Observing Microbes & Microbial Structure Flashcards
Nano meter compared to micrometer
1 micro meter = 1/1000 nanometer
1 micrometer = X meters
10 to the power of -6
1 nanometer = X meters
10 to the power of -9
E. coli size comparison to RBCs
RBCs are bigger
Microscope capable of seeing the smallest thing
Transmission electron microscope
10 picometers
Pathway of light through compound light microscope
Illuminator
condensor
Specimen
Objective lens
Ocular lens
Total magification of compound microscope=
Magnification of objective lens (4-100x) x magnification of ocular lens (10x)
Microscope objective
the different magnification strengths on a microscope
Aperture seettings on a light microscope affect
contrast and resolution
Resolution
The ability to distinguish bw two points or to see fine details
The higher the magnification of the objective the more _____ is required
Light
Longer or shorter waveslengths of light producer greater resolution
shorter
Highest resolution of light microscopes
.2 micrometers (2000x magnification)
What can be seen using a light microscope
Bacteria and fungi
Two forms of light microscopy
Bright field
Dark field
Bright field microscopy
Direct light enter objective lens
Dark objects visible against bright/white background
Light reflected off the specimen
Staining increases contrast
Is staining a part of bright field or darkfield
Bright field
Darkfield microscopy
Opaque disk placed bw light and specimen
- Only light reflected (diffracted) by specimen reaches eyepiece
- Emphasises edges of structures against a dark background
- The background is stained, not the object itself
Fluorescent microscopy
Uses UV light or other short wavelengths of light
Substances absorb UV light and emit visible light (longer wavelengths)
M/os stained with antibodies combined with fluorescent dyes (fluorochromes
How is bacteria identified under a microscope via fluorescent microscopy
To identify unknown bacteria, antibodies specific to the bacteria and fluorochrome are combined and attach to bacteria to light it up
What is lysteria
Organisms that contaminates foods in the fridge section, invades host cells and uses the host cell to propel them to the next cell
Two types of elctron microscopy
TEM: Transmission electron microscopy
SEM: Scanning electron microscopy
What type of images produced by electron microscopy
Black and white
TEM
Ultrathin slice of specimen
2D image of internal structures (The best microscope to see internal structures)
Treatment can cause distortion (Thin slicing)
Magnification of 10,000 - 100,000x
Resolution 2.5 nm
Path of light through TEM
Electron beam through electromagnetic lens - specimen - electromagnetic lens - screen or film
Why do beam of electrons produce higher resolution than light or UV light
shorter wavelength than visible and UV light; therefore, greater resolution
How are specimens stained in electron microscopy
Metal salts (eg. Lead & Uranium salts)
Immuno-staining (antibodies coated with gold particles)
SEM
Electrons produced by electron gun
scans whole surface of specimen (3D image)
- Useful for studying surface structures
Electrons being scattered off specimen are captured by microscope
Less distortion bc no slicing
1000-10000x magnification
Resolution of 20nm
Does SEM or TEM have higher resolution
TEM is 2.5 while SEM is 20nm
What size of specimen is a microscope needed for
1mm or smaller
Stains are used to increase contrast in what kind of microscopy
Electron
Most frequently used differential stain
Gram stain
Characteristics of simple stains
They are basic and can be rinsed with water
Most stains used in microbiology are _______ tyoe of stains
differentialho
how many steps to gram staining
7
Best microscope for visualizing flagella
Electron microscope
Why is staining important
Most Organisms can be colourless, and therefore will be invisible under the microscope
Why are salts used for staining
Bacteria cells are negative and attract the positive ions of salts
How do stains work
One of the ions is colored (chromophore)
WHy are bacteria slightly negative
Bc of they are phospholipids
Chromophore
The stained ion in the salt
Basic dyes (simple stains)
Most common dyes
Chromophore is cation (therefore binding to negative bacteria)
Includes crystal violet, methylen blue, safranin
Acidicdyes
Chromophore is an anion
