Microbiology Task 2
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Uploaded by ChefSandpiperPerson147
Rabeca Plummer
Clinical Microbiology Laboratory C453
4/18/2021
Task 2
Introduction
In this series of experiments, we attempt to categorize and identify different traits of 3 individual bacteria:
Escherichia Coli, Staphylococcus Epidermidis, and Saccharomyces Cerevisiae, using an aseptic technique. The aseptic technique is defined a “
Aseptic techniques refer to any method used to sterilize and maintain the sterility of an object or location, such as an operating theatre or laboratory”
(Greenwood,
2019). This ensures the integrity of the experiment and the results. Failure to use an aseptic technique can skew results and lead to false conclusions. Materials -Large cooking pot
-stove
-tap water
-test tube rack
-paper towels
-isopropyl alcohol
-oven mitt
-marker
-safety gloves
-coffee mug
-test tube clamp
-nutrient agar test tubes
-petri dishes
-Escherichia Coli tablet in vial
-face mask with ear loops
-inoculation loops
-nutrient agar -18ml tubes
-nutrient broth – 5 ml tubes
- safety goggles
-small, graduated pipet – 5ml
-tea candles
-thermometer
-yeast packet
-bucket
-disposable cups
-Lighter or match
-microscope slides
-Barritt’s A Reagent
-Barritt’s B Reagent
-Methyl red Reagent
-MR-VP broth -Scissors
-Fructose powder vial – 0.2g
-Glucose powder -0.2g
-Mannitol powder -0.2g
-Durham test tubes
-Phenol red broth vials – 9mL
-Motility Test agar 0.4% -8mL tubes
-paperclips
Method
Method 1: Pouring Agar Plates
Step 1: Gather all the required materials
Step 2: Label the bottom of 6 petri dishes NA for nutrient agar, do not open the petri dishes
Step 3: Place the test tube rack and agar tubes in cooking pot and add water until the level is higher than the agar level. Step 4: Slightly loosen the tops of the agar tubes to allow air to release. Turn on the stove to bring water to a boil.
Step 5: Monitor agar until it is liquified. When the agar is melted, remove the tubes from the water with an oven mitt and place in a coffee mug filled with hot water.
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Step 6: Pour approximately one tablespoon of alcohol onto your work surface and use a paper towel to spread the alcohol and sterilize the area. Step 7: Open one petri dish onto your work surface, open and pour one half of a test tube of your nutrient agar into the corresponding petri dish. Repeat until all agars have been poured.
Step 8: Allow agar to cool, untouched in the petri dishes until solidified. Plates should remain inverted until inoculated. When agar is cooled and solid, it is ready to be used in an experiment. Step 9: Wash your hands thoroughly with soap and water.
Method 2: Culturing Microbes in Broth
Step 1: Gather supplies.
Step 2: Wash hands thoroughly with soap and water.
Step 3: Put on PPE (gloves, goggles, apron, and face mask)
Step 4: Prepare the work surface by wiping it down with 1:9 bleach water solution.
Step 5: Pour a bucket of bleach for item disposal.
Step 6: Fill a disposable cup with alcohol and place it on your work surface.
Step 7: Place the pipet in the alcohol and squeeze some alcohol into the stem. Leave the pipet in the cup
with the alcohol in the stem.
Step 8: Place the tea candle on the work surface and light the wick.
Step 9: Use a marker to label a nutrient broth tube Escherichia Coli and place it on the work surface.
Step 10: Expel the alcohol from the pipet and shake it dry. Keep the pipet in your hand and don’t allow it to touch anything, as it is now sterile. Step 11: Remove the top from the labelled nutrient broth and pass the rim through the flame of the candle to sterilize it.
Step 12: Place the vial upright on the work surface and repeat with the e coli culture vial.
Step 13: Pipet 0.25mL of nutrient broth into the e coli culture vial, being careful not to touch the sterilized
rim on either vial with your gloves or the pipet. Step 14: Replace the lid on the culture vial and shake until the tablet dissolves. Step 15: Pipet the dissolved tablet and nutrient broth solution into the nutrient broth vial, being careful not
to touch the sterilized rim with the pipet or your gloves.
Step 16: Hold the rim of the nutrient broth vial in the flame again to sterilize it and screw the cap back on.
Set the broth aside.
Step 17: Place the pipet in the alcohol cup, squeeze alcohol into the stem, and leave the pipet in the cup.
Step 18: Repeat steps 9-17 with the Staphylococcus Epidermidis culture vial using a new broth tube labelled Staphylococcus Epidermidis.
Step 19: Open the yeast packet and place ½ teaspoon of the powdered contents into an empty disposable cup.
Step 20: Add ¼ cup warm water to the yeast and swirl until dissolved.
Step 21: Allow the cup to sit for 10 minutes until it begins to froth.
Step 22: Label a new broth tube Saccharomyces cerevisiae. Repeat steps 10-11 with the Saccharomyces cerevisiae broth tube.
Step 23: Pipet 0.25mL of the yeast solution into the nutrient broth tube, being careful not to touch the rim of the tube with the pipet or your gloves.
Step 25: Hold the rim of the nutrient broth vial in the flame again to sterilize it and screw the cap back on.
Set the broth aside.
Step 26: Extinguish the candle.
Step 27: Sterilize the pipet in the alcohol and store with the candle, goggles, mask, and apron for the rest of the experiments.
Step 28: Pour the remaining alcohol in the sink, dispose of the cup.
Step 29: Place the two empty culture vials and the cup of yeast into the bucket of bleach. Remove the items after 3 minutes and dispose of them in the garbage. Pour bleach down the sink. Step 30: Wipe the work surface down with 1:9 bleach water solution.
Step 31: Identify a location in your home where the broth tubes can incubate, upright and untouched, for approximately 48 hours. The location should be room temperature (21°C-25°C), away from heating or air-conditioning vents, out of direct sunlight, and secure from children and pets. An empty cabinet works well. If a countertop is used, place cultures in a box, such as your empty HOL box. Use the thermometer to determine whether the location meets the requirement of 21°C-25°C, if not, identify a new location.
Step 32: Remove PPE. Store apron, mask, and goggle, discard gloves. Wash your hands thoroughly with soap and water.
Step 33: Allow the tubes to incubate for 48 hours. Check for grown by holding the tubes near a light source. (Developed cultures will be either cloudy or have flocculent growth at the bottom of the tube.)
Step 34: If the cultures show no signs of growth after 48 hours, incubate for an additional 24 hours. Method 3: Isolating Individual Colonies
Step 1: Gather the developed cultures of Escherichia Coli, saccharomyces cerevisiae, and e. epidermidis, 3 agar plates and the rest of the required materials from the above list. Step 2: Wash your hands thoroughly with soap and water.
Step 3: Put on your PPE; gloves, goggles, apron, and face mask.
Step 4: Wipe down your work surface with 1:9 bleach water solution. Pour a bucket of bleach for item disposal. Step 5: Fill a small disposable cup halfway with alcohol and place the 3 inoculation loops in the cup, loop
side down. Step 6: Label the agar side of the plate with the name of the microbe it will contain. Step 7: Light the candle and place it on the work surface.
Step 8: Place the agar plate next to the broth tube and remove the plate lid. Step 9: Remove an inoculation loop from the alcohol and shake it dry.
Step 10: While holding the inoculation loop in your hand away from touching anything, uncap the Escherichia Coli broth and pass the rim through the flame to sterilize it. Step 11: Submerge the sterile loop into the broth being careful not to touch the rim with the loop or your gloves.
Step 12: Immediately transfer the broth adhering to the loop into one quadrant of the agar plate using a zig zag motion. Replace the lid of the agar plate.
Step 13: Put the inoculation loop in the alcohol cup.
Step 14: Flame the rim of the broth and close the cap.
Step 15: Remove the inoculation loop from the alcohol and shake to dry.
Step 16: Open the lid of the agar plate and touch the loop to the edge of the inoculated quadrant to transfer the microbes to the next quadrant. Replace the lid of the agar plate.
Step 17: Place the inoculation loop in the cup of alcohol.
Step 18: After 20 seconds, remove the inoculation loop from the alcohol and shake to dry.
Step 19: Repeat steps 16 and 17 until all 4 quadrants have been streaked.
Step 20: Place the used inoculation loop in the bucket of bleach for 30 minutes and then dispose of it in the garbage. Step 21: Repeat steps 8-21 for the Saccharomyces cerevisiae and Staphylococcus Epidermidis samples.
Step 22: Extinguish the candle and store for future use.
Step 23: Pour the remaining alcohol in the sink, dispose of the cup in the garbage.
Step 24: Wipe down work surface with 1:9 bleach water solution.
Step 25:
Identify a location in your home where the inoculated agar plates can incubate agar-side up (inverted) for approximately 48 hours. The location should be room temperature (21°C-25°C), away from
heating or air-conditioning vents, out of direct sunlight, and secure from children and pets. An empty cabinet works well. If a countertop is used, place cultures in a box such, as your empty HOL box. Use the
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thermometer to determine whether the location meets the requirement of 21°C-25°C, if not, identify a new location.
Step 26: Place the broth tubes with the live cultures in them in the refrigerator for future use.
Step 27: Remove PPE. Store apron, mask, and goggle, discard gloves. Wash your hands thoroughly with soap and water.
Step 28: Incubate the plates for 48 hours. (If there is no growth in 48 hours, incubate for an additional 24 hours)
Step 29: Place the developed plates in the refrigerator to preserve the microbes for future use.
Method 4: Methyl Red Testing and Voges-Proskauer Testing Step 1: Clear a work area and gather necessary items from the above list.
Step 2: Allow tubes of MR-VP to warm to room temperature, this may take approximately 30 minutes.
Step 3: Wash your hands thoroughly with soap and water.
Step 4: Don PPE: gloves, face mask, apron, and goggles
Step 5: Prepare the work surfaces by wiping it down with 1:9 bleach water solution.
Step 6: Pour a disposable cup of alcohol and a bucket of bleach.
Step 7: Use a marker to label one MR-VP broth tube Escherichia Coli and the other Staphylococcus Epidermidis.
Step 8: Place 2 pipets in the alcohol cup, squeeze them to ensure that alcohol is in the stem, leave the pipets in the alcohol cup.
Step 9: Light the candle.
Step 10: Gather the Escherichia Coli culture vial. Remove the cap and hold the rim in the flame of the candle to sterilize it.
Step 11: Remove the lid of the labeled Escherichia Coli MR-VP broth tube and flame the rim of the tube in the candle to sterilize it.
Step 12: Remove a pipet from the alcohol, expel the alcohol, and shake to dry.
Step 13: Carefully insert the pipet into the active Escherichia Coli culture and draw up a small amount of broth.
Step 14: Pipe 4 drops of broth into the MR-VP tube.
Step 15: Place pipet into bleach for 30 minutes.
Step 16: Flame the rim of both tubes and replace caps.
Step 17: Repeat steps 10-16 for Staphylococcus Epidermidis.
Step 18: Extinguish the candle.
Step 19: Place MR-VP into your incubation are for 48 hours. Step 20: Replace cultures in the refrigerator.
Step 21: Remove the pipets from the bleach and dispose of in the garbage.
Step 22: Wipe down your area with bleach.
Step 23: Remove PPE. Store apron, mask, and goggle, discard gloves. Wash your hands thoroughly with soap and water.
Step 24: Check the MR-VP tubes after 48 hours, they should be turbid. If not, allow them to incubate for an additional 24 hours.
Step 25: Wipe down your work surface with bleach.
Step 26: Wash your hands thoroughly with soap and water.
Step 27: Don PPE: goggles, face mask, apron, and gloves.
Step 28: Gather materials and MR-VP tubes.
Step 29: Sterilize 4 test tubes with alcohol, dry thoroughly.
Step 30: Label 2 test tubes Escherichia Coli and 2 test tubes Staphylococcus Epidermidis. Place the tubes upright in the test tube holder.
Step 31: Carefully pour half of the inoculated MR-VP broth into each of the corresponding test tubes.
Step 32: Use scissors to remove the tip of the Methyl Red reagent pipet over a garbage can. Use a wet paper towel to cleanse the scissors after and throw away the paper towel.
Step 33: Pipet 7 drops of methyl red into one of the Escherichia Coli test tubes and one of the Staphylococcus Epidermidis test tubes. Step 34: Observe the effects. If the Methyl Red Test is positive, the broth will immediately turn pink or red. Step 35: Record the results. Place the test tubes and all used items in bleach for one hour. Step 36: Use the remaining 2 tubes for the Voges-Proskauer Test.
