Session 1-4

A series of operations have been developed to limit the risk of contaminating these materials during manipulations of isolating pure cultures. These operations are known as _____

Aseptic Technique

What doe maintaining asepsis mean?

the state of being completely free from disease-causing microorganisms like bacteria, viruses, and fungi

What are the general rules for Aseptic Technique?

Work on a clean tabletop. Put all unnecessary items away.

Wear a lab coat; wash hands before performing any manipulations and after you are through.

Disinfect the benchtop with an appropriate disinfectant before you begin working and after you are through.

Focus on your work and avoid being disturbed.

Keep all bottles, tubes, petri dishes, etc closed as much as possible. Keep tubes and bottles upright to avoid spills and contamination.

Flame bottle and tube mouths before and after use.

Sterilize inoculating loops and needles before and after use.

What are the defining features of microbes that are composed of eukaryotic cells?

They have a true nucleus and membrane-bound organelles.

What are defining features of microbes made of prokaryotic cells and what makes prokaryotes so special?

Their DNA is condensed in a region called the nucleoid. They are renowned for their metabolic diversity and an ability to secure a habitat in highly unfavorable conditions.

What are the three kinds of microbe eukaryotes that have a true nucleus?

Algae, Fungi, and Protozoa

What are the two kinds of microbe prokaryotes that have a nucleoid?

bacteria and archaea

What is the phylogenetic relationship between domains Archaea, Eukarya, and Bacteria?

Eukarya and Archaea are more closely related to each other than to Bacteria.

What is the purpose of Kohler illumination?

to maximize resolution, the detail provided by magnification.

What is resolution?

the ability of a viewing instrument (the eye, a camera, a microscope) to distinguish adjacent objects as separate rather than as one larger object

What are the two most important factors that determine resolving power of a microscope?

the quality of the lens system and the wavelength of the source of illumination

What is the relationship between resolution and wavelength?

Resolution varies inversely with wavelength; a shorter wavelength allows for greater resolution.

Describe light microscopes and what is the most common light microscope that is used?

Standard laboratory microscopes are compound microscopes, constructed of two lenses in series that focus rays of light to illuminate the object. High quality lenses give light microscopes resolving powers of about 200 nanometers, a 500- fold increase over that of the eye. This allows the observer to visualize the outlines of bacterial cells and only glimpse at the larger internal structures in cells of plants and animals.

What is total magnification and how is it obtained?

It is the apparent enlargement of an object's image. In a compound microscope, it is calculated by multiplying the power of the objective lens by the power of the ocular (eyepiece) lens (for example: 40x × 10x = 400x)

What is the numerical aperture (NA) of a lens?

its light gathering capacity

Why is immersion oil used on the 100x lens?

Some light is lost when it passes from the specimen on a glass slide through air to the objective lens. This occurs because the refractive index (ability to bend light) of glass and air differ. The air space can be replaced by a drop of immersion oil, which has a higher refractive index, closer to that of glass. When the [100x] objective lens is lowered into the oil, more light is transmitted to the lens.

What is the working distance and what is its relationship with magnification?

Working distance (the distance between the specimen and the objective lens) decreases with higher magnification.

Quality brightfield instruments are parfocal and parcentric.

What does that mean?

A parfocal optical system stays in focus when you switch between different objective lenses.
A parcentric optical system keeps the same center point in the field of view when you change lenses

Match the diagram with its part of a microscope

A: Ocular Lens
B: Revolving
C: Objective Lenses
D: Stage
E: Diaphragm lever
F: Condenser
G: Light Source
H: Field limiting Aperture
I: Base
J: Coarse and fine adjustment knobs
K: Stage Controls
L: Light intensity adjustment
M: Slide Holder
N: Arm

