1. M ICROSCOPE O PERATION AND F OUR Q UADRANT S TREAKING T ECHNIQUE M ANUAL 1

2. O PERATING A MICROSCOPE AND PERFORMING A FOUR QUADRANT STREAK MANUAL B Y : J ACLYN F LORES 2

3. T ABLE OF CONTENTS Introduction……………………….…………………4 Microscope………………………….………………...5 Microscope Troubleshooting……………………….9 Four Quadrant Streaking………………………….10 Four Quadrant Troubleshooting………………….14 Conclusion……………………………………………15 Index……………………………………………….....16 Glossary………………………………………………17 3

4. I NTRODUCTION 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. 4

5. 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. O PERATING A M ICROSCOPE 5

6. The Parts 6

7. 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. MagnificationOcular lens Total Magnification 4x10x40x 10x 100x 40x 100x 10x 400x 1000x 7

8. 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. 8

9. M ICROSCOPE T ROUBLESHOOTING 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. 9

10. F OUR Q UADRANT S TREAKING 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. 10

11. 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. 11

12. 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. 12

13. 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. 13

14. F OUR Q UADRANT S TREAK T ROUBLESHOOTING 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. 14

15. C ONCLUSION 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. 15

16. I NDEX Microscope, 5,6,7,8,9 Microscope Parts, 6 Focusing, 7,8 Four Quadrant Streak, 10,11,12,13,14 16

17. G LOSSARY 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 loose- fitting 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. 17