Name: Hayden Robbins 10-03-2017

Name: Mohammed Al Hashim

Experiment 4
Microscopic Laboratory

OBJECTIVES
1. to learn how to utilize microscope
2. to learn how to perform gram staining

Operating Instruction
There are three objective lenses with the magnification of 10X, 20X, 40X and 1000X (oil immersion). They
can be distinguished by the diameter and length of their lenses. The eye piece has magnification of
10X.
How to determine the total magnification? Multiply the magnification of the lenses by the magnification of
the eye piece.
I. Operation
a) Since the microscope is a precision instrument, always handle it with care, and avoid abrupt
motions of shocks.
b) Avoid exposure to direct sunlight, high temperature, high humidity, dust and vibration.
c) Before bulb or fuse replacement, unplug the power cord from the AC outer.
d) Always ground the microscope to prevent electric hazard.
e) Only use the tension adjustment ring for altering the tension of the coarse adjustment knobs.
f) Don’t contaminate the lens surfaces with dust, fingerprints and etc.
g) Be certain the voltage selector switch on the base plate of the microscope is set to conform
with the local line voltage before use.

II. Maintenance and Storage

a) Use a clean brush or lens tissue paper to clean the lens surfaces. If the lens surfaces are
dirty with oil or fingerprints, wipe them off carefully with gauze moistened with a small amount
of a cleaning medium (alcohol and ether 3:7) or xylene.
b) Do not use organic solutions (e.g. thinner, xylene, ether, alcohol) to wipe painted surfaces or
plastic parts of various components. They should be cleaned with a neutral detergent.
c) Never disassemble each component of the microscope for repair yourself since the integrated
performance may be impaired.
d) When not in use, the microscope should be covered with the dust cover provided or
contained in a storage case, and kept in a placed free from humidity and mold.

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III. Instruction
a) Switching on the bulb
b) Specimen placement
c) Focus
d) Interpupillary distance adjustment
e) Objective change
f) Tension adjustment of the coarse adjustment knobs
g) Locking of the pre-focusing level
h) Use of immersion objective

Bacteria Morphology and Staining Technique

Samples:
 Mixed liquor or Foams

APPARATUS/REAGENT
1) Microscope
2) Glass slides and cover slides
3) Gram staining kit
4) Immersion oil

PROCEDURE
1) Clean slides with 70% ethanol and wipe with KimWipe paper
2) Write down your initial on the side of the slide
3) Add one drop or use the inoculating loop to transfer the sample on the slide. Don’t add to much.
4) Cover with cover slide.
5) Place slide on the platform and turn on the light bulb.
6) Rotate the objective piece to the lowest magnification.
7) Adjust the platform to be closest to the lens (go up).
8) Use coarse adjustment knob to focus by turning toward yourself – Never touch it again after you
see the images. Then, use fine adjustment knob.
9) Change the objective piece to higher magnification. Then, use fine adjustment knob to focus.
10) In order to change the objective piece to 100X, add one drop of immersion oil. Then, rotate the 100X
objective piece to be at the center.

Results

Mixed Liquor or Foam sample

400X

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 200X 1000X

Gram Stain, Modified Hücker Method

Reagents Prepare fresh every 3 to 6 months.
Solution 1 Prepare Solutions A and B separately, then combine them.
Solution A = 2 g Crystal Violet + 20 mL ethanol 95%.
Solution B = 0.8 g ammonium oxalate + 80 mL distilled water.
Solution 2 1 g iodine + 2 g potassium iodide + 300 mL distilled water.
Decolorizing solution Ethanol 95%.
Solution 3 10 mL Safranin O (2.5% w/v in 95% ethanol) + 100 mL distilled water.
Procedures:
1. Prepare thin smears on microscope slides and thoroughly air dry.
2. Stain 1 min with Solution 1; rinse 1 s with water.
3. Stain 1 min with Solution 2; rinse well with water.
4. Hold slide at an angle and decolorize with 95% ethanol added drop by drop (not in a continuous
stream) to the smear for exactly 25 s. Do not over-decolorize. Blot dry using bibulous paper.
5. Stain with Solution 3 for 1 min; rinse well with water; blot dry using bibulous paper.
6. Examine under oil immersion at 1000 magnification with direct illumination (not phase contrast). Blue-
violet is positive; red is negative
7. Test each batch of Gram stains on cultures with known Gram staining reactions.

Results:

Sampl
e Gram: Positive Gram: Negative

Color: Blue/Purple Color: Red/Pink

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Mixed liquor or foam (Gram staining)

200 400X 1000X*

*Camera wouldn’t focus, professor observed at this magnification*

Assignment:
Prepare a report on microscopic component with figure. Upload on Beachboard. (1%)
- In class before leaving, each student will show the instructor that they can use the
microscope. 5 min is provided (1%) individual
- Submit 2 gram straining slides (1%) individual

- Find 5 bacteria in activated sludge (1%)

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