Microbiology BIO 2060: Midterm 1

Yersinia pestis

The bacteria that caused the plague (is gram negative)

Modern science

All natural phenomenon are caused by natural forces, supernatural forces don’t apply

Small animals/rats

Yersinia pestis host

Zacharias Janssen

Made the first compound microscope in the 1500s

Robert Hooke

Achieved 200x magnification, used reflected light

Anton van Leeuwenhoek

Used transmitted light microscopes, could magnify up to 270x. The light comes directly through the sample. Was the first to see individual bacteria/bacterial motility

Animalcules

The name that Anton van Leeuwenhoek gave bacteria

Spontaneous generation/abiogenesis

The idea that lesser organisms could spontaneously produce out of nonliving material

Vital force

Name for the force that inanimate things have as a special life-giving property

Vitalism

Belief that there are vital energy forces that govern our health and wellbeing, not supported by modern science

Francesco Redi

Aimed to prove that the vital force was not the reason for the generation of life; did the cheesecloth experiment

Infusion

The liquid produced when you take plant material and soak it in water

Infusoria

The community of microbes that come off of the plant material after it is soaked in water

John Needham

Did an experiment where he boiled an infusion, cooled it, and then sealed the flasks. Infusoria reappeared (evidence that the vital force could make the infusoria reappear)

Larazzo Spallanzani

Redid Needham’s experiment, but sealed the flask first before boiling it. The infusoria did not come back. There was no air exposure after boiling the infusoria, so this showed that the air is what carries that vital force.

Louis Pasteur

Invented pasteurization (the use of limited heat to kill microorganisms in food).

Performed the S flask experiment (the curve didn’t block the air from reaching the broth, however the curve traps the dust and microbes in the bottom of the curve, preventing it from reaching the broth - therefore the vital force is not doing what everyone thought).

Germ theory

microorganisms are the causative agent of infectious disease

Varro Reatinus

Said the quote “there are bred certain minute creatures which cannot be seen by the eyes, but which float in the air and enter the body through the mouth and nose and cause serious diseases” 2000 years ago, but didn’t have the tools to demonstrate it

Agonstino Bassi

Showed that a mold was responsible for silkworm disease: the elimination of the microbe eliminated the disease

Ignax Semmelweis

Told birthing attendants to wash their hands between patients, and childbirth fever deaths dropped off dramatically. Was the first instance of handwashing in healthcare

Joseph Lister

A surgeon who first cleaned surgical instruments in phenol solutions before using them on a patient. Caused surgical infections to drop off.

Koch’s postulates

Still in effect today, establishes a link between the microbe and the disease.

1) Took blood of a cattle killed by anthrax

2) Found Bacillus anthracis in pure culture

3) Put it into a healthy animal, it got sick and died

4) Recovered B. anthracis in pure culture

Edward Jenner

Introduced the world to the first vaccine. Noticed that people with cowpox rarely got the more serious disease smallpox. Used this to develop a protocol where he would collect fluid from under the scabs of people with cowpox and expose healthy people to that virus with a prick of the skin. Our immune system learns to recognize surface antigens and develops antibodies against them

Rabies

What disease was involved in the first case of a vaccine saving a human life

Paul Ehrlich

Developed a synthetic compound called salvarsan to treat the STD syphilis, caused by spirochete bacteria. Was also effective against sleeping sickness caused by a protozoan

Alexander Fleming

Discovered that a fungus known as penicillium secreted a compound that prevented growth of bacteria. This naturally occurring compound is known as an antibiotic

Antibiotic resistance

Occurs when:

1) the gene conferring that resistance has become established within the lineage of the bacteria

2) overuse and improper use leads to natural selection pressure for the bacteria that have this resistance

3) The bacteria starts to degrade/alter the antibiotic or pump the antibiotic out of the cell

Genetic analysis

The most accurate and efficient way to identify the presence/absence of particular microbes

Paul Berg

Started cloning using plasmids

Stanley Cohen and Herbert Boyer

Set up the first biotechnology company around the use of recombinant DNA in bacteria (Genentech). Could produce human growth hormone from this process

