Day 5 - And the Unknown Bacteria Is...

All staining, streaking, and experiments have led to this moment: figuring out the identity of our bacteria!  We were given several flowcharts from which to determine our bacteria.  The flowchart we used was for lactose-positive bacteria.

We traced the chart to... Enterobacter aerogenes.









We also filled out this chart at the end of lab:













Wow, we are done!  It's hard to believe that 5 days flew by so fast!  We learned a lot and are excited to apply the lab knowledge in our online lecture class!

Day 5 - Oh No, Another Medical Acronym: ELISA

ELISA stands for enzyme-linked immunosorbant assay.  Okay, so what does that really mean?  Basically, it is a test that detects antibodies.  If the test is positive, there are antibodies and that person has been exposed to a disease.  ELISA techniques are also used in pregnancy tests.


Answers to the Pre-Lab Focus Questions:
1.  The immune system protects us from disease by making antibodies against the disease's antigen.
2.  Doctor's use vaccines to use the immune system to protect us from disease.
3.  HIV is an example of a disease that attacks the human immune system.
4.  T-cell defects and autoimmune diseases can prevent the immune system from working properly.
5.  It is important to be able to detect antibodies in people who don't appear sick to be able to treat an illness before it takes over their bodies.  This can also help identify carriers of diseases.
6.  ELISA stands for enzyme-linked immunosorbant assay.
7.  The enzymes are used in this immunoassay to indicate the presence of antibodies.  The enzymes conjugate to the secondary antibody to produce a blue color.  Since the secondary antibody can only remain present in the microplate wells by binding to the antibody, which has bound to the antigen, the presence of the secondary antibody indicates the presence of antibodies.  Thus, the enzymes, which both bind to the secondary antibody and produce a blue color when doing so, indicate a positive result for antibodies.
8.  We need to assay positive and negative control samples as well as our experimental samples to make sure we don't get a false positive or a false negative result.

ELISA test materials:
Yellow tube of patient #10 serum sample
Green tube of purified antigen
Violet tube of positive control
Blue tube of negative control
Orange tube of secondary antibody
Brown tube of substrate
12-well microplate strip, of which we only used 9 wells
Pipet that transfers 50 microliters
Yellow tips
Transfer pipet for wash buffer
70-80 ml wash buffer in beaker
Large stack of paper towels
Marking pencil


Steps of an ELISA antibody test:


1.  Label the outsides of the microplate wells: "+" on three of the wells for the positive control, "-" on three of the wells for the negative control, and "10" on three of the wells for the serum sample.

2.  Using the pipet with a sterile, disposable tip, add 50 microliters of a purified antigen to each of 9 wells of a microplate strip.  Let it sit for 5 minutes, so that the antigens can bind to the walls of the strip.  This happens through hydrophobic interactions.  Dispose of the pipet tip in the biohazard waste bag.

3.  Wash out the excess purified antigen by dumping the fluid onto paper towels, filling the wells with a wash solution, and dumping the wash solution onto paper towels.  Dispose of the paper towels in the biohazard waste bag. 


4.  Add 50 microliters of the serum samples, positive control, and negative control to each of the appropriately labeled microplate wells.  Get a new, sterile pipet tip after every different solution.  We used patient serum sample #10.  Let it sit for 5 minutes to give it time to bind to the antibodies.  Dispose of the pipet tips in the biohazard waste bag.


5.  Wash out the excess purified antigen by dumping the fluid onto paper towels, filling the wells with a wash solution, and dumping the wash solution onto paper towels.  Dispose of the paper towels in the biohazard waste bag. 


6.  Add 50 microliters of the secondary antibody to each of the wells.  Let it sit for 5 minutes to give it time to bind to the antibodies.  Dispose of the pipet tip in the biohazard waste bag.


7.  Wash out the wells as described above, in step 5, but do it TWICE.

8.  Add 50 microliters of the enzyme substrate to each of the wells.  Let it sit for 5 minutes to give it time to bind to the antibodies.  Observe for the appearance of a blue color.  This indicates a positive diagnosis.  Dispose of the pipet tip in the biohazard waste bag.  The "+" wells should turn blue, and the "-" wells should remain colorless.

