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The Light Microscope (PowerPoint download)

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					Staining and Observation of
      Microorganisms
Exercise 10

Objective: To learn proper smear technique

1st Step in good staining is preparation of a good smear

Goals of smearing:
1. To adhere cells to slide (so they are not washed away in staining
   procedures)
2. To insure that shrinkage of cells does not occur
3. To prepare thin smears

Methods:
Smears may be prepared from liquid or solid media (See pg. 88 of lab
   manual)
Air dried
Heat fixed (kills cells)


Always use aseptic technique when preparing smears
              Preparing a smear for staining.
      (The following procedure is used for all of our staining)



1. Flame (sterilize) your
inoculating loop/needle
before and after use.
Heat from base to tip.
Be sure to get the
entire wire red hot.
2. Prepare the smear
  a. If you have a solid
  culture (agar colony), place
  a small drop of distilled
  water on a clean slide. Drag
  the sterile inoculating
  needle tip through the edge
  of an isolated colony.

  b. Gently spread the
  mixture into a circle the
  size of a quarter.




(A loop of liquid culture
can be placed directly on
the slide and spread out.)
3. Let the smear air
dry completely. Do not
apply heat while drying
because this can lyse
the cells.
4. Heat-Fix the smear.
Smears are heat-fixed
by quickly passing the
slide through a flame
(smear side up), two or
three times.
This causes the
microbes to stick to the
slide and not get
washed off during the
staining process.
Exercise 11

Objective: To perform simple staining of bacterial cells (single stain)

Goals of simple staining:
1. To provide contrast for observation with a light microscope
2. To observe morphology (shape), arrangement (e.g. chains, pairs etc.)
    and to view any obvious internal structures (e.g. metachromatic granules)

Methods:
Prepare smears
Stain bacteria provided using methylene blue (see pg. 91 lab manual)


Always use aseptic technique when staining
                                        - - -
Background:
• Bacteria are negatively charged     -       --
                                          - -
• All dyes have color-bearing ions (chromophores)

   Commonly used basic stains (positively charged ions)
     Methylene Blue
     Basic Fuchsin      + chromophores attracted to and stain negatively
     Crystal violet      charged bacterial cell walls

Commonly used acidic stains (negatively charged ions)
    Eosin
    Nigrosin                 - Chromophores repelled by bacteria and
                              coat bacterial surface. (do not penetrate cell)
5. Stain the smear.
Place the slide on a
staining rack over the
sink. Flood the smear
with stain and let it sit
for 60-90 seconds.
Rinse gently and blot
dry.
6. Observe the slide
under low and high-dry
lenses to locate, center,
and focus the image.
Then, place a drop of oil
directly on the stained
smear (no cover slip). Turn
the oil immersion lens into
position and fine focus to
observe the cells.
Coccus (cocci pl.)
Bacillus (Bacilli pl.)
Spirillum (Spirilli pl.)
Answers:
1. Spirillum                                   4
2. Coccus
3. Bacillus
4. Diplobacillus         5
5. Streptobacillus                                 2
6. Diplococcus
                                  6
                                                       1
                     3
                     (shapes and arrangements) seen here.
                       Name the bacterial morphologies
Exercise 12

Goal of negative Staining :To determine morphology,
arrangement and size of bacteria, that may be affected by heat
fixing

GENERAL INSTRUCTIONS AND MATERIALS:
Fixed and/or stained smears from Exercise #10.
Stains, negative: Nigrosin.
Stain: Bacillus megaterium, Staphylococcus aureus, oral organisms


*DO NOT HEAT FIX NEGATIVE STAINS!

* Since bacteria not heat fixed in negative staining this is a useful technique
for:
Obtaining more accurate cell sizes
Observing capsule (dessicated by heat fixing)
Observing spirochetes
Negative Staining
Results of Negative Staining)