Lab Day 3

Results and Conclusions for Lab Day 2

Streak plate culture experiment:

Using the dissection microscope, we found that our bacterial colonies have a circular form. They also have a rose and colorless pigmentation. The colonies covered the entire margin. Also, the organisms were raised and convex. We did not get as good of a result with the pour plate. 

Experiment: Carbohydrate Fermentation 

Materials:

• lactose test tube
• glucose test tube 
• sucrose test tube 
• maltose test tube

What: (pictures shown the next lab day)

We inoculated each test tube with our sample using the aseptic and stab technique. We then set the tubes in the incubator to detect changes for the the next lab day. 

Why: Carbohydrate fermentation tests detect the ability of microorganisms to ferment a specific carbohydrate. Each type of bacteria will use different energy sources depending on the specific enzymes that bacteria possesses. The characteristics feature of the enzyme production in the bacteria enables them to use diverse carbohydrates which aids in identifying the unknown bacteria. 

Experiment:Capsule Stain

Materials:

• crystal violet dye
• blank slide
• slide holder
• microscope

1. We took mixed together a small sample of our bacteria with a drop of water. 
2. We then used another slide to spread the smear and allowed it to air dry.
3. We flooded the slide with crystal violet for one minute and then washed it with water.
4. We were then able to observe the capsule on the microscope.

Why: The capsule stain is a differential stain which selectively stains external capsules surrounding bacterial cells

Experiment: Acid Fast Stain

Materials:

• blank slide
• Methylene blue dye
• carbolfushsin dye
• paper towel
• acid-alcohol

 1. First we mixed the bacteria with a drop of water and heat fixed it.
  2. Then we covered the smear with carbolfuchsin dye. 
  2. Next, we placed a piece of paper towel on top of the dye and dry heated it for 2 minutes.
  4.We then rinsed the slide under water. 
  5. We then decolorized with acid-alcohol for 15-20 seconds.
  6. Next we washed the top and bottom of slide with water and clean the slide bottom well.
  7.The last step was a counter stain with Methylene Blue for 30 seconds.

Why:  The acid-fast stain is a differential stain which distinguishes organisms with waxy cell walls that can resist decolorization with acid alcohol. 

Experiment: Endospore Stain 

Materials:

• beaker
• hot plate
• slide holder
• blank slide
• microscope

1. We heat fixed the bacteria on a microscope slide.

2. Next, we placed the heat fixed slide over a steaming water bath for about 5 minutes while continuing to saturate the slide with malachite green dye. 

3. Then we removed the slide from the water bath and rinsed it with water.

4. The next step was to flood the slide with counter stain safranin and then rinse again.

5. Finally, we were able to view the specimen under oil immersion.

Why: One example of an extreme survival strategy employed by certain low G+C Gram-positive bacteria is the formation of endospores. This complex developmental process is often initiated in response to nutrient deprivation. It allows the bacterium to produce a dormant and highly resistant cell to preserve the cell's genetic material in times of extreme stress. 

Experiment: Hanging Drop

Materials:

• depression slide
• blank slide
• Vaseline
• microscope    

1.First, we put 4 tiny drops of oil on each corner of a clean dry coverslip.

2. Then we aseptically transferred a loop full of culture to the center of a coverslip.

3. We then turned the depression slide upside down. Next, we touched the cover slip gently and the oil and coverslip adhered lightly to the slide.

4. We were then able to test if our bacteria is non-motile or motile using the microscope. 

5. Our bacteria was motile.

Why: to test bacteria's motility 

Experiments: 

Materials:

• starch agar plate
• casein plate
• Lipid plate
• Indole test tube
• Methyl red test tube
• Voges-Proskauer test tube
• Citrate test tube
• Nitrate slant tube
• Triple sugar Ion test tube
• Urea test tube
• Catalase plate
• Gaspak
• Litmus test tube

What: (Pictures shown the next lab day)

We inoculated all of the plates and test tubes. We then put them in the incubator to detect changes for the next lab day. 

Why:

Each different test will help us to discover our unknown bacteria. 

