Reference no: EM133124430

Practical: Microscopy and Aseptic Technique

PART 1: MICROSCOPY

INSTRUCTIONS FOR PART 1: Use of a Microscope
You are required to set up a microscope using the stained slides provided. This activity will be marked and must be successfully completed by the end of the Practical 3: Bacteriology session.

Setting up the Microscope
1. Look at the slide macroscopically and take note of the shape, colour and position of material.
2. Turn in the 4X objective and place the slide on the stage with the coverslip uppermost, on the front of the stage. Gently holding back the spring arm of the mechanical stage, push the slide back into the slide holder and release the arm slowly. The specimen will be held firmly.
3. Turn the brightness control to setting number 5 or halfway.
4. Turn on the electricity at the power point and on the base of the microscope. Adjust the light intensity until it can be seen shining through the condenser.
5. Adjust your seat so that you can look comfortably into the eyepieces of the microscope without stretching upwards or bending forwards.
6. Look down the microscope and adjust the distance between the eyepieces to merge the left and right view fields into one. This can be achieved by pulling the two bodies of the binocular upwards or towards, or sliding the eyepieces nearer or further apart; depending on which microscope model you are operating.
7. Focus individual eyepieces:
a) Look into the right eyepiece (with your right eye) and focus on the slide using the course and/or fine focus of the microscope.
b) Look into the left eyepiece (with your left eye) and focus on the slide using the knurled adjusting ring on the eyepiece tube.
8. Turn in the 10X objective and refocus the microscope if necessary.
9. Close the field iris diaphragm.
10. Rack the condenser up and down until an image of the edge of the field iris diaphragm is as sharply in focus as possible (Figure 1.2).

11. If the image of the field iris diaphragm is not centred, inform a technician who will show you how this can be fixed. Do not play with the adjustment screws yourself at this stage.
12. Open the field iris diaphragm until its image moves just outside the field of view. Close the aperture iris diaphragm (lever on condenser). The image of the specimen becomes darker and more refractile. Now open the aperture diaphragm until the field of view just reaches its maximum brightness. The microscope is now set for Koehler illumination for the 10X objective

Using the 40X Objective
1. After focussing the microscope using the 10X objective carefully revolve the nosepiece to bring the 40X objective into place.
2. Only slight focusing with the fine focusing control should be necessary to bring the specimen into sharp focus. Open the aperture iris (condenser) more, and increase the brightness control to obtain a bright, clear image.
• If the 40X objective has been damaged, it may not be possible to focus. If foxus is not possible after wiping across the lens with a kimwipe, please check this with a technician. If damage is confirmed, skip to 100X instructions.

Using the 100X oil immersion objective
1. After focussing the microscope using the 40X objective carefully revolve the nosepiece anti-clockwise to move to the 4X objective, place a drop of immersion oil on the specimen.
2. Turn the nosepiece anti-clockwise to locate the 100X objective. Do not move the other objectives through the immersion oil.
3. Only use the fine focusing control to focus the specimen. Open the aperture iris (on condenser) more to obtain a bright, clear image.

Removing the Specimen and Cleaning the Microscope
1. Revolve the nosepiece clockwise, so that the objective moves to one side.
2. Remove the slide from the slide holder. At this point it is important to clean the eyepieces, condenser lens, stage, illuminator lens and lastly the objectives, with the 100X objective to be cleaned last.
3. To clean immersion oil off the 100X objective, a KimWipe lens tissue moistened with xylene should be used and should be dried immediately with a dry KimWipe.
4. All microscopes must be returned, cleaned at the end of each use.