Compound microscope

Achieves higher magnification than the dissecting microscope

Contrast

The ability to see the specimen against the background

Resolution

The ability to see 2 points that are close together, but being distinct (“sharpness” of the image)

Quality of the image

Determined by: magnification, contrast, and resolution

Millimetre (mm)

The SI unit used for viewing larger structures under the microscope (10^-3)

Micrometre (um)

The SI unit used for viewing individual cells under the microscope (10^-6)

Nanometre (nm)

The SI unit used for viewing really small structures and wavelengths of light (10^-9)

Ocular lenses

Adjust the difference between these to adjust for the difference between your eyes

Objective lenses

the lenses that are closest to the specimen

Stage

Where you keep the slide bearing the specimen, use knobs to move the specimen around

Condenser lens and iris diaphragm

Allow you to control the quality of the light source

Base

Contains the coarse and fine focusing knobs

Pathway of light

The source → condenser → diaphragm → specimen → objective lenses → body tube → ocular lenses → eye

Ocular x objective lens

Total magnification equation

increases

When magnification increases, the resolution _____

0\.5

Our eyes can resolve things that are ____mm apart

1

Bacteria are about ___mm in at least one dimension

Reflection and refraction

Occur when light travels from one medium to another medium when the two media have a different refractive index

Refraction

When the angle of incidence is different than the angle of refraction

Refraction

Allows us to see the image in the microscope

Resolving power/resolution

The ability of a lens system to be able to resolve two points as being distinct, giving a sharp image, represented by the variable “d”

lambda/(2NA)

Equation for resolving power/resolution (d)

Decreases the value of “d”

1) shorter wavelengths of light from the illumination source (could use filters - can harm contrast)

2) Higher magnification

3) Make the lens closer to the specimen

4) Alter the refractive index in the space surrounding the specimen to match that of the specimen

Oil immersion

Microscope technique where you replace the air between the specimen and the objective lens with a bit of oil. Increases the resolving power, only done at 100x objective as it has a short focal length

Brightfield microscopy

The light from the illumination source is focused on the specimen, the image seen is due to refracted light

Darkfield microscopy

Can place an opaque disk between the illumination source and then condenser lens to increase the contrast. The disk blocks the light so what gets to the specimen is more of a ring of light. Only the refracted and reflected light enter the objective, so there is no background light, making the specimen easier to see

Fluorescence microscopy

Used to identify specific types of microorganisms in a mixed background, uses UV light to detect antibodies specific for the surface proteins on a bacterial species. The antibodies are chemically conjugated to fluorochrome, which emits light when it is hit with UV light. Emission only comes from microbes that have the conjugated antibody stuck to them, therefore you can tell if the microorganism is present or absent

Confocal microscopy

A way of manipulating light at very specific wavelengths that are focused into a specimen at very different depths. The specimen was treated so that different parts of it are stained differently, and the computer can create a 3D image of the specimen

Electron microscopy

Wavelength of electron beam is very small; this microscope doesn’t use light, but rather an electron beam. Resolving power can be 100x better than using visible light

Transmission electron microscopy

Electron beam passes through a very thin section of specimen, allowing detailed image of internal structure. Special stains can increase contrast.

Scanning electron microscopy

Take a specimen coated in an electron dense medium such as gold. The high electron density doesn’t allow the electron beam to penetrate the surface, rather it knocks electrons off the gold coating, and those electrons are collected by sensors to make detailed image

How to change contrast

1) Change the illumination

2) Stain

Staining steps

1) Smear light sample onto slide

2) Heat fix specimen to slide

3) Apply staining technique

Basic dyes

The coloured portion (chromophore) is cationic (+ charge). The negatively charged cells attract the positive charge of the stain. Most common type of dye

Acidic dyes

The coloured portion (chromophore) is anionic (- charge). Dye molecules fall around the cell, leaving the cell unstained and the background dark. Do not need to be heat fixed onto the slide (prevents distorted shape/size)

Nigrosin, negative stains

Examples of acidic dyes

Crystal violet, safranin, simple stains

Examples of basic dyes

Differential/special stains

Target specific features that differ between different bacterial types. Uses mordants, counterstains, and heat

Mordant

Enhances the effect of a dye

Gram stain

Targets a difference in the cell wall of bacterial cells (thin or thick?)

