Biol 1118 Lec Lab Reviewer

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BIOL 1118 Lecture

The Five “I” s of Microbiology


1) Inoculation- introduction of a sample into a sterile culture media
2) Incubation- allowing cultures to grow under favorable growth conditions using an incubator
3) Isolation- separating one bacterial species to another using streak plating or pour plating techniques
4) Inspection- observation of different characteristics by naked eye (macroscopic) or under a microscope
(microscopic)
5) Identification- identifying the genus and species of the sample using biochemical tests, DNA-based
methods, immunological testing
The Microscope
 Microscope- instrument used to visualize micrometers or micron (μm) size objects such as microorganisms
and cells, made up of lenses for magnification
o Magnification – ability to enlarge objects
o Resolving power – ability to show detail

• Total magnification= power objective x power of ocular lens


• Magnification in most microscopes results from interaction between visible light waves and curvature of the
lens.
o Refraction- angle of light passing through convex surface of glass changes

• Oil immersion lens- requires the use of oil to prevent refractive loss of light
• Types of microscopes
o Light microscope- uses visible light, 400-750 nanometers (nm), magnification between 40-2000x
o Electron microscope- uses electron beams, electron waves are 100,000 times shorter than the waves
of visible light, magnification is between 5,000- 1,000,000x
Parts of a Microscope
1) Eyepiece – Also known as the ocular. This is the part used to look through the microscope. Its
standard magnification is 10x with an optional eyepiece having magnifications from 5X to 30X.
2) 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,
each lens has its own magnification power.
3) Nose piece – 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.

4) Adjustment knobs – These are knobs that are used to focus the microscope. There are two types of
adjustment knobs i.e., fine adjustment knobs and coarse adjustment knobs.
5) Stage – This is the section in which the specimen is placed for viewing. They have stage clips that hold
the specimen slides in place. The most common stage is the mechanical stage, which allows the
control of the slides by moving the slides using the mechanical knobs on the stage instead of moving
them manually.
6.) Aperture – This is a hole on the microscope stage, through which the transmitted light from the source
reaches the stage.
7.) Microscopic illuminator – This is the microscope’s 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.
8.) 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.
9.) Diaphragm – It’s 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.
10.) Condenser focus knob – This is a knob that moves the condenser up or down thus controlling the
focus of light on the specimen.
11.) Stage control – It controls how far the stages should go preventing the objective lens from getting too
close to the specimen slide which may damage the specimen. It is responsible for preventing the
specimen slide from coming too far up and hitting the objective lens.
Types of Light Microscopes
• Brightfield
o most widely used; specimen is darker than the surrounding field; live and preserved stained
specimens
• Darkfield
o brightly illuminated specimens surrounded by dark field; live and unstained specimens

• Phase contrast
o transforms subtle changes in light waves passing through the specimen into differences in light
intensity, best for observing intracellular structures
• Differential Interference Contrast (Nomarski Interference Contrast)
o uses interference patterns to enhance contrast between different features of specimens, useful in
distinguishing structures within live specimens, no staining
• Fluorescence
o uses fluorescent dyes that emit visible light when bombarded with shorter UV rays, can distinguish
between living and dead cells
• Confocal Laser Scanning
o uses laser beam of light that scan at various depths in the specimen

Types of Electron Microscopes


• Transmission Electron Microscope (TEM)
o transmit electrons through the specimen, darker areas represent thicker, denser parts and lighter
areas indicate more transparent, less dense parts
o can resolve subcellular structures such as organelles and interior of membranes and molecular
structures such as DNA
o cells are embedded in a plastic resin and dehydrated using different concentrations of ethanol
o thin sections (20-100 nm thick) are cut using ultramicrotome
o samples are fixed to copper wire or carbon-fiber grids and stained with uranyl acetate or osmium
tetroxide, which contain electron dense heavy metal atoms
• Scanning Electron Microscope (SEM)
o provide detailed three-dimensional view, bombards the surface of a whole, metal-coated specimen
with electrons while scanning back and forth over it
o can resolve surfaces of larger specimens like bacterial cells
o samples are mounted on a wire grid and sputter-coated with gold or palladium before viewing

