Microbiology Lab Techniques and Microscopy

Flashcards about lab techniques and microscopy

Purpose of the streak method of isolation

Spreads sample so individual cells are scattered across the agar surface.

CFU (Colony Forming Unit)

The cell or group of cells that produces a colony when transferred to plated media.

T-streak Method

Divides the plate into 3 sections, no advantage over quadrant.

Zigzag streak Method

Performed for low concentration samples or when dealing with pure cultures.

Quadrant Streak Method

Divides the plate into 4 sections while diluting the sample on a solid surface.

Proper loop pressure for streak method

Light pressure while dragging the loop across the agar surface at an angle.

Thioglycollate Broth

An enriched liquid medium that promotes growth of a wide variety of fastidious and nonfastidious microorganisms.

Application of Anaerobic Jar

To grow and study anaerobic and/ or microaerophilic bacteria in a standard incubator.

Resazurin

Allows for the visual detection of gaseous oxygen.

Obligate aerobes

Require oxygen to conduct aerobic respiration and therefore only thrive at the top of the medium.

Facultative anaerobes

Grow throughout the medium, but will be denser near the top of the medium because of the higher ATP yield from aerobic respiration compared to fermentation or anaerobic respiration.

Aerotolerant anaerobes

Indifferent to oxygen levels, these microbes live uniformly throughout the medium.

Microaerophiles

Grow in the middle region of the medium.

Obligate anaerobes

Even small amounts of oxygen are lethal, only grow toward the bottom of the medium.

Bright-field microscopy

simplest, most common way we observe microscopic organisms in the microbiology lab. Specimen appears as a darker object set against a bright background

Compound light microscopes

Provide two levels of magnification that compounds (increases) the magnification of the final image.

Resolution (microscopy)

Clarity of an image produced by a lens, the ability of a lens to distinguish between two pints in a specimen. High resolution microscope is desirable

Magnification (microscopy)

Final magnification = (Objective lens magnification) × (Ocular lens magnification)

Iris diaphragm

Allows you to control how much light illuminates the specimen, the more open the more light.

Condenser (microscopy)

Focuses and aims the light onto the specimen.

Coarse knob (microscopy)

Moves the stage up and down.

Getting the microscope ready for use

• Carry the microscope with two hands—one on the arm and one under the base.

  Place it on a flat, stable surface near a light source or plug it in.

  Turn on the light source or adjust the mirror if using natural light.

  Rotate the nosepiece to select the lowest power objective (usually 4×).

  Adjust the diaphragm to allow enough light through the stage.

Focusing the microscope without immersion oil

• Place the slide on the stage and secure it with clips.
  
  

• Use the coarse focus knob to bring the stage up while looking from the side until it is just below the objective lens.
  
  

• Look through the eyepiece and slowly lower the stage using the coarse knob until the image appears.
  
  

• Use the fine focus knob to sharpen the image.
  
  

Center the specimen and switch to a higher power lens (10× or 40×) if needed.

• Focusing the microscope with immersion oil

-(Used only with the 100× oil immersion objective)

Focus the specimen using the 40× lens and fine focus it clearly.

Rotate the nosepiece halfway between 40× and 100×.

Place one small drop of immersion oil directly on the slide over the specimen.

Carefully rotate the 100× oil immersion lens into place—it should touch the oil.

Use the fine focus knob only to bring the image into sharp focus.

Do not use the coarse focus knob—it can damage the slide or lens.

When finished, clean the 100× lens and slide with lens paper and lens cleaner.