Step 37: Remove the tip of the Barritt’s Reagent A pipet with scissors. Cleanse scissors with a wet paper towel and throw away the paper towel.
Step 38: Add 12 drops of Barritt’s Reagent A into each tube, mix by swirling.
Step 39: Remove the tip of the Barritt’s Reagent B pipet with scissors. Cleanse scissors with a wet paper towel and throw away the paper towel.
Step 40: Add 4 drops of Barritt’s Reagent B into each test tube. Shake the tubes gently for 30 seconds to expose the contents to oxygen. Step 41: Place the test tubes in the test tube holder for 30 minutes.
Step 42: After 30 minutes, observe the results. A positive Voges-Proskauer Test is indicated by the formation of a red surface layer. Step 43: Record your results.
Step 44: Place test tubes in bleach for one hour.
Step 45: Remove test tubes from bleach and clean thoroughly for a future experiment.
Step 46: Throw away chemical pipets in the garbage.
Step 47: Wipe down work surface with 1:9 bleach water solution.
Step 48: Remove PPE, keep apron, goggles, and face mask for future use, discard gloves.
Step 49: Wash hands thoroughly with soap and water.
Method 5: Catalase Testing
Step 1: Gather supplies.
Step 2: Wash hands thoroughly with soap and water.
Step 3: Don PPE (gloves, mask, apron, and goggles)
Step 4: Disinfect work surface 1:9 bleach water solution.
Step 5: Pour alcohol in a disposable cup and bleach in a bucket. Step 6: Place 2 inoculation loops in alcohol cups, loop side down. Place 1 pipet in the alcohol and squeeze
to ensure that alcohol is in the stem, leave the pipet in the alcohol cup.
Step 7: Label one blank microscope slide Escherichia Coli and one blank microscope slide Staphylococcus Epidermidis with a permanent marker. Draw a dime-sized circle on the center of each slide.
Step 8: Remove one inoculation loop from the alcohol and shake it dry.
Step 9: Open the lid of the Escherichia Coli culture plate and collect several colonies with the inoculation loop. Step 10: Transfer the collected colonies to the circle on the slide labeled Escherichia Coli.
Step 11: Remove a pipet from the alcohol, expel the alcohol, and shake to dry. Pipet a single drop of hydrogen peroxide onto the sample and observe for the formation of oxygen bubbles. The absence of bubbles is a negative test result. Step 12: Repeat steps 8-11 for Staphylococcus Epidermidis.
Step 13: Soak the slides and inoculation loops in the bleach bucket for 20 minutes and then dispose of them in the garbage.
Step 14: Wipe down your work area with 1:9 bleach water solution.
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Step 15: Remove PPE. Store apron, mask, and goggle, discard gloves. Wash your hands thoroughly with soap and water.
Method 6: Carbohydrate Fermentation Testing
Step 1: Gather all your materials from the above list.
Step 2: Allow the tubes of phenol red broth to warm to room temperature for approximately 30 minutes.
Step 3: Pour alcohol in a disposable cup and bleach in a bucket.
Step 4: Wash your hands thoroughly with soap and water.
Step 5: Don PPE (mask, gloves, apron, and goggles)
Step 6: Disinfect work area with 1:9 bleach water solution. Step 7: Use a permanent marker to label 6 phenol red vials as follows: -Saccharomyces cerevisiae- Glucose
-Staphylococcus Epidermidis- Glucose
-Saccharomyces cerevisiae- Fructose
-Staphylococcus Epidermidis- Fructose
-Saccharomyces cerevisiae- Mannitol
-Staphylococcus Epidermidis- Mannitol
Step 8: Place the 6 Durham tubes and 2 dropper pipets in the cup of alcohol
Step 9: Light the tea candle
Step 10: Remove the lid from the Saccharomyces cerevisiae Glucose vial and pass the rim over the flame to sterilize.
Step 11: Transfer half the glucose powder to the vial. Step 12: Pass the rim of the vial over the flame again to sterilize and recap. Shake the vial until the powder dissolves.
Step 13: Repeat steps 10-12 with the remaining 5 vials and their correlating powders.
Step 14: Remove the Durham tubes from the alcohol and allow to dry thoroughly.
Step 15: Insert one Durham tube into each vial, opening side down.
