Identification of a Bacteria

IDENTIFICATION OF A BACTERIA Room Copy

PROCEDURE

I. STUDY OF BACTERIAL CULTURES (Days 1,2,3)
DAY 1:
1. Obtain five sterile Petri dishes. Never open a Petri dish until necessary. If you accidentally open
     one tell the teacher, so it is not used later. Obtain a wax pencil for labeling each dish. Use the      following titles. (NOTE – Always write on the bottom of
     the dish since the top can be moved.)
2. Label each dish as follows:
     #1 -> Air
     #2 -> Fingers
     #3 -> Unknown bacteria spread into “S”-shaped Streak
     #4 -> E. coli spread into "field"
     #5 -> L. lactis & S. marcences mixture spread into “Full” Streak
     Also write your name or initials on all the dishes.
3. Take all the dishes to where the hot liquid agar is. Raise one side of each dish just enough
     to pour in some agar. It is usually easiest to pour agar in when the dishes are near the edge of the      table. (NOTE - You do NOT need to pour agar in until the whole dish is covered.) Pour some agar
      in and GENTLY swirl the dish as it sits on the table. If done properly this will spread the agar out      over the entire bottom of the dish without splashing any out on the table. The agar dishes now need      to be carried back to your lab tables without tipping them. Do not move the dishes for      approximately 10 to 15 minutes to allow them to solidify. (Sometimes more time is needed.) When      solid the agar will not creates ripples like water if shaken. From this point on these dishes will
     be referred to as agar plates.
4. Place Plate #l in front of you. Once the agar is solid open the dish to the air for 15 - 20
     minutes. Now put the lid back on.
5. Tape Plate #1 closed with masking tape, and place it IN the incubator for 24 hours.
6. Place Plate #2 in front of you. One partner should raise one side of the lid just enough to place      his/her finger SOFTLY on one side of the agar and hold it there 5-10 seconds. Now the other lab      partner should raise the other side of the lid just enough to place his/her finger on the other side of      the agar. Each partner should put a mark or initials where they touched the agar.
7. Tape Plate #2 closed with masking tape, and place it IN the incubator for 24 hours.
8. Place Plate #3, a bunsen burner, and a wire loop in front of you. (Note - the wire loop is easily      sterilized by placing it directly into the flame of a bunsen burner. BE CAREFUL!)
9. Light the bunsen burner and sterilize the wire loop.
10. Obtain a culture of Unknown bacteria growing in a liquid broth.

FROM THIS POINT ON IT IS IMPORTANT THAT STUDENTS CLOSELY FOLLOW ASEPTIC TECHNIQUES. REVIEW THESE TECHNIQUES AS SHOWN IN DIAGRAM #1.

11. Using the aseptic techniques shown in Diagram #1, use the wire loop to remove the Unknown      bacteria from the broth. Spread this bacteria across the plate using the“S”-shaped version of the      streak technique shown in the Introduction.
12. Tape Plate #3 closed with masking tape, and place it ON TOP of the incubator for 24 hours.
13. Place Plate #4, a bunsen burner, and a bacterial spreader in front of you. (Note - the bacterial      spreader is easily sterilized by placing it into alcohol and then quickly into the flame of a bunsen      burner. BE CAREFUL!)
14. Light the bunsen burner and sterilize the bacteria spreader.
15. Obtain a culture of E. coli in a liquid broth.
16. Using the aseptic techniques shown in Diagram #1, use a pipette to remove the E. coli bacteria      from the culture. ( NOTE -> It is not necessary to flame the pipette before and after each use.) Use      the pipette to draw out approximately .2 ml of broth. Quickly open one side of the Petri dish and      place the broth in the middle of the plate. Remember where you placed the drop of broth since that      size of drop is hard to see.
          NOTE -> THE DROP OF BACTERIA SHOULD BE SPREAD WITHIN 30-60 SECONDS AFTER BEING PLACED ON THE AGAR                           TO PREVENT IT FROM SOAKING INTO THE AGAR BEFORE IT CAN BE SPREAD.
17. GENTLY place the sterilized spreader onto the plate near the drop of broth. Move the spreader in
      a circular motion to quickly spread the bacteria across the entire surface of the plate.
18. Tape Plate #4 closed with masking tape, and place it IN the incubator for 24 hours.
19. Place Plate #5, a bunsen burner, and a wire loop in front of you.
20. Light the bunsen burner and flame the wire loop to sterilize it.
21. Obtain a mixed culture of "Lactococcus lactis" and "Serratia marcescens".
22. Using the aseptic techniques shown in Diagram #1, use the wire loop to remove the
     L. lactis/S. marcescens mixture of bacteria from the broth. Spread this bacteria across the plate      using the “Full” version of the streak technique shown in the Introduction.
23. Tape Plate #5 closed with masking tape, and place it ON TOP of the incubator for 24 hours.
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DAY 2:
     Use aseptic techniques to fill 3 sterile test tubes approximately ½ half of a special type of agar called motility agar. This agar is less dense thus allowing some types of bacteria to move through it. Set the agar tubes nearby so that they can solidify while you make the observations of the agar
plates requested below.

