Documente Academic
Documente Profesional
Documente Cultură
I. Introduction
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.
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
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