Med Micro Readings

Darkfield microscopy

Light objects are visible against a dark background

Opaque disk placed in condenser

Only light reflected off the specimen enters the objective lens

Used to visualize Treponema palldum

phase contact microscope

Splits a beam of light into 2 types of light,

- direct

- reflected

brings them together to form an image of the specimen; it allows the detailed observation of living organisms, especially the internal structures

Hooke's contribution

First to describe cells using a microscope.

Coined the term "cell"

Idenfitied cell as the basici unit

Scientific Nomenclature

Two-name system for naming organisms

The first name is the Genus; it is italicized and capitalized

The second name is the specific epithet (species name); it is italicized, but no cap

Types of microorganisms

Bacteria

Archaea

Fungi

Protozoa

Algae

Viruses

Multicellular animal parasites

Bacteria characteristics

- unicellular and prokaryotic

- 3 shapes: Cocci (circular), Bacillus( Rod), and Spiral

- enclosed in carbohydrate wall & petidoglycan

- Replicate via binnary fission

* bacteria obtain nutrience through mostly organic matter, but some can photosynthesis

Archea characteristics

unicellular, are often extremophiles, living in harsh environments (high temp, high salinity, no light). and often using alternative sources of energy such as chemosynthesis.

- prokaryotic but lack peptidoglycan wall

- these are not known to cause disease to humans

Fungi

An organism that absorbs nutrients from the environment.

- can be singel celled or multicellular

- eukaryotic

- most cannot carry out photosynthesis

-

Mold

Forms visible masses known as mycelia, which are composed of long filaments called hyphae)

Protozoa characteristics

-eukaryotic

- unicellular

- lack a cell wall

- sexual or asexual reproduction

they can either live as free entities or as parasites

- If parastitic will absorb nutrients from host

- some can utlize photosynthesis

Algae

a very simple plant without stems or leaves that grows in or near water.

- Photosynthetic Euks

- Asexual or sexual reproduction

- cell walls are made of cellulose

viruses characteristics

-acellular

-nucleic acids with protein coat

-has capsid

-can only be seen with mircoscope

- Viruses require a host to reproduce

Spontaneous Generation Theory

Hypothesis stating that life could arise from nonliving matter.

Supported by John Needham Experiment

- Boiled nutrients broth and poured it into covered flask

- Microbes grew

Conclusion:

Claimed a mircobes arose spontaneously from a "vital force" in the broth. What really happened was the jar was not sterilzed prior

Van Leeuwenhoek

First to see organisms under a microscope

- This contributed to the cell theory

Biogenesis Theory

Theory that states living things can only come from other living things

Supporting experiments include:

- Redi's Meat Jars

- Spallazoni's sterialized jars

- Pastuers S-shaped flasks

Francesco Redi

This scientist disproved spontaneous generation by showing that maggots do not spontaneously arise from decaying meat.

Placed decaying meat under 3 conditions:

- Open jar, tightly sealed jar, gauze covered jar

- the maggots only appeared in the open jar

This showed that large organisms do not arsie spontaneously

Lazzaro Spallanzani (1765)

repeated Needham's experiments to show that organisms entered the broth from air

- The nutrience broth was boiled in the flask and then it was sealed

- one set allowed air in and the other set didn't

Demonstrated that no mircoorganisms grew in the sealed thoroughly boiled flask

Louis Pasteur

A French chemist, this man discovered that heat could kill bacteria that otherwise spoiled liquids including milk, wine, and beer.

His biogenesis experiment: Boiled nutrience broth in a S-shaped neck flask which allowed fresh air in but prohibited microbes

The broth remained sterile as long as the neck remained intact.

Conclusion:

Disproved spontaneous generation definitively for microorganisms, establishing that living cells arise only from pre-existing cells

Germ Theory of Disease

the principle that microorganisms cause disease

Experiments

- Pasteur: Microbes cause the " spoilage of wine"

- Se

Compound Light Microscope

microscope that allows light to pass through a specimen and uses two lenses to form an image

condenser lens

located immediately below the specimen, focuses light from the light source onto the specimen

objective lens

the part of a compound light microscope that is located directly above the specimen and that magnifies the image of the specimen

ocular lens

Eyepiece of a microscope

this adds a 10x magnification

Path of light through a compound microscope

light source; condenser; specimen; objective lens; ocular lens

Total Magnification

objective lens x ocular lens

- Remember to start at the lowest objective lens and work up

Resolution of a microscope

measures direct clarity or ability to distinguish small but close objects

This is the ability of the microscope to distingish fine + small details.

Specifcally it is the ability of the lens to distingish two points that are a specific distance apart.

For example, a mircoscope w/ a resolving power of 0.4nm can tell points at least 0.4nm apart

refractive index

a measure of the light-bending ability of a medium

- If we can change the refractive index of specimens it can help get a clearier more sharp image

- we can stain the speciman to obtain a sharper contrast between the medium and speciman. When the ray changes direction the angles change increasing the contrast

Also remember to preserve the direction of the light rays, we use immersion oil at high resolution

Differential Interference Contrast Microscopy

Uses two beams of polarized light.

The combined images look as if the cell is casting a shadow on one side.

This adds contrasting colors to the speciman

The resolution of DIC microscope is higher than a standard phase microscope

Fluorescence Microscopy

Uses UV light

Fluorescent substances absorb UV light and emit visible light

Cells may be stained with fluorescent dyes (fluorochromes)

Immunofluorescence

a method of tagging antibodies with a fluorescent dye to detect or localize antigen-antibody combinations

confocal microscopy

a light microscope that uses fluorescent stains and laser to make two- and three-dimensional images

Transmission Electron Microscopy (TEM)

An electron microscope in which a beam of electrons is transmitted through a specimen and focused to produce an image.

