Micro Lab Final - Short Answer

What is a halotolerant microbe? How do halotolerant microbes differ from halophiles?

A halotolerant microbe is a microbe that is able to survive at high salt concentrations but do not require high salt conditions for growth. A halophile differs from a halotolerant microbe because halophiles do require a high salt concentration for growth and would require high salt conditions for growth.

What is a colony forming unit and how is it used to infer microbial numbers in a sample?

A colony forming unit is a measure that is used in microbiology to determine the number of bacteria present in a sample that can multiply under controlled growth conditions. Plates that are viable are can be used to accurately estimate the the total number of microogranisms on a plate (microbial numbers).

What is a colony and how does a colony relate to a bacterial cell? Why are colonies important in the study of microbiology?

A colony is a mass of microorganisms that originate from a single cell. A colony relates to a bacterial cell because the cells that makeup the colony are clones of the original cell. Meaning they are all genetically the same. Colonies are important in the study of microbiology because they allow scientists to isolate a single bacterium to study that bacteria. Studying colonies can also help identify bacteria

Are bacteria easier to observe on a plate or in a broth? How do the bacteria look on the plate as compared to the broth?

Bacteria are easier to observe on a plate than on a broth. Observing bacteria in a clear container makes it easier to observe and allows us to see the morphology of the bacterium (size, shape, elevation and margin), whereas when observing the bacteria in broth you can only identify if the bacteria is turbid or if there is flocculent microbial growth in the bottom of the nutrient broth tube.

Based on your pH testing results, can you categorize either of the microbes as acidophiles, neutrophiles, or alkaliphiles? Explain your answer in relation to Data Table 2.

Based on my pH testing results I can categorize both microbes as neutrophiles. Since both the S. epidermis and S. cerevisiae in my experiment had growth in all of the pH environments of the experiment environments they have a tolerance for alkaline and acidic environments.

How do the experimental results relate to the metabolic pathways used by E. coli and S. epidermidis?

Based on the experimental results, both S. epidermis and E. coli tested positive for the methyl red test and only S. epidermis tested positive for the Vogues-Proskauer test it can be concluded that the metabolism of S. epidermis is more efficient than the metabolism of E. coli. They both undergo mixed-acid fermentation but only S. epidermis undergoes Butanediol fermentation (fermenting glucose).

Why are biochemical tests used to identify microbes?

Biochemical tests are designed to identify various metabolic properties of different bacteria species and can lead to unambiguous identification of an organism. Biochemical tests can shorten the time required to identify microbes, reduce costs and can more accurately identify microbes.

Based on the results from your experiment, what can you conclude about the metabolism of E. coli and S. epidermidis?

Both E. Coli and S. Epidermis were positive for catalase showing that they are aerobic microbes. Microbes with aerobic respiration result in metabolites that include oxygen ions and peroxides. They are chemically reactive and can cause damage to cell structures.

What is catalase? What types of microbes are more likely to produce catalase?

Catalase is an enzyme that neutralizes the harmful effects of hydrogen peroxide. Catalase mediates the breakdown of hydrogen peroxide into oxygen and water. Aerobic microbes are more likely to produce catalase, observed by the presence of rapidly forming oxygen bubbles when hydrogen peroxide is introduced. While anaerobic microbes generally lack the catalase enzyme.

Is Crystal Violet a basic substance or an acidic substance? Is Congo red a basic or an acidic substance? Describe how you were able to arrive at this conclusion and relate your findings to your experimental observations.

Crystal violet is a basic stain. Congo red is an acidic substance. I was able to come to this conclusion because crystal violet was used in the direct stain. Only basic dyes are used in direct stains, which only color the cell's cytoplasm and leave the background colorless. Congo red was used in the negative stain. Acidic dyes are only used in negative stains.

A microbiology student Gram-stained the yeast, S. cerevisiae, producing a sample with cells colored purple, pink, brown, and green. Explain why this organism produced results that deviate from typical purple or pink Gram-stained cells.

Gram staining spots the differences in peptidoglycan in the cells. Due to a variation in the cell wall of S. cerevisiae it does not contain a lipid layer and it is both gram-positive and gram-negative, resulting in the sample with cells colored purple, pink, brown and green.