FROM PREVIOUS DAY:
1. Locate Plate #1. Locate three obviously different colonies. Use a stereomicroscope or magnifying      lens to better view each of these colonies. Locate Diagram #2 in the Introduction pages and use it      to describe colony shape, elevation, and appearance for each of your colonies. List each colony's      description in Table #1 provided on the data sheet. Now you can answer Questions #1-5.
2. Locate Plate #2. Select three obviously different colonies on Plate #2. Use a stereomicroscope or      magnifying lens to better view each of these colonies. Locate Diagram #2 in the Introduction pages      and use it to describe colony shape, elevation, and appearance for each of your colonies. List each      colony's description in Table #2 provided on the data sheet. Now you can answer Questions #1-4.
3. Locate Plate #3. Look for any individual colonies on Plate #3. Use a stereomicroscope or
      magnifying lens to better view each of these colonies. Locate Diagram #2 in the Introduction
      pages and use it to describe colony shape, elevation, and appearance for each of your colonies.      List each colony's description in BOTH Table #5 and Table #10 provided on the data sheet. Now      you can answer Questions #1-4 under table #5.
4. Locate Plate #4. Look at the general surface appearance of the bacteria on Plate #4. Describe the      general appearance of the bacteria, and complete a drawing of the bacterial growth present of the      plate in Table #3 on the data sheet. Now you can answer Questions #1-3.
5. Locate Plate #5. Look for any individual colonies (should be 2 different types) on Plate #5. Use a      stereomicroscope or magnifying lens to better view each of these colonies.
     a. First describe the general appearance of the bacteria, and complete a drawing of the bacterial           growth present on the plate in Table #4 on the data sheet.
     b. Next locate Diagram #2 in the Introduction pages and use it to describe colony shape,           elevation, and appearance for each of your colonies. List each colony's description in Table #5           provided on the data sheet. Now you can answer Questions #1-8 under table #4.

YOU MAY NOW DISPOSE OF PLATES #1 & #2.

NOTE -> WHEN FINISHED WITH EACH PLATE, SPRAY INTO IT WITH BLEACH OR ALCOHOL TO KILL THE BACTERIA, PUT THE LID BACK ON, AND THROW IT IN THE TRASH.

MOTILITY TEST
1. Obtain the 3 solid agar tubes you made previously. Label the tubes as shown below.
     Tube #1 -> L. lactis
     Tube #2 -> S. marcescens
     Tube #3 -> Unknown bacteria
     Also write your name or initials on all the tubes.

FROM THIS POINT ON IT IS IMPORTANT THAT STUDENTS CLOSELY FOLLOW ASEPTIC TECHNIQUES. REVIEW THESE TECHNIQUES AS SHOWN IN DIAGRAM #1.

2. Place Tube #1, a bunsen burner, and a wire needle in front of you. (Note - the wire needle is easily      sterilized by placing it directly into the flame of a bunsen burner. BE CAREFUL!)
3. Light the bunsen burner and sterilize the wire needle.
4. Obtain a culture of L. lactis in a liquid broth.
5. Place the wire needle into the L. lactis culture, and then immediately inoculate the bacteria into      Tube #1 by stabbing the needle approximately 2/3 of the way to the bottom of the agar. Look at      Diagram #3 in the Introduction to review this procedure. Store Tube #1 IN the incubator for 24      hours in a container (such as a beaker) to hold it upright.
6. Place Tube #2, a bunsen burner, and a wire needle in front of you. (Note - the wire needle is easily      sterilized by placing it directly into the flame of a bunsen burner. BE CAREFUL!)
7. Light the bunsen burner and sterilize the wire needle.
8. Obtain a culture of S. marcescens in a liquid broth.
9. Place the wire needle into the S. marcescens culture, and then immediately inoculate the bacteria      into Tube #2 by stabbing the needle approximately 2/3 of the way to the bottom of the agar. Look
      at Diagram #3 in the Introduction to review this procedure. Store Tube #2 ON TOP of the      incubator for 24 hours in a container (such as a beaker) to hold it upright.
10. Place Tube #3, a bunsen burner, and a wire needle in front of you. (Note - the wire needle is easily      sterilized by placing it directly into the flame of a bunsen burner. BE CAREFUL!)
11. Light the bunsen burner and sterilize the wire needle.
12. Obtain a culture of the Unknown bacteria in a liquid broth.
13. Place the wire needle into the Unknown bacteria culture, and then immediately inoculate the      bacteria into Tube #3 by stabbing the needle approximately 2/3 of the way to the bottom of the      agar. Look at Diagram #3 in the Introduction to review this procedure. Store Tube #3 IN the      incubator for 24 hours in a container (such as a beaker) to hold it upright.
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DAY 3:
FROM PREVIOUS DAY:
1. Locate Tube #1. Examine the growth of the bacteria looking particularly for any cloudiness located      away from the stab itself. It is usually helpful to hold the tubes up to the light to better view any      bacterial growth present. Determine if the bacteria displays motility (+) or no motility (-) and enter      this data into the spaces provided in Table #6.
2. Locate Tube #2. Examine the growth of the bacteria looking particularly for any cloudiness located      away from the stab itself. It is usually helpful to hold the tubes up to the light to better view any      bacterial growth present. Determine if the bacteria displays motility (+) or no motility (-) and enter      this data into the spaces provided in Table #6.
3. Locate Tube #3. Examine the growth of the bacteria looking particularly for any cloudiness located      away from the stab itself. It is usually helpful to hold the tubes up to the light to better view any      bacterial growth present. Determine if the bacteria displays motility (+) or no motility (-) and enter      this data into the spaces provided in both Table #6 and in Table #10.
4. Now you can answer Questions #1-4 under table #6 in the data sheet.

