Lab #2

Review of Lab #1 Properly putting away the microscope: Light off Wrap the cord Stage all the way down On lowest objective (4X) Iris diaphragm open Clean any oil on 100X using LENS paper. Also, clean other objectives with lens paper Clean ocular lenses! DO NOT ROTATE THE HEAD WITH THE OCULAR LENSES! Show bibulous paper/ blotting paper vs lens paper

Review of Lab #1

Review of Lab #1 Bacillus, chains Bacillus, singles

Review of Lab #1 Cocci, clusters Cocci, chains

Review of Lab #1 Cocci, tetrads

Review of Lab #1 Prokaryotes: no nucleus (bacteria and archae) Eukaryotes: nucleus and membrane bound organelles Protozoa: Giardia, Amoeba, Trypanosoma Unicellular Fungi: Pencillium Unicellular algae: Chlorella Viruses Giardia Show bibulous paper/ blotting paper vs lens paper Amoeba Trypanosoma

Lab #2 – 3. Ubiquity of Microbes Microbes are found in every environment Each environment has a unique collection of residents Your petri plate Observe 3 different colonies on your petri plate Describe each colony  fill out table on pg. 17 Use pg. 19 for reference Are microbes ubiquitous? Are they diverse? Each colony is a population of clones derived from a single dividing cell Microbiologists study isolated colonies Colonies are described on the basis of size, shape, margin, elevation, texture, and pigment A lot of diversity in microbes

Colony morphology

Lab #2: 4A. Aseptic Technique Aseptic technique: a technique utilized by microbiologists to prevent contamination – only the desired organism is inoculated in the growth medium Includes: Washing hands Tying back your hair Disinfecting your work station Turning on the flame Sterilizing your tools (loop, needle, etc)

Lab #2: 4A. Aseptic Technique Growth media contains all nutrients: CHNOPS, minerals, and vitamins Many different types of growth media: Liquid media: Broth Solid media: prepared by adding a solidifying agent called agar Petri plate Slant Tall/ deep Microbes can be transferred from any one form of media to another Media is sterilized using an autoclave or a filter before use

Types of growth media Broth Slant Petri plate Tall/ deep

Autoclave

Lab #2: Aseptic Technique Tools (pg. 22): Loop – broth, slants, petri plates Needle – Talls/ deeps Swab – petri plates Today you will learn the proper use of a loop and a needle Each student: Choose one of the following organisms: B. subtilis E. coli K. pneumoniae S. epidermidis Inoculate organism onto a Tryptic Soy Agar slant (use loop), Motility Media (use needle), and thioglycollate broth (use loop)

Lab #2: Gram staining Type of a differential stain  separate major groups of microorganisms based on retention or loss of the initial dye Grams stain: divides bacteria into 2 major groups: Gram positive Gram negative One group retains the initial dye of the specific gram staining procedure and the second group loses the initial dye in the staining procedure Discovered by a Danish biologist, Hans Christian Joachim Gram. http://www.medizin-forum.de/prostatitis/gram.jpg

How does Gram staining work? Gram positive cells have a thicker layer of peptidoglycan in their cell wall as compared to gram negative cell wall The staining dye crystal violet forms a large complex with iodine  this complex stays in the thick cell wall of G+ bacteria  cells don’t decolorize Gram negative cells are decolorized when treated with Gram’s alcohol  the counter stain, safranin, is then taken up by gram negative cells http://www-micro.msb.le.ac.uk/video/Gram.html

Preparation of a smear Smear (pg 31 & 32): Suspension of cells in water which is dried on a slide Should be light and uniform in appearance for best results! Madigan 10th edition, Figure 4.3, pg 58

Gram Staining: Procedure (Pg. 34) 1 min 30-45 sec 5-10 sec You may save your slides after preparing and/or viewing Madigan, 10th edition, figure 4.4, page 59

Gram positive cocci in cluster (masses) Gram positive bacilli http://www.kcom.edu/faculty/chamberlain/Website/gallery.htm http://www2.mf.uni-lj.si/~mil/bakt4/jpeg/35.jpg Gram negative rods in singles Mix of gram positve and gram negative organisms http://microvet.arizona.edu/Courses/MIC420/lecture_notes/ecoli/ecoli_gram.html http://www.cat.cc.md.us/courses/bio141/lecguide/unit1/prostruct/grmix.html

Lab #2: Gram staining Read pages 31 – 32 – know common terms Each student gram stain two organisms: B. subtilis & K. pneumoniae E. coli & S. epidermidis Go around class to view the other two organisms Record results on Pg. 35 Use a separate slide for each gram stain