Health and Medicine - Nanogold
Observation of Bacteria
To understand an infectious disease, one has to study
microbes intensely. This is essential to identifying which
specific microbe causes a disease. Among the many
microbes one might find living in a sick patient's body,
only one is usually causing the disease, and the rest are
usually harmless. Sometimes distinguishing one micro-
organism from another requires the use of dyes or stains
that highlight various structural features of the organism.
Many stains colour some kinds of microbe, while leaving
others alone. The Gram stain (actually a series of staining and washing techniques) is often
used to help classify bacteria. It is used to distinguish between gram-positive and gram-
negative bacteria, which have distinct and consistent differences in their cell walls. The
difference between gram-positive (blue) and gram-negative (pink) bacteria lies in the ability
of the cell wall of the organism to retain a stain called crystal violet.
When bacteria are heat-fixed and stained they tend to clump together. At high dry power
individual cells often can't be detected because the space between cell walls is within the
limits of resolution of the microscope due to diffraction. To properly view stained bacteria it
is necessary to use oil immersion microscopy.
Most bacterial species are rod-shaped or round (known as cocci), although some are
curved, spiral-shaped, or irregularly shaped. Bacterial cells form clumps which have specific
arrangements; paired, in chains, in clusters or simply random.
1. To observe bacterial cells from a live specimen under a light microscope.
2. To learn the gram stain technique for the identification of different bacterial species.
3. To complete accurate scientific drawings of bacterial cell arrangements.
Materials and Equipment
Immersion Oil for Microscopes
Prepared slides of Bacteria
Yoghurt Culture: better if it is close to its use-by date
Small test-tube or vial
Crystal Violet stain
Gram’s Iodine Stain
95% ethanol solution
PART A: Observation of Bacterial Cells under oil-immersion.
1. Obtain the prepared slides of Staphylococcus species and Streptococcus species.
2. Mount the slide with smear up and focus at low power on the etching. Move the slide so
that the lens is over the smear itself. Now move the objective away from the slide until the
upper surface comes into focus.
3. A bacterial smear at low power looks like a patch of dirt. Focus on the mess, first at 40x
then at 100x and finally 400x, all in bright field mode. Note that you can see some detail at
400x, but the shapes and colours of the bacteria are somewhat distorted.
4. Move the 400x lens out of the way, place a drop of immersion oil directly on the smear
where the objective was, and swing the oil immersion lens in place. Move the fine focus up
and down slightly to ensure that the lens is in contact with the oil. Now watch the end of the
objective and bring it as close to the slide surface as you can without touching it. Note in
which direction you must focus to move the objective away from the slide, look in the
eyepieces, and slowly rotate the fine focus control until the image is focused.
5. Complete scientific drawings of both specimens and describe the shape and arrangement
Note: To remove immersion oil from a slide without damaging the smear, lay a piece of lens
tissue on the slide, add a drop or two of alcohol and draw the lens tissue across the slide.
Repeat if necessary.
PART B: Examination of bacteria from fresh yoghurt cultures.
1. Clean slides and cover slips for dust and other particles.
2. Place a very small portion of plain yogurt onto the slide.
3. Add one drop of water to the yogurt and place the cover slip on top.
4. Under low power, find a section where the yogurt is very thin; this is where you will find
the bacteria. Sketch view on worksheet.
5. Switch to high power (400X for most microscopes) for a better view of the bacteria.
Sketch view on worksheet.
6. Find a small vial and clean it thoroughly. If a collection vial is unavailable a plastic cup
will do. Make sure ALL soap is completely rinsed off.
7. Put a small amount of yogurt in the container and put it aside in a dark, relatively
warm area. Leave undisturbed for at least 24 hours.
8. After the time has passed, take a small sample with a toothpick and place on a slide. If
the sample seems too thick, dilute with a drop of water.
9. Place a cover slip on top and observe the bacteria at low power (100X) to find a good
place to start looking. The diaphragm setting should be very low (small) because
these bacteria are nearly transparent.
10. Switch to the highest power to identify the bacteria according to arrangement.
11. Identify any bacteria you might find.
12. Clean the collection vials and slides thoroughly after usage.
PART C: Gram Stain Technique
1. Collect a very small amount yoghurt on the surface of a clean glass slide
and add a small drop of water. Mix the two together and spread over a
small area with a toothpick. The aim is to get a very thin smear on your
2. Allow the film to air dry.
3. Fix the dried film by passing it briefly through the Bunsen flame two or three
times without exposing the dried film directly to the flame. The slide should
not be so hot as to be uncomfortable to the touch.
4. Flood the slide with crystal violet solution for up to one minute. Wash off
briefly with tap water (not over 5 seconds). Drain.
5. Flood slide with Gram's Iodine solution, and allow to act (as a mordant) for
about one minute. Wash off with tap water. Drain.
6. Remove excess water from slide and blot, so that alcohol used for
decolourization in the next step is not diluted. Flood slide with 95% alcohol
for 10 seconds and wash off with tap water. (Smears that are excessively
thick may require longer decolourization. This is the most sensitive and
variable step of the procedure, and requires experience to know just how
much to decolorize). Drain the slide.
7. Flood slide with safranin solution and allow to counter-stain for 30 seconds.
Wash off with tap water. Drain and blot dry with bibulous paper. Do not rub.
8. All slides of bacteria must be examined under the oil immersion lens.
9. Draw what you see on the worksheet. Note the colour and arrangement of
Results and Discussion
1. Draw what you see under the microscope on your results sheet. Describe the
arrangement and shape of each of the cells examined.
2. Why do bacteria have different shapes and cell wall structures?
3. Why do scientists stain cells?
4. Explain why it is necessary to examine bacterial cells under oil immersion.
5. Would you expect to see internal structures in a bacterium? Explain.
6. How do Scientists classify bacteria?
7. Imagine we had an electron microscope in the lab. Explain how the images
of the bacteria would be different.
Briefly summarise and write a conclusion for this experiment.
PART A: Observing Bacteria Cells
PART B: Observing Bacteria Cells in Yoghurt
PART C: Gram Stain Technique