Used for negative staining (bacteria remains colourless while background is stained)
3 types of stains
simple
differential
special
Simple stains
Stains the cell
Increases contrast
Includes crystal violet, methylen blue, safranin
Differntial stains
Using more than one type of dye
Differentiate bw organisms or structures
Gram stain MOST FREQUENTLY USED (Differentiates gram neg bacteria from gram positive)
acid fast stains (bw acid fast and non acid fast)
Special stains
(isolates specific groups)
Used to differentiete structures
Capsule (negative since capsules remain unstained, instead halo appears around them), endospore (stain to detect presence of), flagella stains
Gram staining
A type of differential staining
- Gram positive bacteria: stainined purple (Crystal violet/iodine)
- Gram negative bacteria: stained red by safranin (counterstain)
Steps of gram staining
Application of crystal violet (wait 60 seconds)
Rinse
Add iodine (mordant = reactive to crystal violet to cause greater adhesion to the stain)
Rinse off excess mordant
Alcohol wash (decolorization)
To wash off any dye not strongly held to cells
Application of safranin (counterstain)
Those that lost the crystal violet will be stained red; however, those already purple will be unaltered by the additional pinkish/red dye
Purple cells = gram positive
Pink/red cells = gram negative
Gram positive cells
More peptidoglycan
When dye added, the crystal violet attaches throughout the layer peptidoglycan layer TO THE PLASMA MEMBRANE WHICH IS NEGATIVE
When decolorizer is added, peptidoglycan is compressed and locks in the dye in
Gram negative cells
Cell wall Made up of two membranes
In bw two membranes is thin single peptidoglycan layer
Crystal violet attaches to outer membrane and some trickles into peptidoglycan
Decolourizer results in gram violet dye escaping outer lipid layer
What colour is a gram positive stain
Purple of blue
What colour is the gram negative stained cells
Red/pink
Important gram positive bacteria
Corynebacterium diphtheriae
Staphylococcus aureus
Streptococcus pyogenes
Streptococcus pneumoniae
Gardnerella vaginalis (gram variable)
Important gram negative bacteria
Escherichia coli
Neissseria gonorhoeae
Gardnerella vaginalis (Gram varariable)
Stains used in acid fast staing
Red and blue
Acid fast bacteria appear what colour under staining
Red
Non acid fast bacteria appear what colour under staining
BLue
What identifies a bacteria as acid fast
Acid fast bacteria have thick outer lipid layer
Mycolic acid
Examples of important acid fast bacteria
Mycobacterium tuberculosis (TB)
Mycobacterium leprae (leprosy)
Process
Process of acid fast staining
- Primary stain is carbolfuschin (red dye) - CARBOLFUSCHIN stains the mycolic layer
- Decolorized - by acid alcohol
- Anything without mycolic layer dye is washed off
- Counter stain methylene blue is used (Stains the plasma layer)
- Non-acid fast stained blue
What is the primary stain in acid fast staing
Carbolfuchsin (red dye),
Who proposed three domain system
Carl Woese
three domains
Bacteria, Archaea & Eukarya
Three distinctly different cell groups based on ribosomal RNA (rRNA)
Basic defintion of bacteria
Pathogenic and non pathogenic prokaryotes
Basic def of archaea
Prokaryotes live in extreme environments, carry out unusual metabolic processes
Extreme halophile and hyperhermophiles (habitatish)
Basic def of Eukarya
animals, plants, fungi;
Characteristics of bacteria
Prokaryotic
Contains peptidoglycan
Straight carbon chains
Sensitive to antibiotics
Characteristics of Archaea
Prokaryotic
Does not contain peptidoglycan
Varies in composition
Not sensitive to antibiotics
Characteristics of Eukarya
Vary in composition
Not reactive to antibiotics
Prokaryotes vs Eukaryotes
Prokaryotes
One circular chromosome, not membrane bound
No histones
No membrane bound organelles
Peptiodclycan cell walls (bacteria)
Divide by binary fission
Eukaryotes
Paired chromosomes
Histones (DNA wrapped around proteins called histones)
Membrane bounded organelles
Polysaccharide cell wall
Division via mitosis
LARGER than prokaryotes
Structures found in all bacteria
Plasma membrane
Cytoplasm
Ribosomes
Nucleoid w/ DNA
Can bacteria be infected by viruses
Yes
Basic shapes of bacteria
Coccus, bacillus, spirillum (rigid), spirochetes (flexible) ALSO intermittent (something in bw two structures)
Shapes determined by cell wall structure
Flagellin
Forming helical chains that make up the filament in the flagellum
Difference bw flagellum in eukaryotes vs prokaryotes
Eukaryotic flagella is covered by a membrane
Pathogenic bacteria can be identified by
differences in flagerllar proteins
Basal body similar to what in a car
Driveshaft
Cell division in Cocci bacteria
Can divide on many planes
Diplococci
Streptococci (Chain of spherical cells)
Staphylococci (CLuster of spherical cells)
Cell division in bacillus
Cell division can involve just one plane, hence fewer arrangements:
Pairs: Diplobacilli
Chains: Streptobacilli
Coccobacillus:
Vibrio
Curved shapes
Spirullum
Helical shape (corkscrew)
- Rigid structure
Spirochetes
Helical shape but flexible
Propelled by an internal axial filament
Other shapes bacteria can take
Star or rectangualr
Monomorphic
Maintaining a single state (most bacteria are this)
Pleoorphic
Having many shapes, usually if lacking a cell wall
Two meanings of bacillus
can be genus AS WELL AS shape
Do all bacteria have a cell wall
A few of them are pleomorphic and thus DO NOT
Main external structures of prokaryotes
Pilia and fimbria
Glycocalyx
Flagella
GLycocalyx
(outside structure mostly contained of carbs)
External to the cell wall
Extracellular polysaccharide
Usually sticky
Capsule: neatly organized and tightly packed, preventing phaogcytosis. MAKES THE bacteria INVISIBLE to phagocytes
Slime layer: organized and loose
Two parts of glycocalyx
Capsule and slime layer
Flagella
Made of chains of flagellin
Attached to a protein hook
Anchored to the wall and membrane by the basal body
What can alter the speed and direction of rotation of flagella
Bacteria
4 kinds of flagella arrangements
Peritichous (Attatchment around the perimeter)
Monotrichous
Lophotrichous(several bunched arising from one end) and polar
Amphitrophous (Flagella at both ends)
What is a run for a a bacteria with a flagella
Movement of bacteria by flagellar propulsion (counterclockwise)
also called a random walk
Flagella tumbling
Flagella tumbling: When one flagellum spins in the other direction (Clockwise) causing the whole rhythm to be mixed up and random tumbling
Biased random walk
Resulting from a chemotxis agent, the movement of a cell toward an attractive agent that results in less tumbling than a random walk
Chemotaxis
Using chemicals as a way to direct cells somewhere
Random walk determined by rotation of the flagella
Run is moving in one direction (counterclockwise)
Tumbling is spinning into a new direction (Caused by clockwise)
Spirochete
Spirochete bacteria have their flagella embedded in hte middle of the membrane
Fimbriae and pili
Fimbraie allow attachment
Pili (Pilus) are used to transfer DNA from one ell to another (Longer than fibraie)
Fimbriae is one of the way diseases are spread
UTIs can begin by fimbriae of bacterial cells attaching in geneital area
Which part of the phospholipid bilayer is hydrophobic
The fatty acid tails
Axial filaments are composed of
Endoflagella
Streptobacillus
Chains of rods
Correct order for gram staining
Primary stain
Mordent
Decolorize
Counterstain
What is the difference bw the condensor and the diaphragm on the microscope
Diaphragm controls amount of light entering condensor
Condensor focuses light thorugh specimen
Objects smaller than _____ must be observed through an electron microscope
0.2 micrometers
Disadvantages of TEM
No 3D aspect
specimens must be fixed, dehydrated, and viewed under a high vacuum to prevent electron scattering. These treatments not only kill the specimen, but also cause shrinkage and distort
Smear
a thin film of material containing the microorganisms is spread over the surface of the slide
Benefits of negative staining
valuable for observing overall cell shapes, sizes, and capsules because the cells are made highly visible against a contrasting dark background
Dostrotions mimized bc no fixing necessaru
Mordant
a chemical is added to the solution to intensify the stain
Malachite green stain is used
To detect the presence of endospores in bacteria
Is crystal violet a basic or acidic dye>
Basic
Microscope that produces colour images without staining using two beams of light
DIC
Affect of Gram’s iodine on gram positive cells
Forms complexes with crystal violet aking it more difficult for it to wash out
How does the age of a culture affect gram staining?