9.  Dispose of the microplate wells in the biohazard waste bag.




Result:  Our sample was a positive result.  The two "+" wells turned blue, the three "-" wells remained colorless, and the three "10" wells with our sample turned blue.  Thus, patient serum sample #10


Answers to the Post-Lab Focus Questions:
1.  Our serum did have antibodies to the disease, because it turned blue, thus indicating a positive result.
2.  If we tested positive for antibodies, it means that we have been exposed to the disease.
3.  There could be several reasons for a positive test when we do not actually have the disease: we are carriers, we are immune even though we are exposed, or the ELISA test was faulty or contaminated.
4.  We assayed our sample in triplicate to ensure that the results were accurate.  Multiple positive results ensured the reliability of the results.
5.  When we added the positive serum samples to the wells, the serum antibodies conjugated with the antigens on the sides of the wells.  If the serum had been negative, there would be no antibodies to stick to the antigens on the sides of the wells.
6.  We needed to wash the wells after every step to get rid of excess antigens or antibodies that would remain in solution and mess up results.  We wanted only the antigens that adhered to the wells' walls and the antibodies that conjugated with them to remain.
7.  When we added the secondary antibody to our positive serum sample, it adhered to the antigen-antibody complex that had already formed between the antigen on the well's walls and the primary antibody.  If our serum had been negative, the secondary antibody would not have adhered to anything, because it could only adhere to the antigen-antibody complex.  Then antigen-antibody complex would not be present if the serum were negative, since there would be no primary antibodies.  Thus, with a negative serum sample, the secondary antibody would just be washed away with the wash buffer.
8.  At our local pharmacy, we can buy pregnancy tests, illegal drug tests, and air quality tests, among others.

 

Day 5 - Selection and Differentiation

Different types of agar plates can encourage the growth of one type of organism, while inhibiting the growth of other types of organisms.  This is called selection media, since it selects a certain type of microorganism to grow on it.  Agar media that produce different characteristics for different types of organisms is called differential media, since it differentiates between different organisms.

Selection and differentiation can be combined in the same plate.  For example, the mannitol salt plate is selective for Staphylococcus spp., but it differentiates S. aureus from other Staphylococcus species.

We used several different selective/differential media in the lab.  Here is what we did:

Blood Agar Plate:
This test selects fastidious bacteria in clinical specimens, and it differentiates among bacteria that lyse red blood cells.  It differentiates between alpha-hemolysis, which is partial degradation of RBC and produces a greenish hue around the colonies; beta-hemolysis, which is complete destruction of RBC and produces a colorless zone around the colonies; and gamma-hemolysis, which is no lysis of RBC.

Materials:
Blood agar plate
Our unknown bacteria in broth culture

Steps:
1.  Label the blood agar plate with our group number. We shared a blood agar plate with group 3, so we divided the plate in half and labeled our half with a "4."
2.  Use aseptic technique to inoculate the blood agar plate from our broth culture.
3.  Incubate the plate upside-down at 35 degrees for 24 hours.
4.  Examine the results.
5.  Discard the plate properly.

Results:

Our bacteria is on the right side of the plate
Positive for growth

Our bacteria is on the left (we flipped the plate over for a different view)

Discussion:  Our bacteria is positive for growth, so it can grow in clinical specimens.  The hue of our colonies is not green, nor is it colorless around the colonies.  Thus, our bacteria must be gamma-hemolytic, meaning that our bacteria does not destroy red blood cells.




Mannitol Salt Agar:
This plate is selective for bacteria that can tolerate salt (7.5% NaCl, to be exact).  These types of bacteria include Staphylococcus, Micrococcus, and Enterococcus.  The mannitol in the mannitol salt agar plate makes it differential between Staphylococcus aureus and other salt-tolerant bacteria.  The mannitol salt media differentiates by turning yellow for S. aureus, because the mannitol fermentation produces an acid that turns the medium yellow.

