Name: Francis Gideon P.

Tagnong Date Performed:

Section: Fish 154 Lab, W 1:00-4:00 Date Submitted: October 10, 2019

Experiment 4 - Staining Techniques

I. Introduction

The correct identification of microorganisms is of fundamental importance to microbial

systematists as well as to scientists involved in many other areas of applied research and industry (e.g.
agriculture, clinical microbiology and food production). Most bacteria are without color, transparent
and have refractive index similar to that of the aqueous fluids in which they are suspended.
Staining is technique used in microscopy to enhance contrast in the microscopic image. Stains
and dyes are frequently used in biological tissues for viewing, often with the aid of different
microscopes. The Gram stain procedure was originally developed by the Danish physician Hans
Christian Gram to differentiate pneumococci from Klebsiella pneumonia.
Owing to the small size of bacteria little structural details can be seen with the ordinary light
microscope unless the organisms are stained. Microscopes are of little use unless the specimens for
viewing are prepared properly. Microorganisms must be fixed & stained to increase visibility,
accentuated to see specific morphological features, and preserved for future use.
This specific experiment introduced us to the different stains and staining techniques observed
in the microbiology laboratory. Likewise, the experiment showed us how to prepare bacterial smear,
and perform various staining techniques such as the simple staining, Gram staining, and endospore
staining.
Staining helps in the identification of bacteria, specifically, in Gram staining because it classifies
the bacteria into Gram negative or Gram positive. Likewise, staining helps in revealing certain
bacterial structures, giving a clearer view of the bacteria in the microscope. Lastly, endospore staining
helps in the identification of the vegetative and the spore bearing bacterial cells.

II. Materials and Methods

The materials and equipment needed for the conduct of the activity were prepared beforehand.
Prior to the beginning of each experiment, the countertops inside the laboratory were disinfected with
tissue wipes doused in 70% alcohol.

A. Preparation of bacterial smear

The following materials are used in the preparation of bacterial smear: clean glass slides,
labelling tape, distilled water, inoculating loop, burner and forceps, and bacterial colony sample.
A clean glass slide was prepared which was labelled on the far end of the slide with the group
number and staining method used. An oblong mark was made on the back side of the slide for the area
of bacteria introduction later. Using an inoculating loop, a small drop of distilled water was placed in
the center of the slide.

Using an inoculating loop, a colony was taken out in the bacterial colony sample (test tube) and
was transferred to the slide with distilled water i.e. the introduction of bacteria to the slide. The glass
slide was repeatedly passed a safe distance above the flame for fixation. A specific precaution was
taken to avoid breaking of glass slides due to prolonged exposure to high temperature i.e. the fire.
Lastly, the loop was sterilized using the flame from the burner.

B. Simple staining

With the prepared bacterial smear, simple staining was done by exposing the former smear to
drops of crystal violet dye for one minute and was rinsed with water by letting the water flow from
the top of the smear. Simultaneously, proper tilting of the slide was observed in the rinsing process.
It was then blot dried using a tissue paper and was viewed under the microscope using the Low Power
Objective, High Power Objective and Oil Immersion Objective. Under the OIO, the observed bacteria
were drawn.

C. Gram Staining

For this type of staining, another bacterial smear was prepared. Staphylococcus aureus and
Escherichia coli were namely the two bacteria used for this type of staining. Subsequently, the smear
was flooded with crystal violet for one minute. After 1 minute, the excess stain was washed off gently
with water. The smear was flooded with an iodine solution for thirty seconds.
To decolorize the smear, absolute alcohol was used by dropping it one at a time into the smear
while holding the slide at an angle. The decolorization time span for the several groups were different
but a general rule of under 15 seconds was observed. The smear was immediately rinsed with water.
Safranin was used as a counterstain and was treated to the smear for 30 seconds. The smear was
rinsed afterwards and was air-dried once again. The stained smear was then viewed under the
microscope using the Low Power Objective, High Power Objective and Oil Immersion Objective. Under
the OIO, the observed bacteria were then classified into either being Gram-positive or Gram-negative.

