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Microbiology Week 3 Notes - Only 1 lecture due to snow day

by: Toni Franken

Microbiology Week 3 Notes - Only 1 lecture due to snow day MICR 3050

Marketplace > Clemson University > Microbiology > MICR 3050 > Microbiology Week 3 Notes Only 1 lecture due to snow day
Toni Franken
GPA 3.97
General Microbiology
Dr. Whitehead

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About this Document

These notes cover the lecture from week 3 of Microbiology with Dr. Whitehead at Clemson University. The material includes information from Chapter 2 regarding Electron microscopes and staining and ...
General Microbiology
Dr. Whitehead
Class Notes
MICR, MICR 3050, 3050, Microbiology, microbio, Basic Microbiology, Principles of Microbiology, Clemson, Clemson University, Whitehead, Dr. Whitehead, Electron Microscope, Microbes
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This 4 page Class Notes was uploaded by Toni Franken on Sunday January 24, 2016. The Class Notes belongs to MICR 3050 at Clemson University taught by Dr. Whitehead in Spring 2016. Since its upload, it has received 53 views. For similar materials see General Microbiology in Microbiology at Clemson University.


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Date Created: 01/24/16
MICR 3050 Notes Set 5 01202016 Dr Whitehead Clemson University Chapter 2 Microscopes and Staining Procedures Focal length and working distance The direction in which focal length works is different than working distance Focal length is the distance from the center of the lens to where the light focuses The focal point is not at the object but instead back towards the eye The working distance on the other hand is towards the object Reminder in a compound microscope there are two lenses so two focal points which work together In addition the eye acts as a focal point Working distance is the distance from the end of the lens usually the objective lens to the object being observed Working distance and focal distance DO tend to coordinate as in if one is shorter the other will be shorter Electron Microscopes We usually use electron microscopes to see fine details of bacteria or to see viruses most viruses are entirely too small for a light microscope 0 Light microscope magnification is limited by the wavelength of physical light When using electron microscope instead of using wavelengths of light we re using waves of electron beams We can get electron beam wavelengths thousands of times shorter than that of light wavelengths They replace light as the illuminating beam This leads to a much higher resolution and a much higher amount of magnification O Magnification capabilities of an electron microscope Magnification is greater than 100000X and resolution is Snm 0 There are two types of electron microscopes 0 Transmission Will be able to see through objects as electrons pass through Can use to see internal structures 0 Scanning Will be able to see the surface of the object as electrons pass over the surface 0 Transmission Electron Microscope TEM When using a TEM we re usually looking at a very thin slice of a specimen Electrons scatter when they pass through thin sections of a specimen The transmitted electrons are under a vacuum Denser regions in a specimen scatter more electrons making them unable to pass through and appear darker If an area is less dense it will allow more electrons through and will appear lighter You can get an idea of internal structures with a TEM Must use a machine called an ultratome to slice thin specimens and also use a stain to get a finer picture 0 Scanning Electron Microscope We look at how electrons re ect off the surface of an object to create an image You ll see where they re absorbed versus re ected It produces a realistic 3D image of specimen s surface features In addition the specimen is often coated with a metal gold is common to help electrons re ect from the surface You can t see internal structures but can get 3D images Usually black and white color can be added later Preparation and staining of specimens Purposes Increases visibility particularly for brightfield microscopes A bacterium unstained relative to the bright field around it is difficult to Visualize so the staining enables us to see them better There are certain types of stains that are used that let you see speci c morphological features that you wouldn t otherwise be able to see Help clarify said features Ex There is a stain that colors agella almost impossible to see them with a compound microscope unless you use a acular stain Can also be used for preservation It fixes the specimen to keep and look at time and time again Fixation Purpose Preserves internal andor external structures and xes them in position Heat is a common fixative Organisms are usually killed and firmly attached to microscope slides Heat fixation routine use with bacteria and archaea It also helps to preserve the overall shapemorphology of the organism but not much else Most internal structures