Review classification within the biological world Understand
characteristics used to identify microorganisms Learn how to
prepare a Gram stain Purpose of lab 13:
Slide 3
Three Lineages of Life: Domain Bacteria All living organisms
are classified into 1 of 3 domains: Prokaryotes: No true nucleus No
membrane-bound organelles Cell Wall composed of peptidoglycan
Reproduce asexually by budding and fission Very small (1 - 10
m)
Slide 4
Three Lineages of Life: Domain Archaea Prokaryotes: No true
nucleus No membrane-bound organelles NO peptidoglycan in cell wall
Reproduce asexually by budding and fission Very small (1 - 10 m)
Extreme environments (high temperatures)
Slide 5
Identification of Bacteria Bacteria: Small in size Lack
discernable internal features Methods of identifying bacteria:
Macroscopic examination: Colony color, shape, and odor Microscopic
examination: Cell shape Cell surface
Slide 6
Bacterial ID : Microscopic Examination : Cell Shape
Unflagellated bacteria are classified according to 1 of 3 major
shapes:
Microscopic Examination: Cell Shape: Bacillus Bacillus Single
Bacillus (fusiform) Streptobacillus (chain) Single Bacillus
Slide 9
Microscopic Examination: Cell Shape: Spirillum Spirillum Single
Spirillum
Slide 10
I. EUBACTERIA Examine bacteria cultures both macroscopically
and microscopically. A. Macroscopic Observations of Bacteria
Colonies Pick two colonies from the plates provided, each from a
different source. Prepare a table (Table I) that summarizes these
characteristics for each colony Whole Shape Of Colony Edge/Margin
Of Colony Color Opacity Of Colony Transparent (clear), opaque,
translucent (almost clear, but distorted visionlike looking through
frosted glass), iridescent (changing colors in reflected light)
Elevation Of Colony Surface Of Colony Smooth, glistening, rough,
dull (opposite of glistening), rugose (wrinkled) Consistency
Butyrous (buttery), viscid (sticks to loop, hard to get off),
brittle/friable (dry, breaks apart), mucoid Emulsifiability Of
Colony Is it easy or difficult to emulsify? Does it form a uniform
suspension, a granular suspension, or does not emulsify at all?
Odor Absent or present? If it has an odor, what does it smell like?
characteristics
Slide 11
I. EUBACTERIA C. Prepared Microscope Slides of Bacteria
Prepared slides of various types of bacteria will be made
available. You should examine them, noting and diagramming their
characteristics. Bacillus subtilisStaphylococcus
epidermidisEscherichia coli Neisseria sicca
Slide 12
Bacterial ID : Microscopic Examination : Cell Surface Nearly
all prokaryotes have cell walls External to their cell membrane
Domain Bacteria (eubacteria), PEPTIDOGLYCAN GRAM STAIN: One of the
most valuable tools for identifying eubacteria Separates eubacteria
into 2 groups based on differences in their cell walls Gram
Positive Gram Negative
Slide 13
Gram staining a Important Technique A staining technique used
to classify bacteria Bacteria are stained with gentian violet and
then treated with Gram's solution After being decolorized with
alcohol and treated with safranine and washed in water, those that
retain the gentian violet are Gram-positive and those that do not
retain it are Gram-negative 13
Slide 14
Microscopic Examination : Cell Surface : Gram Positive
Gram-positive Large amounts of peptidoglycan in cell wall
Peptidoglycan traps the violet dye (crystal violet) These cells
stain purple
Slide 15
Microscopic Examination : Cell Surface : Gram Negative
Gram-negative Thin peptidoglycan Its located in a periplasmic gel
between the cell membrane and an outer membrane The violet dye is
easily rinsed out The red counter-stain (safranin) is retained
These cells stain pink
Slide 16
Organizing the Staining Bottles Dr.T.V.Rao MD 16
Slide 17
Slide 18
1. Place one needle of solid bacterial growth or two loops of
liquid bacterial growth in the center of a clean slide.
Slide 19
2. If working from a solid medium, add one drop (and only one
drop) of water to your specimen with a water bottle. If using a
broth medium, do not add the water.
Slide 20
3. Now, with your inoculating loop, mix the specimen with the
water completely and spread the mixture out to cover about half of
the total slide area.
Slide 21
4. Place the slide on a slide warmer and wait for it to dry.
The smear is now ready for the staining procedure.
Slide 22
Gram-staining Procedure Gram-staining is a four part procedure
which uses certain dyes to make a bacterial cell stand out against
its background. The specimen should be mounted and fixed on a slide
before you proceed to stain it. The reagents you will need to
successfully perform this operation are: Crystal Violet (the
Primary Stain) Iodine Solution (the Mordant) Decolorizer (ethanol
is a good choice) Safranin (the Counterstain) Water (preferably in
a squirt bottle)
Slide 23
Before starting, make sure that all reagents, as well as the
squirt-bottle of water, are easily accessible because you won't
have time to go get them during the staining procedure. Also, make
sure you are doing this near a sink because it can get really
messy. Wear a lab coat.
Slide 24
STEP 1: Place slide on rack. Flood with crystal violet (~1 min)
Let stand 60 seconds Rinse with water (5 sec)
Slide 25
STEP 2: Flood with iodine (~1 min) Rinse with water (5 sec) At
this point, the specimen should still be blue-violet. Proceed to
STEP 3.
Slide 26
STEP 3: Rinse with water (5 sec) Add the ethanol dropwise until
the blue-violet color is no longer emitted from your specimen
Slide 27
STEP 4: Flood with safranin (~1 min) Let stand 60 seconds Rinse
with water (5 sec) Gram positive cells will remain blue-violet in
appearance. Gram negative bacteria take on a pink color..
Slide 28
After you have completed steps 1 through 4, you should blot the
slide gently with bibulous paper or allow it to air dry before
viewing it under the microscope. DO NOT RUB THE SMEAR!
Slide 29
Examine the results
Slide 30
Gram staining not a fool proof procedure Excessive heat during
fixation Low concentration of crystal violet Excessive washing
between steps Insufficient iodine exposure Prolonged
decolourization Excessive counterstaining
Slide 31
Observe (400X), draw and label: Part II: CYANOBACTERIA A.
GleocapsaB. OscillatoriaC. Anabaena