. A Gram-positive culture more than 24 hours old contains a substantial fraction of old cells with a thin peptidoglycan layer and may Gram stain pink.
How much of the culture should be sampled?
The loop should barely swipe the surface of an isolated colony to ensure that the smear is made to the correct level of thickness.
What will happen if broth is not resuspended prior to sampling>
most cells will settle at the bottom of the tube, leaving few cells at the top of the tube.
How is preparing a solid culture different from a liquid one
During smear preparation from solid cultures, a droplet of water must first be added to the slide and then mixed with the cells.
What is used to view internal cell structures in their native state
phase-contrast microscope
Which of the more high powered microscopes uses visible light?
differential interference contrast microscope
Which microscope allows visualization of atoms
scanning tunneling microscope
How can intracellular detail be viewed in a LIVING cell
Two-photon microscope
The order of making a smear
Make it
Fix it
Stain it
What is fluoresence microscopy
microorganisms stained with a fluorochrome are examined under a fluorescence microscope with an ultraviolet or near-ultraviolet light source, they appear as luminescent, bright objects against a dark background.
How does fluorecent microscopy work
Antibodies for a specific bacteria combine with fluorochrome and then bind to the bacteria, lighting it up
Scanning Acoustic Microscopy
basically consists of interpreting the action of a sound wave sent through a specimen
What does fixing a specimen imply
by passing it through the flame of a Bunsen burner several times, smear side up, or by covering the slide with methanol for 1 minut
Forgetting to fix a specimin
Will result in it washing away
Gram positive bacteria react to dye how
By retaining purple colour
Are gram positive or negative bacteria more resitient to antibiotics
Gram negative
What is alcohol used for in the gram staining process
Decolorization
Which type of stain is used to identify the bacterial agents responsible for tuberculosis and leprosy?
Acid fast staining
What is an endospore
special resistant, dormant structure formed within a cell that protects a bacterium from adverse environmental conditions
Longer, outmost region of flagella
Filament
Flagellin
Globular protein arranged in several chains that intertwine and form a helix around a hollow core.
Hook of the flagella
Slightly wider part of flagella attatching it to basal body
Basal body of flagella
anchors the flagellum to the cell wall and plasma membrane
How is fimbrae relevant to pathogens?
Fimbriae is one of the way diseases are spread
UTIs can begin by fimbriae of bacterial cells attaching in geneital area
A bacteria is determined to be virulent by the fimbriae
Example of bacteria with large capsule
Streptococcus pneumonia has a large capsule
purpose of capsule
The capsule makes the bacteria invisible
Can prevent antibiotics from entering bacteria
Protects bacteria
Purpose of cell wall
Gives bacteria its shape
Prevents osmosis lysis
Osmosis
Water moving from high to low conc across cell membrane
Is water conc higher inside or outside the cell
Outside, bc of dissolved solutes inside the cell; therefore water flows in
Cell wall in gram + bacteria
Composed of peptidoglycan
Repeating NAG-NAM units (long chain of polysachharides)
Crosslinked by peptides, teichoic acid (holds the wall together), and lipteichoic acid (anchors the wall to the plasma membrane)
Cell wall in gram - bacteria
Cell wall made of pepdidoglycan and outer membrane in G-
Cell wall (Outer membrane + peptidoglycan layer) = protection from phagocytes, complement, and antibiotics
Porins (made of proteins) form channels from inter membrane to outer to allow for easier crossing of the cell wall
Penicillin most affective against which type of gram bacteria
Gram positive bc penecillin affects peptidoglycan
Blocks peptidoglycan cross-linking of cell wall
Lysozymes effect on bacteria cells
Breaks peptidoglycan
Sleep in the eyes is often dead bacteria cells
Penecillin affect on gram - bacteria
Pepditodglycan layer sndwhiched, so penicillin cannot easily get there to affect it
Penecillin is hydrophilic, and the inner layers of membrane are hydrophobic
Important Gram + bacteria
Corynebacterium diphtheriae
Staphylococcus aureus
Streptococcus pyogenes
Streptococcus pneumoniae
Gardnerella vaginalis
Important Gram - Bacteria
Escherichia coli
Neissseria gonorhoeae
Gardnerella vaginalis
Saccharide
Sugar
What differentiates Gram - bacteria
Lipopolysaccharide within gram negative walls
Three parts of Lipopolysaccharide
lipid A buried in the outer membrane (is an endotoxin)
Core polysaccaride
Connects to the O polusaccharide tail
Endotoxin vs exotoxin
Endotoxin is only released to become a toxin when the cell breaks down (destroyed)
Exotoxins are only produced by Gram + that are released to the outside without the bacterias death
Which type of gram bacteria are antibiotics potentially dangerous for treating?