Describe the difference between Gram-negative and Gram-positive bacteria. Which type of bacteria are most often treatable with antibiotics and why?

Gram-positive bacteria cells have a thick outer cell wall that is composed of peptidoglycan. Teichoic acids can also only be found in gram-positive bacteria. The size of the gram-positive cell wall is 20-80nm in thickness. Gram-negative bacteria cells have a thin cell wall and only a single layer of peptidoglycan between the inner and outer cell membrane. The outer cell membrane of gram-negative cells contains lipopolysaccharides which are not found in gram-positive cells. The gram-negative cell wall is 2-8nm in thickness. Gram-positive bacteria is often more treatable with antibiotics because gram-positive bacteria has a more superficial cell wall composed of peptidoglycan. It is easier for antibiotics, like penicillin to reach the cell wall of gram-positive bacteria than the gram-negative bacteria where the cell wall is under a strong layer of the plasma membrane.

How do you think the experimental results would have differed if you performed this experiment with your foot and toes instead of your hand and finders? Explain your answer.

If I had performed this experiment with my foot and toes instead of my hand and fingers I think there would have been less transient flora because I generally wear closed toed shoes with socks so I would assume my foot and toes touch less surfaces resulting in acquiring less bacteria than my hands and fingers which are exposed to many surfaces and bacteria through out the day.

Why is it important to calculate the diameter of the field when first using the microscope?

It is important to calculate the diameter of the field when first using the microscope because you can use it to determine the the approximate size of an object you are examining at the given magnification.

Identify three environmental influences on microbial growth. How does each affect microbial distribution?

One environmental influence on microbial growth is pH, which is the measurement of the relative acidity of a solution. Microbes grow in very specific environments. Some microbes can grow in a very acidic or alkaline solution, some microbes can grow in a neutral solution while some microbes have adapted to grow in pH extremes. Another environmental influence on microbial growth is oxygen. Oxygen availability can affect microbial distribution. Oxygen is required in order for metabolism for some microbes but can be toxic for others. Another environmental influence on microbial growth is temperature. Temperature can limit the distribution of many organisms through enzymes. Enzymes serve as catalysts for metabolic reactions and typically function in a narrow temperature range.

Based on the nature of the two agents tested, which one should be the most effectiveagainst microbial growth? Research each agent (bleach and mouthwash) as necessary to support your answer.

Out of the agents that were tested bleach should be the most effective against microbial growth because it contains chlorine. Chlorine is one of the main ingredients of bleach and kills bacteria and viruses by breaking down chemical bonds in their molecules. Chlorine causes enzymes to not function properly, when the enzymes cannot function properly the bacteria will die. Mouthwash is least effective of the two agents because its main ingredients are phenol and alcohol. The concentrations of alcohol and phenol in mouthwash is too low to kill microorganisms.

Research and compare Staphylococcus and Streptococcus. A. Describe the morphology of each bacerial type. Indicate a common disease caused by each. B. What type of plate would you use to culture both Staphylococcus and Strptococcus? Why would this plate be useful? C. What type of plate would you use to select for Staphylococcus? Why would this plate be useful?

Staphylococcus is a spherical cocci, which forms regular grape-like clusters. They can occur singly in pairs, tetrads or in pairs and is gran-positive. To culture Staphylococcus you would plate it on Mannitol Salt Agar (MSA) which has a high concentration of salt allowing Staphylococcus to grow but inhibits growth of other organisms. Streptococcus is a spherical or cocci shaped bacteria that is often grown in pairs or chains when grown in liquid media and is gram positive. For both Streptococcus and Staphylococcus you would use a blood agar plate. This plate is useful because it allows both Staphylococcus and Streptococcus to grow and blood agar plates can be used to differentiate organisms.

Direct counts of cells in liquid samples can be performed using a Petroff-Hausser counting chamber. Research this method and describe how it compares to the viable plate count method of determining the number of CFU in a sample.

The Petroff-Hausser counting chamber is used as a direct method to determine the number of bacterial cells in a culture or liquid medium. The Petroff-Hausser counting chamber is also known as a hemocytometer, a special microscopic slide. Once the slide is in place, bacterial cells in each square can be counted under a microscope. The viable plate count is an indirect method that is conducted once dilutions of a liquid culture is placed onto an agar plate. The CFUs are counted without a microscope and then the original cell number is calculated.

. What is the major advantage of a differential stain, such as the Gram stain, over a simple stain? Was this observed in your experiment?

The advantage of a differential stain over a simple stain is the differential stain allows you to observe cell morphology and structural components of the cell, while the simple stain only allows you to observe cell shape, size and arrangement. Yes this was observed in my experiment. In experiment one just one stain was used (simple stain, just crystal violet). In experiment two I worked with 4 stains to create a differential stain which allowed me to determine if a cell was gram-negative or gram-positive.

What is the purpose of a broth in a microbiology laboratory?

The purpose of a broth in microbiology is to provide a growth medium for bacteria by giving it a constant and steady amount of nutrients that allows the bacteria to reproduce quickly.

Define the three major types of bacterial shapes. Which of these did you observe in the exercise?

The three major bacterial shapes include coccus, bacillus and spirilla. Coccus are spherical or round-shaped bacterium with a diameter of approximately 0.5um. Bacillus are cylindrical or rod shaped bacterium 0.5um to 20um in length. Spirilla are helical or spiral-shaped bacterium approximately 15um in length.

Compare the colonies that developed from the skin and urine samples to the E. coli and S. epidermidis colonies that developed on the same media. Do either of your samples appear to be E. coli or S. epidermidis. Explain your answer.

The urine sample did not grow on any of the media. The skin samples were much smaller and had very little growth compared to the E. coli and S. epidermis colonies that grew very quickly and seemingly exponentially on the same plate. The same observation was made for the skin colony that grew on the MAC plate. None of my skin or urine samples appear to be E. coli or S. epidermis because there are observable differences in the cell morphology of the samples.

A new microbiology student failed to properly sterilize the pipet between creating E. coli and S. epidermidis cultures from the tablets in the HOL kit. How would this mistake be recognized when the mixed culture is streak plated? How could the student create a pure culture from the mixed culture streak plate?

This mistake would be recognized when the bacterial colony forms. If the culture was a pure culture (just the E. coli or the S. Epidermis) the bacterial colony morphology would all be the same. In this instance, since a contaminant was was introduced to the plate it would be recognized when the bacterial colony forms because the morphology of the bacteria will be different for the S. epidermis and the E. Coli. Each of the microbes shape, color, pattern and elevation will be distinguishable. The student could create a pure culture from the mixed culture streak plate by separating the mixed cultures into individual colonies of only one microbe in order to be successfully organized.

The optical microscope is regularly used to identify pathogenic microbes. In 1918 the Spanish Flu infected almost one third of the world and was thought to be caused by bacteria. However, a virulent virus was the cause of the Spanish Flu.From what we have learned throughout this lab on microscopy, why weren't scientists able to identify the Spanish Flu with an optical microscope?

Unlike many bacteria, the virulent virus that was the cause of the Spanish Flu was too small to be seen by the optical microscope. Many light microscopes allow us to see cells clearly but viruses are very small and therefore unable to be seen by light microscopes including the optical microscope.

How could you tell if one of the colonies on your pure plate was a contaminant from the environment and did not come from your original culture?

When examining the colonies on the pure plate the morphology should be the same. If one of the colonies on the pure plate did not come from the original culture it would be considered a contaminated culture. A contaminant in the plate will look different from the original culture in the plate. You would be able to observe two or more different color, texture or growth patterns.

If you allowed your dilution tubes to incubate for 24 hours before plating them, do you think the results of the experiment would be impacted? Assume that unlimited resources are present in the tubes. Explain your answer.

Yes if I allowed my dilution tubes to incubate for 24 hours before plating them I think the results of the experiment would be skewed. Assuming unlimited resources are present in the tubes, the yeast would multiply and when plated would result in an increase in CFUs in each plate.

Was the streak plate method effective at diluting the population size in each of the plates? How could your methods be improved in the future?