Materials:
Mannitol salt agar plate
Our unknown bacteria in broth culture

Steps:
1.  Label the manitol salt agar plate with our group number. We shared a mannitol salt agar plate with group 3, so we divided the plate in half and labeled our half with a "4."
2.  Use aseptic technique to inoculate the mannitol salt agar plate from our broth culture.
3.  Incubate the plate upside-down at 35 degrees for 24 hours.
4.  Examine the results.
5.  Discard the plate properly.

Results:

Our bacteria is on the right side of the plate
Positive growth with a very slight amount of mannitol fermentation

Discussion:  Our bacteria grows on this medium, so it can tolerate salt.  Our bacteria can also ferment mannitol, which is a verification of previous test, but it cannot ferment it strongly.  This means that our bacteria is not Staphylococcus aureus. 




MacConkey Agar:
This test is selective for gram-negative enteric bacilli, and it differentiates between them via the bacteria's ability to ferment lactose.

Materials:
MacConkey agar plate
Our unknown bacteria in broth culture

Steps:
1.  Label the MacConkey agar plate with our group number. We shared a MacConkey agar plate with group 3, so we divided the plate in half and labeled our half with a "4."
2.  Use aseptic technique to inoculate the MacConkey agar plate from our broth culture.
3.  Incubate the plate upside-down at 35 degrees for 24 hours.
4.  Examine the results.
5.  Discard the plate properly.

Results:

Our bacteria is on the left side
Positive for growth

Discussion:  Our bacteria grows on this medium, so it is a gram-negative bacillus.  This verifies our results from the staining procedures.  Based on the color, our bacteria can also ferment lactose.  This is another verification of earlier tests.



Phenyl Ethyl Alcohol Agar:
This test is selective for gram-positive bacteria.  This test verifies whether or not our bacteria is gram-positive or gram-negative.

Materials:
Phenyl Ethyl Alcohol agar plate
Pure culture of our unknown bacteria on an agar slant

Steps:
1.  Label the agar plate with our group number and test type
2.  Use aseptic technique to inoculate the phenyl ethyl agar plate with our bacteria
3.  Incubate at 35 degrees for 24 hours
4.  Examine the results
5.  Discard the plate properly

Results:

Note: we are group 4, on the right side of the agar plate.

Discussion:  Our bacteria did not proliferate much in this culture.  This indicates that our bacteria is gram-negative, which verifies the gram stain we did that gave that same result.



Eosin Methylene Blue (EMB):
This test is selective for gram-negative bacteria.  It differentiates between those bacteria that can ferment lactose and/or sucrose, and those that cannot.  Gram-negative bacteria that produce significant acid, via the fermentation of the sugars, produce a green sheen.  Those bacteria that produce less amounts of acid yields a pink color.  Bacteria that cannot produce acid are colorless or the color of the medium. 

Materials:
EMB agar plate
Pure broth culture of our unknown bacteria

Steps:
1.  Label the EMB agar plate with our group number and type of test
2.  Use aseptic technique to inoculate the agar plate.
3.  Incubate at 35 degrees for 24 hours.
4.  Discard the plate properly.

Results: 

Our bacteria is on the right.
It produced pink colonies.

Discussion:  Based on the production of pink colonies, our bacteria is gram-negative and can ferment lactose and sucrose.  However, our bacteria does not produce much acid. 

Day 4 - How Cleaning Agents and Essential Oils Affect Our Unknown Bacteria

This experiment is to show how cleaning agents and essential oils affect our unknown bacteria. 

Materials:
Agar plate streaked with our unknown bacteria
Lysterine mouthwash
Lysol
Ginger essential oil
Lime essential oil
Clove essential oil
5 small circles of paper
Sterilized forceps
Pipet
Disposable ends for pipets


Steps:
1.  Using sterilized forceps, put 5 small pieces of paper on the agar plate: on the top, right, left, bottom, and center of the agar plate. 
2.  Draw 5 microliters of Lysterine into the pipet and expel onto one of the paper circles.
3.  Dispose of the pipet tip in the biohazard waste bag and get another sterile tip.
4.  Draw 5 microliters of Lysol into the pipet and expel onto one of the paper circles.
5.  Dispose of the pipet tip in the biohazard waste bag and get another sterile tip.
6.  Draw 5 microliters of ginger essential oil into the pipet and expel onto one of the paper circles.
7.  Dispose of the pipet tip in the biohazard waste bag and get another sterile tip.
8.  Draw 5 microliters of lime essential oil into the pipet and expel onto one of the paper circles.
9.  Dispose of the pipet tip in the biohazard waste bag and get another sterile tip.
10.  Draw 5 microliters of clove essential oil into the pipet and expel onto one of the paper circles.
11.  Dispose of the pipet tip in the biohazard waste bag.
12.  Incubate the agar plate at 35 degrees for 24 hours.
13.  Measure and record the diameters of the clear circles around the paper circles.



