Staining Secondary Positive Negative

Mechanism Steps Primary Stain Mordant Decolorizer Picture

Technique Stain Outcome Outcome
Basic dye (e.g.,
1. Prepare smear Limited
Simple crystal violet, Enhanced visibility
2. Apply primary stain - - - differentiation of
Staining methylene of microorganism
3. Rinse; Blot dry; Examine structures
blue, safranin)
1. Heat fix the slide: click on the Bunsen burner,
Based on differences in pass the slide gently two or three times (1-2
cell wall components: secs) through the flame. Do not overheat - this
Gram (+): thick will cause distortion of the cells.
peptidoglycan cell wall; 2. Flood slide with crystal violet (1 min)
lack an outer membrane 3. Rinse w/ H2O
Gram Ethanol or Gram-positive: Gram-negative :
4. Flood slide w/ Iodine (1 min) Crystal violet Iodine Safranin
Staining acetone Blue/Purple Pink/red
Gram (-):thin 5. Rinse w/ H2O
peptidoglycan cell wall; 6. Decolorize with alcohol for (5-10 secs)
outer membrane 7. Rinse w/ H2O
containing 8. Flood the slide with safranin (1 min)
lipopolysaccharide 9. Rinse with H20
10. View slide under the microscope
1. Apply primary stain (carbolfuchsin)
2. Heat gently
Acid-alcohol
3. Rinse Acid-fast bacteria Non-acid-fast
Acid-Fast Carbolfuchsin (e.g., Methylene
4. Apply acid-alcohol (decolorizer) - (e.g., bacteria remain
Staining (basic) hydrochloric blue
5. Rinse Mycobacterium) colorless
acid in alcohol)
6. Apply counterstain (methylene blue)
7. Rinse; Blot dry ; Examine
1. Prepare smear
2. Apply primary stain (malachite green)
Malachite Highlights Vegetative cells
Endospore 3. Heat gently
green (water- - Water Safranin endospores within may be less
Staining 4. Rinse
soluble) bacterial cells distinct
5. Apply counterstain (safranin)
6. Rinse; Blot dry; Examine
1. Apply primary stain (e.g., India ink or Congo
red) Capsules may be
Capsule India ink or Highlights
2. Wash - Water or saline Crystal violet difficult to
Staining Congo red bacterial capsules
3. Apply counterstain (e.g., crystal violet) visualize
4. Rinse; Blot dry; Examine
 Test Purpose
In order to perform many of the tests necessary to
identify a microorganism, it is essential to isolate the
Streak Plate Test
organism in pure culture. Remember — biochemical
tests are ONLY valid when a pure culture is used.
To detect the presence of the enzyme catalase in
bacteria, which breaks down hydrogen peroxide into
water and oxygen.
Catalase Test
The enzyme, catalase, is produced by bacteria that
use oxygen, and protects them from the toxic by-
products of oxygen metabolism.
To differentiate between Staphylococcus aureus
(positive) and other staphylococci (negative) based
Coagulase Test
on the ability to produce coagulase, an enzyme that
causes blood plasma to clot.
Steps
1. Sterilize the loop in the flame and allow it to cool.
2. Pick an isolated colony from the agar plate and spread it over
approximately 1/4 of the plate using close, parallel streaks.
3. Flame and cool the loop (easiest way is to gently touch it to an
uninoculated edge of the agar).
4. Turn the plate 90 degrees and lightly sweep the loop 1-2 times through
the inoculated area, then streak into the next quadrant
without overlapping previous streaks.
5.Flame and cool the loop.
6. Repeat #4, streaking into the 3rd quadrant of the plate
7. Flame and cool the loop
8. Repeat #6, streaking the remainder of the plate.
9. Incubate the plate under appropriate conditions.
1. Add a 1-2 drops of 3% hydrogen peroxide (H2O2) to the bacterial
culture.
2. Place a small amount of bacterial culture on a clean microscope slide.
3. Mix, dispose tooth pick
4. Observe for the production of oxygen bubbles.
1. Drop water on slide
2. Add a small amount of bacterial culture to a tube of rabbit plasma.
3. Mix bacteria into water
4. Dispose toothpick
5. Add 1-2 drops of rabbit plasma
6. Rock slide, watch clumping
Positive Result Negative Result
Plate w/ growth in all the areas of the plate that were
struck with the loop.
Each quadrant = lower density of bacteria (successive
dilution of inoculum)
4th quadrant = isolated colonies
Bubbling (production of oxygen bubbles) indicates the No bubbling (no production of oxygen bubbles)
presence of catalase. indicates the absence of catalase.
Staphylococci Streptococci
Catalase-positive bacteria include strict aerobes as Catalase-negative bacteria may be anaerobes, or
well as facultative anaerobes, although they all have they may be facultative anaerobes that only
the ability to respire using oxygen as a terminal ferment and do not respire using oxygen as a
electron acceptor. terminal electron acceptor (ie. Streptococci).
Clot formation indicates a positive result for coagulase
production.
Coagulase is an enzyme produced by S. aureus that
No clot formation indicates a negative result for
converts (soluble) fibrinogen in plasma to (insoluble)
coagulase production.
fibrin. S. aureus actually produces two forms of
coagulase, soluble and bound. This benefits the
bacteria in the host in that bacteria coated w/ fibrin
may be protected from the host immune system.

Alpha hemolysis: Greenish discoloration around

To determine the ability of bacteria to lyse (break
Hemolysis Test down) red blood cells, which can be classified as
alpha, beta, or gamma hemolysis.
1. Streak bacterial culture on a blood agar plate. - No greenish discoloration (for alpha hemolysis).
colonies.
2. Incubate the plate at 37°C. - No clear lysis (for beta hemolysis).
Beta hemolysis: Clear, complete lysis around colonies.
3. Observe the type of hemolysis around bacterial colonies. - No change in agar (for gamma hemolysis).
Gamma hemolysis: No hemolysis, no change in agar.

Bacteria that are oxidase-negative may be

To aid in identification of bacteria that produce
cytochrome c oxidase, an enzyme of the bacterial
electron transport chain.
Oxidase Test
When present, the cytochrome c oxidase oxidizes the
reagent (tetramethyl-p-phenylenediamine) to a
purple color. When the enzyme is not present, the
reagent remains reduced and is colorless.
1. Remove DrySlide frompacket.
2. Pick an isolated colony w/ sterile toothpick.
3. Rub bacteria onto slide, watch for color change
4. dispose toothpick & Dry Side
Positive = purple-black
Note: All bacteria that are oxidase positive are
aerobic, and can use oxygen as a terminal electron
acceptor in respiration. This does NOT mean that they
are strict aerobes.
anaerobic, aerobic, or facultative; the oxidase
negative result just means that these organisms
do not have the cytochrome c oxidase that
oxidizes the test reagent. They may respire using
other oxidases in electron transport. Bacterial
genera characterized as oxidase-positive include
Neisseria and Pseudomonas. Genera of the
Enterobacteriaceae family are characterized as
oxidase negative.