• Ultramicrotome used for the preparation of ultrathin sections of samples for TEM
Principles of Staining Techniques
• Stains (Dye)
o salts composed of negative or positive ion
o one of which is colored (chromophore)
o dyes create contrast by imparting a color to cells or cell parts
o Basic dyes - with positive charges on the chromophore, binds with negatively charge surface
(methylene blue, crystal violet, safranin)
o Acidic dyes - with negative charges on the chromophore, binds with positively charge surface, (india
ink or nigrosin)
• Positive staining – surfaces of microbes are negatively charged (due to teichoic acid) which
attract basic dyes
• Negative staining – microbe repels dye, the dye stains the background
Types of Staining
• Simple stains – one dye is used; reveals shape, size, and arrangement
• Differential stains – use a primary stain and a counterstain to distinguish cell types or parts
o Gram stain - differentiates between gram-positive and gram-negative bacteria, dyes- crystal violet,
safranin
o Acid-fast stain (Ziehl-Neelsen stain)- differentiates cells with mycolic acid present in the cell wall
(Nocardia, Mycobacteria), (dye- carbol fuchsin)
o Endospore stain - uses heat to drive the primary stain into the endospore, (dye malachite green)

• Special stains – reveal certain cell parts not revealed by conventional methods
o Capsule stain- uses acidic dyes like eosin and nigrosin which stains the background and leave the
cells colorless
o Flagellar stain- stain such as carbol fuchsin and mordant such as tannic acid can be used to
increase the diameter of the flagella
Gram Staining Principle and Procedure
• developed by Hans Christian Gram
• a differential staining technique which uses more than one dye, crystal violet as primary stain and
safranin as the secondary stain, which distinguishes gram-positive and gram-negative bacterial cells
• it uses iodine solution as a mordant, which forms insoluble complex with the crystal violet stain and
increases its affinity to the thick peptidoglycan layer of gram-positive bacteria
• during the decolorizing step, alcohol dissolves the lipids in gram-negative cell walls and increases its
permeability allowing the crystal violet-iodine complex to be washed out of the cell
• the secondary stain, safranin, will be absorbed by the gram-negative bacterial cells in the final step,
which will appear red
• gram-positive bacterial cells will retain the crystal violet stain, which will appear purple
• Specimen Preparation for Optical Microscopes
o Wet mounts and hanging drop mounts– allow examination of characteristics of live cells- motility,
shape, and arrangement
o Fixed mounts- are made by drying and heating a film of specimen, this smear is stained using dyes
to permit visualization of cells or cell parts
• Fixation
• process by which the internal and external structures of the cells are preserved and fixed in position
o Heat fixation- by gently flame heating
o Chemical fixation- is used to protect fine cellular structures using ethanol, acetic acid,
formaldehyde, glutaraldehyde
Tools Used in Bacterial Identification
• Biochemical Tests
o tests used for the identification of bacterial species based on the differences in the biochemical
activities, examples:
o Starch hydrolysis Test- used to observe amylase production in a bacterial culture indicated by a
clearing zone around the colonies
o Lipid Hydrolysis Test- used to test ability of bacterial culture to hydrolyze lipids, positive reaction is
observed by the formation of clearing zone
o Gelatin Hydrolysis Test- used to identify bacteria which can produce extracellular proteolytic
enzymes (gelatinases) that liquefy gelatin, an animal protein, positive reaction is observed from the
liquefaction of the nutrient gelatin medium
• Analytical Profile Index (API)
o is a miniaturized panel of biochemical tests compiled for identification of groups of Gram-negative
Enterobacteriaceae
o the kit contains 20 miniature biochemical tests for manual identification up to the species level
o bacterial suspension is used to rehydrate each well and the strips are incubated

o during incubation, metabolism of the substrate produces color changes which will
o indicate positive or negative reaction
o all positive and negative reactions are compiled to generate a profile number
o the 7-digital code obtained from the reaction results is used to identify the sample using the API
catalog or apiweb (online)
• Biolog Microbial ID
o allows identification of gram-positive and gram-negative bacteria in a single panel
o it analyzes the ability of the cell to metabolize all major biochemicals at the same time determining
other important physiological properties such as pH, salt, lactic acid tolerance, reducing power, and
chemical sensitivity by creating a metabolic fingerprint from discrete test reactions performed within a
96-well clear plate
o culture suspensions are tested with a panel of pre-selected assays, then incubated, read and
compared to extensive databases of environmental organisms, human pathogens, animal and plant
pathogens
Analysis of Nucleic Acids

