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1. Microbiology Laboratory Safety Rules
1. All materials and clothes other than those needed for the laboratory are to be kept away from
the work area.
2. A lab coat or other protective clothing must be worn during lab. The lab clothing is not to be
worn outside of the laboratory.
3. Clean the lab table before and after lab with the disinfectant solution provided.
4. Wash hands before leaving lab.
5. Any item contaminated with bacteria or body fluids must be disposed of properly.
Disposable items are to be placed in the BIOHAZARD container. Reusable items are to be
placed in the designated area for autoclaving prior to cleaning. Sharps are to be disposed of in
the appropriate container.
6. Reusable items should have all tape and marks removed by the student before being
autoclaved.
7. Because organisms used in this class are potentially pathogenic, aseptic technique must be
observed at all times. NO eating, drinking, application of cosmetics or smoking is allowed.
Mouth pipetting is not allowed.
8. Cuts and scratches must be covered with Band-Aids. Disposable gloves will be provided on
request.
9. Long hair should be tied back while in lab.
10. All accidents, cuts, and any damaged glassware or equipment should be reported to the lab
instructor immediately.
11. Sterilization techniques will involve the use of punsen flames that are fire and burn hazards.
Keep all combustibles away from the flames. Do not leave inoculating loops or needles propped
in the flame.
12. Microscopes and other instruments are to be cared for as directed by the instructor.
13. It is the responsibility of the student to know the location and use of all safety equipment
in the lab (eyewash, fire extinguisher, etc.)
14. Cultures may not be removed from the lab. Visitors are not allowed in the lab.
15. Doors and windows are to be kept closed at all times.
16. For the best lab experience, read labs before coming to class. Make notes as necessary.
Wait for a laboratory introduction by the instructor before starting work.

2. Microscopy
Introduction:
Microorganisms are too small to be seen with the naked eye so a microscope must be used to
visualize these organisms. While a microscope is not difficult to use it does require some
practice to develop the skills necessary to use the microscope to its maximum capabilities.
Bacteria and other cellular microorganisms are measured in micrometres (µm) or 1 x 10-6
meters. Viruses are even smaller and are measured in nanometres (nm) or 1 x 10-9
m.
When carrying a microscope always use both hands. One should be on the arm of the
microscope and one should be under the base of the microscope.
Discussion:
There are several types of microscopes but the only one used in this laboratory is the compound
light or bright-field microscope. Individual microscopes will vary depending on the
manufacturer but all microscopes have the same basic features.
These microscopes are known as compound microscopes because there are two magnifying
lenses in the microscope. One magnifying lens is in the ocular and one is in the objective. Each
contributes to the magnification of the object on the stage. The total magnification of any set
of lenses is determined by multiplying the magnification of the objective by the magnification
of the ocular. The nosepiece rotates allowing the objectives to change and thus change the
magnification of the microscope.
The stage is where the slide is placed. The stage adjustment knobs allow the slide to be moved
easily. Light provides the illumination for the specimen. To control the amount of light
reaching the eye the iris diaphragm may be opened or closed using the lever just under the
stage. On low magnifications less light is need than on higher magnifications. Too much light
on low magnification may mask the specimen, particularly something as small as a bacterial
cell.
The coarse and fine adjustment knobs are used to focus on the specimen. When a slide is on
the stage there is a space between the objective and the slide. This space is known as the