Results:

Our unknown bacteria

Diameters:
Lysterine mouthwash - 0 mm
Lysol - 0 mm
Ginger essential oil - 0 mm
Lime essential oil - 0 mm
Clove essential oil - 8 mm


Discussion: 
Our bacteria is slightly sensitive to clove oil.  Our bacteria is resistant to Lysterine, Lysol, ginger oil, and lime oil.

 

 

Day 4 - How Antibiotics affect MRSA and Our Unknown Bacteria

This experiment is to show how different antibiotics affect MRSA and our unknown bacteria.

Materials:
Agar plate streaked with MRSA
Linezolid
Vancomycin
Ginger essential oil
Cinnamon essential oil
Oxacillin
Agar plate streaked with our unknown bacteria
Penicillin
Novobiocin
Neomycin
Tetracycline
Amoxycillin
Pipet
Bunsen burner
Forceps
Small beaker of alcohol

Steps:
1.  Sterilize the forceps by dipping them in alcohol and then burning off the alcohol.
2.  Take linezolid and place at the top of the MRSA agar plate.  Sterilize the forceps.
3.  Take vancomycin and place at the left of the MRSA agar plate.  Sterilize the forceps.
4.  Take ginger oil and place at the right of the MRSA agar plate.  Sterilize the forceps.
5.  Take cinnamon oil and place at the bottom of the MRSA agar plate.  Sterilize the forceps.
6.  Take 5 microliters of oxacillin in a pipet with a sterile, disposable end, and place it at the center of the MRSA agar plate.
7.  Incubate at 35 degrees for 24 hours.
8.  Take penicillin and place at the top of the MRSA agar plate.  Sterilize the forceps.
9.  Take novobiocin and place at the left of the MRSA agar plate.  Sterilize the forceps.
10.  Take neomycin and place at the right of the MRSA agar plate.  Sterilize the forceps.
11.  Take tetracycline and place at the bottom of the MRSA agar plate.  Sterilize the forceps.
12.  Take 5 microliters of amoxicillin and place in the middle of the MRSA agar plate. 
13.  Incubate at 35 degrees for 24 hours.
14.  Measure the diameter of the clear circles around the antibiotics.  This helps determine how effective the antibiotics are.


Results:

MRSA

MRSA

Our Unknown Bacteria

Diameters for MRSA:
Linezolid                         27 mm
Vancomycin                    10 mm
Ginger essential oil           8 mm
Cinnamon essential oil   19 mm
Oxycillin                          0 mm

Diameters for our unknown bacteria:
Penicillin     - 0 mm
Novobiocin - 15mm
Neomycin    - 0mm
Tetracycline - 18 mm
Amoxicillin - 11mm


Discussion:
For MRSA, we combined antibiotics and essential oils.  Our unknown bacteria will be tested with essential oils in the next experiment.
MRSA is sensitive to linezolid and cinnamon oil.  It is slightly sensitive to vancomycin and ginger oil.  It is resistant to oxycillin. 
Our unknown bacteria is sensitive to tetracycline.  It is also rather sensitive to novobiocin and amoxicillin.  It is resistant to penicillin and neomycin. 

 

 

 

 

 

Day 4 - Yogurt

Since we both love yogurt, this experiment was especially intriguing to both of us.  Who knew that making yogurt is so easy?