Step 16: Remove a pipet from the alcohol, expel the alcohol, and shake to dry. Step 17: Remove the lid of the active Saccharomyces cerevisiae vial and flame the rim to sterilize.
Step 18: Pipet 4 drops of active culture into each of the phenol red tubes labeled Saccharomyces cerevisiae.
Step 19: Place the pipet in the bleach bucket for 20 minutes.
Step 20: Flame the Saccharomyces cerevisiae vial and the phenol vials’ rims and close the lids. Step 21: Repeat steps 16-20 for Staphylococcus Epidermidis. Step 22: Place the 6 inoculated phenol red broths and 2 active culture broths in your incubation location for 12 hours.
Step 23: Wipe down the work area with 1:9 bleach water solution.
Step 24: Remove the pipets from the bleach and throw away in the garbage.
Step 25: Remove PPE. Store apron, mask, and goggle, discard gloves. Wash your hands thoroughly.
Step 26: After 12 hours, observe each of the broths for color changes. If no color changes have occurred, allow for 6 more hours of incubation. Step 27: Wipe down your work area with 1:9 bleach water solution and pour a bleach bucket.
Step 28: Wash your hands thoroughly with soap and water.
Step 29: Don PPE (gloves, goggles, mask, and apron).
Step 30: Gather the broth tubes, observe for color changes, and gas bubbles.
Step 31: Record results and categorize each result as follows: fermenter with acid production only, fermenter with acid and gas production, or non-fermenter.
Step 32: Soak the phenol vials in the bleach bucket for one hour before placing them in the garbage.
Step 33: Wipe down your work area with a 1:9 bleach water solution.
Step 34: Remove PPE. Store apron, mask, and goggle, discard gloves. Wash your hands thoroughly with soap and water.
Method 7: Motility Testing
Step 1: Gather all materials needed.
Step 2: Wash hands thoroughly with soap and water.
Step 3: Don PPE (gloves, mask, apron, and goggles)
Step 4: Wipe down work area with 1:9 bleach water solution.
Step 5: Pour alcohol into a disposable cup and bleach into a bucket.
Step 6: Straighten 2 paperclips and place them into the cup of alcohol. Step 7: Use a permanent marker to label one Motility tube Escherichia Coli and the other Motility tube Staphylococcus Epidermidis.
Step 8: Light the tea candle.
Step 9: Remove the lid from the Escherichia Coli Motility media tube and run the rim of the tube in the flame to sterilize. Step 10: Remove one paperclip from the alcohol and shake to dry completely.
Step 11: Remove the lid from the Escherichia Coli culture plate and collect several colonies with the paperclip. Step 12: Stab the paperclip into the Motility agar and then carefully remove the paperclip in the same pathway. Step 13: Flame the rim of the Motility media tube to sterilize and replace the lid of the tube and the culture.
Step 14: Place the paperclip in the bucket of bleach for one hour.
Step 15: Repeat steps 9-14 for the Staphylococcus Epidermidis.
Step 16: Place the Motility tubes and cultures in your incubation location for 48 hours.
Step 17: Wipe down your work area with 1:9 bleach water solution.
Step 18: Remove paperclips from bleach after 1 hour and dispose of in the garbage.
Step 19: Remove PPE. Store apron, mask, and goggle, discard gloves. Wash hands thoroughly with soap and water.
Step 20: Observe the Motility tubes for growth, if after 48 hours, no growth has occurred, incubate for an additional 24 hours.
Step 21: Wipe down work area with 1:9 bleach water solution. Pour bleach bucket.
Step 22: Wash hands thoroughly with soap and water. Step 23: Don PPE (apron, gloves, goggles, and mask)
Step 24: Gather Motility tubes and observe for growth, turbidity, and definition of the original stab line. Clear agar and a defined slab line are a negative result.
Step 25: Soak the Motility tubes in bleach bucket for one hour and then place them in the garbage.
Step 26: Wipe down area with 1:9 bleach water solution. Step 27: Remove PPE. Store apron, mask, and goggle, discard gloves. Wash hands thoroughly with soap and water.
Results
In the Methyl Red test, my Escherichia Coli sample resulted in a pink/orange color which translated to a positive test result, my Staphylococcus Epidermidis also showed a pink/orange color and indicated a positive test result. (See image 4). In the Voges-Proskauer test my Escherichia Coli tube and my Staphylococcus Epidermidis tube both had a red line on top, this indicated a positive test result.