Microscope part:
Ocular lens/eyepiece

Magnifies the image. In BIOL 302L, ocular lens is 10x

Microscope part:
Revolving Nosepiece

Rotates objective lenses into viewing position

Microscope part:
Mechanical Stage Controls

Moves slide holder/specimen to view different regions of the specimen

Microscope part:
Slide holder

Holds slide in place and adjust with stage controls

Microscope part:
Stage

Holds slide and opening to allow light pass to slide

Microscope part:
Coarse Adjustment Knob

Brings the specimen into focus with the 4X and the 10X objective

Microscope part:
Fine Adjustment Knob

Brings the specimen into focus with the 40X and 100X objective

Microscope part:
Condenser

Focuses the light on the specimen

Microscope part:
Diaphragm Lever

Controls the amount of light passing through the condenser

Microscope part:
Light source

Illuminates the specimen

Microscope part:
Light Intensity Adjustment

Controls the light intensity

Microscope part:
Arm

Supports the head and stage

Microscope part:
Base

Supports the microscope

Before returning the microscope to its designated space in the storage cabinent:

remove any slide

clean the optical system

rotate the lowest power objective into position

center the mechanical stage

wrap the light cord around the base, and cover.

How does a fluorescence microscope work?

This variation of the brightfield microscope uses a mercury arc vapor lamp as a source of ultraviolet light. A fluorophore, or a chemical compound with known fluorescent activity, can be attached to a sample for visualization. When ultraviolet light hits the fluorophore, the electrons in the molecule become “excited.” When they return to their natural energy state, they release energy in the form of light, which we can then visualize in the fluorescence microscope. A filter is between the objective and ocular lenses, which allows only the fluorescence emitted from the sample to reach the eyepiece. A special condenser is used so that the background field is dark, thereby resulting in a high contrast fluorescent image. The color that we visualize after the electrons emit light depends on how much energy the electrons absorb, which depends on their location in the valence shell. It can be predicted using a graph called an absorption and emissions profile, which plots relative intensity of fluoresced light against wavelength.

Why are Fluorescence Microscopes used in BIOL 302L?

Fluorescent microscopes are often used in laboratories to visualize localization of certain compounds within cells, since fluorophores with specificity for certain molecules can be designed.

What is the difference between Confocal microscopy and fluorescence microscopy?

Confocal microscopy is different in that it uses point illumination. By illuminating only the point of focus, unwanted scattered light is reduced. In addition, light from the specimen passes through a pinhole aperture that blocks light not coming from the focal point. Reducing stray light from sources not at the focal point improves resolution. This technique also allows 3D images of the sample to be built from viewing thin sections, which is useful for looking at thick samples and preserving spatial relationships.

How does a phase contrast microscope work?

Living cells can best be viewed with a phase-contrast microscope. The optics of the microscope translate differences between the refractive indices of cellular components and the surrounding medium into differences in light intensity. The phase contrast microscope is very useful for looking at small cells at lower magnification (40X).

How does a darkfield microscope work?

In this microscope, the condenser is designed to prevent light from passing to the objective unless it strikes the specimen. Thus the background is dark. The high contrast between background and the specimen makes it possible to view living unstained specimens.

What are the two kinds of electron microscopes?

Transmission electron microscope (TEM) and the scanning electron microscope (SEM)

What is shared between transmission electron microscope (TEM) and the scanning electron microscope (SEM)?

Both contain magnetic lenses that focus a beam of electrons on the specimen. Electrons used in this fashion generate a wavelength that may be 100,000 times shorter than that of visible light. As a result, electron microscopes have resolving powers as much as 400 times that of light microscopes and 200,000 times that of the human eye. Electron microscopy gave scientists their first look at the world of viruses.

How does a transmission electron microscope (TEM) work?

The TEM bombards a thin specimen with electrons. Depending on their composition, the components of the specimen transmit, absorb or deflect the electrons. The image produced on a photographic plate is a visual translation of this interaction of electrons with the specimen. The transmission electron microscope exposes many secrets of subcellular structure. Samples are usually fixed in a resin and then very thin slices are observed allowing us to see a cross section of a cell (2D).

How does a scanning electron microscope (SEM) work?

It is designed to generate dramatic images of the surface that provide information about depth and surface structure (3D). In this microscope an electron beam is moved back and forth over the surface of a metal-coated specimen causing the emission of secondary electrons from the specimen. The secondary electrons produce the stunning images characteristic of scanning electron microscopy.

Contrast “magnification” and “resolution.”

Magnification makes small objects appear larger, while resolution is the clarity and ability to distinguish two separate points as distinct objects.

Which lens is used with immersion oil? Does immersion oil improve magnification or resolution? Explain.

Immersion oil is used with the 100x oil immersion objective lens (yielding a total magnification of 1000x when paired with a 10x ocular lens). Immersion oil does not improve magnification, it improves resolution (and image clarity/brightness).