ANtibiotics can be dangerous to give to those with Gram - bacteria infections bc toxins can be released
Mycoplasmas
Type of microorganism with an atypical “cell wall”
Lack cell walls (THEREFORE UNAFFECTED BY ANTIBIOTICS)
Pleomorphic
Sterols in plasma membrane (Cholestrol etc. help keep membrane structure intact)
Acid fast cell walls
Like Gram-positive
Waxy lipid (mycolic acid)
Cells grow slowly bc of wax layer
Mycobacterium, nocardia
Parts of phospholipid bilayer
Outer is hydrophilic heads
Inner is hydrophobic (tails)
Excludes many things that are hydrophilic
Associated proteins
Associated proteins with phospholipid bilayer
Perpheral proteins: Not deep/tightly bound, can be stripped from the membrane
Integral proteins: Imbeddded in the membrane, only remobved through destroyign the membrane structure
Transmembrane proteins: Cross the membrane (porins), allow for stuff to cross the membrane (transport proteins, channels etc.)
Carbohydrates in microorganisms
Located on the outside of the membrane, attached to proteins OR lipids
Used for energy
Cell communication (When cell communication stops it results in cancer cells)
Cell identification
What are granules in the cytoplasm for?
Primarily phosphate and energy reserves
Endospores
Highly durable dehydrated dormant cells (withstands boiling for up to 20 hours)
Allows cells to survive adverse conditions
Formation of endospores called
Sporation
Endospore germination
AN ENDOSPORE returning to a vegetative (growing) cell
One endospore gives rise to one bacteria cell
Medical significance of endospores
Baciculls antrhacis - the cause of anthrax
Clostridium (gram positive therefore it is an exotoxin)
Major kinds of endospore cholstridium
Tetanus (contracts muscles)
Botulism (relaxes muscles) - used for BOTOX
Gas gangrene
C. difficile
What protein composes flagella in eukaryotes vs prokaryotes
Tubulin in eukaryotes
Flagellin in prokaryotes
How can pathogens be identified by their flagellum?
Differences in their flagellar proteins
Glycoproteins
Proteins attatched to carbohydrates
Pili are used for
Transfer of DNA and motiility
Best description of how streptomyocin attacks bacterial cells
Streptomycin targets the 70S ribosome and inhibits protein synthesis
Why are most Gram-negative bacteria resistant to the actions of penicillin?
Penicillin is unable to pass through the outer membrane of the cell wall.
What part of E. coli are responsible for the symptoms of a UTI
Fimraie
A gram-positive bacterium suddenly acquires resistance to the antibiotic methicillin. This trait most likely occurred due to acquisition of new genetic information through
conjugation
How do spirochetes and spirilla differ?
Spirilla have external flagella, but spirochetes have axial filaments.
Which kind of gram cells have porins?
Gram - bacteria
Explain Acid fast staining
What does the Gram Positive cell wall NOT protect the bacteria from
Does not prevent cell lysis in hypertonic environment
A bacterium that an grow in the presence or absence of oxygen produces energy by
Glycolysis, Krebs cycle, and oxitative phosyphorilation