DNA-DNA hybridization
o when two organisms share many identical or highly similar genes, their DNAs are expected to
hybridize
o DNA–DNA hybridization therefore is useful for differentiating between organisms as a complement
to small subunit rRNA gene sequencing
 GC Ratios
o the GC ratio is the percentage of guanine (G) plus cytosine (C) in an organism’s genomic DNA
o if two organisms’ GC ratios differ by more than about 5%, they have few DNA sequences in
common and are therefore, unlikely to be closely related
Bacteria
 Phylogenetic analysis using 16S ribosomal RNA revealed distinct domain of Bacteria
 Ribosomal RNA is present in all organisms and can be used for analyzing evolutionary relationships
 The three domains of life are Domain Bacteria, Domain Archaea, and Domain Eukarya
General Morphology of Bacteria
• Bacteria are prokaryotes- no membrane-bound nucleus and organelles
• Bacteria shares general morphology with archaea
• Cell wall- functions as a protective layer and responsible for the cell shape
• Plasma membrane- serve as barrier and separates cell from the external environment
• Cytoplasm- composed of complex solution of organic molecules
• Ribosomes- where protein synthesis occurs
• Chromosomes- consists of circular double-stranded DNA (contains genetic information) located in the
area of the cell called nucleoid
• Plasmid- which consists of extrachromosomal DNA
• Capsule- allows for attachment and protects cell from dehydration and attack by phagocytic cells
• Flagella- (singular flagellum) used for locomotion
• Pili- (singular pilus) used for attachment to surfaces

 Prokaryotes have three basic cell shape- (a) cocci or spherical, (b) bacilli or rod-shaped, and (c) spirilli or
spiral-shaped
Bacterial shape is genetically and environmentally determined
• Stella humosai- star-shaped bacteria, marine bacteria
• Haloarcula quadrata- rectangular-shaped bacteria, marine bacteria
Cell Envelope
• External covering outside the cytoplasm
• Composed of two layers- cell wall and cell membrane (phospholipid bilayer)
• Two main groups of Bacteria are classified based on the difference in cell wall
• Gram-positive bacteria- thick peptidoglycan and cell membrane
• Gram-negative bacteria- outer cell membrane (which contains lipopolysaccharides or LPS), thin
peptidoglycan layer, and cell membrane
• Phospholipid bilayer- two layers of phospholipids which surrounds the cell, hydrophilic exterior (water-
loving) and hydrophobic interior (water-hating)
• Ester-linked lipids
Endospores

 Some Gram-positive bacteria produce endospores (sporulation) which are highly resistant to extreme
temperatures and chemicals
 Endospores are inactive resting stage of bacterial cells capable of returning to vegetative growth
(germination) in favorable environments
 Gram-positive genera which form endospores
o Bacillus
o Clostridium
o Sporosarcina

Flagellar Arrangements
1. Peritrichous- flagella dispersed over the surface of the cell
2. Monotrichous- single flagellum at one end
3. Lophotrichous- small bunches at one end
4. Amphitrichous- flagella at both ends
Modes of Nutrition
 Autotrophs
o Photoautotrophs- derives energy from light (Purple sulfur bacteria, Cyanobacteria)
o Chemoautotrophs- derives energy from inorganic substances
 Heterotrophs or chemoorganotroph
o Parasite- lives and feeds in a living host which may cause disease or illness
o Saprobe- decomposers that feeds on dead organic matter

Modes of Reproduction
• Asexual reproduction through binary fission
• Binary fission- parent cell divides into two identical daughter cells
• Horizontal Gene Transfer (HGT)- transmission of genetic information on the same generation and not
from parents to offspring
o Transformation- uptake of DNA (genetic material) from external environment
o Conjugation- transfer of DNA through direct cell to cell contact mediated by conjugation pilus
o Transduction- transfer of DNA mediated by a virus particle (bacteriophage)

Habitat and Ecology


• Lives everywhere even in extreme environments
• Temperature Adaptation
o Psychrophiles- thrives in temperature between 0°C-20°C
o Mesophiles- thrives in temperature between 10°C-50°C
o Thermophiles- thrives in temperature between 45°C-80°C

Oxygen Adaptation
o Obligate aerobes- grows in normal atmospheric oxygen and handle toxic oxygen by-products
o Obligate anaerobes- can live in environment without oxygen, killed by oxygen
o Facultative anaerobes- aerobes which can live without oxygen
o Aerotolerant anaerobe- cannot use oxygen for respiration and not killed by oxygen
o Microaerophile- aerobes which requires small amount of oxygen but does not grow under anerobic
conditions
 Microbial Taxis- bacteria responds to stimuli from the environment
o Chemotaxis- response to chemicals, chemoattractant (attracts bacterial cells), chemorepellent
(repels bacterial cells)
o Phototaxis- response to light
o Aerotaxis- response to oxygen
o Osmotaxis- response to ionic strength
o Hydrotaxis- response to water
o Magnetoxis- response to magnetic field
o Scotophobotaxis- response to changes in illumination

