Ferimi Bajado

BIO83 A

Kindly do the following:

1. Write down the procedure of preparing a:
 
  A. Wet smear sample
1. Place a drop of fluid in the center of the slide
2. Position sample on liquid, using tweezers
3. At an angle, place one side of the cover slip against the slide making
contact with outer edge of the liquid drop
4. Lower the cover slowly, avoiding air bubbles
5. Remove excess water with the paper towel

    B. Fixed smear sample

1. First, use a wax pencil to draw a circle on the microscope slide to separate
each type of bacteria that is going to be sampled.
2. In order to be able to clearly see individual bacteria, a sample of a
bacterial colony must be mixed into water or physiological saline. This
helps to evenly spread out the bacterial sample.
3. Place a drop of water into the wax circle that has been created on the
slide.
4. Using a sterilized and cooled inoculation loop, obtain a very small sample
of a bacterial colony.
5. Gently mix the bacteria into the water drop.
6. In order to heat fix a bacterial smear, it is necessary to first let the
bacterial sample air dry.
7. Then either place the slide in the slide holder of a microincinerator, or
pass the dried slide through the flame of a Bunsen burner 3 or 4 times,
smear side facing up.
8. Once the slide is heat fixed, it can then be stained.
 
  C. Gram staining procedure
1. Apply a smear of bacteria on to a slide. Air dry and then heat fix by
passing it through a flame a few times. Make sure you air dry the bacteria
before heat fixing.
2. Add about 5 drops of Hucker’s Crystal Violet to the culture. Let stand for
one minute. Bacteria will stain purple. Wash briefly with water and shake
off excess.
3.Add about 5 drops of iodine solution to the culture. Let stand for 30
seconds, wash briefly with water and shake off excess.
4. Tilt slide and decolorize with solvent (acetone-alcohol solution) until
purple color stops running. Be careful not to over-decolorize. Wash
immediately (within 5 seconds) with water and shake off excess.
5. Add about 5 drops of Safranine O. Let stand for one minute, wash briefly
with water and shake off excess.

2. Differentiate in terms of the following: (Point of

Differentiation)
    A. Method of preparation
Examined, with 10x
eyepiece lens
magnification, under
High Power Objective
(400x total
magnification) or Low
Examined, with 10x
eyepiece lens
magnification, under
High Power Objective
(400x total
magnification) or Low
Examined, with 10x
eyepiece lens
magnification, under
High Power Objective
(400x total
magnification) or Low
Examined, with 10x
eyepiece lens
magnification, under
High Power Objective
(400x total
magnification) or Low
Examined, with 10x
eyepiece lens
magnification, under
High Power Objective
(400x total
magnification) or Low
    B. Manner of examining the smear
    C. Field of examination (what objectives are used)
A. Method of B. Manner of C. Field of
preparation examining the examination
smear (what objectives
are used)

A. Wet smear - Fewer steps/ Can be viewed -Examined, with

sample less under a 10x eyepiece
complicated light microscope lens
than the two - Can be viewed magnification,
procedures entirely, under
- Wet mount or with a specific High Power
method field of Objective
- Makes use of view (depending (400x total
a on what you’re magnification)
coverslip studying on the or Low Power
specimen) Objective (100x
- Examines, total
finds, and magnification)
quantifies cell
organelles
B. Fixed - Uses a spreader -Observed in -Viewed under
smear sample slide to four Oil
spread the separate Immersion
sample (blood quadrants and Objective
smear) averaged to (1000x total
- Uses the heat know the magnification
fixing Bacterial Index when
method (bacteria) multiplied with
(bacteria) and - Examines the the 10x
chemical fixing blood eyepiece
(blood smear in the magnification)
smear) zone of using a quality
- Includes morphology, microscope.
staining for a found in the (bacteria
clearer view of feathered edge and blood
the sample’s of the smear)
valuable specimen.
 organelles Individual - Views the
- Air-dries the cells are monolayer
slides examined, and part of the
before applying their smear as the
stains morphology is cellular blood
characterized elements
and are more
record spread and
easily be
studied in this
field
C. Gram -A detailed and -Look at areas - Observed
staining complex that are under oil
procedure procedure one cell thick immersion
- Uses a only; objective for
primary stain observation of higher
(1st), a mordant thick areas magnification,
(2nd), a will give as
decolorizing variable and the cells
agent (3rd), and often incorrect examined are
a secondary results. small (100x
counterstain White blood times 10x =
(4th) – staining cells and 1000x total
steps macrophages magnification)
- Identifies should stain - Identifies the
gram-positive Gram-negative, gram-
(purple) and/or whereas positive and
gram- squamous gram-
negative (red, epithelial cells negative
pink) cells are Gram- components of
- Uses the water- positive the heat-fixed
wash specimen
method

Sources:
https://www.microscopemaster.com/microscope-slides.html
https://www.microscope.com/education-center/how-to-guides/grams-
stain/
https://www.studocu.com/ph/document/silliman-
university/pharmacology/mandatory-assignments/jakiran-jr-activity-2-
ph-bio-sci-23-lab-c/9203042/view
https://www.scienceprofonline.com/microbiology/how-to-prepare-
microscope-slide-of-bacteria.html