Microbiology Lecture Notes Chapter 4

Microscopy, Staining, and Classification

 

I.                    Units of Measure

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

2. 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

Disadvantages
Type of Microscope Means of Magnification Advantages 
 

Light Microscope      

 
 

Phase Contrast Microscope      

Try the simulation

       
Fluorescent Microscope

 
 

Electron Microscope      

Try the simulation

 

Tunneling Microscopes      

 
 
III. Staining Microbes  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 Teichoic acid Lipopolysaccharides
Components Peptidoglycan (outer and inner
membrane)
Gram Stain Crystal Violet Safranin
 Feature Gram Positive Gram Negative
Penicillin More Sensitive More Resistant
Sensitivity
Tetracycline More Resistant More Sensitive
Sensitivity
Lysozyme Sensitive Resistance
Sensitivity
Toxins Exotoxins Endotoxins
Tolerance to High Low
Drying
Typical Spore- forming Many rods
Bacteria rods, Many cocci 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 -
 

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        

IV.     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)
                2. Reproduction = binary fission (no sex, no genetic variation except for
mutations)
                3. 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
           Criteria for Classification of Prokaryotes
 Cultural Microscopic Cellular Growth Metabolic Molecular
Morphology Morphology Components Characteristics Pathways Genetics

Location in Cell Shape Cell Wall Atmospheric Carbon DNA base

Broth requirements requirements  ratio

Colony Cell Size Gram Stain pH tolerance Nitrogen DNA

Appearance requirements sequence

Pigmentation Arrangement Capsule Temperature Sulfur RNA

requirements requirements sequence
  Cultural Microscopic Cellular Growth Metabolic Molecular
Morphology Morphology Components Characteristics Pathways Genetics

   Internal Structures    Symbiotic lifestyle Fermentation Probes

  Accessory    Antibiotic sensitivity Respiration PCR

Structures

         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.