D. Endospore staining

As with the previous types of staining, another bacterial smear was prepared for the endospore
staining. Since endospores are highly resistant to normal staining procedures, only a small quantity
of malachite green was dropped on the bacterial smear. The quantity was only enough to cover the
whole smear.
 The smear was then put above the water bath for the span of 5 minutes. Simultaneously, more
stain was added whenever the smear dried up. Excess malachite green was rinsed off with water.
Then, the counterstain safranin was added and was left for the span of one minute. Afterwards, the
excess counterstain was drained, and the smear was rinsed with water.
The slide was then observed under the microscope using the Low Power Objective, High Power
Objective and Oil Immersion Objective. Under the OIO, the observed bacteria were analyzed according
to its parts and characteristics in the pre-laboratory lecture given by the instructor.

III. Results

Figure1. Simple stain bacteria viewed under Oil

Immersion Objective

Figure2. Gram stain bacteria viewed under Oil

Immersion Objective

Figure3. Endospore stain bacteria viewed under

Oil Immersion Objective
IV. Discussion
Bacterial sample used in the preparation of the bacterial smear is Staphylococcus aureus. The
smear was used to perform the simple staining. Figure 1 shows the bacteria observed under the OIO.
Simple stain is used to determine the shape, size and arrangement of the bacteria. Basic stains such as
the crystal violet will readily give up a hydroxide ion or accept a hydrogen ion, which leaves the stain
positively charged. Since the surface of most bacterial cells is negatively charged, these positively
charged stains adhere readily to the cell surface. Simple staining is only used to characterize the bacteria
and not to classify them.
For the preparation of bacterial smear for Gram staining and endospore staining, unidentified
species of bacteria were used. This is for the purpose of classifying the sample into Gram positive or
negative; endospore or vegetative cell.
Gram staining is a differential staining technique that differentiates bacteria into two groups:
gram-positives and gram-negatives. The procedure is based on the ability of microorganisms to retain
colour of the stains used during the gram stain reaction. Figure 2 shows a Gram negative bacterium.
Gram negative bacteria have multi-layered peptidoglycan cell wall which retain the crystal violet stain
and stay dark violet or violet. Gram positive bacteria have thin single layered peptidoglycan cell wall
and usually turn pink to red when counterstained by Safranin. Crystal Violet dissociates in aqueous
solutions into CV+ and Cl – ions. These two ions then penetrate through the cell wall and cell membrane
of both Gram-positive and Gram-negative cells. The CV+ ions later interacts with negatively charged
bacterial components and stains the bacterial cells purple. Iodine (I– or I3-) acts as a mordant and as a
trapping agent. A mordant is a substance that increases the affinity of the cell wall for a stain by binding
to the primary stain, thus forming an insoluble complex which gets trapped in the cell wall. In the Gram
stain reaction, the crystal violet and iodine form an insoluble complex (CV-I) which serves to turn the
smear a dark purple color. At this stage, all cells will turn purple.
Alcohol or acetone dissolves the lipid outer membrane of Gram negative bacteria, thus leaving
the peptidoglycan layer exposed and increases the porosity of the cell wall. The CV-I complex is then
washed away from the thin peptidoglycan layer, leaving Gram negative bacteria colorless. On the other
hand, alcohol has a dehydrating effect on the cell walls of Gram positive bacteria which causes the pores
of the cell wall to shrink. The CV-I complex gets tightly bound into the multi-layered, highly crosslinked
Gram positive cell wall thus staining the cells purple. The decolorized Gram negative cells can be
rendered visible with a suitable counterstain, which is usually positively charged Safranin, which stains
them pink. Pink colour which adheres to the
Gram positive bacteria is masked by the purple of the crystal violet
An endospore stain is used to distinguish between the vegetative cells and the endospores. A
primary stain (malachite green) is used to stain the endospores. Because endospores have a keratin
covering and resist staining, the malachite green will be forced into the endospores by heating. In this
technique heating acts as a mordant. Figure 3 shows the bacteria treated with malachite green stain and
are viewed under the OIO. Endospore staining was performed in this part. The green bacteria are spore
bearing bacteria and the red one is the vegetative cell.

VI. References
Amrita Virtual Lab Collaborative Platform (2014) Endospore Staining and Simple Staining Techniques.
Retrieved from http://amrita.vlab.co.in/?sub =3&br ch=73&sim =208&cnt=1
Rollins, David M. (2000). Gram Positive and Gram Negative Bacteria. Retrieved from
http://www.life.umd.edu/classroom/bsci424/BSCI223WebSiteFiles/GramPosvsGr
amNeg.htm