MICROSCOPE OPERATION AND FOUR QUADRANT
STREAKING TECHNIQUE MANUAL
1
OPERATING A MICROSCOPE
AND PERFORMING A FOUR
QUADRANT STREAK MANUAL
BY: JACLYN FLORES
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TABLE OF CONTENTS
Introduction……………………….…………………4
Microscope………………………….………………...5
Microscope Troubleshooting……………………….9
Four Quadrant Streaking………………………….10
Four Quadrant Troubleshooting………………….14
Conclusion……………………………………………15
Index……………………………………………….....16
Glossary………………………………………………17
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INTRODUCTION
The required lab manual in the course Microbiology
2041 is very useful in many ways. Unfortunately,
the students in the lab may need more guidance
than the manual provides. Students in the course
are having difficulties to operate the microscope
correctly and understanding how to put it in full
focus. Also, the students aren’t obtaining a great
understanding on how to do a four quadrant streak
correctly. These issues won’t occur if the students
have more information regarding these tasks. This
manual will provide the information necessary for
the students to be able to understand and
successfully complete these tasks.
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OPERATING A MICROSCOPE
In microbiology, the main topic we focus on are
microorganisms. In order to identify a
microorganism, we must first observe its
morphology. By doing so, we can limit the
possibilities of its identity to fewer numbers. The
first part has all to do with the microscope. Without
the microscope, we would not be able to observe
microorganisms. In order to observe
microorganisms we must first know how to operate
a microscope.
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The Parts
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Magnification
There are 4 magnifications to a microscope. The magnification is
written for each objective. In addition to this, the ocular lens
(eyepiece) has a magnification. The total magnification is the ocular
magnification times the objective magnification.
Magnification
Ocular lens
Total
Magnification
4x
10x
40x
10x
10x
100x
40x
100x
10x
10x
400x
1000x
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Focusing on Specimens




1. Always start with the lowest objective (40x). Most of the
time you will be able to see something on this magnification. Use
the Coarse Knob to focus. The image may be very small and
undetailed, but you can not focus on the specimen on the higher
levels without doing it at the lowest first. If you see nothing, try
moving the slide around until you see something even if it’s
blurry. Once you think you’ve found something, focus in on it.
2. Once you've focused on the 40x, switch to the 100X. Use
the Coarse Knob to refocus on the image. Focusing on this level is
what will allow you to focus on the next level.
3. Switch to 400x . Use the Coarse Knob to refocus on the image.
Focusing on this level is what will allow you to focus on the next
level.
4. Now finally switch to 1000x. (If you have a thick slide, or a
slide without a cover, do NOT use the high power objective). At
this point, ONLY use the Fine Adjustment Knob to focus
specimens.
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MICROSCOPE TROUBLESHOOTING
Sometimes people run into problems with their microscopes.
Here are some common questions and answers that people have
with microscopes.
1. The image I see is too dark.
Adjust the amount of light the diaphragm is giving off.
2. I keep seeing the same spot on my image. Even when I move
the slide it still remains in the same exact spot.
Your lens could be dirty. Use lens paper and clean the leans of the
ocular and objective lens.
3. I can’t see the specimen on the highest magnification.
You always want to follow the steps provided before trying to see
something on the highest magnification. Try going back and
refocusing on each level.
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FOUR QUADRANT STREAKING
Once you’ve used the microscope to observe the
microorganisms’ morphology, it is now time to do a
four quadrant streak. By doing so, you can observe
other characteristics of the specimen such as
amount of growth, color, and amount of growth on
a certain type of magnification.
. The main goal is to get pure colonies to grow on
the agar plate.
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Procedure
Label an agar plate with an A, B, C, and D on
four equal sections of an agar plate.
 Loosen the cap of the bottle containing the
specimen and hold it in your left hand.
 Hold an inoculating loop in your right hand.
 Flame the loop and let it cool down.
 Lift the test tube containing the specimen.
 Remove the cap off the test tube with the pinky
of your right hand.
 Flame the opening of the test tube.
 Insert the loop into the culture broth and
withdraw.
 Flame the neck of the test tube again.

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Replace the cap of the test tube using the pinky
of your right hand. Place the test tube on the
rack.
 Lift the lid of the Petri dish containing the agar.
 Hold the loop along the surface of the agar.
Smear the specimen backwards and forwards
across the small area of the agar labeled A.
 Remove the loop and close the Petri dish.
 Flame the loop again and allow it to cool.
 With the cooled loop, streak the plate from area
‘A’ across the surface of the agar in three or four
parallel lines to area ‘B’. Make sure that a small
amount of the culture is carried over.

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Remove the loop and close the Petri dish.
 Flame the loop again and allow it to cool. Turn
the dish again and streak from ‘B’ across the
surface of the agar in three or four parallel lines
into area ‘C’.
 Remove the loop and close the Petri dish.
 Flame the loop again and allow to cool. Turn the
dish and streak the loop across the surface of the
agar from ‘C’ into the section of the plate labeled
‘D’.
 Remove the loop and close the Petri dish. Flame
the loop again.
 Tape the plate closed and incubate the plate
upside down for about 24 hours.

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FOUR QUADRANT STREAK TROUBLESHOOTING
There are sometimes mistakes made while trying to successfully
perform a four quadrant streak. Here are some common questions
and answers that people ask in regards to a four quadrant streak.
1. My culture grew all over the place and I don’t have any pure
colonies.
More than likely you forgot to flame the inoculating loop in between
each time you did a streak into a different quadrant.
2. When I put the inoculating loop on the agar I hear a sizzling
noise.
You need to allow the inoculating loop to cool down before making
contact with the agar.
3. More than one thing grew on my agar plate.
You may have not used aseptic technique completely and because of
that your agar plate may have gotten contaminated. Just do it again.
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CONCLUSION
With the information provided in this manual you
can now start the basics of becoming a successful
microbiologist! You now know how to operate a
microscope and perform a four quadrant streak and
are well on your way to identifying lots of different
types of microorganisms. You can grow and observe
the microorganisms in no time.
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INDEX
Microscope, 5,6,7,8,9
Microscope Parts, 6
Focusing, 7,8
Four Quadrant Streak, 10,11,12,13,14
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GLOSSARY
Microbiology- the branch of biology dealing with the structure,
function, uses, and modes of existence of microscopic organisms.
Microorganism- any organism too small to be viewed by the unaided
eye, as bacteria, protozoa, and some fungi and algae.
Specimen- a sample of a substance or material for examination or
study.
Morphology- the branch of biology dealing with the form and
structure of organisms.
Petri Dish- a shallow, circular, glass or plastic dish with a loosefitting cover over the top and sides, used for culturing bacteria and
other microorganisms.
Magnification- a measure of the ability of a lens or other optical
instrument to magnify, expressed as the ratio of the size of the
image to that of the object
Agar- a culture medium having an agar base.
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