Representative Bacteria
1. Bacillus subtilis
o Gram-positive bacteria
o Rod-shaped cells (bacillus)
o Endospore-forming
o Peritrichous flagella

2. Staphylococcus aureus
o Gram-positive bacteria
o Spherical-shaped (coccus)
o Non-motile
o Non-spore forming
o Produce staphylococcal toxins which cause food poisoning

3. Escherichia coli
o Gram-negative bacteria
o Non-spore forming
o Peritrichous flagella
o Cause food poisoning
o Used as competent cell in recombinant DNA technology (genetic engineering)

4. Spirillum volutans
o Gram-negative bacteria
o Spiral-shaped cells
o Amphitrichous flagella

5. Cyanobacteria
o Also called blue green algae
o Gram-negative bacteria
o Aquatic and photosynthetic
o Nostoc, Oscillatoria, Spirulina
o Involved in carbon fixation

Bacteria Product
Antibiotics
Streptomyces sp. (Streptomycin, Tetra
cyclin, Erythromycin))
Lactobacillus sp. Lactic acid
Acetone, rubbing
Clostridium acetobutylicum
acetone
Microbial plastic (poly-
Bacillus sp.
hydroxybutyrate or PHB)
BIOL 1118 LAB
Biosafety is the use of precautionary measures to avoid becoming infected with potentially infectious
microorganisms and contaminating the environment.
• General safety procedures must be followed to ensure a safe and accident-free working environment
for all researchers who work in the laboratory.
Biosafety Levels (BSLs)
• there are four Biosafety Levels (BSLs)
• each biosafety level has unique containment controls based on the degree of pathogenicity, disease
severity, microorganism transmissibility, and nature of the work
• each level has its own procedures for laboratory practices, the use of safety equipment, and the
construction of facilities

Biosafety
Level Pathogenicity Example
(BSL)
BSL – 1 Non-pathogenic microorganisms Escherichia coli
BSL – 2 Moderate pathogenic Staphylococcus aureus
Pathogenic microorganisms which can cause potentially lethal Mycobacterium
BSL – 3
disease through respiratory transmission tuberculosis
Microorganisms are dangerous and pose high risks of aerosol-
BSL – 4 Zaire ebolavirus
transmitted infections that are fatal and without treatment or vaccines

BSL – 1 BSL – 2 BSL – 3 BSL - 4


Laboratory Aseptic BSL-1 plus restricted BSL-2 plus medical BSL-3 plus change clothing before
Practice techniques, access to laboratory when surveillance of researchers, entering the laboratory, shower
experiments work is being conducted and immunization for the upon exiting, decontaminate all
performed on lab microorganisms they work materials before exiting
bench or table with
Safety Personal BSL-1 plus face shields, BSL-2 plus respirators, and BSL-3 plus all experiments must
equipment protective experiments performed all works performed within be performed within Class III
equipment (PPE): within a biological safety a biological safety cabinet biological safety cabinet (BSC),
lab gown, gloves, cabinet (BSC), autoclave (BSC) and wearing full body, air-
eye protection for decontamination and supplied, positive pressure suit
proper waste disposal
Facility Sink for hand BSL-1 plus self-closing BSL-2 plus, hand-free sink BSL-3 plus all experiments must
construction washing, doors to doors, sink and eyewash and eyewash near the exit, be performed within Class III
separate working are readily available exhaust air cannot be biological safety cabinet (BSC),
space with the circulated, laboratory must and wearing full body, air-
rest of the facility have sustained directional supplied, positive pressure suit
airflow from the clean
areas towards the
contaminated areas,
entrance to the lab must be
through two sets of self-
closing and locking doors

 Laboratory safety procedures and aseptic techniques are utmost importance to prevent any accidents, getting
infected or contaminating the environment.

DOs

1) Wear personal protective equipment inside the laboratory at all times (lab gown, closed shoes, eye goggles,
hand gloves).
2) Always pay attention to the biohazard symbol (to avoid disease transmission) and warning signs (to minimize
risks of exposure to harmful chemicals).
3) Tie long hairs.
4) Before and after experiments, wash your hands with soap.
5) Disinfect workbench before and after use with disinfectant (70% ethyl alcohol).
6) Place beaker with alcohol to dispose pipette tips.
7) Decontaminate materials and glass wares used with microorganisms.
8) Always use disinfectant to clean up any spills (70% ethyl alcohol).
9) Clean and disinfect glassware and slides that have been contaminated with bodily fluids (blood, urine, saliva,
feces, semen, vaginal secretions, and breast milk).
10) When not in use, turn off electrical equipment.
11) Maintain a clean and tidy working environment.
DON’Ts

12) Don’t perform unauthorized experiments.