When using the 100X objective to view samples on our BIOL 302L microscopes, what is the total magnification of the sample?

Objective Lens × Ocular Lens
(100x) x (10x) = 1000x

What important discovery did the German scientist Robert Koch in microbiology?

Developing a solid nutritional medium for isolating pure cultures, a single organism and its reproduced progeny. Only with pure cultures can the properties of individual types of microorganisms be examined and understood.

What is a colony?

During isolation of pure cultures, microbes are spread across the solid plating surface and separated from each other. When these individual organisms reproduced, they formed colonies that represent the progeny of a single bacterium (colony-forming unit or CFU); each colony is a pure culture.

What solidifying agent in plates replaced gelatin?

Agar, a more stable algal product, replaced gelatin.

What are the two pure culture techniques used?

Streak plate and spread plate

How does streak plates work?

In the streak plate, cells are dragged farther and farther apart from each other on the surface of the plate so, at some point in the streak, single cells and their offspring can form a distinct colony. Because the streak plate involves a kind of dilution on the surface of the plate via repetitive streaking with a sterile loop, it can be performed with very dense cultures.

How does spread plates work?

Spread plates need to be performed with cultures that are diluted enough to give a countable number of colonies. (using glass rod).

Why is it difficult to view bacteria on a microscope and what can be done to mitigate this issue?

Even with the microscope, bacteria are difficult to see unless they are treated in a way that increases contrast between the organisms and their background. The most common method to increase contrast is to stain part or all of the microbe.

What are the two classifications of staining methods that are used routinely with bacteria?

These methods may be classified as 1) nonspecific and 2) specific

How do nonspecific stain work?

Nonspecific stains will react with all microbes in an identical fashion. They are useful solely for increasing contrast so that morphology, size and arrangement of organisms can be determined.

How do specific stains work?

Specific stains give varying results depending on the organism being treated. These results are often helpful in identifying the microbe.

Generally, what are the four main categories of bacterial morphology?

rod-shaped (bacillus; plural bacilli), spherical (coccus; plural cocci), curved (vibrio), and spiral (spirillum or spirochete).

Match the letters with bacterial morphologies

A: diplobacilli
B: streptobacilli
C: coccobacilli
D: Vibrio (curved rod)
E: Spiral bacteria: spirilla (rigid shape, external flagella) or spirochetes (flexible shape, periplasmic flagella)
F: diplococci
G: Streptococci
H: Tetrad
I: Sarcia
J: Staphylococci

What is a basic stain?

a stain that is cationic (positively charged) and will therefore react with material that is negatively charged. The surface of bacteria at neutral pH is somewhat negatively charged and will therefore attract basic stains.

What are examples of basic stains?

crystal violet, safranin, basic fuchsin and methylene blue

What is an acidic stain?

stains that have negatively charged chromophores and are repelled by the bacterial surface. They stain the background and leave the microbe transparent.

What are examples of acidic stains?

Nigrosine and Congo red

When a single stain is used, the procedure is known as a ____?

Simple Stain

What does it mean to prepare a smear?

it means to prepare a thin layer of the specimen immobilized on a slide before staining.

What is fixation?

improves attachment of the sample to the slide, it also preserves cell structures and inactivates enzymes that might damage cell morphology.

What are the three ways a sample can be immobilized onto a slide

Air drying helps to immobilize a sample onto a slide, but for best adherence, the sample should be fixed.
Fixation can be accomplished using heat (passing the slide through a flame) or chemical fixatives. Heat fixation is simple and quick, but chemical fixation may be preferred when observing delicate structures within cells.

What is the three gradient of the Winogradsky column?

oxygen, sulfide, and light

Match the order of the microbial zones of the Winogradsky column?

A: Algae, Cyanobacteria, Aerobic heterotrophs
B: H2S- Oxidizers, Facultative anaerobes (though can be found throughout).
C: Purple non-sulfur, photoheterotrophs
D: Purple sulfur bacteria
E: Green sulfur bacteria
F: Sulfate reducers, fermentative heterotrophs

How do purple sulfur and non-sulfur phototrophs work?

