1. Class 2 Review of Class One - Microscopy and Introduction to Aseptic Technique and Media
Mrs. Sidelsky

2. Key Words Parfocal and parcentric( paracentric)
Parfocal means that once you have focused on low power you should be close to focus when you increase the magnification
Parcentric – If your point of interest on a slide is centered on low power, it should remain in a central position.

3. Magnification
Increase the size of microorganisms by using lenses to affect the light
A maximum of 1000x can be attained by using a light microscope without sacrificing the clarity of the image
1000X means that the object is magnified by 1000 times its actual size

4. Managing light Three ways to adjust light Iris diaphragm
Condenser lens
Diopter( adjust light intensity)

5. Coarse and fine adjustment
Use coarse adjustment only with scanning and low
Use fine adjustment only with high and oil
Never use fine adjustment with scanning – you should not need it

6. Resolution Resolution is the clarity and accuracy of the image.
When light is produced by the lamp underneath the stage it can enter the lens system through the aperture or opening from different directions
High and oil immersion have the smallest distortion of images and the highest clarity, because the light rays enter the lens system almost perpendicular to the stage.

7. How large are bacteria ?
Bacteria range in size from 0.1 um to 600 um( microns)
Mycoplasma are very small, but there are also nanobacteria
Epulopiscium fisheloni is the largest bacteria

8. Oil Immersion
In order to view prokaryote cells, it is necessary to use high magnification.
Start your focus on scanning and low. Find the best portion of the slide for study.
You want to choose a place where the cells are space so you can study the shape and arrangement

9. Bacterial cells – shape and arrangement

10. Oil Immersion (continued)
When you increase the magnification remember to adjust the lighting
You may need more light on higher magnifications
When you have focused on high power and your image is clear, turn the revolving nosepiece between high and oil
Place a drop of oil on the slide and turn your oil immersion lens gently into the drop
Remember to use the fine adjustment

11. Aseptic
Work to protect yourself and contain organisms under safe working conditions
Prevent contamination of cultures from external sources so that microorganisms in the Petri Dish can be identified and characterized.

12. Part Two Introduction to Microbiological Techniques
Objective One-To be able to work with aseptic technique
Goals -
- To be able to handle media and cultures
- To be able to transfer microorganisms from one culture to another

13. Terminology Media – Solid – Agar
Trypticase Soy Agar( TSA) - Enriched Media
Nutrient Agar( NUT)
Enriched Media( many)
Media – broth
TSA broth
NUT Broth
Fermentation tubes- sugars

14. Equipment Inoculating Loop Inoculating Needle Flaming the loop
Petri Dish
Streak Plate
Slant
Deep
Broth

15. Media

16. Flaming- prevents contamination of culture
Hold Inoculating loop
Insert in flame until loop glows red
Allow to cool

17. Transfer of broth to broth
Steps for Transfer of Broth to Broth
Hold loop or needle with dominant hand( right )
Flame the loop
Hold culture tube in left hand
Remove red cap with pinkie of right hand
Flame mouth of culture tube 
Place loop into broth( water)
Flame mouth of culture tube and close
Open culture tube with broth( should be labeled)
Dip loop into new broth and mix
Flame mouth of tube and close
Flame loop
Place to the side of your rack

18. Broth to slant
1. Wrap fingers of non dominant hand around the culture tube containing broth for transfer
    2. Using the pinkie finger of your dominant hand twist the red cap from the tube.  Hold in your pinkie and do not place it on the counter
3.     Pass the mouth of the culture tube across the flame
4.     Direct the inoculating needle into the broth.
5.     Flame the mouth of your broth culture tube and replace the cap.  Place it in your rack
6.     Pick up the slant in your non dominant hand
   

19. Part 2 Twist off the red cap 8. Flame the mouth of the slant tube
9.     Direct the inoculating needle into the tube and “ stab” the agar in the base( butt)
10. Withdraw on the entry line and when you reach the surface make a simple streak along the face.
11.  Flame the mouth of the tube and replace the cap.
12. Flame your inoculating needle and replace in your rack.

20. Broth to streak plate Procedure for Streaking a Plate for Isolation:
  1.  Flame the loop and wire and streak a loopful of broth as at A in the diagram.   2.  Reflame the loop and cool it.   3.  Streak as at B to spread the original inoculum over more of the agar.   4.  Reflame the loop and cool it.   5.  Streak as at C.   6.  Reflame the loop and cool it.   7.  Streak as at D.   8.  Label the plate and incubate it inverted.    
Go To Results of Streak Plate Lab Procedures  

21. Streak plate

22. Growth of organism over plate