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MICROBIOLOGY
LAB MANUAL

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Lab Index Page 1 Page 3 Lab 2

Lab 1
Microscopy


CONTENT

1) Compound Light Microscope

Basic Theory of Light Microscopy
Examples of Compound Light Microscopy
Microscopic Observations
Light Microscopy Questions
Virtual Light Microscopy
Virtual Light Microscopy Questions

2) Electron Microscopy

Examples of Electron Microscopy
Virtual Electron Microscope
Virtual Electron Microscopy Questions

What Do I Need To Hand In For This Lab?

Sketches

Compound Light Microscope
Bacteria Types (3)
Fungi
Pond Mix

Questions

Light Microscopy Questions 1-4
Virtual Light Microscopy Questions 1-4
Virtual Electron Microscopy Questions 1-4

Cover Page

Lab 1 Cover Page


Types of Microscopes

Light Microscope (LM)

The models found in most schools, use compound lenses and light to magnify objects. The lenses bend or refract the light, which makes the object beneath them appear closer.

Stereoscope

This microscope allows for binocular (two eyes) viewing of larger specimens. (The spinning microscope at the top of this page is a stereoscope)

Scanning Electron Microscope (SEM)

This microscope allows scientists to view a universe too small to be seen with a light microscope. SEMs dont use light waves; they use electrons (negatively charged electrical particles) to magnify objects up to two million times.

Transmission Electron Microscope (TEM)

This microscope also uses electrons, but instead of scanning the surface (as with SEM's) electrons are passed through very thin specimens.


1) Compound Light Microscope

The microscope has been one of the key instruments used by the biologist for hundreds of years.  A number of improvements were made to light microscopes during the first two or three centuries after their invention. Since the turn of the century, however, there have been no significant improvements. There have been continuous advances in the methods of preparing and analyzing specimens. There have also been modifications to the light microscope which permit new methods of analysis (phase contrast, fluorescence, confocal laser scanning, etc.) and the introduction of the electron microscope which can push microscopic analysis to the molecular level.

Link to a more thorough discussion of the history of microscopy

A) Basic Theory of Light Microscopy

Magnification

The main purpose of a microscope is to magnify and increase the visibility of a small object. The magnification of a lens is always engraved on it. You will be using a compound microscope which has a system of lenses. The total
magnification of the scope is a product of the magnifications of the objective lens and the eyepiece (or ocular lens). For example, using a low-power objective (magnification = 3.4X) and a standard eyepiece (magnification = 10X), the total magnification is 34X.

Link to a more thorough discussion of magnification

Resolution

Resolution is an important attribute of a microscope. The limit of resolution of an optical system is the minimum distance by which two objects can be separated and still be perceived as distinct. Two points placed closer than this limit will be seen as one. Greater resolution allows one to see an object more sharply and to make out internal detail.

 Link to a more thorough discussion of resolution

Illumination

Adequate lighting will allow you to obtain the best resolution possible. Illumination must be adjusted for each objective every time a change is made. The adjustments which will affect illumination on your microscope involve changing the iris diaphragm. The iris diaphragm is used to match the aperture (opening) to that of the objective. It should not be used to control the intensity of illumination. With some unstained or transparent specimens, it may be necessary to close the iris slightly to improve contrast. This is always done at the expense of resolution.

Link to a more thorough discussion of illumination

SKETCH 1
**Sketch a compound light microscope and label the following:
Eyepiece, Objective, Stage, Fine Adjustment, Coarse Adjustment, Diaphragm

 

Click Here to Access a Form to Perform Sketch 1 (MS WORD)

Click Here to Access a Form to Perform Sketch 1 (PDF)


Figure 1.1
Compound Light Microscope
 

Figure 1.2 Diaphragm
 

Figure 1.3 Objective
 

Figure 1.4 Ocular (Eye Piece)
 

Figure 1.5 Stage
 

Link below to quiz yourself on the parts of a compound light microscope. You do not need to submit the answers for the quiz with your lab report.

http://www.biologycorner.com/microquiz/index.html


B) Examples of Compound Light Microscopy

Figure 1.6
St
aphylococcus aureus (bacteria)

Figure 1.7
Fungal hyphae (fungus)
 

Figure 1.8
Amoeba (protist)
 

Figure 1.9
Green algae
 



Figure 1.10
Mycobacterium leprae (stained dark blue)
are shown above in human tissue
 

Mycobacterium leprae, the bacterium that causes leprosy was discovered in 1873 by Gerhard Henrik Armauer Hansen (Bacteria
Genomes 2008). Also known as Hansens disease, leprosy has a long history of associated stigma, which prevents people from seeking medical treatment and often thwarts public health intervention to curb further spread of the disease. For this reason, public health interventions should include efforts to reduce stigma via an educational awareness program and training of local health officials.

Leprosy is a human disease caused by the bacillus Mycobacterium leprae (Figure 1.10). M. leprae is an acid-fast bacterium. As one of the slowest growing bacteria known and its inability to grow independently, successful in vitro cultivation has never been achieved. Although found in the same genus as the tuberculosis bacterium (Mycobacterium tuberculosis), the two diseases cause different symptoms.

