151 lec 8

• LIGHT MICROSCOPY

  • Analytical tool used for magnifying specimens
  • Has potential to view objects at 1000 times their original size
• Light Source - produces light; can be focused by a condenser lens
• Objective Lens - creates a magnified image that is inverted
• • Eyepiece / Ocular Lens - further magnifies the image which the eye receives
  • Additional optical components can be added to the light path to correct the orientation of the image
• COMPOUND MICROSCOPE

  • Utilizes multiple lenses
• • Total magnification is calculated by multiplying the magnification of the objective lens by the magnification of the ocular lens.
• • Eyepiece Reticle 
  • Help estimate the size of objects under the microscope
  • Scale that is projected over the image
  • At higher magnification, the tick marks in the eyepiece reticle will represent smaller distances than when viewed at lower magnification.
• As the objects become clearer, the resolution increases and the shortest observable distance between them decreases.
• • Bacteria
  • Living microorganisms
• • Bacteria with thick peptidoglycan layer will stain purple (layer cannot be penetrated easily by decolorizer)
• • Gram Negative - reddish pink / red
  • Has thin peptidoglycan, loses purple color to the decolorizer
  • Safranin counterstain causes the reddish pink stain as it binds to lipopolysaccharide layer on the outside of the cell.
• Capsule Staining

  • Used to visualize external capsules that surround some types of bacterial cells
• • Capsule present → non-ionic composition and tendency to repel stains → simple staining methods won’t work → uses Negative Staining Method
• • Negative Staining Method
  • Stain background with an acidic colorant (ex. Congo Red); background should appear darkly stained under the microscope
  • Stain bacterial cells with crystal violet
• Negative Staining Method

  • Bacterial cells with capsule: Capsule will appear as a clear halo around the cells
• Endospore Staining

  • Also known as Schaeffer-Fulton Method
  • Determines if bacteria being studied forms spores
• • Endospores
  • Dormant, tough, non-reproductive
  • Ensures survival of bacteria through periods of environmental stress (extreme temp and dehydration)
  • Difficult to stain with standard techniques and impermeable to many dye
• Endospore StainingProcess:

  1. Apply malachite green stain to bacteria fixed to a slide.
  2. Wash the slide with water.
  3. Apply counterstain (Safranin).

  • Vegetative Cells - will appear pinkish-red

  Bacterial cells w/ Endospores - retain the green color of the stain; appear bluish-green in a colony under the microscope
• SERIAL DILUTIONS AND PLATING

  • Serial dilution and plating techniques are used to reliably quantify bacterial load and isolate individual colonies. 
• SERIAL DILUTIONS

  • A process through which the concentration of an organism is systematically reduced by successive resuspension in fixed volumes of liquid diluent
• SERIAL DILUTIONS

  • Usually, the volume of the diluent is a multiple of 10 (ex. 1:10)  to facilitate logarithmic reduction of the sample organism
• • Streak plating and spread plating enable the isolation and enumeration of bacteria within a sample, respectively
• • As different sections of the plate are streaked, crossing from the previous sample only once, the sample is spread more thinly.
  • This means that you may only need to streak from one dilution to achieve individual colonies in the later sections.
• Spread Plating 

  • Main goal is the enumeration of the bacteria in a sample. 
• 
  

  • Typically, since the bacterial numbers in the mix sample are unknown, a spread plate is made for each of the dilutions.
• Spread Plating 

  • Any plates with colony counts fewer than 30 should be discarded since small counts are subjected to greater error.
  • Similarly, any counts over 300 should be discarded because colony crowding and overlapping can lead to underestimation of colony count.
• • Clonal Colony
  • When a bacterium in an agar plate undergoes multiple rounds of asexual reproduction.
• PURE CULTURE AND STREAK PLATING

  • Getting a single bacterium from mixed samples such as soil or stool can be very difficult.
  • One loopful can contain one trillion individual bacteria.
  • Even if a zig-zag pattern is used to streak the sample onto a agar plate to obtain single colony, the number of agar plates needed can circle the perimeter of Liberty Island.
• Streak Plating Procedure

  • The agar plate is visually divided into 5 parts with the first 4 fragments coming from the circumference of the plate and the 5th being the plate center.
  • When incubated, these discrete bacteria multiply to produce isolated clonal colonies of daughter cells which are also known as Colony Forming Units or CFUs.
  • The CFUs are then harvested and restreaked to ensure that subsequent work involves only a single bacterial type referred to as pure culture.

  
• The initial streak plate may contain colonies originating from cells of different bacterial species or different genetic make-up, depending upon the sample purity.
• • Through subsequent isolation of a single colony where all units are derived from a common mother cell, the 2nd streaking procedure generates a relatively clonal bacterial population that is suitable for further characterization or inoculation into broth.
• • This ability to colonize vastly different niches is due to their adaptability and vast metabolic diversity, which allows them to utilize a wide variety of molecules for energy generation.
• • The massive array of diversity leads to the phenomenon that <1% of the bacterial species on the planet are considered culturable.
  • This is only possible due to an understanding of their specific metabolic and environmental needs
• • Performing manipulations of media and environment in the laboratory allows:

  1. Researchers to experiment to find optimal conditions for culturing a species of interest
  2. Enables enrichment - the process of changing conditions to select for specific species from a mixed culture
• • Generalists: 
  • Able to tolerate a wide variety of states or environment
  • Such organisms may grow readily under laboratory conditions.
  • They may also be prevented from growing if given an extreme habitat.
• • Fastidious Organisms:
  • Can be culturable but only when specific conditions are met
  • e.g. Neisseria sp. or Haemophilus sp. require media containing partially broken down red blood cells and high CO2 concentration which may also discourage the growth of other species
• • Extremophiles: 
  • Named for their preference for extreme conditions
  • Can be very low temperatures due to absent oxygen conditions
  • Can be very high temperatures due to presence of high salt
• INDICATOR MEDIA

  • To further enrich an organism of interest, some media types contain indicators which give insight into the metabolism of the organism.
• Mannitol Salt Agar (MSA)

  • Inhibits the growth of organisms sensitive to high salt
  • Gram-negative bacteria typically can’t survive on MSA because it is toxic to most gram-negative species.
• Mannitol Salt Agar (MSA

  • Gram-positive Staphylococcus genus are able to thrive.