PRACTICAL MICROBIOLOGY Dr. Waleed Khalid

Laboratory Safety Rules, Microscope and Preparation of smear

Laboratory Safety Rules There are specific safety rules that are needed to be followed while working in microbiology lab. These safety rules include : 1-Wear a lab coat in the lab.

2-Eating, drinking and smoking are forbidden in microbiology lab. 3-Thoroughly wash your hands with soap and water before and after lab. 4-Clean the lab bench with disinfectant before and after lab.

5-Dispose of all contaminated materials and slides in specific containers. 6-Bacterial loop has to be sterilized by flame before and after use.

LIGHT MICROSCOPE - A basic microscope consists of two lenses. The uppermost lens, called the ocular lens which is the part through which a person looks. The lower lens is the objective lens. Usually, several objective lenses are localized on a turret, allowing rapid changing of objective lenses. The eyepiece tube holds the ocular and objective lenses in place. Most microbiological specimens are mounted on glass slides and placed on the stage.

Procedure : 1 - Clean your lenses with lens paper. 2 - Set the microscope on the scanning lens (red lens (4*)). 3 - Focus using the coarse adjustment.

4 - Change to low power lens (yellow lens (10*)) and focus. 5 - Switch to high power lens (blue lens (40*)). Only use the fine adjustment knob. 6 - Switch the objective to half way between the high power and the oil immersion lens (black and white) (100*) . 7 - Place a drop of oil on the slide. 8 - Turn oil immersion lens into the oil. 9 - Check your image and only use fine to adjust.

PREPARATION OF SMEAR :- Preparation of fixed smear :- A - From fluid material : ( such as broth culture , urine, sputum , pus , purulent exudates …, etc) 1 - Sterilize the loop in benzene flame , and let it to cool.

2 - Using a septic technique , withdraw a loopful of the specimen and spread it on the center of a clean slide to form a somewhat thick film of 1-2 cm in diameter , then re-sterilize the loop.

3 - Allow the film to dry by air without heating. 4 - The film is fixed on the slide by passing it 3 times through the benzene flame, allow the slide to cool before staining.

B - From solid material: ( such as a culture on agar i.e colonies ) : 1 - Sterilize the loop in benzene flame and let it to cool. 2 - Place a loopful of a clean water ( tap water can be used ) on the center of a clean slide. 3 - By the resterilized loop, transfer a small portion of the colony to the drop of water, emulsify thoroughly and spread the mixture evenly on the slide to form a thin film of 1-2 cm of diameter. 4 - Dry and fix as mentioned above

AIM OF FIXATION: 1 - Kill the microorganism 2 - Make the microorganism stuck to the surface of the slide 3 - Make the microorganism more permeable to the stain 4 - Prevent the microorganism from going autolytic changes.