Flow Chart

Experiment 1

1. Sterilization of the inoculating loop:

1) Take up the inoculating loop by the handle and
hold the loop down.
2) Carefully flame the loop, holding it first in the
coolest part of the flame (yellow), then in the hot,
blue cone until it glows red.
a) Heat the entire inoculating wire and the
lower portion of the handle, without letting it
become too hot to hold.

Allow the inoculating wire to enter the flame at approximately a 45° angle to the
flame. Allow the loop to cool for a few seconds without touching anything

2. Transfer from a broth culture:

1) Pick up a broth culture of one of the organisms in your
other hand.Still holding the loop like a pencil, but more
horizontally, use the little finger of the hand that is
holding the loop to remove the test tube closure (slip-on
cap).
2) Keep your little finger curled around this cap once it is
free. Do not let the cap touch anything. Do not
place it on the bench.

3. - Pass the neck of the open tube quickly through the burner
flame two or three times.
- Do not overheat the test tube by holding it in the flame.
- Prevents cool air which may contain contaminants from entering the pure
culture.

4. - Insert the loop into the open tube (holding

both at a 45° angle). Touch the loop only (not the
handle) to the broth and remove a loopful of the bacterial suspension.
- Keep it steady and do not touch anything (it is loaded with bacterial cells)
Re-flame the mouth of the tube again and replace the cap. Put the tube back in
the rack.

HINT: When holding the "loaded" loop, simply place the cap without excessive
effort. When the tube is back in the test tube rack you push the cap down further
on the tube

5. Simple staining of bacterial cells:

1) Place the loop of bacterial broth on a microscope slide.
2) Repeat the procedure in steps 2 through 4 to add a
second loopful of the same culture to the first loopful on
the slide.
3) With the loop, spread the liquid into a dime-sized spot.
Several smears of different organisms can be made on
the same slide by following steps #1 through #4
(remember to sterilize your inoculating loop each time
you use a new bacteria).
4) Reheat the entire inoculating wire to kill any remaining
bacteria before returning it to its stand.

6. - Allow the smears to air dry. Do not speed the drying

process by holding them near the flame as it will distort
the cells' shapes.
- Always wait until the slide is dry before heat-fixing.

7. - "Heat fix" the organisms to the slide after they have been
air-dried by quickly passing the slide through the flame
(smear side up) two or three times.
- Properly "heat-fixed" slides can be handled easily; if
they are too hot to handle, you have heated them too
much. Unless fixed on the glass slide, the bacterial
smear will wash away during the staining procedure.
Heat fixing coagulates the bacterial proteins and fixes
them on the slide.
8. Place the air-dried, heat-fixed slides on the staining rack over the small
disposable pan on your bench.

10. - Using Loeffler's methylene blue stain, cover the bacterial smears with 1-3
drops of stain.
- Leave the stain in contact with the smear for one to two minutes. Do not
allow it to dry out.

11. - After 1 to 2 minutes, pour off the excess stain into the discard pan.
- Rinse the slides into the discard pan by using a gentle stream of water
from the squirt bottle provided.
- Keep washing until the water draining from the slides becomes colourless

12. - Remove all excess water from the slides by GENTLY blotting dry
between paper towels. DO NOT RUB!
- The dried slides are now ready for microscopic examination. Keep them
until you have learned the parts and proper use of the microscope
 Flow Chart
Experiment 2

Microscopic Examination of a Slide Preparation

1. - Remove the cover of the microscope and Gently

position it in a clear area on the lab bench directly in front of
you by grasping the handle of the arm with one hand and
supporting the base with the other hand.
- Before you turn on the lamp, set the light intensity
knob to the lowest setting.
- Plug the microscope in and switch the lamp on.

2. Take a few moments to familiarize yourself with the

location of the various parts of the microscope (Method
A).
- If you have difficulty locating a part or are unclear
regarding its use, ask your lab instructor for help
before proceeding.