It is hypothesized that M. leprae infects a new host by way of skin or upper respiratory tract, but most experiments suggest the latter as the more likely possibility. M. leprae causes a chronic disease of the peripheral nerves, skin and mucosal membranes of the body and has an incubation period of about 3-5 years. If initial symptoms are left untreated, then permanent damage may result in many parts of the body including the eyes and outer extremities.



Figure 1.11 Human Cheek Cells
(The nucleus is the darker, spherical organelle near the center of the cell)

C) Microscopic Observations

I) Bacteria

Observe the following image which illustrates the three different types of bacteria. Use the links given below to view microscopic slides of each of the three shapes of bacteria.

SKETCH 2
**Identify the different shapes of bacteria in your sketch as: Baccilus, Cocci, Spirillum

Click Here to Access a Form to Perform Sketch 2 (MS WORD)

Click Here to Access a Form to Perform Sketch 2 (PDF)

Baccillus (Rod shaped)

Cocci (Spherical shaped)

Spirillum (Spiral shaped)

Click here to see rod shaped bacteria

Click here to see spherical shaped bacteria

Click here to see spiral shaped bacteria


Figure 1.12 Types of Bacteria


II) Fungi

The most evident structure for most fungi is the spore bearing mushrooms of fungi. However, the defining character of fungi are the hyphae . Hyphae are cylindrical, branching tubes in which the cytoplasm of the fungus is found. Food is absorbed through the walls from the surrounding fluids or medium. The life cycle of fungi involve a spore (two are shown in Figure 1.13) settling and then the hyphae begin to grow, branching and growing outwards. The cytoplasm tends to migrate to the growing tips of the fungi and interconnected hyphae may extend over many yards. Leading some to suggest that fungi are the largest organisms on earth.

SKETCH 3
**Sketch hyphae and spores seen in the figure to the right.
 

Click Here to Access a Form to Perform Sketch 3 (MS WORD)

Click Here to Access a Form to Perform Sketch 3 (PDF)

 


Figure 1.13
Fungal hyphae / spore
 


III) Pond Mix

Video of a 100X microscopic view of a drop of pond water. Click on the arrow to view the video.

The variety of organisms you see here would be typical of most freshwater ponds. The small spherical organisms seen floating about are bacteria. The large and small, fast moving, green organisms that are darting about are paramecium feeding on bacteria. The hat shaped organisms are vorticella again feeding on bacteria. The darker greenish brown masses are bacterial colonies and algae. The green rectangular organisms are green algae. The long, thin strands scattered throughout the sample are blue-green algae.

SKETCH 4
**Observe the video and sketch some of the variety of living organisms present
 

 

An alternative link can be found below:

http://www.youtube.com/watch?v=gd3UOqxabuk

Click Here to Access a Form to Perform Sketch 4 (MS WORD)

Click Here to Access a Form to Perform Sketch 4 (PDF)


Some Interesting Movies Using Light Microscopy
This is optional

Go to this site to see a movie of bacteria on a mites leg

Bacteria on a Mites Leg

Click on the links below to see a Water Bear moving under a microscopic power of 250X

Water Bear 1

Water Bear 2

Water Bear 3

An amoeba engulfs a food item in a process called phagocytosis at a magnification of 400x

Amoeba 1

Pseudopodia, or false feet, extend and retract as this amoeba moves across the microscope field at a magnification of 100x

Amoeba 2

An up close and personal look at the internal workings of a paramecium at a magnification of 400x

Paramecium 1

A group of swimming paramecia, looking more like a bunch of bumper cars as they collide and rebound off of one another

Paramecium 2

Following the mitotic process, the surfaces of the new daughter cells seem to bubble wildly as if they were suddenly placed under high heat and were being boiled alive. Watch a few of the cells divide as you observe them.

Human Lung Cells in a Petri Dish


QUESTIONS
D) Light Microscopy Questions

1) What is the function of the diaphragm?
2) Describe the advantage of having a microscope of highest resolution.
3) Calculate the magnification of the lens system of the following:
     
a) Ocular-10X Objective-10X
     
b) Ocular-10X Objective-43X
     
c) Ocular-10X Objective-1X
     
d) Ocular-10X Objective-2X
4) What is the most important attribute of a microscope?

Click Here for a MS WORD version of the questions

Click Here for a PDF version of the questions


E) Virtual Light Microscopy

VIRTUAL LAB

 

**Go to the following site to link to a virtual light microscope. At the virtual microscope site you will need to perform the tutorial so that you learn how to use the microscope. Click on the GETTING STARTED link on the upper left side.  After learning how to use the microscope and viewing the speciments, answer the questions given below on Virtual Light Microscopy.

VIRTUAL LIGHT MICROSCOPE

A) Perform the tutorial (Getting Started) so that you learn how to use the microscope
B) Choose to view the cheek smear slide
C) You will be using the 4X,10X and 40X objective lens powers
D) Use the focus and illumination slide bars to see the cheek cells better.
E) Answer the questions on your experiences at this site below.