CATALASE TEST:
1. Locate Plate #5, a wire loop, a bunsen burner, a microscope slide, Hydrogen peroxide, and a      medicine dropper. (Note - the wire loop is easily sterilized by placing it directly into the flame of a      bunsen burner. BE CAREFUL!)
2. Light the bunsen burner and sterilize the wire loop.
3. Look at Plate #5 and locate 1-2 large individual colonies of L. lactis (yellowish-white).
     Open one side of the agar plate slightly and use the wire loop to remove these colonies of L. lactis.
4. Place these colonies of L. lactis onto a slide. With a medicine dropper add 1-2 drops of Hydrogen      peroxide directly onto the bacteria on the slide.
5. Observe the bacteria on the slide closely for bubbling. The appearance of bubbling indicates the      presence of catalase and is labeled a (+) result. No bubbling indicates no catalase present and is      labeled a (-) result. Determine if the bacteria display a (+) or (-) result and enter this data into the      spaces provided in Table #7.
6. Light the bunsen burner and sterilize the wire loop again.
7. Look at Plate #5 again and locate 1-2 large individual colonies of S. marcescens (reddish-pink).      Open one side of the agar plate slightly and use the wire loop to remove these colonies of S.      marcescens.
8. Place these colonies of S. marcescens onto a slide. With a medicine dropper add 1-2 drops of      Hydrogen peroxide directly onto the bacteria on the slide.
9. Observe the bacteria on the slide closely for bubbling. The appearance of bubbling indicates the      presence of catalase and is labeled a (+) result. No bubbling indicates no catalase present and is      labeled a (-) result. Determine if the bacteria display a (+) or (-) result and enter this data into the      spaces provided in Table #7.
10. Light the bunsen burner and sterilize the wire loop again.
11. Look at Plate #3 again and locate 1-2 large individual colonies of Unknown bacteria. Open one
      side of the agar plate slightly and use the wire loop to remove these colonies of Unknown bacteria.
12. Place these colonies of Unknown bacteria onto a slide. With a medicine dropper add 1-2 drops of      Hydrogen peroxide directly onto the bacteria on the slide.
13. Observe the bacteria on the slide closely for bubbling. The appearance of bubbling indicates the      presence of catalase and is labeled a (+) result. No bubbling indicates no catalase present and is      labeled a (-) result. Determine if the bacteria display a (+) or (-) result and enter this data into the      spaces provided in both Table #7 and in Table #10.
14. Now you can answer Questions #1-4 under table #7 in the data sheet.

REMAINING TIME:
     Any time remaining will be used by the teacher to review staining techniques in preparation for the next 2-3 days. If time permits begin the simple staining process.
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II. STUDY OF INDIVIDUAL BACTERIA (Days 4,5,6)

DAY 4:
SIMPLE STAINING:

FROM THIS POINT ON IT IS IMPORTANT THAT STUDENTS CLOSELY FOLLOW ASEPTIC TECHNIQUES. REVIEW THESE TECHNIQUES AS SHOWN IN DIAGRAM #1.

