Microbiology Lab

Lab 2

Aseptic technique is essential to prevent contamination because of the ubiquity of microorganisms

Culturing Bacteria:

• Studying bacteria requires isolating desired bacterial species from environmental samples containing mixed cultures

  • You want a PURE culture

  • You need to employ aseptic technique to minimize contamination

• Culture media types:

  • Liquid: for growth studies or biochemical analysis

    • Used to grow cells and get a larger biomass

  • Solid: liquid media containing solidifying agent (agar)

    • Solid media is optimum for obtaining pure cultures

Colony Morphology of Specific Bacterias:

• Mycobacterium smegmatis - fried egg appearance due to waxy cell membrane (mycolic acids)

• Streptomyces griseus - dry texture, has mold-like appearance

• Proteus mirabilis - colonies spread (highly motile microbe)

• Serratia marscens - forms red colored pigment (when grown below 30ºC)

• Bacillus subtilis - soil odor; endospore forming bacteria

• Pseudomonas aeruginosa - produces pigments; blue-green (pyocyanin) & yellow-green (pyoverdine)

How to describe growth on solid media:

• Form:

  • Punctiform - Small, dot-like colonies

  • Circular

  • Filamentous - Colonies that appear as thread-like structures

  • Irregular - Colonies with an uneven or undefined shape

  • Rhizoid - Colonies with root-like extensions

  • Spindle - Colonies that resemble a spindle or elongated shape

• Elevation:

  • Flat

  • raised

  • convex

  • pulvinate

  • Umbonate - Colonies with a raised, rounded center. 

  • Crateriform - looks like there’s a crate in the middle

• Margin

  • Entire

  • Undulate - A wavy or rippled edge

  • Lobate - An edge with rounded projections or lobes

  • Erose

  • Filamentous - An edge that is thread-like or needle-like

How to describe liquid media:

• Turbidity - the cloudy appearance of liquid media due to the presence of bacteria

• Pellicle - a floating biofilm

• Sediment - can form at the bottom when bacteria grow

• Follicle - uneven chunky growth

Lab 1

6 Major Groups of Microorganisms

• Prokaryotes:

  • bacteria

  • archaea

• Eukaryotes

  • Algae

  • Protists

  • Fungi

• Viruses

Magnification is required to view an individual cell in a colony

• Micrometers (um) and nanometers (nm) are metric units typically used in reference to microbe size and to the size of their structural components

Eukaryotic cells: 50-100 um

Bacteria: 1-10 um

Virus: .01-.1 um

Refraction of light by lenses result in magnification

• Condenser

• Objective lens

• Ocular lens

Total magnification = objective lens magnification x ocular lens magnification

Resolution - refers to clarity/sharpness of the image

• Limit of resolution - the minimum distance the two can be to still be viewed as separate points; also called resolving power

• The distance apart needs to be less than the LOR. If equal to or more than, then the two points are seen as a single blur

Optimum resolution depends on:

• The wavelength of light used

• Optimum amount of light; high contrast (staining)

• Numerical aperture - refers to the light gathering ability of the objective lens and condenser

Lenses with high N.A. values better able gather refracted light rays

• Less light refracted, more enters lens

Classifying Microbes:

• based on metabolism, cell ultrastructures, DNA, RNA, & proteins

• Eukaryotic microbes: protozoans, algae, fungi

• Prokaryotic microbes: bacteria, archaea

3 Domains of life:

• Bacteria

• Archaea

• Eukarya

Lab 3

Microorganism Growth Requirements:

• Proper nutrition

• Oxygen or other gases must be available, as required

• Moisture is necessary

• Appropriate pH

• Proper temperature

• Contamination must be prevented and medium must be free of interfering bioburden

CHONPS are necessary for growth:

• Carbon

• Hydrogen

• Oxygen

• Nitrogen

• Phosphorous

• Sulfur

Also water, trace metals, and vitamins

Carbon and nitrogen are the most important

Complex vs. Defined Medium:

• Defined - the exact chemical composition is known

• Complex - contains one or more complex nutrients of undefined composition

  • We don’t know the exact ingredients that make it up