are going to be destroyed Too much heat is not good and will denature proteins 0 Process spread bacteria on a thin film over the slide and allow it to air dry so the water doesn t boil when heating After it has dried you pass it through the ame brie y and then stain and rinse it before Viewing Chemical xation used with larger more delicate organisms and lets us see preserved internal structures Use it a lot with tissue samples and histology Will still kill the organismscells but will not destroy structures as readily Common one used is formaldehyde as well as acetic acid and ethanol Dyes and simple staining Purpose Dyes allow us to make internal and external structures of a cell more Visible by increasing contrast with the background All dyes have two common features 0 Have the ability to bind to the cells 0 They all have a chromophore group the chemical groups that impart the color to the cell There are two general classes of dyes 0 Basic dyes Dyes with positive charges that bind to negatively charged structures like proteins nucleic acids and the surface of most bacterial cells Some examples are methylene blue basic fuchsin chstal Violet and safranin often used in gram staining 0 Acidic dyes Negatively charged dyes that bind to positively charged structures like cytoplasm red blood cells and collagen Sometimes used in eukaryotic structures Eosin acid fuchsin Simple stains You take a single dye and you usually just use it to look for the presence of cells and their general shape Usually you make a smear let it dry heat fix it stain it rinse of excess stain and examine Crystal Violet and methylene blue are common simple stains You look for cell shape and the organization of the cells in a chain clumped grapelike Cell size Differential Staining More commonly used in microbiology You use more than one dye to preferentially stain features Allows us to differentiate between bacteria types or to see different structures within bacteria Used to detect the presence or absence of the structures Microorganisms can be divided into groups based on their staining properties 0 Gram staining Very good way to check purity of the culture in other words see on a superficial level if you re growing what you think you are and to see if you have a single or a mixed culture This is the most widely used of the staining procedures Usually divides bacteria into two groups based on differences in cell wall strucutres 39 Grampositive very thick peptidoglycan cell wall Baccillus species are gram positive 39 Gramnegative a thinner wall of peptidoglycan E coli are gram negative I Almost all bacteria can be defined as gram positive or gram negative There ARE exceptions The man who developed this procedure had the last name was Gram 0 Gram Staining Procedures 1 The very first thing you ll do is create a smear and heat fix it The rst dye you add is crystal violet a purple dye Anything that takes up crystal Violet will look purple In this process it is known as the primary stain Then you ll rinse that slide ALL bacteria present will now be purple at the end of step one 2 Next will be to add iodine It acts as a mordant or a substance that helps the dyes bind better to the organisms You let the iodine sit for a minute and then rinse with water All bacteria will still appear to be purple and are not yet differentiated 3 Next is a harsher wash step where you usually use a combination of ethanol and acetic acid that clears the color from gramnegative cells while grampositive cells hold onto the purple color 4 Finally we ll use a secondary stain in this case safranin which is a pink dye It will stain anything that is not stained by crystal Violet Your gram positives are still purple and your gram negative cells are now pinkred This only works for bacteria because it is all based on peptidoglycan It can do everything that simple staining can do see cell size shape and arrangement 0 Acid fasting A much more speci c type of staining particularly useful for staining members of the genus Mycobacterium They are an exception in the bacterial world due to the high lipid content in the cell walls mycolic acid This makes them incredibly hydrophobic which prevents most normal dyes from binding to them It also gives them a measure of antibiotic resistance 1 Start with a primary dye called fuchsin a pink dye You can t just ood the slide with it to get Mycobacterium to take it up You have to force them to take it up with heat and phenol After step 1 of acid fasting pretty much everything is going to look pink 2 The next step is a wash step where you use acidi ed ethanol to wash color from everything but the Mycobacterium At the end of this step mycobacterium will be pink everything else is clear 3 Finally will use methylene blue as a countersecondary stain safranin also considered one to stain everything else blue Fun fact Copralites are fossilized feces which can be studied in a microbiology setting Her husband thought about getting her copralite jewelry


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