MCB3020 Week 1: History & Microscopy

Main themes

Understanding basic life processes and the application of that understanding to benefit humans

All cells

Metabolism, growth, evolution

Some cells

Differentiation, genetic exchange, communication, and/or motility

Microorganism interactions

Enzymes, control/influence of ecosystems by changing the physical and chemical properties of their habitats such as removing nutrients from the environment and excretion of waste product

Early earth

Anoxic, cyanobacteria were the first oxygenic lifeform on earth

Microbial mass

Nutrients; carbon, phosphorous, nitrogen

1900 leading causes of death

infectious diseases; flu, pneumonia, tuberculosis

Modern day leading causes of death

nonmicrobial diseases; heart disease, cancer, stroke

Gut high to low microbe count

Large intestine, small intestine, stomach with lower microbe count being more acidic (stomach) with the higher microbe count being neutral (large intestine)

Microbial impact on humans

Disease, food, agriculture, energy/environment, biotechnology

Pasteur

Discovered fermentation is a biological process not chemical, proved all cells come from preexisting cells, developed vaccines for anthrax and rabies

Pasteur’s experiment

Swan neck flask, microorganisms get trapped inside of the bend and there’s no contamination. When spilled, microorganisms enter and the liquid is contaminated.

Koch

Developed techniques for obtaining pure cultures (one microbe)

Koch’s first postulate

The suspected pathogen must be present in all cases of the disease and absent from healthy animals

Koch’s second postulate

The suspected pathogen must be grown in pure culture

Koch’s third postulate

Cells from a pure  culture of the suspected pathogen must cause disease in a healthy animal

Koch’s fourth postulate

The suspected pathogen must be reisolated and shown to be the same as the original

Cons of Koch’s postulates

Developed before the knowledge of viruses, animal models aren’t always available to test or can’t cultivate outside host body (pure culture) including cholera or rickettsia’s.

Beijerinck

Developed enrichment culture technique by manipulating nutrient and incubation conditions

Winogradsky

Developed the concept of chemolithotrophy

Chemolithotroph

Obtains energy from the oxidation of inorganic compounds such as sulfur or nitrogen

Chemoorganotroph

Obtains energy from the oxidation of organic compounds

Chemoautotroph

Obtains energy using inorganic energy sources such as hydrogen sulfide, sulfur, ferrous iron, and ammonia with most of these being extremophiles

Oxidation

In this chemical process, oxygen is gained

Reduction

In this chemical process, oxygen is taken away

Fixation

In microbiology this is done to preserve biological tissues or cells for examination

Applied microbiology

Medical microbiology and immunology with roots in Koch’s work. Agricultural and industrial microbiology with roots in Beijerinck and Winogradsky’s work

Basic microbiology

Aquatic and marine microbiology from soil microbiology, and microbial ecology

Distribution of microorganisms

From highest to lowest; marine subsurface, terrestrial subsurface, surface soil, oceans, all other habitats

Hooke

First to use a microscope and describe microbes

Van Leeuwenhoek

First to describe bacteria, described as wee animacules

Cohn

Founded the field of bacteriology and discovered bacterial endospores

Light microscope

This type of microscope is used to look at intact cells under low magnification and are composed of an ocular, objective, stage, condenser, focusing knobs, and a light source

Resolution

Determined by the wavelength of light used

Phase-contrast

Improves contrast of a sample without the use of a stain, results with dark cells on a light background, allows visualization of live samples

Dark-field

Similar to phase-contrast, results with light cells on a dark background, excellent for observing motility

Fluorescence

Used to visualize specimens that fluoresce, fluorescent dye can be used like DAPI and widely used in microbial ecology

Contrast

This can be improved by using a different type of microscopy, such as phase-contrast or dark-field

Total magnification

Product of the magnification of the two sets of lenses; 100x, 400x, 1000x

Gram stain

To perform this, spread culture in thin film over slide, dry in air, pass slide thru flame to heat fix, thinly flood slide with stain, air dry, then place drop of oil on slide to view with 100 objective lens

In more detail, flood the heat-fixed smear with crystal violet for a minute then add iodine solution for a minute. Decolorize with alcohol for 20 seconds; true gram-positive cells will remain purple while the gram-negative ones are colorless. Counterstain with safranin for 1-2 minutes, gram-negatives will appear red/pink.

Gram stain

In this type of staining, different kinds of cells are different colors due to the differences in cell wall thickness

Gram-positive

In gram-staining, the cells turn purple

Gram-negative

In gram-staining, the cells turn red/pink

DIC

Gives structures such as endospores, vacuoles, and granules a 3D appearance

AFM

A stylus is placed close to a specimen and measures weak repulsive forces between it and the specimen. A computer generates an image based on said repulsion.

CSLM

Uses a computerized microscope with a laser to generate a 3D image, laser focuses on single layers of the specimen and are then compiled to make a 3D image.