Microbiology Lecture Notes Chapter 4

Microscopy, Staining, and Classification

 

I.                    Units of Measure http://learn.genetics.utah.edu/content/begin/cells/scale/

A.     Review of Chemistry

B.    Do the practice questions in the book and on the CD.

II.                  Microscopy

A.     Light Microscopy

 

B.    Darkfield Microscopy- Syphilis

Used with organisms that are difficult to stain. Displays the organism but is not helpful with internal anatomy.

 

C.    Phase Microscopy- Phase Contrast Microscopes - Use the fact that components within the cell have different refractive natures. This results in shades and shadows that reveal structures more clearly. Phase contrast was helpful in exploring the internal anatomy of single cells.

 

http://micro.magnet.fsu.edu/micro/gallery/radiolarians/radiolarians.html

D.    Fluorescent Microscope and Confocal - using specific antibodies to stain an organism and specifically identify it.

 

E.     Electron Microscope

  1. TEM transmission electron microscope

  1. SEM scanning electron microscope

 

F.     Probe Microscopy

http://invsee.asu.edu/Invsee/invsee.htm

         

Review the information available at the Nobel Prize website on the various types of microscopy http://www.nobel.se/physics/educational/microscopes/1.html

Type of Microscope

Means of Magnification

Advantages 

Disadvantages
 

Light Microscope

 

     
 

Phase Contrast Microscope

Try the simulation

     
 

Fluorescent Microscope

 

     
 

Electron Microscope

Try the simulation

     
 

Tunneling Microscopes

 

     

 

III. Staining Microbes 

Microscopy

Warm up

    A. Simple Stain

    B. Differential Stains

        1. Gram Stain - Based upon chemical differences in the cell wall of bacteria

               a. Gram positive cell walls - Thick peptidoglycan layer (durable, resistant to drying, and chemicals such as alcohol, susceptible to Penicillin and lysozyme)

                b. Gram negative cell walls (thin peptidoglycan, but thick lipoprotein layer/phospholipid layer - this layer represents an endotoxin)

Feature

Gram Positive

Gram Negative

Major

Components

Teichoic acid

Peptidoglycan

Lipopolysaccharides

(outer and inner membrane)

Gram Stain

Crystal Violet

Safranin

Penicillin

Sensitivity

More Sensitive

More Resistant

Tetracycline

Sensitivity

More Resistant

More Sensitive

Lysozyme

Sensitivity

Sensitive

Resistance

Toxins

Exotoxins

Endotoxins

Exotoxins

Tolerance to Drying

High

Low

Typical Bacteria

Spore- forming rods, Many cocci

Many rods

Few cocci

 

         2. Acid Fast Stain - indicates the presence of fatty acids in the cell wall that resist the decolorizing process, even with acid alcohol. An important stain for the genus Mycobacteria - Mycobacterium tuberculosis, M. leprae etc. Other acid fast organisms are Cryptosporidia, Nocardia etc.

            3. Endospore Stain -

 

 

 

 

 

 

 

 

 

 

 images/cdcphil/banthracisspores.jpg

Photo credit: Larry Stauffer, Oregon State Public Health Laboratory.

            4. Flagellar Stain -

            5. Capsular Staining

            6.  Fluorescent and Immunofluorescent staining - Advantages include the ability to very specifically stain items by tagging them with antibodies. Disadvantages include the necessity of a fluorescent scope for reading, and potential eye damage.

Live cells fluoresce green                         Auramine O staining Mycobacteria        

  1.     Prokaryotic Cells - are very simple with few anatomical components, few shapes and few abilities. They survive by reproducing a rapid rates.

                A. Shapes of bacteria - cocci, rods (bacilli), spirilla (vibrio, spirilla, & spirochete)

                B. Reproduction = binary fission (no sex, no genetic variation in the chromosome, except for mutations)

                C. Arrangement - based upon division - these cell orientations are the result of genetic controls and are characteristic for species or genera of bacteria

      

Arrangements are so characteristic some genera are named after the arrangement e.g. Streptococci, Staphylococci

Bacterial Cell Shapes Review

    D. Classification and Classifying Bacteria

Dichotomous Keys   

 

 

 

          Criteria for Classification of Prokaryotes

 Cultural

Morphology

Microscopic Morphology

Cellular

Components

Growth Characteristics

Metabolic Pathways

Molecular

Genetics

Location in Broth

Cell Shape

Cell Wall

Atmospheric requirements

Carbon requirements

DNA base

 ratio

Colony Appearance

Cell Size

Gram Stain

pH tolerance

Nitrogen

requirements

DNA

sequence

Pigmentation

Arrangement

Capsule

Temperature requirements

Sulfur

requirements

RNA

sequence

 

 Internal Structures

 

 Symbiotic lifestyle

Fermentation

Probes

 

Accessory

Structures

 

 Antibiotic sensitivity

Respiration

PCR

 

 

 

 

 

 

 

 End Products

 

Chapter 4 Microscopy, Staining, and Classification

Objectives - After finishing this lecture the students should be able to

 

  1. Use metric measurements, such as microliter (μl), milliliter (ml), liter (L), microgram (μg), gram (g), kilogram (kg), millimeter (mm), centimeter (cm), cubic centimeter (cc), and meter (m).
  2. Use the metric abbreviations with the correct measurement tool.
  3. Define the important aspects of microscopy and identify the anatomical parts on a microscope (as described in the lab).
  4. When given an image of a microbe and access to resources, determine the type of microscope used and explain why that technique was best for the given image.
  5. Analyze the components stained in each of the differential staining techniques and integrate this with the medical importance of that cell component.
  6. When given a situation determine the appropriate stain to use and evaluate the results.
  7. Identify the Gram reaction, cell shape, and arrangement of bacterial images.

 

Lecture 5

 

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Date last updated 08/07/2011
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