Yesterday we tested antibiotics and oils to see how they were effective against our bacteria.
Antibiotics:
1. Penicillin (P10): no effect
2. Erythromycin (E15): no effect                                                           2
3. Chlomiphenic (C30): Effective- 1.5cm diameter                         1   5   3
4. Novoblociv (NB30): no effect                                                           4
5. Augmentin: no effect

Oils:
1.Clove: somewhat effective-1cm diameter
2. Cinnamon: most effective- 1.5cm diameter                                  2
3. Thyme: 2nd most effective-1.2cm diameter                            1   5    3
4. Eucalypte: least effective-0.8cm diameter                                    4
5. Ginger: not effective

Yesterday we also tested for food purity.  We created 4 wells in a petri dish and added extract and albumin to them.
1. Hamburger extract
2. Goat Anti-horse Albumin                         2          3
3. Goat Anti-bovine Albumin                             1
4. Goat Anti-swine Albumin                               4
After leaving the dish in room temperature overnight, we observed a reaction between the hamburger extract and Anti-bovine Albumin.  The reaction is the white semicircular streak seen below between 1 and 3:

Next we tested a sample to determine if HIV was present. First, we added 50ul of the purified antigen into 9 wells and waited 5 minutes.  Then we washed the unbound antigen out of the wells.  After that we added 50ul of the positive control to the three "+" wells, and 50ul of the negative control to the three "-" wells.  Then we transfered 50ul of serum sample to the 3 remaining wells.  After 5 more minutes, we washed the wells out again.  Then we added 50ul of secondary antibody into all of the wells.  After 5 more minutes, we washed the unbound antibody out of the wells.  We then transfered 50ul of enzyme substrate into each of the 9 wells and observed changes in color. 

#microbiologylovers!

For the last week we performed different tests in order to figure out our unknown bacteria. These are the tests with our results that we performed:

Hydrolytic (digestive) Enzymes
Starch: negative- the bacteria has no enzymes to use starch
Lipid: positive- the bacteria had a slight clear zone indicating it can break down lipids.  This means the bacteria secretes hydrolytic enzymes called lipases.
Casein: negative- there was no clear zone indicating that the milk protein, casein, was not used.
Gelatin: positive-there was some liquid still present after refrigerating the test tube

Utilization of Carbohydrates
Sucrose, Lactose, Manitol: negative- there is no change in color, there are no bubbles.
Glucose: negative- there is a slight change in color, there are no bubbles present.
Methyl Red Test (Mixed Fermentation): negative- no change in color, no acid end products
Voges-Proskauer Test (Butanediol Fermentation): negative- did not use butanediol fermentation
Citrate Utilization Test: positive- blue color, bacteria did grow, the pH indicator brothymol blue turns blue in an alkaline pH. This means bacteria utilizes citrate.

Respiration Tests
Nitrate Reduction Test: positive- it did not change color because nitrate was converted to ammonia. It did not turn a color and this means that nitrite is not present.

Degradation of Amino Acids
Indole (Tryptophan Degradation) Test: negative- did not turn red, tryptophan was not used as a source of energy.

Miscellaneous Tests
Triple Sugar Iron (TSI) Agar Test: The alkaline slant was red, and the alkaline butt was also red.  This means that none of the sugars were fermented so it is not a member of the enterobacteria.  Also, no carbohydrate was fermented and glucose was not used.
Urea Hydrolysis: negative, the urea remained yellow because it was not used.
Litmus Milk Reactions: Negative, when the bacteria was placed in the litmus milk, no gas, clot, or changed happened. It was an alkaline reaction.
Motility Testing: Positive, the bacteria was in a test tube moved up the test medium after sitting overnight in the incubator, indicating that it was motile.