Materials:
Milk
A big glass measuring cup
Cultured yogurt
Styrofoam cups
Plastic spoon
Aluminum foil

Steps:
1.  Boil some of the milk in the measuring cup in the microwave.
2.  Pour the milk into the cups.
3.  Mix in a little bit of the cultured yogurt into each cup.
4.  Pour some cold, not boiled milk into another Styrofoam cup and mix in some yogurt.
5.  Loosely cover all Styrofoam cups with aluminum foil so that air can get underneath the foil.
6.  Let the cups incubate overnight.
7.  Check the yogurt to see if it formed.
8.  Eat!

Results:  Well, we accidentally forgot to put the yogurt in the incubator overnight, so the yogurt sat on the counter all night.  When we arrived in the laboratory in the morning, we put it in the incubator.  By the end of the lab, about 4.5 hours later, the yogurt had not curdled.  This is probably due to the yogurt sitting out all night.

Day 4 - Viruses

Viruses multiply by taking over a host cell, using that cell to multiply their genetic material (DNA or RNA) and then spreading to nearby cells.  Viruses can kill their host cells.  Yet viruses are hard to kill because they use our own cells, and our body will not attack its own cells.  Viruses against a type of bacteria can kill that bacteria once it finishes using it for multiplying. 

 

In the lab, we test for viruses not through presence of viral colonies, but through absence of bacterial colonies.  For example, if we wanted to test for a virus that harms a certain kind of bacteria, we would inoculate an agar plate with that bacteria and put some of the virus on the same agar plate.  If the bacteria does not appear where the virus is, we know that the virus is present.  We illustrated this in the lab through the following experiment.

Materials:
Agar plate
Agar slant of bacteria
3 different viruses in tubes
Disposable sterile pipet tips

Steps:
1.  Inoculate the agar plate with the bacteria using aseptic technique.
2.  Use a sterile pipet tip to write one's initials on one part of the inoculated agar plate.  Dispose of the tip in the biohazard waste bag.
3.  Repeat step 2 twice, each with a different virus.  For each virus, write on a different part of the agar plate.
4.  Incubate the agar plate for 24 hours. 
5.  Observe results.

Results:


Discussion:
There are viruses, because they used the bacteria on the agar plate to multiply. 

Day 4 - Effect of UV Light Sterilization

There are different ways to sterilize things.  One way is through UV light radiation. 

Materials:
2 agar plates
Beaker of about 300 mL distilled water
UV light wand
4 different kinds of unknown bacteria in broth cultures from our four lab groups
Pipets with disposable sterile tips





Steps:
1.  Label the agar plates.  On one write "before" and on the other write "after."

2.  Inoculate the "before" agar plate with the distilled water.

3.  Using pipets, put 5 microliters of each kind of bacterial broth in the beaker of water.  Mix well.  Dispose of the pipet tips in the biohazard waste bag.


4.  Use the UV wand to sterilize the water.  The wand is automatically set for the correct amount of time for the amount of water we have.



5.  Inoculate the "after" agar plate with the sterilized water.

6.  Incubate the two agar plates at 35 degrees for 24 hours.

7.  Observe results.







Results:  No bacterial growth on either agar plate.

Discussion:  Neither agar plate had bacterial growth.  For the "after" sterilization, this is because the sterilization worked, killing the bacteria.  For the "before" agar plate, there is no growth because we used distilled water, which has no bacteria in it.  We had intended to use normal tap water for the "before" agar plate, which would likely have had bacteria in it.  It is Steubenville, after all.  The city is not exactly known for clean water.  If we had used tap water, we would probably have gotten colonies on the agar plate.  But we did not get colonies because we used distilled water.

Day 4 - Extra Tests on Our Unknown Bacteria

Today we did some additional tests to determine some properties of our bacteria.

Oxidase Respiration Test:
This test determines whether our bacteria has cytochrome oxidase, which helps with electron transport during respiration.  Cytochrome oxidase helps attach electrons to oxygen or to nitrogen when oxygen is not available.  But not all bacteria that grow in oxygen have cytochrome oxidase.

Materials:
Oxidase reagent
Piece of filter paper
Pure culture of our unknown bacteria on an agar plate
Petri dish lid

Steps:
1.  Clean the top of the Petri dish lid with alcohol so it is sterile. 
2.  Set the piece of filter paper on the lid and saturate it with the oxidase reagent.
3.  Using aseptic technique, transfer some our bacteria to the saturated filter paper.
4.  Observe for the appearance of a purple color.

