Republic of the Philippines
CEBU TECHNOLOGICAL UNIVERSITY
MAIN CAMPUS
Website: http://www.ctu.edu.ph E-mail:
[email protected] Phone: +6332 402 4060 loc. 1109
COLLEGE OF ARTS AND SCIENCES
Name:_____________________________________ Date:___________________
Year and Section:___________________________ Instructor:_____________________
ACTIVITY 8
DIFFERENTIAL STAINING
Objectives:
At the end of the activity, the student shall be able to:
a. explain the principles of gram and acid-fast staining.
b. enumerate the components of gram stain and acid-fast stain.
c. perform bacterial smear preparation and subsequent differential (gram and acid-
fast) staining.
d. identify the staining reactions of different important bacteria.
e. discuss the medical importance of gram staining and acid-fast staining in clinical
specimens.
Materials:
a. Cultures of Staphylococcus species and Moraxella catarrhalis
b. Gram stain set-up: glass slides, alcohol lamp, wire loop/needle, blotting paper, slide marker,
stains: gentian violet, Gram’s iodine, acetone alcohol, safranin
Procedure:
Part I. Preparation of a Bacterial Smear
1. Get a clean glass slide and gently heat one side to remove grease. Hold the slide along the
edges to prevent recontamination with grease from the fingertips.
2. Divide the slide into two equal parts and label with the organism used. With a marker draw
on the bottom of the slide 2 circles with the size of a ten centavo coin. Place the bacterial
smear within the circles.
3. Sterilize the wire loop or needle until red hot by holding it at a 450 angle to the blue
portion of the flame, then heat half of the handle proximal to the wire loop/needle.
4. Allow the sterilized wire loop or needle to cool before getting inoculum. If the source of the
specimen is growing on solid media, place a loopful of distilled water on the center of the
circle. Touch an isolated colony using the sterilized wire needle and emulsify in the drop
of water on the slide. Smears from broth or liquid media are made directly on the slide by
taking one loopful of the culture and spreading it on the center of the circle drawn on the
slide.
5. Place the prepared smear on the table top and allow to air dry. Air drying preserves the
morphology of the organisms. Do not dry by blowing nor heating the slide. Flaming will
distort the cell morphology.
6. Fix the smear with heat by passing the slide smear side up, over a flame 3-5 times. Heat
fixation a) kills any bacteria that may still be alive, b) facilitates stain penetration, and c)
fixes cells to the slide so they do not wash off easily when stained.
7. Perform the desired staining technique.
8. Gently blot dry with filter paper, paper towel or absorbent paper and then examine under
OIO.
Republic of the Philippines
CEBU TECHNOLOGICAL UNIVERSITY
MAIN CAMPUS
Website: http://www.ctu.edu.ph E-mail: [email protected]
Phone: +6332 402 4060 loc. 1109
COLLEGE OF ARTS AND SCIENCES
Part II. GRAM STAIN (Hucker’s method)
1. Divide the slide into 2 equal parts and label Staphylococcus spp. and Moraxella
catarrhalis on each side. Place thin bacterial smears of said cultures on the
corresponding part of the slide.
2. Hold one end of the slide with a clothes pin. Flood the smear with gentian/crystal violet
solution for 1 minute. Wash with tap water.
3. Flood the smear with Gram’s iodine for 1 minute. Wash with tap water.
4. Tilting the slide, decolorize the smear with acetone alcohol until a faint violet color flows
off from the slide. Wash with tap water.
5. Counter stain with safranin for 30 seconds. Wash with tap water.
6. Blot dry and examine under OIO.
Result:
Draw organisms stained with gram stain. Indicate the staining reactions.
Gram stain reaction Gram stain reaction
Staphylococcus species Moraxella catarrhalis
Conclusion:
Republic of the Philippines
CEBU TECHNOLOGICAL UNIVERSITY
MAIN CAMPUS
Website: http://www.ctu.edu.ph E-mail: [email protected]
Phone: +6332 402 4060 loc. 1109
COLLEGE OF ARTS AND SCIENCES
Questions:
Gram Staining:
1. State the “general rule” on Gram staining reaction of bacteria.
2. Give at least 2 bacteria that that will not take up the gram stain.
3. What is the role of the counterstain in Gram Staining?
4. What is the role of the counterstain in Gram Staining?
5. Why is Gram Staining a valuable tool in Microbiology?