Stains: Methylene blue | Crystal violet | Carbol fuchsin
Staining time: approximately 1 minute for all 3 stains

1. Place a microscope slide, a bunsen burner, and a wire loop in front of you. (Note - the wire needle
      is easily sterilized by placing it directly into the flame of a bunsen burner. BE CAREFUL!)
2. Light the bunsen burner and sterilize the wire loop.
3. Obtain a culture of E. coli in a liquid broth.
4. Use the wire loop to make a smear approximately the size of a dime. Heat fix the smear so that
     the slide is ready to be stained.
5. Place the completed slide over the sink of the lab table and add several drops of one of the stains.      Allowing the stain to sit for the appropriate staining time.
6. Gently wash the smear with a slow stream of tap water from the sink faucet to remove excess
      stain. During this step the slide should be held parallel to the stream of water, thereby reducing
      the loss of organisms during this step.
7. Using bibulous paper, GENTLY blot dry, but do not wipe the slide.
8. Examine the slide under the oil immersion lens of a microscope.
          (Note -> Show the slide to the teacher BEFORE adding oil to the slide to make sure you                           are looking at the bacteria.)
9. Draw the appearance of the E. coli in the spaces provided on the Data Sheet in Table #8.
10. Clean the slide so it can be reused.
11. Again sterilize the wire loop.
12. Obtain a culture of L. lactis in a liquid broth.
13. Repeat steps 4-9 using a second stain and the bacteria L. lactis.
14. Clean the slide so it can be reused.
15. Again sterilize the wire loop.
16. Obtain a culture of Unknown bacteria in a liquid broth.
17. Repeat steps 4-9 using a third stain and the Unknown bacteria.
18. Now you can answer Questions #1-6 in the data sheet.
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DAY 5:
GRAM STAINING:

FROM THIS POINT ON IT IS IMPORTANT THAT STUDENTS CLOSELY FOLLOW ASEPTIC TECHNIQUES. REVIEW THESE TECHNIQUES AS SHOWN IN DIAGRAM #1.

FROM THIS POINT ON IT IS IMPORTANT THAT STUDENTS CLOSELY FOLLOW THE GENERAL STAINING TECHNIQUES INCLUDING BOTH PREPARATION OF SMEARS AND HEAT FIXATIONS. REVIEW THESE TECHNIQUES AS SHOWN IN DIAGRAM #5.

1. Place a microscope slide, a bunsen burner, and a wire loop in front of you. (Note - the wire needle
      is easily sterilized by placing it directly into the flame of a bunsen burner. BE CAREFUL!)
2. Light the bunsen burner and sterilize the wire loop.
3. Obtain a culture of E. coli in a liquid broth.
4. Use the wire loop to make a smear approximately the size of a dime. Heat fix the smear so that the      slide is ready to be stained.
5 Add several drops of crystal violet to the smear and let stand for 1 minute.
6. Gently wash the smear with a slow stream of tap water from the sink faucet to remove excess stain.      During this step the slide should be held parallel to the stream of water, thereby reducing the loss      of organisms during this step.
7. Add several drops of Gram's iodine mordant to the smear and let stand for 1 minute.
8. Gently wash the smear again using the same procedure as in step 5.
9. Add several drops of ethyl alcohol to the smear. This will decolorize (removes the crystal violet) the      smear. Caution: Do not over decolorize. Add the alcohol drop by drop for about 10-20      seconds.
10. Gently wash the smear again using the same procedure as in step 5.
11 . Add several drops of safranin to the smear. This will counterstain the smear (give it a contrasting      color from the crystal violet). Leave the safranin on for approximately 45 seconds.
12. Gently wash the smear again using the same procedure as in step 5.
13. Using bibulous paper, GENTLY blot dry, but do not wipe the slide.
14. Examine the slide under the oil immersion lens of a microscope.
          (Note -> Show the slide to the teacher BEFORE adding oil to the slide to make sure you                           are looking at the bacteria.)
15. Draw the appearance of the E. coli in the spaces provided on the Data Sheet in Table #9.
16. Clean the slide so it can be reused.
17. Sterilize the wire loop again.
18. Obtain a culture of Bacillus cereus in a liquid broth.
19. Repeat steps 4-15 using the bacteria B. cereus.
20. Clean the slide so it can be reused.
21. Sterilize the wire loop again.
22. Obtain a culture of Unknown bacteria in a liquid broth.
23. Repeat steps 4-15 using the Unknown bacteria.
24. Now you can answer Questions #1-4 in the data sheet.

IDENTIFICATION OF UNKNOWN BACTERIA:
1. Look at the data you’ve collected in Table #10 for your Unknown bacteria.
2. Compare this data to the chart entitled “Bacteria Identification Expected Results” located in the lab Introduction.
3. Determine which bacteria described in the chart “BEST FITS” (has the most matches) with the Unknown bacteria. Use this information to identify your unknown bacteria.
4. Now you can answer Questions #1-5 in the data sheet.