Use for Dark-Field microscope

to examin living microorganisms that are invisable in brightfield microscopy and do not stain easily or are distorted by frequent staining

- Live, unstained microorganisms

- View ones typically distorted by staining

Phase Contrast Microscopy Use

To facilitate detailed examination of the internal structure of living specimens

Detailed examination of the internal structures of living organisms

Use of differential interference constract microscope

To provide three-dimensional images

- generating brightly colored psuedo-3D images of lving cells & internal structures

Use of a fluorescence microscope

For flourscent antibody techiniques to rapidly detect and identify microbes in tissues or clinical specimens

use of a transmission electron microscope

Observing the internal ultrastructure of cells under high magnification and resolution

To examine viruses or the internal ultrastructure in thin sections of cells

- Very high magnification!

- produces structure 2D of internal features

Use of scanning electron microscope

Observing the surface of a sample

To study the surface feature of cells and viruses

- This is used for the external surface

- produces 3D image of external structures like Cilia or Flagella

Use of Confocal Microscope

To obtain a two or three dimensional image of a cell for biomedical application

Glycocalyx functions

- in cell adhesion

- recognition and immunity; it prevents phagocytosis

It can be capsule form or slime form

Gram negative flagella

It contains two pairs of rings

- outer pair of rings ancored to various portions of the cell wall

- inner pair of rings anchored to plasma membrane

gram postive flagella

Only the inner ring is present

Bacteria Flagella

contain a filament composed of flagellin, a basal body that anchors and rotates the flagellum, and a hook that connects the two

The movement is generated by the rotation of the basal body. It can go clockwise or counter clockwise

Runs and tumbles

Atrichous flagella

bacteria that lack flagella

peritrichous flagella

flagella that cover the surface of a cell

monotrichous flagella

single flagellum at one end

lophotrichous flagella

multiple flagella at one end

amphitrichous flagella

flagella at both poles of the cell

Archaella

•Archaeal motility structure

•Made of glycoproteins archaellins

•Anchored to the cell

•Archaella (singular: archlaellum) rotate like flagella

axial filaments

-also called endoflagella

-in spirochetes. -anchored at one end of a cell. -rotation causes cell to move

Brightfield characteristics

- Speciman is darker against white, bright background

- Not good for objects less than 0.2 µm

Darkfield characteristics

Specimen: appears bright and luminous against an entirely black background

- Thin glowing outlines will be against pitch black

Phase contrast characteristics

Background: Flat, neutral gray.

Specimen: Sharply defined internal structures with characteristic bright or dark halos around the cell perimeter.

Dead giveaway: Unstained, living cells with distinct glowing fringes or rings around the edges.

Differential interference contrast (DIC) characteristics

Background: Often gray or soft pastel colored.

Specimen: Looks like an embossed, 3D relief sculpture with distinct shadows cast across the cell surface, often with prism-split colors.

Dead giveaway: Appears pseudo-3D and brightly colored despite being unstained.

Transmission electron microscopy characteristics

Format: Flat, 2D slice.

Specimen: High-magnification internal ultrastructure (ribosomes, organelle membranes, viral envelopes, DNA filaments).

Dead giveaway: Looking inside a cut cell or viewing tiny viruses in black and white (or artificially tinted).

scanning electron microscopy characteristics

Format: Dramatic 3D perspective.

Specimen: Intact outer surfaces, showing external textures like cilia, flagella, cellular grooves, or biofilms with realistic shadows.

Dead giveaway: Looks like a photograph of an intact object taken with a camera, showing full external depth.

Diplococci

Spherical bacteria that grow in pairs and cause diseases such as pneumonia.

Streptococci

bacteria that form a chain

Tetrads

4 cocci in a square

Sacrine

Cube like Cocci

Staphylococci

a group of about 30 species of bacteria that form irregular groups or clusters resembling grapes

Diplobacilli

A double bacillus, two being linked end to end to each other

Streptobacilli

rod-shaped bacteria occurring in chains

Coccobacilli

very short rods

Semmelwies

-Brilliant individual in the 19th century

-Stressed the importance of washing hands before conducting surgery

- Advocated for handwashing bc of observatiions

Robert Koch

German doctor who identified the bacterium that caused tuberculosis

Proved bactereium cause disease

Koch's Postulates steps

1. The same pathogen must be present in every case of the disease.

2. The pathogen must be isolated from the diseased host and grown in pure culture.

3. The pathogen from the pure culture must cause the disease when it is inoculated into a healthy, susceptible laboratory animal.

4. The pathogen must be isolated from the inoculated animal and must be shown to be the original organism.

basic dyes

cationic, positively charged chromophore

Bind negative molecules; nucleic acids

- crystal violet

- methylene blue

- malachite green

- safranin

This will be used in postive staining

acidic dyes

anionic, negatively charged chromophore

- eosin

- acid fuchsin

- nigrosin

This is used in negative staining

normal mircobiota

permanently colonize the host and do not cause disease under normal conditions

Simple stains

one dye is used; reveals shape, size, and arrangement

- typically a basic dye

- mordant can be used to increase affinity of stain to speciman and coat structure like flagellum

acid fast stain

carbolfuchsin used to stain Mycobacterium species

A staining procedure for identifying bacteria that have a waxy cell wall.

A differential stain used to identify bacteria that are not decolorized by acid-alcohol

Negative staining

microbe repels dye, the dye stains the background

- Capsule can be identified as clear, colorless halo

Bacterial capsules are composed water-soluble polysaccharides to they repel stains and wash off in water

flagella staining

mordant applied to increase thickness of flagella

Mordant on flagella

Carbolfuchsin simple stain

endospore stain

A differential stain used to detect the presence and location of spores in bacterial cells.

Malachite green will be used to stain endospores, and safrin will be applied to rest