Selective and/or Differential Media
Blood Agar Plate: Bacteria appeared clear and whitish, indicating there was no lyses which means it was gamma.
Eosin Methylene Blue (EMB) Agar: Negative; this test involves enteric vs. non-enteric bacteria, and which bacteria do and do not ferment lactose.  Our bacteria did not grow or produce a green metallic sheen, which means it was negative.
Mannitol Salt Agar: Negative, our bacteria did not grow.  A positive test would have resulted in yellow growth.
MacConkey Agar: Negative, nothing grew.  This test allows you to isolate Gram negative bacteria.  Crystal violet will inhibit gram positive bacteria.  Also you will know whether or not your bacteria produces lactose.  Gram negative will ferment lactose (it will release acid that will have red colonies).  Only gram negative will grow and acid will change the color to red.
Phenylethyl Alcohol (PEA) Agar: Negative, ours did not grow because it is gram negative.  PEA isolates gram positive by inhibiting gram negative bacteria. 

After completing all of these tests, we have concluded that our unknown bacteria is (alkaline) Pseudomonas fluorescens. We concluded that this was our bacteria by following the chart we received in class. First, we know that the bacteria is gram-negative bacilli (rod shaped). Then, we followed the chart down to where the bacteria is negative for the lactose test and negative for the glucose test. Then, we followed the chart down to where the bacteria is positive for nitrate reduction and then alkaline for the litmus milk test. Therefore, according to the chart our unknown bacteria is Pseudomonas fluorescens as confirmed correct by our professor. 

Day 6

Today in lab, we observed that our unknown bacteria is an obligate aerobe and it is motile.

In this test tube, bacteria only grew on top. This means that the bacteria can only grow where there is oxygen. The bacteria didn’t grow on the bottom of the tube because it would require the bacteria to grow in carbon dioxide instead of oxygen. The technical term for our bacteria is an obligate aerobe.


For this second test tube, the bacteria was stabbed with an inoculating needle into a semisolid agar. This test proves that the bacteria is motile because the bacteria appears to be growing out from the line of inoculation. In the case of motility, the cells swim away from the stab and a diffuse “cloud” of growth extends from the stab. This is what we observe in the test tube.



Then, we completed more tests in order to figure out the type of bacteria we are observing.


The first four tests we completed were starch, gelatin, casein and triglyceride hydrolysis test. We performed the tests in order to determine the bacteria’s ability to hydrolyze carbohydrates, proteins, and fats by testing if the bacteria has specific enzymes.

Casein is the major protein in milk. This test is used to assess the production of proteinases.

Gelatin is an animal protein. This test is used to assess the production of gelatinase.

Starch is a carbohydrate. This test is used to assess the production of amylase.

Triglyceride is a lipid. This test is used to assess the production of liapases.

Next, we completed two different tests called methyl red test, and voges-proskauer test. The purpose of the MR test, is to determine the ability of the bacteria to ferment glucose through mixed acid fermentation. The purpose of the VP test is to determine the ability of the bacteria to ferment glucose through butanediol fermentation.

Next, we completed three more tests called Triple Sugar Iron AgarTest, Litmus Milk Reactions Test, and Indole (Tryptophan Degradation) Test. The purpose of the TSI test is to determine the bacteria’s ability to ferment glucose, lactose and sucrose and to produce H2S. The purpose of the Litmus Milk Reactions is the differentiate the ability of the bacteria to utilize lactose, protein, and litmus in litmus milk. The purpose of the Indole test is to determine the ability of the bacteria to split amino acid tryptophan into indole and pyruvic acid.

Lastly, we put carbon dioxide on one of the inoculated plates. When doing this, bubbles formed and this means that cataylase is present in our bacteria.

Day 5

Today we observed the slide we created yesterday. 
1. Acid-Fast
       The acid-alcohol decolorized the Zeihl-Neelsen stain.  Therefore, the bacteria took the color of   the blue from the methylene blue which is the counter stain of the Zeihl-Neelsen stain.  This bacteria is non-acid fast since the bacteria on the slide is stained blue.

2. Endospore Stain
     The cells are bacilli (rod-shaped), negative gram stain.  There are no endospores because the bacteria are stained red, took the safranin stain. There are not green circular shapes within the individual cells. It did not keep the malachite green stain so there are no endospores. 

3. Capsule Stain
        There are no capsules on this bacteria.  The safranin has stained the rod-shaped bacteria because the bacteria appears red.  There is not a thick black outline around the individual cells which would indicate the presence of a capsule.
The bacteria are the small red spots in the picture below:

Day 4

Today we prepared 3 different types of stain: Acid-Fast, Endospore, and Capsule stain.  We began with the capsule stain.  In order to prepare this, we made a Negative stain where we placed nigrosin at the end of a clean slide.  Then we added a small amount of bacteria into the drop, mixed it, and spread it across the slide.

