Name: SAMONTE, Dennise Ann R.

Date:
Section: NUR203 Score:

Exercise No. 5
The Gram Staining
In the previous exercise, microorganisms were stained by the simple method and they
picked up whatever stain they used. No differentiation in staining property was shown. This
Gram Stain is one of the oldest and most useful methods of staining bacteria. In this method, the
bacteria are classified as Gram positive if they retain the first stain after treatment with mordant
and decolorizer and Gram negative if they pick up the secondary stain. This staining property
shown is attributed to the difference in their surface chemical structure.
Objectives:
1. The student must be able to know and perform the methods and techniques of isolation
and identification of bacteria.
2. The student must be able to know the principle involved in Gram staining.
3. The student must be able to enumerate the reagents in Gram stain and give the purpose
of each.
4. The student must be able to classify the bacteria as to Gram positive or Gram negative.
Materials:
Glass slides
Microscope
18 hours slant cultures of Escherichia coli, Staphylococcus epidermidis and Bacillus
subtilis
Immersion oil
Reagents:
Crystal violet
Gram’s iodine
95% Ethyl Alcohol
Safranin
Procedure:
1. Prepare smear from each culture.
2. Cover the smear with crystal violet and allow the stain to act for one minute. Wash with
water.
3. Place Gram’s iodine for one minute. Wash with water.
4. Decolorize with 95% ethyl alcohol until free stain has been washed off. Wash with water.
5. Counterstain the smear with safranin for 30 seconds. Wash with water.
6. Blot dry and examine with the LPO and HPO magnification.
7. Draw your results.

*Google pictures of the microorganisms to answer this portion

LPO Magnification of E. coli HPO Magnification of E.coli

 LPO Magnification of S. epidermidis HPO Magnification of S. epidermidis

LPO Magnification of S. aureus HPO Magnification of S. aureus

Experiment worksheet on Gram Staining

I. Determine the use of the following reagents in gram staining:
1. Crystal violet
2. Gram iodine
3. 95% ethyl alcohol
4. Safranin
Reference:

II. List down 10 Gram positive and 10 Gram negative bacteria

Gram positive bacteria Gram negative bacteria

Reference:
 Exercise No. 6
The Acid Fast Stain

The acid-fast organisms resist decolorization with acid alcohol due to its high mycolic acid
content in the cell wall thus retaining the primary stain, carbol fuchsin. Non-acid fast organisms
on the other hand are easily decolorized with acid alcohol thereby taking up the color of the
counterstain. Heat and/or solvents needed to drive the stain into the cell wall of the Mycobacteria.
Acid fast organisms are hard to stain but once stained, difficult to decolorize.

Objectives:
1. The student must be able to know and perform the methods and techniques of isolation
and identification of bacteria.
2. The student must be able to know the principle involved in Acid fast staining.
3. The student must be able to enumerate the reagents in Acid fast staining and give the
purpose of each.
4. The student must be able to classify bacteria as to Acid fast or Non-acid fast
Materials:
Sputum sample
Wire loop
Alcohol lamp
Microscope
Reagents:
Aqueous Carbol fuchsin
Acid alcohol
Methylene blue
Procedure:
1. Prepare a smear from a sputum sample on a clean glass slide.
CAUTION: AVOID SPILLING CONTENTS OF THE BOTTLE ON THE TABLE/ON
YOUR THINGS. PRACTICE STRICT ASEPTIC PRECAUTIONS. STERILIZE LOOP
AFTER USING.
2. Dry and fix smear by passing the slide over a flame.
3. Stain the slides following Ziehl-Neelsen method (Hot Method).
a. Flood the smear with carbol fuchsin and heat gently using alcohol lamp. Apply the
lighted tip to the undersurface of the slide. DO NOT ALLOW TO DRY OR BOIL.
STAIN MUST BE ADDED IF NECESSARY. The stain is allowed to act for 4 to 5
minutes with continuous heating.
b. Wash slide with tap water and decolorize with acid alcohol until no more stain goes
with the alcohol.
c. Wash with tap water and counterstain with methylene blue for one minute.
d. Air or blot dry and examine under LPO, HPO and OIO.
e. Draw your results.
*Google possible results and draw it
Result:
Sputum under LPO Sputum under HPO Sputum under OIO

*** ACID FAST- red bacilli against a blue background; NON ACID FAST- blue bacilli against a blue background
 4. Stain the slides using Kinyoun’s Method (Cold Method)
a. Prepare a smear.
b. stain with Kinyoun’s carbol fuschin for 3 minutes. DO NOT HEAT.
c. wash gently in running water
d. Decolorize with acid alcohol until no more color appears in the slide (about 2
minutes)
e. Wash gently in running water
f. Counterstain with Malachite green for 30 seconds
g. Wash gently in running water.
h. Air or blot dry and examine under LPO, HPO and OIO.
i. Draw your results.
Result:
Sputum under LPO Sputum under HPO Sputum under OIO

***ACID FAST- red bacilli against a green background; NON-ACID FAST - blue bacilli against a green background

Worksheet on experiment on Acid fast staining

I. Differentiate the acid fast staining methods based on its use
Acid fast staining method Use

1. Ziehl neelsen Method

2. Kinyoun Method

3. Pappenheim’s method

4. Baumgarten’s method

5. Gabbet’s Method

II. Differentiate the acid fast staining methods based on its components
 Ziehl Kinyoun’s Pappenheim Baumgarten Gabbet
neelsen

1 stain

Mordant

Decolorizer

2 stain

Results

Reference: