Transcript Class 2 Review of Class One - Microscopy and Introduction to

Class 2
Review of Class One Microscopy
and
Introduction to
Aseptic Technique and
Media
Mrs. Sidelsky
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 light
Iris diaphragm
b. Condenser lens
c. Diopter( adjust light intensity)
a.
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
Bacterial cells – shape and
arrangement
Oil Immersion (continued)
 When you increase the magnification
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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 – broth
TSA broth
NUT Broth
Fermentation tubes- sugars
Equipment
 Inoculating Loop
 Inoculating Needle
 Flaming the loop
 Petri Dish
 Streak Plate
 Slant
 Deep
 Broth
Media
Flaming- prevents contamination
of culture
 Hold Inoculating
loop
 Insert in flame until
loop glows red
 Allow to cool
Transfer of broth to broth
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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
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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
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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
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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
Streak plate
Growth of organism over plate