PART 2: ASEPTIC TECHNIQUE

INSTRUCTIONS FOR PART 2: Aseptic Techniques

Use of Wire Loop
Subculture Serratia marcescens provided on nutrient agar (NA) plates onto:

a) 1 X nutrient agar plate using the streak plate method

1. Label the base of the agar plate with your name, the date and the organism that you are subculturing.
2. Place the agar plate for subculturing and the previously cultured agar plate upside down on the bench so that the lids are on the bench.
3. Sterilise your wire loop as follows:
Adjust the flame of the Bunsen burner to produce a flame with a short, blue, central zone. Hold the metal handle of the loop at 45 degrees and place in the hottest part of the flame, which is just above the blue cone, until the whole length of the wire is glowing red. Allow to cool before use.
4. Using the loop pick a single colony of bacteria from a previously cultured plate.
5. Replace the lid of the previously cultured agar plate.
6. Hold the agar plate for subculturing within the zone of sterility leaving the lid on the bench.
7. Gently rub the inoculated loop across the plate spreading the bacteria over part of the plate as illustrated below (Figure 1.3) and continue the streaking as depicted, sterilizing the loop in between.
8. Place your agar plate in the container provided to be incubated at 37°C for 24 hours.

b) 1X slope of nutrient agar (solid medium)
1. Label the slope medium lid with your name, the date and the organism that you are subculturing.
2. Place the previously cultured agar plate upside down on the bench so that the lids are on the bench.
3. Hold the previously cultured agar plate within the zone of sterility leaving the lid on the bench.
4. Using a sterile loop pick a single colony of bacteria from a previously cultured plate.
5. Replace the agar plate lid.
6. Remove the lid of the slope medium bottle and retain in the hand avoiding contamination (Figure 1.4).
7. Sterilise the top of the bottle; by waving it through the hottest part of the flame.
8. Gently rub the inoculated loop back and forth gently over the agar slope; from bottom to top in a zigzag pattern.
9. Sterilise the top of the bottle again.
10. Replace the lid of the sample bottle.
11. Re-sterilise the loop.
12. Place your slope in the container provided for incubation at 37°C for 24 hours.

c) 1 X nutrient broth
1. Label the liquid medium lid with your name, the date and the organism that you are subculturing.
2. Place the previously cultured agar plate upside down on the bench so that the lid is on the bench.
3. Hold the agar plate within the zone of leaving the lid on the bench.
4. Using a sterile loop pick a single colony of bacteria from the plate.
5. Replace the agar plate lid.
6. Remove the lid of the liquid medium bottle and retain in the hand avoiding contamination (Figure 1.4).
7. Sterilise the top of the bottle by waving it through the hottest part of the flame.
8. Move the inoculated loop back and forth within the liquid medium, fairly vigorously to remove all bacterial cells from the wire loop.
9. Sterilise the top of the bottle.
10. Replace the lid of the sample bottle.
11. Re-sterilise the loop.
12. Place your slope in the container provided for incubation at 37°C for 24 hours.

Use of Disposable Pipette
1. Label the liquid medium lid with your name, the date and the organism that you are subculturing.
2. Remove the lid of the liquid medium bottle and retain in the hand avoiding contamination.
3. Sterilise the top of the bottle; by waving it through the hottest part of the flame.
4. Transfer one drop of Escherichia coli suspension into the medium, using a plastic disposable pipette. Microbial growth should occur after incubation.

Keeping Media Sterile
1. Label 2 bijoux of uninoculated nutrient broth with your name and date.
2. Using a plastic disposable pipette, transfer a small amount of medium back and forth between the 2 bijoux 6 times.
3. Place your media in the container provided for incubation at 37°C for 24 hours. No microbial growth should occur after incubation.

Part 3: Microorganisms on Everyday Objects
1. Using a sterile swab, moistened with sterile water, swab an object within the laboratory. For example; pens, books, jewellery, door handles or bench surfaces.
2. Label the base of a nutrient agar plate with your name, date and the location that the swab was taken from.
3. Place the agar plate upside down on the bench so that the lid is on the bench.
4. Hold the agar base within the zone of sterility leaving the lid on the bench.
5. Move the swab over the entire surface of the agar plate. This can be achieved by streaking the swab back and forth across the plate working up and down several times. Turn the plate 90 degrees and repeat rubbing the swab back and forth and up and down across the entire plate. Turn the plate 45 degrees and streak a third time (Figure 1.5).
6. Place your media in the container provided for incubation at 37°C for 24 hours. No microbial growth should occur after incubation.

Attachment:- Practical Microscopy and Aseptic Technique.rar