Gram positive bacteria

Have a thick cell wall/stain purple in the gram stain

Gram negative bacteria

Have a thin cell wall/stain red in the gram stain

Gram stain steps

1) Stain the fixed smear with crystal violet, then rinse off the excess dye. The cells will stain purple

2) Apply a mordant (iodine). Enhances the effect of the dye by binding to the crystal violet and creating a CV-iodine complex

3) Remove CV-I complex using a decolourizing agent such as alcohol. The complex gets removed from gram negative cells but not gram positive cells, so the gram positives remain purple

4) Counterstain with safranin to stain the gram negative cells red

Negative

Rod bacteria TEND to be gram ___

Positive

Coccal bacteria TEND to be gram ____

Acid fast stain

Stain oriented towards bacteria that have waxy mycolic acids in their cell wall (most notably the genus Mycobacteria - causative agents of tuberculosis and leprosy)

Acid fast stain steps

1) Stain bacteria with carbol fuchsin.

2) Heat the slide to enhance penetration of the stain into the bacterial cell wall. If the cell wall contains mycolic acids, it will bind the carbol fuchsin tightly.

3) Use an acid-alcohol rinse, if mycolic acids were present, the rinse would not remove the stain and the cells remain red. If mycolic acids were absent, the rinse would remove the stain.

4) Counterstain the unstained cells with methylene blue

Endospores

Sessile cells that are created by two genera of bacteria (bacillus and clostridium) in response to environmental pressure. A copy of replicated DNA is isolated in a small portion of the cell, and the plasma membrane and cytoplasm surround the DNA. A layer of peptidoglycan is laid down, called the spore coat. Remains sessile until triggered to become vegetative again

Endospore stain

Stain that looks for the formation of endospores

Endospore stain steps

1) Malachite green applied to a heat fixed slide

2) Heat to help the dye penetrate the thick endospore wall

3) Rinse off the excess stain with water (will also remove the stain from vegetative cells)

4) Counterstain the vegetative cells with safranin. Endospores will be green, and vegetative cells will be red

Flagella stain

Thicken the flagella with a mordant (potassium alum, mercuric chloride, and tannic acid). When the thickness has built up, can stain the flagella with carbol fuchsin (CAREFULLY as they are thin and can break off)

Glycocalyx

Allows bacteria to attach to a substrate. Is composed of carbohydrates, glycoproteins, and glycolipids

Prokaryote

Means “pre-nucleus”

Eukaryote

Means “true nucleus”

Bacteria and archaea

The 2 domains of prokaryotes

Coccal

Spherical form of bacteria. Often form groupings, also found in tetrads, and groups of 8 known as sarcinae

Bacillus

Rod form of bacteria

Coccobacillus

Form of bacteria that is in between “round” and “not round”

Curved bacteria

Form of bacteria that has 3 subgroups: vibriod, spirillum, and spirochete

Pleomorphic

Term that describes the fact that the form of cells within some species can be somewhat fluid

Inner mitochondrial membrane

Where does the electron transport chain occur in eukaryotes

Plasma membrane

Site of electron transport chain in prokaryotes

Thermophilus

Bacterial genus that like high temperatures, have a higher degree of saturation in the phospholipids

Pseudomonas

Bacterial genus that likes low temperatures, lower degree of saturation in the phospholipids

Aquaporin

A type of transporter protein that regulates the flow of water in/out of the cell

Lysed

What happens to a cell in a hypotonic solutions

Shrivels

What happens to a cell in a hypertonic solution (slows metabolically and ___). Is the reason why salty brine and high sugar are good preservatives

NAG and NAM

The two amino sugars that make up the long strands of polysaccharides that make up the cell wall of prokaryotes

Peptides

Strands of amino sugar polysaccharides are connected by short ____ that make up side chains and cross bridges in the cell wall

Peptidoglycan

Determines the thickness of the cell wall of bacteria