Using this negative stain, we prepared a capsule stain by covering the spread smear with safranin.  Then we rinsed off the excess stain and blotted off the excess water. 

Next we prepared an acid-fast stain.  First, we fixed a bacteria smear on the slide.  We put bibulous paper on the slide and saturated it with Ziehl-Neelsen carbolfuchsin while holding it over boiling water for 3 minutes.

  After the slide cooled, we removed the paper and rinsed the slide with water.  We then decolorized the slide with acid-alcohol and immediately rinsed it with water.  Then the covered the smear with methylene blue for 2 minutes and removed the excess dye with water.

The final stain was the endospore stain.  We took a slide fixed with a bacteria smear, covered it with bibulous paper, and for 5 minutes added malachite green stain.  We did this while holding the smear over boiling water. 

Then we rinsed the slide for 30 seconds and covered the smear with safranin for 60 seconds.  To finish, we rinsed the excess safranin off the slide and blotted the water to dry it.

These are the 3 completed slides:

Lesson of the day: don't spill the dye!!!



Day 3

Today we removed our broth streak plate from the incubator.  The bacteria is yellow with small white colony spots. Most of the colonies are flat, but some areas are slightly raised. It smells awful!

Next, we observes the bacteriophages in the spread plates that we created yesterday.  Our instructor traced "FUS" into the bacteria with the bacteriophages.  One of the spread plates had a visible "FUS" outline.  However, the other bacteria colony did not grow.  The outline was caused by the bacteriophages (viruses that target bacteria) killing the bacteria that it was spread on. 

We then created a gram stain for both the broth bacteria and the lightswitch bacteria. To set up the gram stain, we did a bacterial smear by transferring bacteria from the streak plates to a slide and fixing it there with water and heat.  For the gram stain, we  stained bacteria with crystal violet stain and Gram's iodine then decolorized it with ethanol.  Then we covered the bacteria smear with safranin and viewed it under the microscope using the oil emersion lens. 
Here is the safranin staining the gram stain:

The bacteria from the broth streak plate was lightly colored red.  The red coloration indicates that the bacteria is gram negative.  In the picture below, the bacteria is difficult to see, but the red stains indicate bacteria.

The bacteria from the lightswitch was colored deeply red indicating that it is gram negative bacteria.
Notice the circular shape of the bacteria. 

 

Day 2

Yesterday we prepared a streak plate of the bacteria from the lightswitch.  We sterilized the inoculating loop, touched it to the original bacteria colony, and spread it across the streak plate, four times. This procedure isolates bacteria colonies so they can be seperately observed. We then placed it in the incubator overnight. 
Today we removed our lightswitch bacteria from the incubator and observed the growth on the spread plate. The bacteria colonies grew much larger throughout the spread plate, in the streak pattern we performed yesterday. 
Colony Observation: Circular, cream-colored colonies that are slightly raised.  The edges are entire.

Yesterday we also recieved an unknown bacteria in a test tube.  Using a sterilized inoculating loop, we transfered the bacteria to broth and placed it in an incubator at 25 degrees celsius. 
The more transparent tube is the broth with the bacteria and the cloudier one is the original bacteria sample:

Today, we observed the colony growth in a pellicle form, meaning that the bacteria colony formed on the surface of the broth. It was white and circular as seen on the surface below:

We then did a hanging drop slide to observe the colony under the microscope.  In this procedure, we placed a drop of the bacteria-in-broth solution on a coverslip and attached that to a depression slide.  Using oil immersion, we observed the bacteria moving.  In the video below, the bacteria are the small white ovals "swimming" around:

At the end of lab, our instructor demonstrated how bacteriophages kill bacteria cells.  Bacteriophages are bacteria-specific viruses.  He spread bacteria from broth into a petri dish, then traced a design into the bacteria using the bacteriophages. Then we placed it in the incubator.  According to the specificity of bacteriophages to bacteria, we expect to see no bacteria colony growth where the bacteriophages were placed.