These microbes are living by photosynthesis, but they do not oxidize water to produce oxygen. The purple non-sulfur phototrophs oxidize organic material with photosynthesis and the purple sulfur bacteria oxidize hydrogen sulfide to produce sulfur and/or sulfate using photosynthesis. These organisms often accumulate elemental sulfur granules in their cells and produce a variety of reddish purple pigments.

How do green sulfur bacteria work?

They can tolerate high levels of hydrogen sulfide and only need low levels of light. They are also photosynthetic and oxidize hydrogen sulfide to sulfur and/or sulfate.

How do sulfate reducing bacteria work?

These anaerobic organisms are generally heterotrophs, consuming organic material for energy, but they breathe with sulfate instead of oxygen.

What is a negative/background stain and how is it performed?

When a stain, such as an acid dye, cannot penetrate the outer layers of a microbe, the cell will appear transparent on a colored background. This stain is called a negative or background stain and is performed by mixing the dye with a suspension of bacteria on a slide and spreading the mixture into a thin layer for viewing.

What is an example of negative/background stain?

Capsule Stain

What is a capsule?

is a well-organized, protective outer layer of sugar polymers or proteins that surrounds the cell envelope of many bacteria.

What are capsules made out of?

polysaccharide (or polypeptide)

What are the three purposes of capsules?

1.) Prevents Desiccation (drying out)
2.) Resists phagocytosis (evade white blood cells)
3.) Aids attachment.

What are the two stains used in Capsule stain and how will the capsule appear?

The acidic dye, Congo Red will stain the background, and a basic dye, Maneval’s stain will stain the bacterial cell. The capsule appears as a colorless layer between the bacterium and the background.

Which stain is never heat fixed and why?

A capsule stain is never heat-fixed because heat destroys, shrinks, or distorts the delicate polysaccharide or polypeptide capsule surrounding the bacterial cell.

What kind of stain method is the gram stain?

It is a differential stain method. The Gram stain categorizes bacteria into two groups based on their reaction to the stain, mainly due to differences in the thickness of the cell wall.

What are the steps in gram stain?

1.) Primary (basic) stain with Gram’s Crystal Violet (CV). Stains all cells purple/violet. (1 min)
2.) Mordant - Gram's Iodine (I) (CV-I complex formation)
3.) Decolorization - Gram’s alcohol (Washes CV-I complex in gram (-) due to thin cell walls, does not easily wash gram (+) due to thick cell wall) (10-20 sec)
4.) Counterstain - Safranin (20 sec). (stains any decolorized cells pink-red. will not change the color of the cells that retain the crystal violet.)

What will gram positive bacteria look like after a gram stain?

will appear violet at the end of the procedure, since the thick cell wall allows the bacteria to retain the crystal violet-iodine (CV-I) complex during the decolorization step

What will gram negative bacteria look like after a gram stain?

since the thin cell wall of Gram negative bacteria allows the CV-I complex to be washed out during the decolorization step, these cells will appear pink from the counterstain at the end of the procedure.

Is Penicillin more effective against Gram positive bacteria or Gram negative bacteria?

penicillin is rarely effective against gram negative bacteria, but often is the drug of choice against gram positive bacteria. (Penicillin must penetrate the bacterial cell wall in order to interfere with cell wall synthesis. The Gram negative outer membrane usually prevents entry of penicillin.)

Why might it be necessary to dilute bacteria who have a high population density like (10^9/ml)?

in order to isolate single organisms, estimate their numbers, or prepare smaller populations for analyses.

Fill in the blank
Dilutions are generally performed in multiples of _A_ or _B__.

A: two
B: ten

Fill in the blank
To determine the actual number of organisms in each tube, a __ ml sample can be removed and plated.

0.1

Fill in the blank
The ideal number of colonies to count is in the range of _A_ to _B_ colonies per plate

A: 30
B: 300

Fill in the blank
If there are more than 300 colonies on a plate, in this course we will call that “too numerous to count” (_A_).

A: TNTC

What is the formula for calculated the # of expected CFUs plated?

CFUs = [Final Concentration] x (Volume plated mL)

What is the formula for calculating dilution?

Dilution = (x) / (x+y)

What are the two formulas for calculating total dilution factor (TDF)?

TDF = DF1 x DF2 x DF3 x …….
TDF = [Original Conc.] x [Final Conc.]