.

IRIS DIAPHRAGM
- It is also known as the IRIS.
- It is found under the stage of the microscope and its primary role is to control the amount of
light that reaches the specimen.
- It’s an adjustable apparatus, hence controlling the light intensity and the size of the beam of
light that gets to the specimen.

ILLUMINATOR
- This is the microscopes light source, located at the base. It is used instead of a mirror.
- It captures light from an external source of a low voltage of about 100v.

CARE AND HANDLING OF THE MICROSCOPE

1. Always use both hands to carry the microscope, one holding the arm, one under the base.
2. Before each use, examine the microscope carefully and report any unusual condition or
damage.
3. Keep the oculars, objectives, and condenser lens clean. Use dry lens paper only.
4. At the end of each laboratory period in which the microscope is used, remove the slide from
the stage, wipe away the oil on the oil-immersion objective, and place the low-power objective
in vertical position.
5. Replace the dust cover, if available, and return the microscope to its box.
LAB 3
SPECIMEN COLLECTION, TRANSPORT, & PROCESSING

PRECAUTIONS
• Collect specimen before any antimicrobial agent has been administered.
• If the patient has already taken antibiotics, say, penicillin, administer penicillinase to
inactivate penicillin or simply dilute the specimen.

• Collect specimen at the right stage of the disease (acute stage):

• Enteric infection – bacteria are present in greater numbers in acute or diarrheal stage
• Typhoid fever
• blood culture on the 1st week of infection
• stool/urine culture on the 2nd week of infection
• serological test on the 3rd week

 Collect specimen at the right site of infection.

 Collect specimen with as little or no contamination as possible.

MAJOR APPROACHES TO AVOID AREAS WITH NORMAL FLORA

• Cleanse the area with disinfectant.

• Bypass the area with normal flora.
• Culture for specific pathogen only.
• Quantitate culture result by making colony count.
• Sufficient quantity of specimen.
• Prompt delivery to the laboratory.

HANDLING OF SPECIMEN
• Certain specimen can be placed in a transport medium:
• Transgrow
• Amie’s Medium
• Carry & Blaire (Stool Samples)
• If urine is not examined right away, it may be refrigerated but it must be examined within 24
hours.
• Stool & sputum could also be refrigerated but it must be examined within 2-3 hours.
  CSF must be processed immediately.
• If not, incubate for not more than 12 hours or stand at room temperature for not
more than 1 hour
• Refrigeration is contraindicated because certain CSF isolate are quite sensitive to low
temperature like N. meningitidis & H. influenzae
• In case of viral culture, refrigerate specimen immediately. If held for more than 24
hours, freeze specimen at –70C

SPECIMEN PRIORITIZATION
• Laboratory staffing and specimen load may have a significant impact on timely processing
• A four – level scheme for prioritizing specimen based on the critical nature of the specimen
or potential for specimen degradation

Level Description Specimens

1 Critical / Invasive Amniotic fluid Blood
Brain
CSF

Heart valves Pericardial fluid

2 Unpreserved Body Fluids not listed in level 1 Bone
Drainage from wounds
Feces

Sputum Tissue
3 Quantitation Catheter tip
Required Urine
Tissue for quantitation
4 Preserved Feces in preservatives Urine in preservatives Swabs in holding
medium (aerobic and aerobic)

CLINICAL SPECIMENS

BLOOD
General Considerations In Blood Collections:
• Ideal time to collect blood is shortly before the expected rise in temperature
• A single negative blood culture does not rule out bacteremia
• There must be 3-4 blood cultures taken within 24-48 hours in order to establish diagnosis
• Obtain 2 simultaneous blood cultures in 2-3 different sites

- Collect blood aseptically.

• Use alcohol then 2% tincture of iodine, let dry for 1 minute, then remove iodine afterwards
with alcohol

- Ideal volume is 10ml which is to be added to the culture media

• It is important that blood is prevented from clotting by the addition of an anticoagulant

 A good anticoagulant is liquid which contains SPS (Sodium Polyanethol Sulfonate

 Although both heparin and citrate could be used as anticoagulants, they are toxic to
most bacteria & inhibitory to their growth.
 SPS must be at 0.025% - 0.05% (w/v) concentration
 If the patient is on penicillin, add penicillinase to the medium (but it is not effective for
methicillin, cloxacillin & nafcillin which are penicillinase resistant penicillins)
• At times, addition of penicillinase may prove to be unnecessary because dilution of
penicillin in blood by broth renders it non-inhibitory.
Procedure:
• Mix blood sample with media.
• Incubate at 35-37C.
• Examine periodically by Gram stain & subculture.
• Hold for 2 weeks before reporting for negative (21 days for Brucella).
• Indications of a (+) blood culture:
a. Turbidity
b. Hemolysis
c. Colony or pellicle formation
d. Presence of gas (bubbles)
 BLOOD COMPONENTS
• Organisms in fatal transfusion reactions are usually psychrophilic gram (-) bacilli such as
Pseudomonas spp. except P. aeruginosa.
• Incubate specimen at 4C, 22C, 35-37C