3. working distance. The coarse adjustment knob will cause the working distance to visibly
change while the fine adjustment knob is for final, fine focusing.
The ability to see things using a microscope is limited by the resolving power of the
microscope. The resolving power of a microscope is the distance two objects must be apart and
still beseen as separate and distinct. For the light microscope this is 0.2µm. Objects closer
together than 0.2 µm will not be distinctly seen. Increasing the magnification will not make the
objects more distinct, just bigger.
Each objective has the magnification of the objective written on the objective.
The magnification of the ocular is also inscribed on the ocular. Low magnifications are used
for quickly examining the slide to find an appropriate area to examine. Higher magnifications
allow the examination of a particular object on the slide.
When you look through the ocular you will see a lighted circle. This is known as the field of
view or the field. While looking through the microscope, move the iris diaphragm lever and
notice how the brightness of the light changes. As you move the objectives to provide increased
magnification you will look at a smaller section of the slide. Be sure you move the object you
want to view into the centre of the field before moving to the next objective.
These microscopes are parfocal. Once you have focused on an object using one objective the
object will be approximately in focus on the next objective. Use of the fine focus knob will
sharpen the focus.
Procedure for Focusing
1. Obtain a slide.
2. Use the coarse adjustment knob to obtain maximum working distance.
3. Place the slide on the stage. The slide should fit into the slide holder but is not
placed under the slide holder. Use the stage adjustment knob to move the slide
over the hole in the stage.
4. Rotate the low power (10X) objective in place.
5. Use the coarse adjustment knob to obtain the minimum working distance.
Develop the habit of watching this process to be sure the objective does not crash into the slide.

4. 6. Look through the ocular. Adjust the light with the iris diaphragm lever if necessary. Slowly
turn the coarse adjustment knob until something comes into focus. Use the fine adjustment
knob to sharpen the focus.
7. Using the stage adjustment knob move the slide around until you find an area you wish to
examine more closely. Move the slide until the object you wish to examine is in the centre of
the field.
8. Rotate the high power objective into place. Use the fine adjustment knob to sharpen the
focus. Do not use the coarse adjustment knob. Adjust the light using the iris diaphragm lever
if necessary.
9. Rotate the high power object halfway to the next position. Place a drop of immersion oil on
the slide, then rotate the oil immersion objective into place. The objective should be immersed
in the oil on the slide. Use the fine adjustment knob to sharpen the focus. Adjust the light using
the iris diaphragm lever if necessary.
10. When finished viewing the slide use the coarse adjustment knob to maximize the working
distance and remove the slide from the stage. If you want to look at another slide, begin the
process over. If you are finished with the microscope clean the microscope and return it to
storage.
Procedure for Cleaning a Microscope
1. Turn off the light and unplug the cord. Store the cord appropriately.
2. Using the coarse adjustment knob to obtain maximum working distance and remove the slide
from the stage.
3. Using lens paper clean all the lenses starting with the cleanest first—ocular, low power, high
power and oil immersion. Use lens cleaner if necessary.
4. Clean any oil off of the stage using lenswipes or paper towels.
5. Rotate the scanning objective into place. Use the coarse adjustment knob to obtain minimum
working distance.
6. Return the microscope to the appropriate storage area.

6. Hand washing laboratory:
Aim:
To demonstrate the presence of different organisms on our hands and other parts of the body
in order to increase the awareness of hand washing importance in preventing the health care
associated infections.
Objectives:
Screening hands for presence of organisms using nutrient agar.
Hands will be screened before and after washing, with or without drying them.
Students will also be asked to screen the anterior nares area.
Hand washing policy is available for students (referenced from Health Protection Scotland).
Materials:
Nutrient agar.
Soap, alcohol gel, tissue paper and cotton swabs.
37°C incubators.
Methods:
Students will be divided into two groups, A and B.
For both groups:
Streak your fingers (as demonstrated) on the agar plate before washing your hands and label
the plate appropriately.

7. Group A:
Wash your hands using soap and water, and then streak them on the agar plate before drying
your hands out.
Wash and dry your hands using the provided tissue, then streak the fingers on a labelled plate
as appropriate.
Group B:
Wash your hands using the provided alcohol gel then streak your fingers on a labelled plate
as appropriate.
Both groups:
Take nasal swab and streak it on a labelled plate as appropriate.
Incubate all today’s plates at 37°C overnight.
Record your results next day.
Prof. Hamed Alzoubi