Post on 30-Dec-2015
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.
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
Managing light
Three ways to adjust lighta. Iris diaphragmb. Condenser lensc. Diopter( adjust light intensity)
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
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.
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
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
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
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.
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
Terminology
Media – Solid – Agar Trypticase Soy Agar( TSA) - Enriched
Media Nutrient Agar( NUT) Enriched Media( many) Media – brothTSA brothNUT BrothFermentation tubes- sugars
Equipment
Inoculating Loop Inoculating Needle Flaming the loop Petri Dish Streak Plate Slant Deep Broth
Flaming- prevents contamination of culture
Hold Inoculating loop
Insert in flame until loop glows red
Allow to cool
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
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
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.
Broth to streak plate Procedure for Streaking a Plate for Isolation:
Procedure: 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