THROAT & NASOPHARYNGEAL CULTURE

• a streptococcus is the most abundant normal flora
• group A streptococcus is the most common pathogen
• culture must include anaerobic conditions for  streptococcus
• nasopharyngeal swab is the recommended specimen for isolation & detection of
carriers
Media:
• H. influenzae - enriched CAP or BAP with Staphylococcus streak across the inoculum
• N. meningitidis - enriched CAP or Thayer- Martin agar
• B. pertussis - Charcoal Cephalexin medium

• Ideal specimen: early morning sputum (deep cough material or sputum)

• Suitable sputum for culture: <10 epithelial cells & >25 pus cells/hpf

Procedure:
• Make a direct smear for:
• Gram staining = for general bacterial flora
• Acid Fast staining = for AFB
• Use concentration technique to
isolate AFB
• Inoculate to appropriate culture media

URINE CULTURE

• Ideal specimen: Midstream, clean-catch, early morning urine

• Process within 1 hour of collection or refrigerate not longer than 24 hours or use a special
preservative
• GS of uncentrifuged specimen is done for rapid screening procedure to detect UTI.
• Observe under OIO for the presence of bacteria. Presence of numerous squamous
cells indicate vaginal or urethral contamination.
  Inoculate the specimen into applicable culture media like BAP, EMB & MAC using a
calibrated loop (0.01 or 0.001 ml).
• Incubate overnight. Count the actual number of colonies multiplied by the calibration of
the loop to obtain the number of bacteria/ ml of urine.
• Colony count of 100,000 bacteria/ml indicates infection.
• If there is no growth for the 1st 24 hours, continue examining for another 24 hours before
reporting negative.

For Pour Plate method:

• Prepare a 1:100 dilution of urine with sterile water.
• Transfer 0.1ml of this dilution to a petri dish.
• Add cooled melted agar. Mix well incubate.
• Count the colonies & multiply by 1000 = bacteria/mL

CEREBROSPINAL FLUID
• Volume: 1ml obtained by lumbar puncture
• Procedure:
• Examine immediately or incubate not longer than 1 hour
• Centrifuge. Make smears for GS and India ink
• Culture specimen for H. influenzae, N. meningitidis, S.
pneumoniae.
• Incubate & examine for at least 72 hours before reporting as negative

STOOL CULTURE
• Culture the specimen as soon as possible, or refrigerate specimen.
• Put rectal swab in enrichment broth or transport medium.

GS is not usually done but helps in the identification of etiologic agents:

• If it reveals typical gram (+) cocci in clusters, it is suggestive of Staphylococcal infection.
• If gram (-) comma shaped bacilli: Vibrio or Campylobacter
• If large numbers of gram (+) bacilli: Clostridium difficile
• If gram (-) bacilli: Salmonella or Shigella

DAY TO DAY PROCEDURE IN PROCESSING STOOL SPECIMEN:

1ST DAY
• Inoculate the specimen to differential media (EMB, Mac or BLM) or selective media (SSA,
HEA, ALD)
• Inoculate also into enrichment medium (selenite F, tetrathionate, Hayna CN, APW)
• Incubate inoculated medium overnight

2ND DAY
• Examine differential media for growth of LFs & NLFs
• Subculture & do biochemical tests
• If there is no growth, inoculate culture from the enrichment medium into EMB or Mac or
BLM.
• Incubate overnight

3RD DAY
• Note the patterns of biochemical reactions. If suggestive for Salmonella, Shigella or Vibrio,
perform serological typing
• If growth occurs after doing step 2 of the 2nd day subculture into biochemical test media.
• Incubate overnight then perform first step of day 3

GENITAL CULTURES
• Specimen: cervical (female), urethral (male), rectal & throat swabs
• Use media appropriate for organisms sought
• STDs include infections caused by:
• T. pallidum
• N. gonorrheae
• C. trachomatis
• G. vaginalis
• C. albicans
• HSV

WOUND CULTURE
• Direct GS
• Prompt anaerobic, as well as aerobic cultivation of wound swabs & aspirates

THE END !!!!!!!!!!!!!!!!!!!!!!!!!!! : D GOODLUCK