Lab Day 4

Lab Day 4

Results and Conclusions for Lab Day 3:

• Glucose: Positive, 
• Lactose : Positive (because there were gas bubbles)
• Sucrose: Negative
• Maltose: Negative
• Therefore our unknown can ferment glucose and lactose

• Starch Agar: Negative, therefore our unknown bacteria does not hydrolyze starch.
  

• Casein: Negative,therefore our unknown bacteria does not hydrolyze casein.
  
   

• Lipid (Fat): Negative
• 
• Gelatin Liquefaction: Negative, therefore our unknown bacteria does not hydrolyze gelatin.
  
• Indole: Negative, therefore our unknown bacteria does not use tryptophan as a source of energy.
  
• Methyl-Red: Negative, therefore our unknown bacteria does not ferment glucose via mixed-acid fermentation.
  
• Voges-Proskauer: Positive, therefore our unknown bacteria can ferment glucose via butanediol fermentation.
  
  
  
• Citrate: Negative, therefore our unknown bacteria cannot utilize citrate as its sole source of carbon and energy.
  
• Nitrate: Negative, therefore our unknown bacteria is unable to reduce nitrate ions to either nitrite ions or nitrogen gas.
  
• Triple Sugar Ion (H2S Production): Negative for H2S production and positive for ability to ferment glucose.
  

(our tube is on the right and isbeing compared to an uninoculated tube which is on the left)

• Urea: Negative, therefore our unknown bacteria cannot hydrolyze urea.
  
• Catalase: Positive, therefore our unknown bacteria uses the enzyme catalase to quickly break down H2O2 into water and O2
  
• Oxidase: Negative, therefore our unknown bacteria does not have cytochrome oxidase
  
• Litmus: Positive
  
   Left: our litmus test after one day incubation.
  

Right: our litmus test (middle) after one more day of incubation.

Experiment: Selective and/or Differential Media

Materials:

• Blood Agar plate
• EMB Agar plate
• Mannitol Salt Agar plate
• MacConkey Agar plate
• Phenylethyl Alcohol (PEA) Agar plate
• working stock of unknown bacteria
• inoculating loop

What we did and why:

What: (pictures will be shown in the results and conclusions in the next post)

1. we inoculated the blood agar plate

2. we inoculated the EMB plate

3. we inoculated the mannitol salt plate

4. we inoculated the MacConkey plate

5. we inoculated the phenylethyl pl-e

6. we placed them all in the incubator to grow overnight.

(results will be posted in next post)

Why: we did this experiment to isolate and differentiate from different types of bacteria (which will be specified in the results and conclusions in the next post).

Learning how to perform these tests will give us the ability and knowledge that we need to identify and know what to look for in bacteria.

Experiment: Testing Antibacterial Medicines

Materials: 

• Mueller-Hinton agar plate (2)
• individual antibiotic disks in cartridges
• forceps
• sterile cotton swabs
• working stock of our unknown bacteria

What we did and why (pictures will be shown in the results and conclusions of next post)

What: 

1. first we divided both of the agar plates into 4 quadrants per plate so 8 all together

2. next we inoculated the agar plates with our unknown bacteria from our working stock

3. we placed 8 different antibiotic disks, each one had their own quadrant 

4. we labeled each quadrant with the name of the antibiotic that was in and placed the plates in the incubator.
We labeled them:
Red
1. Neomycin
2.Norobicin
3.Penicillin
4. Streptomycin
Blue
1.Erythromycin
2.Tetracycline
3.Amoxocillin
4.Oregano

Why:We did this to determine the sensitivity of our unknown bacteria to several antibiotic medications. This will also help us in the future to show us what makes a bacteria resistant or sensitive

Lab Day 2

Results and Conclusion for Lab Day 1:

As expected, our hands 

had less bacteria

on them after we washed them.

The results from our environmental sample where inconclusive. Our quest to find bacteria on a clean plate didn't work out so well. Surprisingly, the clean plates in the cafeteria were actually clean. 

Experiment 1: Environmental Sample take 2

Materials

• sterile swab
• clean agar plate
• environmental sample

1. We put our brains together and decided to take a sample from a place that would actually have bacteria(the fountain outside of Coda).

