1. Lab #2

3. 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

4. Review of Lab #1

5. Review of Lab #1
Bacillus, chains
Bacillus, singles

6. Review of Lab #1
Cocci, clusters
Cocci, chains

7. Review of Lab #1
Cocci, tetrads

8. 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

9. 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

10. Colony morphology

11. 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)

12. 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

13. Types of growth media
Broth
Slant
Petri plate
Tall/ deep

14. Autoclave

15. 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)

16. 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.

17. 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

18. 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

19. 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

20. Gram positive cocci in cluster (masses) Gram positive bacilli
Gram negative rods in singles
Mix of gram positve and gram negative organisms

21. 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