Result:  We did not observe a purple color, so we had a negative test result.

Discussion:  Based on the result, our bacteria does not contain cytochrome oxidase.  This does not mean that our bacteria does not grow in oxygen.  Our bacteria just does not have this enzyme.




Hydrogen Peroxide:
Organisms that use oxygen as the electron acceptors in cellular respiration have an enzyme called catalase, which breaks down hydrogen peroxide.  This reaction produces the bubbles that are seen when hydrogen peroxide is poured on an infected wound.  Anaerobic bacteria do not have catalase, since they do not use oxygen.  Thus, the hydrogen peroxide will kill those bacteria, because they cannot break it down.



Materials:
Pure culture of our unknown bacteria on agar plate
Hydrogen peroxide








Steps:
1.  Uncover our agar plate and pour enough hydrogen peroxide to cover the surface.
2.  Watch to see if bubbles form.

Result:  Positive result, since bubbles formed.





Discussion:  Since we have a positive result, our bacteria can use oxygen in respiration.

 

 

Day 3 - Is that Cotton I Smell?

Hooray!  Today we learned how to do some lab techniques that nurses perform as their tasks.  Of course, the lab tests were not glamorous, but we had a lot of fun!  After performing the lab tests, we streaked the samples we obtained on the proper agar plates.

Throat Swab: To test for bacterial strep throat.

Materials:

Disposable gloves

Sterile cotton-tipped applicator

Sterile tongue depressor

Blood agar plate

Steps:

1.  Wash hands and put on gloves.

2.  Gather sterile equipment which should be nearby.  Be sure that the parts of the sterile equipment that will touch the patient do not touch anything else.

3.  Use the tongue depressor to flatten the patient's tongues while quickly running the cotton-tipped applicator over the arch near the tonsils in the back of the patient's mouth.

4.  Streak the blood agar plate with the throat sample on the cotton-tipped applicator.

5.  Incubate the blood agar plate at 35 degrees for 24 hours.

6.  Dispose of the cotton-tipped applicator and tongue depressor in the biohazard waste bag.  Throw away gloves and wash hands.

Results:



View on the table                                   Under Microscope



Discussion:  Since there is no glowing yellowish color around the colonies, it is not beta-streptococcus (full lysis).  There are alpha- and gamma-streptococcus (partial and no lysis).

Nasal Swab: To test for MRSA (Methicilline-resistant staphylococcus aureus).

Materials:

Disposable gloves

Sterile cotton-tipped applicator

Sterile saline solution

Mannitol salt agar plate

Steps:

1.  Wash hands and put on gloves.

2.  Gather sterile equipment which should be nearby.  Be sure that the parts of the sterile equipment that will touch the patient do not touch anything else.

3.  Dip the cotton-tipped applicator into the saline solution.

4.  Place thumb on the top of the patient's nose so that the cotton-tipped applicator collects a better sample.  Run the cotton-tipped applicator a couple of times around the circumference right inside of the patient's nose.  Repeat for the other nostril.

5.  Streak the mannitol salt agar plate with the nasal sample.

6.  Incubate the mannitol salt agar plate at 35 degrees for 24 hours.

7.  Dispose of the cotton-tipped applicator in the biohazard waste bag.

8.  Dispose of gloves and wash hands.

Results:

Under microscope

Discussion:

Staphylococcus grew, but it is not staphylococcus aureus, because the agar medium did not turn yellow.

Urine Streak:  To test for E. Coli in the urine.

Materials:

Closed container with urine specimen

Disposable gloves

Pipet with disposable sterile tip

MacConkey agar plate

Inoculating loop

Steps:

1.  Wash hands and put on gloves.

2.  Open container of urine and use the pipet with the disposable sterile tip to withdraw a drop of urine.

3.  Put the drop of urine on the MacConkey agar plate.

4.  Streak the agar plate with the inoculating loop using aseptic technique.

5.  Incubate the MacConkey agar plate at 35 degrees for 24-48 hours.

6.  Flush the extra urine and dispose of the disposable pipet tip in a biohazard waste bag.

7.  After incubation, examine the plate for bacterial colonies.

8.  Discard the plate in the proper place for sterilization.

Results:







Discussion:  Nothing grew on the agar, so there is no E. Coli in the urine.