 2. Next we inoculated the agar plate with the sterile swab and placed in the incubator. 

Stay tuned for tomorrow's lab.

Why: The purpose of this experiment was to become aware of all of the bacteria that is in the 

environment.

Experiment 2: Preparing Stock Cultures 

Materials: 

• 2 slant tubes- nutrient agar
• Nutrient broth culture
• inoculating loop 
• Bunsen burner

1. Using Aseptic technique, we inoculated a working and reserve stock with our sample on slant agar test tubes. We then put them in the incubator.

2.  We also inoculated the nutrient broth with our sample using the stab technique and put it in the incubator.

Why: Depending on the growth requirements of the bacteria and the information that the scientist hopes to gain, various types of growth media are available for culturing bacteria. The two main types of bacterial growth media used are liquid nutrient broth and solid agar. Each has their specific advantages and disadvantages for growing bacteria.

Experiment 3: Streak Plate Culture  

Materials:

• agar plate
• inoculating loop
• 4 quadrants labeled on a piece of paper

1. We set the agar plate up correctly with the different quadrants labeled.

2. Next, we inoculated each quadrant with a thin sample and put it in the incubator.

Why: In order to help us to determine the pathogen and find a pure bacterial colony, we needed to use the streak plate to isolate the bacteria. This technique is used to separate the individual cells so that, when they multiply, each will form a discrete colony. This is to ensure that only one type of organism will be present. 

Experiment 3: Pour plate culture 

 Materials:

• edvotek
• beaker
• 70% ethanol
• L shaped tool
• Bunsen burner 
• broth culture 

1. Using the edvotek, we took 200 micro liters of the broth culture 

 2. Then, Hannah gracefully dispensed the sample into the pour plate.

4. We then immersed our L shaped tool into the ethanol and held it over the flame to ensure that our plate remained sterile.

 5. Next, I used the sterile tool to spread the sample on the pour plate. I then set the petri dish in the incubator. 

Why: To find a pure bacterial colony, the same reason we used the streak plate. 

Experiment 4: Gram Stain

Materials:

• blank slide
• crystal violet stain
• slide holder
• Bunsen burner 
• bibulous paper 
• microscope

1. First we added a drop of water to the slide and put a small amount of the culture on it

2. Next, we smeared the culture and water in a circular motion, with the red marker as our boundary.

3. We then briefly put the slide over the Bunsen burner to aid in drying the slide. 

4. Then, we added the crystal violet stain over the fixed culture and let it stand for 60 seconds. 

5. Once the 60 seconds was up, we rinsed off the crystal violet with water and applied Grams Iodine to the slide and let it stand for 60 seconds. 

6. We then rinsed the slide with water and blotted it with bibulous paper to remove the excess water. 

7. Finally, we examined our finished slide under the microscope. Our bacteria was Gram positive. 

Why: The gram stain is the most important staining procedure in microbiology. It is used to differentiate between gram positive and gram negative organisms. Gram positive and Gram negative cell walls are distinguished from each other by the differences in the cell walls. Our bacteria was Gram positive. 

Lab Day 5

Lab Day 5

Results and Conclusions for Lab Day 4:
Selective and/or differentiation experiment:
Blood Agar: Negative (no hemolysis), therefore our unknown bacteria is Gamma-hemolytic

EMB plate: Negative,  therefore our unknown bacteria is gram-pos.

Mannitol Salt: Negative, therefore our unknown bacteria cannot tolerate 7.5%NaCl and is Non-pathoogenic.

MacConkey plate: Negative, therefore our unknown bacteria is gram-pos. and non-lactose fermenting.

PEA agar plate: Positive, therefore our unknown bacteria is gram-pos.

Antibiotics experiment:
Red
1. Neomycin-12mm, therefore it is resistant
2. Norobicin-14mm, therefore it is resistant
3. Penicillin-31mm, therefore it is sensitive
4. Streptomycin-16mm, therefore it is sensitive
Blue
1. Erythromycin-20mm, therefore it is intermediate
2. Tetracycline-30mm, therefore it is sensitive
3. Amoxocillin-25mm, therefore it is sensitive
4. Oregano-10mm, therefore it is resistant

Experiment: AIDS Testing