You can use the page link below to access a labeled image of the microscope

Click Here to View a Labeled Virtual Microscope


QUESTIONS
F) Virtual Light Microscopy Questions

1) How many individual cells can you count at the following objective powers of magnification?
      a) 4X
      b) 10X
      c) 40X
2) If the eyepiece has a 10X power, what is the total magnification when you observe cells at objective power of 40X?
3) At what power are you able to discern the nucleus of the cheek cells? (The nucleus is the large, darker organelle located near the center of the cell)
4) Describe what happens if there is too much illumination.

Click Here for a MS WORD version of the questions

Click Here for a PDF version of the questions


Please Find Below Links to Other Virtual Light Microscopy Sites

http://micro.magnet.fsu.edu/primer/virtual/magnifying/index.html

http://micro.magnet.fsu.edu/primer/virtual/translational/index.html

http://micro.magnet.fsu.edu/primer/virtual/focusdepth/index.html


2) Electron Microscopy

Two types of electron microscopes have been developed over the past half century: the Transmission Electron Microscope (TEM) and the Scanning Electron Microscope (SEM). These instruments 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. 

The TEM bombards a thin specimen with electrons. Depending on their composition, the components of the specimen either 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 gave scientists their first look at the world of viruses, invisible by light microscopy, and today permits us to see molecules and atoms. 

The SEM is quite different from the TEM. It is designed to generate three-dimensional images of surface detail. This microscope moves an electron beam 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.

Video of how an electron microscope works. Click on the arrow to view the video.

 

An alternative link can be found below:

http://www.youtube.com/watch?v=fToTFjwUc5M

 

 

 

 

Figure 1.14
Transmission
Electron Micrograph (TEM)
 

Figure 1.15
Scanning Electron Micrograph (SEM)
 

A) Examples of Electron Microscopy


Figure 1.16
Transmission Electron Micrograph
of Polio Virus
 

Figure 1.17
Transmission Electron Micrograph
of Ebola virus
 

Figure 1.18
Scanning electron micrograph of
HIV grown in cultured lymphocytes

 

Figure 1.19
Scanning electron micrograph of
Treponema pallidum
(The causative agent of syphilis)

 

Video of numerous electron microscopic views of organisms we live with every day. Click on the arrow to view the video.

 

An alternative link can be found below:

http://www.youtube.com/watch?v=dBU6-XK2v54

 
 

B) Virtual Electron Microscope

To observe (resolve) objects smaller than 0.2 m requires the utilization of Electron Microscopy (EM). Rather than using visible light, electron microscopes focus a beam of electrons on a very thin section of biological material that has been chemically preserved (fixed) and embedded in plastic. Electrons have a much shorter wavelength than the photons of visible light used in LM. Since resolving power is inversely related to wavelength, modern electron microscopes can resolve objects of approximately 0.2 m. It is this tremendous increase in resolution that has allowed biologists to discern the precise details of cell structure. Although a powerful tool, only chemically preserved cells can be observed with EM. The routine observation of living cells by electron microscopes is a goal yet to be achieved.

The type of electron microscopy described above is generally referred to as Transmission Electron Microscopy (TEM). In TEM, the beam of electrons passes directly through the sample except where the electrons are deflected by atoms of heavy metals (lead and/or uranium) that have been used to "stain" the specimen; the transmitted electrons are focused onto photographic film where the image is visualized and recorded.

A variation on this approach is Scanning Electron Microscopy (SEM). In SEM, the electron beam scans the surface of a sample that has been coated with a thin layer of gold. The beam of electrons excites the atoms of the sample causing them to eject electrons which are collected and converted into an image that is displayed on a monitor. The image that is produced has a great depth of field and thus appears to be three dimensional. SEM is used to reveal the surface details of various types of cells.

VIRTUAL LAB

**Go to the following site to experiment with a virtual scanning electron microscope. Answer the questions on Virtual Electron Microscopy given below.
VIRTUAL ELECTRON MICROSCOPE

A) There are three specimens to view shown on the left side
B) You can use the MAGNIFY button on the machine to zoom in on your specimen
C) Answer the questions on your experiences at this site below.


QUESTIONS
C) Virtual Electron Microscopy Questions

1) How many bacteria do you think are on the image?
2) Describe the different shapes of the bacteria that are visible.
3) Which cell is the largest between the macrophage and the bacteria?
4) Describe the shape of the bacteria.
5) Why do you think the virus has so many spikes on it?
6) Does the electron microscope allow a higher degree of magnification than the light microscope? Why?

Click Here for a MS WORD version of the questions

Click Here for a PDF version of the questions


A Link to Another Virtual Electron Microscope

http://education.denniskunkel.com/Java-SEM-expert.php

Something Interesting About Measurements

View the Milky Way at 10 million light years from the Earth. Then move through space towards the Earth in successive orders of magnitude until you reach a tall oak tree just outside the buildings of the National High Magnetic Field Laboratory in Tallahassee, Florida. After that, begin to move from the actual size of a leaf into a microscopic world that reveals leaf cell walls, the cell nucleus, chromatin, DNA and finally, into the subatomic universe of electrons and protons.

Measurements Movie


END LAB 1


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