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In the Catalase test the Escherichia Coli sample showed no oxygen bubbles on the slide, this indicated a negative result. The Staphylococcus Epidermidis sample showed oxygen bubbles on the slide, indicating a positive result. In the Motility Test, both the Escherichia Coli and the Staphylococcus Epidermidis samples had mushy and messy agar with growth in them, this indicated a positive result.
In my Carbohydrate Test, Staphylococcus Epidermidis showed a pink tube with no bubble for glucose and mannitol, a fermenter result. Staphylococcus Epidermidis showed a yellow tube with no bubble for fructose, a fermenter result. Saccharomyces cerevisiae showed a yellow tube with no bubble for both the fructose and the glucose, a fermenter result. Saccharomyces cerevisiae showed a pink tube with a bubble for the mannitol, a fermenter with gas production result. Discussion
I believe that my Escherichia Coli culture was successful and the methyl red test and Voges-Proskauer test results were accurate. My Staphylococcus epidermidis culture may have been compromised in some way because my Methyl red test came back positive however, upon conducting some research, I concluded that was an incorrect result. (Aryal, 2019) My Escherichia Coli result for the Voges-Proskauer test was identical to my Staphylococcus Epidermidis result and this implied I had also gotten an incorrect result, as proven by the same website I had found the previous information in. My Catalase test followed suit of the previous test by producing one incorrect test result, the Escherichia
Coli test should have been positive as well as the Staphylococcus Epidermidis. (Aryal et al., 2018)
My Motility Test was compromised because the agar was missing, I used nutrient agar at my instructor’s
suggestion but then, I misunderstood the images in the instructions and poured agar plates instead of leaving it in the tube. Escherichia Coli should have tested positive for Motility but Staphylococcus Epidermidis should have tested negative.
My Carbohydrate Testing was verified by my research. I believe that my results were the correct conclusions and that my experiment was successful. (Chujo et al., 2015)
All in all, I believe it is very difficult to conduct successful experiments in a home environment without the supervision of trained staff who know what they are doing and without a sterile environment that can be clear of dogs and children. Resources
Greenwood, M. (2019, February 26). Aseptic techniques in microbiology. Retrieved April 18, 2021, from https://www.news-medical.net/life-sciences/Aseptic-Techniques-in-
Microbiology.aspx
Welcome to Microbugz - Methyl Red & Vogues-Proskauer Test. (n.d.). https://www.austincc.edu/microbugz/mrvp_test.php#:~:text=When%20methyl%20red
%20is%20added%20to%20MR%2DVP%20broth%20that,is%20a%20negative%20MR
%20result
.
Aryal, S. (2019, August 15). Biochemical Test and Identification of Staphylococcus epidermidis
. Microbiology Info.com. https://microbiologyinfo.com/biochemical-test-identification-
staphylococcus-epidermidis/
. Aryal, S., D, R., s, Getahun, N., Ogeisia, J. M., Keyu, Mcwaters, J., Annais, Touchette, B., Rahman, S. S. U., Wallace, B., & Blake. (2018, September 26). Biochemical Test and Identification of Escherichia Coli
. Microbiology Info.com. https://microbiologyinfo.com/biochemical-test-and-identification-of-e-coli/. Chujo, M., Yoshida, S., Ota, A., Murata, K., & Kawai, S. (2015, January 1). Acquisition of the Ability To Assimilate Mannitol by Saccharomyces cerevisiae through Dysfunction of the General Corepressor Tup1-Cyc8
. Applied and Environmental Microbiology. https://aem.asm.org/content/81/1/9
.
Image 1: Escherichia Coli Growth
Image 2: Saccharomyces Cerevisiae Growth
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Image 3: Staphylococcus Epidermidis Growth
Image 4: Methyl Red Test
Image 5: Voges-Proskauer Test
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Image 6: Escherichia Coli Catalase Test
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Image 7: Staphylococcus Epidermidis Catalase Test
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Image 8: Motility Test Image 1
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Image 9: Motility Test Image 2
Image 10: Carbohydrate Test Image 1
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Image 11: Carbohydrate Test Image 2
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