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 Steps Positive Result Negative Result
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 Plate w/ growth in all the areas of the plate that were
uninoculated edge of the agar). struck with the loop.
In order to perform many of the tests necessary to
4. Turn the plate 90 degrees and lightly sweep the loop 1-2 times through
identify a microorganism, it is essential to isolate the
Streak Plate Test the inoculated area, then streak into the next quadrant Each quadrant = lower density of bacteria (successive
organism in pure culture. Remember — biochemical
without overlapping previous streaks. dilution of inoculum)
tests are ONLY valid when a pure culture is used.
5.Flame and cool the loop.
6. Repeat #4, streaking into the 3rd quadrant of the plate 4th quadrant = isolated colonies
7. Flame and cool the loop
8. Repeat #6, streaking the remainder of the plate.
9. Incubate the plate under appropriate conditions.
Bubbling (production of oxygen bubbles) indicates the No bubbling (no production of oxygen bubbles)
To detect the presence of the enzyme catalase in presence of catalase. indicates the absence of catalase.
bacteria, which breaks down hydrogen peroxide into 1. Add a 1-2 drops of 3% hydrogen peroxide (H2O2) to the bacterial
water and oxygen. culture. Staphylococci Streptococci
Catalase Test 2. Place a small amount of bacterial culture on a clean microscope slide.
The enzyme, catalase, is produced by bacteria that 3. Mix, dispose tooth pick Catalase-positive bacteria include strict aerobes as Catalase-negative bacteria may be anaerobes, or
use oxygen, and protects them from the toxic by- 4. Observe for the production of oxygen bubbles. well as facultative anaerobes, although they all have they may be facultative anaerobes that only
products of oxygen metabolism. 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.
1. Drop water on slide
To differentiate between Staphylococcus aureus 2. Add a small amount of bacterial culture to a tube of rabbit plasma.
Coagulase is an enzyme produced by S. aureus that
(positive) and other staphylococci (negative) based 3. Mix bacteria into water No clot formation indicates a negative result for
Coagulase Test converts (soluble) fibrinogen in plasma to (insoluble)
on the ability to produce coagulase, an enzyme that 4. Dispose toothpick coagulase production.
fibrin. S. aureus actually produces two forms of
causes blood plasma to clot. 5. Add 1-2 drops of rabbit plasma
coagulase, soluble and bound. This benefits the
6. Rock slide, watch clumping
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 1. Streak bacterial culture on a blood agar plate. - No greenish discoloration (for alpha hemolysis).
colonies.
Hemolysis Test down) red blood cells, which can be classified as 2. Incubate the plate at 37°C. - No clear lysis (for beta hemolysis).
Beta hemolysis: Clear, complete lysis around colonies.
alpha, beta, or gamma hemolysis. 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 anaerobic, aerobic, or facultative; the oxidase
cytochrome c oxidase, an enzyme of the bacterial Positive = purple-black negative result just means that these organisms
electron transport chain. 1. Remove DrySlide frompacket. do not have the cytochrome c oxidase that
2. Pick an isolated colony w/ sterile toothpick. Note: All bacteria that are oxidase positive are oxidizes the test reagent. They may respire using
Oxidase Test
When present, the cytochrome c oxidase oxidizes the 3. Rub bacteria onto slide, watch for color change aerobic, and can use oxygen as a terminal electron other oxidases in electron transport. Bacterial
reagent (tetramethyl-p-phenylenediamine) to a 4. dispose toothpick & Dry Side acceptor in respiration. This does NOT mean that they genera characterized as oxidase-positive include
purple color. When the enzyme is not present, the are strict aerobes. Neisseria and Pseudomonas. Genera of the
reagent remains reduced and is colorless. Enterobacteriaceae family are characterized as
oxidase negative.