13) Don’t use equipment without permission and proper training.
14) Don’t eat, drink, smoke, bring food, and apply cosmetics inside the laboratory.
15) Don’t play inside the laboratory.
16) Don’t pipette by mouth.
17) Don’t touch broken glass wares with bare hands.

 Biohazard - It issues a warning on lab equipment that may contain biohazardous materials such as blood
samples.
 Carcinogen Hazard - It is concerned with human carcinogens such as methylene chloride, formaldehyde, and
benzene. This sign indicates that you must wear appropriate personal protective equipment.
 Corrosive Hazard - Strong chemicals that corrode your skin and other materials. A single drop of the corrosive
agent can cause significant damage. When working with corrosive substances, wear protective equipment at all
times.
 Electrical Hazard - Electrical hazards in the lab can cause burning and even death. When not in use, devices
labeled as electrical hazards should always be turned off.
 Explosive Hazard - It refers to laboratory chemicals with explosive properties.
 Flammable Hazard - Chemicals have the tendency to ignite and cause fire. Should be stored properly, away from
flames, sparks, and oxidizing substances.
 General Warning - It is a warning that hazardous materials are present in the lab.
 Glassware Hazard - It is a physical hazard with the potential to become a health hazard if contaminated with
toxic or infectious substances.
 High Voltage - A symbol represents lightning, which can cause serious injury or even death.
 Hot Surface - It warns of the dangers of burns from hot surfaces. This symbol is commonly found on laboratory
equipment that generates heat, such as lab ovens and autoclaves.
 Ionizing Radiation - It denotes the presence of ionizing radiation, which carries energy and is capable of
liberating and ionizing electrons from molecules/atoms. Examples include x-ray machines, accelerators, and
beam cannons.
 Low Temperature - It refers to a cryogenic hazard within the lab, such as cold storage areas where chemicals
such as liquid nitrogen are kept.
 Non-Ionizing Radiation Hazard - It warns about non-ionizing radiation sources such a ultraviolet, infrared, visible
light, radiofrequency, and microwave.
 Oxidizing Hazard - They can transfer oxygen to flammable substances by transferring oxygen to another
chemical substrate. They should be kept separate from flammable materials. When working with oxidizing
agents, wear gloves and eye protection.
 Toxic Material Hazard - It is a generic sign for toxic/poisonous substances that, if inhaled, absorbed, or
swallowed, can cause severe harm.
 UV Light Hazard - It warns of potential dangers such as skin redness and ulceration. Long-term exposure to UV
light can result in skin cancer.

Biohazard Carcinogen Non-Ionizing


General Warning Hot Surface
Hazard Radiation Hazard UV Light Hazard

Corrosive Electrical Hazard Glassware


Hazard Ionizing Radiation Oxidizing Hazard
Hazard

Explosive Flammable Low Temperature Toxic Material


High Voltage
Hazard Hazard Hazard
PLUGGING

o Test tubes and flasks containing media are plugged with cotton before sterilization to prevent the entrance of
microorganisms. This should be carefully done to avoid any post-process contamination. The cotton plug should snugly fit,
not too tight that it will break the test tube or too loose that it might easily fall. There should be no opening between the
cotton plug and the mouth of the test tube or flask because this may serve as avenues for contamination. The cotton plug
allows air to enter the tubes or flasks, but filters microorganisms so that only sterile air is present inside.

WRAPPING

o Petri dishes and pipettes are wrapped in paper to prevent contamination after they have been brought out of the
autoclave. In addition, petri dishes are protected from movement during the sterilization process that might result in
breakage.

CULTURE MEDIA

o Preparations containing the needed substances


o The cultivation of microorganisms in vitro, requires the provision of a substrate and necessary substances that will be used
by the microorganism for its metabolism, growth, and reproduction.

Culture media are classified according to:

1) composition,
2) consistency, and
3) selectivity.

Media classification according to composition can either be empirical or natural (e.g., peptone, vegetable infusions) and synthetic or
defined (organic or inorganic). Based on consistency, media are classified as liquid (broth), solid (slant, plate), and semi-solid (soft
agar). According to selectivity, media are categorized as general or non-selective (e.g. nutrient agar), enrichment or selective (e.g.,
lactose broth) and differential (e.g. EMB agar).

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