Day 3 - Odds and Ends: Miscellaneous Tests

We performed some miscellaneous tests to discover some different properties of our bacteria. 

TSI (Triple Sugar Iron) Agar Test:
This test helps differentiate between different types of gram-negative bacilli, by testing the bacteria's ability to ferment glucose, lactose, and sucrose and to produce H2S.

Materials:
TSI agar slant tube
Pure culture of our bacteria on an agar slant
Inoculating needle

Steps:
1.  Label TSI tube with our group number and test type.

2.  Using aseptic technique, inoculate the TSI agar slant from our bacteria's agar slant.  For this inoculation, we stabbed the bacteria into the TSI agar slant and then as we withdrew the needle, we streaked side to side along the surface of the agar. 

TSIA Test

3.  Incubate the tube at 35 degrees for 24 hours.

4.  After incubation, examine the tube for color changes, gas appearance, and black precipitate. 

5.  Discard the tube properly.



Results:


Discussion:  We have a positive result.  Our bacteria produced acid in the slant and butt of the tube, along with gas.  Based on this, our bacteria uses glucose, sucrose, and lactose.


Urea Hydrolysis Test:
This test determines whether our bacteria can hydrolyze urea.  If so, the urea will be hydrolyzed into carbon dioxide and ammonia, which will raise the pH of the medium.  So a pH indicator will enable us to figure out if our bacteria hydrolyzes urea.


Urea

Materials:
Urea-containing broth
Pure culture of our bacteria on an agar slant

Steps:
1.  Label the urea tube with our group number and type of test.

2.  Use aseptic technique to inoculate the urea broth.

3.  Incubate the urea tube at 35 degrees for 24 hours.

4.  After incubation, examine the tube for color change.  A bright pink color indicates a positive result.

5.  Discard tube properly.

Results:


Discussion:  We had a negative result, so our bacteria cannot hydrolyze urea into carbon dioxide and ammonia.  We incubated the culture longer to verify these results.


Litmus Milk Reactions: 
Since bacteria differ in their abilities to digest lactose, protein, and litmus in litmus milk, we will use this to help determine the identity of our bacteria.  There are 4 possible results: 1) acid production through fermentation of lactose (indicated by lighter-colored medium), 2) acid curd (red at top) with litmus reduction, 3) peptonization with alkaline reaction, 4) alkaline reaction, and 5) no reaction.





Litmus Milk

Materials:
Litmus milk tube
Pure culture of our bacteria on an agar slant

Steps:
1.  Label the litmus milk tube with our group number and type of test.

2.  Use aseptic technique to inoculate the litmus milk tube from our bacterial agar slant.

3.  Incubate the inoculated tube at 35 degrees for up to 7 days, checking every 24 hours.

4.  Discard the tube properly.


Results:


Discussion:  We have a positive result!  Our tube medium was a lighter color and soft curds formed.  This means that our bacteria ferments lactose into lactic acid (hence the color change) and forms curds in the process.



Motility Testing:
This test indicates whether our bacteria are motile.





 

Motility

Materials:
Tube with motility test medium
Pure culture of our bacteria in broth culture

Steps:
1.  Label the tube with our group number and type of test.

2.  Use aseptic technique to dip inoculating needle into our bacterial broth culture and stab our bacteria into the center of the motility test medium.

3.  Incubate at 35 degrees for 24-48 hours.

4.  After incubation, examine the motility tube.  If bacterial growth spreads out from the stab, it is a positive result for motility.

5.  Discard the tube properly. 


Results:


The uniform and cloudy appearance is due to our bacteria moving out from the center.
The original deep stab is not visible.

Our positive result (left) with another group's negative result (right)

Discussion:  Our bacteria likes to move!  It is definitely motile.  This verifies the hanging-drop slide we saw under the microscope, in which we saw our bacteria swimming.