EXPERIMENT 2
MICROSCOPIC ANALYSIS OF MICROORGANISM
Figure 1: A Gram staining of Staphylococcus aureus (Source:
https://en.wikipedia.org/wiki/Gram_staining)
TOPIC OUTCOMES:
Student should be able to
1. To familiarize with the equipment and materials needed to observe microorganism
2. To understand the components, usage and safety procedure of using microscope
3. To understand and differentiate micrscopic characteristic of microorganism
MATERIAL
- Microorganism (s) -Agar plates containing Escherichia coli, Bacillus subtilis and X
microbe should be streaked beforehand by each group.
- Wire loop (1 for each group)
- Microscope (will be provided)
- Immersion oil (will be provided)
- Slide glass (10 for each group)
- 50-mL 70% Ethanol (1 Bottle each group)
- Staining reagents (methylene blue, safranin dye, indian dye, crystal violet; will be
provided)
METHODOLOGY
Draw a schematic diagram of a working microscope in front of you and label them.
Make sure you submit this before starting the experiment.
1) Preparation of bacterial smears OR heat fixation
Figure 2: Bacteriological smear: A – transfer of bacteria by bacteriological loop, B –
homogenisation of bacteria with water, C – bacteriological smear. (source:
http://mmp.vfu.cz/frvs2011/?title=ukoly-biologie-vse&lang=en)
a) Each group should have their own agar plates containing microorganisms needed.
The microorganisms should be an overnight culture.
b) Take one slide glass and place one loopful of sterile distilled water and place it on
the center of the slide. All should be done ASEPTICALLY.
c) Transfer a small amount of bacterial colonies (preferably one single colony) into
the water and spread the mixture into thin layer (see Figure 2).
d) By holding the slide in one end, quickly pass the smear over the flame of Bunsen
burner twice to three times.
e) Let it cool and proceed with the microbial staining steps.
CAUTION: TOO MUCH SMEARS WILL CAUSE DIFFICULTIES IN MICROSCOPIC
OBSERVATION.
2) Microbial staining
2.1 Simple staining
a) Take out clean slide glasses and place a drop of water (refer to heat fixation steps)
to make smears on the slides.
b) After the slides are cooled, add one drop of methylene blue stain and let it sit for
one minute.
c) Rinse the slide with distilled water and wipe with tissue paper at the sides of the
stained part to get rid of excess water,
d) Examine it under the microscope using oil immersion lens and draw your
observation.
2.2 Negative staining
a) Prepare a smear on the slide glasses, but this time do not heat-fixing it, instead let
it air dry.
b) Place one drop of indian ink onto the smears.
c) Spread the solution across the slide using the edge of another slide as a spreader
by slanting it 45 degree and move to the top slide in one motion to produce smears.
You should obtain a thin layer.
d) After the smears are air-dried, observed the shape of the microorganisms under
the microscope using high power objective lens (x40) and oil immersion power
objective lens (x100).
e) You should observe the morphology of the microorganisms and should be to draw
the shapes.
2.3 Gram staining
a) Prepare smears without heat-fixation. Air-dried the slides.
b) Add crystal violet solution to color all cells for one minute.
c) Rinse the slide gently with a slow running tap water or water dispenser.
d) Add one drop of iodine solution and let it sit for one minute before rinsing with
water as previously.
e) Add decoloriser solution or 100% Ethanol drop wise until the crystal violet color is
merely seen. Do not apply the alcohol too long.
f) If it is gram positive, it will show vivid of blue color retaining the primary color
before. If it is gram negative, the smears turn to colorless.
g) Observed the slides, under high power objective lens and oil immersion power
objective lens.
QUESTIONS
1. In your report, discuss the reason to prepare new microbial culture beforehand
and not to use old microbial cultures (more than 24 hrs).
2. Write a brief description on how to observe fungi under a microscope?