3. - Raise the condenser lens to its highest position

beneath the stage of the microscope.
- Also check to verify that the iris diaphragm is "half
open"

4. - Rotate the scanning objective (4x) by rotating the revolving nosepiece

using the knurled ring. (NEVER rotate the nosepiece using the objectives.
Always use the knurled ring to rotate!).
- Usually, the microscope will be stored with the stage raised, but if not use
the coarse adjustment knob to raise the stage till it is at the top of the travel
(will not rise further). Most microscopes have stops that prevent the
objective lens from touching the slide.

5. - Place a prepared slide on the stage with the stained smear surface facing
upward.
- Pull back the lever of the mechanical slide holder and secure the slide flat
against the stage, then release the lever. By turning the appropriate
 mechanical stage knobs, move the slide until the stained smeared area is
located over the center of the light source.

6. The Basic Rule of Focus is to FOCUS AWAY from the specimen.

- Without looking through the eyepieces (viewing the microscope from the
side), note the distance between the objective lens and slide while moving
the stage upwards, bringing the objective as close as possible to your slide
- Then while looking through the eyepieces turn the focus knob the other
way, increasing the distance between the objective and the slide until your
specimen is in focus.

7. - Look through the ocular and adjust the light intensity to a level that is
comfortable. At this time you may want to adjust the eyepieces by folding or
unfolding them to decrease or increase the distance until you see one illuminated
circle.
- While looking through the ocular, slowly lower the stage with the coarse
adjustment knob until the image of the smear comes into approximate
focus.

8. With the 4X objective in place and the iris diaphragm

set at 4, using The Basic Rule of Focus, bring the
specimen into focus.

9. Rotate the nosepiece to the 10x objective and set the

iris diaphragm to 10. Refocus slowly with the coarse
adjustment knob.

10. To move from the sharply focused low magnification objective to 40x, you
need
Only rotate the nosepiece until the next objective lens is locked in place.
- Set the iris diaphragm correspondingly to 40. The image can be brought to
sharp focus by rotating the fine adjustment knob.
100X Objective: This lens must only be used on prepared slides of bacteria or
very thin wet mounts with coverslips (as the working distance is very small).
11. - To move from the sharply focused high-dry objective to the oil immersion
objective (100x), rotate the high-dry lens (40x) halfway so that no lens is directly
above the smear.
- At this point, place one small drop of immersion oil directly over the smear
in the center of the area illuminated by the condenser.
- Gently rotate the oil immersion objective into the oil. If the oil immersion
objective ever comes in contact with the slide before it is in its proper
locked position, call your demonstrator for advice**. The tip of the lens
becomes immersed in oil without touching the slide
FOCUS ONLY USING THE FINE FOCUS ADJUSTMENT.
- DO NOT return to using 40X objective with a slide that has immersion
oil on it.
- Clean the lens thoroughly with lens cleaner immediately after use. Be
careful not to drag the 40X objective through the oil. If oil has contacted the
40X objective clean immediately!!
 Flow Chart
Experiment 3
1) Observe each of your prepared slides using the oil immersion objective.
- Start first with the provided demonstration material until you are
accustomed to using the microscope, then observe your prepared
slides.

2) Make sketches of the bacterial types, trying to maintain relative size in

your drawings.

3) NOTE: Remember making careful sketches enhances effective

observation. You need not be an artist to draw what you see. But when
making your drawings, pay special attention to the size relationships
between bacteria.
- For example, how big are the bacteria relative to each other and to
other species?
- Try to emphasize this in your drawing.
- Also pay attention to spatial relationships.
- For example, where is one bacterium in relation to others?
- Are they all together in chains or single?
- You may want to use words such as singly, pairs, chains,
tetrads or clusters to express cellular arrangement.

4) Calculate the magnification of each specimen in your drawing. (make

an estimate).
- To do this measure the length of the specimen in your drawing with a
millimeter ruler.
- Multiply this figure by 1000 to convert millimeters to micrometers
(1000 pm = 1mm).