Yes the streak plate method was effective at diluting the population size in each of the plates because each of my plates formed isolated colonies. This was my first time using a streak plate method and one of the ways my methods can be improved in the future is by working more from the middle of the plate to maximize the amount of surface area used.

Did you note any color changes on the MAC plate? What can be concluded from this observation?

Yes, the E. coli colonies on the MAC plate changed to appear pink/red. I can conclude from this observation that E. coli is a gram-negative bacteria, as MAC is a selective medium that inhibits growth of gram-positive bacteria and selects for gram-negative bacteria. I can also conclude that E. coli is lactose positive due to the color change to red/pink.

Were there any samples that showed no growth? If so, list the samples and media. Given that microorganisms are everywhere, would you expect to see growth on every plate? Why or why not?

Yes, the samples that showed no growth were skin on the EMB plate, urine on all plates and S. epidermis on the MAC and EMB plates. I would not expect to see growth on every plate because because the selective media's (EMB and MAC) select for gram-negative bacteria and inhibit growth of gram-positive bacteria.

Describe the similarities and differences between the cheek cell wet mount and dental plaque wet mount

Both the cheek cell wet mount and the dental plaque wet mount are best viewed at the 600X magnification. The cheek cells are eukaryotic cells since they are easily shed it's easy to obtain them for observation under the microscope. In the cheek slide smear you can clearly see the nucleus through the microscope. The dental plaque cells are biofilm and are composed of mostly bacteria. Since dental plaque is composed of bacteria it is a prokaryotic cell, meaning unlike the cheek cell the dental plaque is lacking a nucleus and other membrane bound organelles.

List five safety precautions you used to protect yourself while culturing microbes.

Five safety precautions I used to protect myself while culturing microbes is wearing PPE (gloves, mask, eye goggles and apron), clearing the workspace of unnecessary items, cleaning the work area with bleach before and after the experiment, properly washing my hands before and after an experiment and placing all used items in bleach or a 10% bleach solution and disposing of items properly.

S. cerevisiae occurs in nature on ripe fruit. S. epidermidis naturally occurs on human skin. How do your experimental results from Exercises 1 and 2 relate to the natural environment of each microbe?

In my experimental results from exercise 1 I found that S. cerevisiae prefers an isotonic (equal) environment and is not a halophile as it does not require a high salt concentration for growth, which was concluded since growth was reported in the 1% solution. In experiment 2 I was able to conclude that S. cerevisiae is a neutrophile, meaning it grows best at a pH level of 7. S. cerevisiae would occur in nature on ripe fruit because that specific environment is suitable for them, providing food in the form of sugar. In my experimental results from exercise 1 I found that S. epidermis is halotolerant, which means it is able to survive at high salt concentrations. S. epidermis is also not halophilic. In exercise 2 I found that S. epidermis is also a neutrophile. S. epidermis would naturally occur on the human skin because it needs an environment that is wet and humid, which is available on places of the human bdy, like the armpits.

Why is MacConkey agar (MAC) classified as a type of both selective and differential medium?

MacConkey agar (MAC) is classified as both a selective and differential media because it is designed to differentiate enterics based on their ability to ferment lactose. As a selective medium MAC contains bile salts and crystal violet dye which inhibit the majority of Gram-positive bacteria, selecting for Gram-negative bacteria. As a differential media MAC contains lactose which E. coli and Enterobacter can metabolize which produces a pH change.

Why are the methyl red and Voges-Proskauer tests often performed together?

Methyl red and Voges-Proskauer tests are often performed together to differentiate bacteria within the family Enterobacteriaceae. Both tests use the same broth which contains glucose, peptones and buffers to culture microbes.Comparing the results of these two tests can provide identification of bacteria within the Enterobacteriaceae family.

What is mixed-acid fermentation? How is methyl red used to determine if a microbe has undergone mixed-acid fermentation?

Mixed-acid fermentation is an anaerobic metabolic process that produces lactic acid, acetic acid, ethanol, carbon dioxide and hydrogen gas. Methyl red is used to determine if a microbe has undergone mixed-acid fermentation by turning red at acidic pHs between 4.4 and 6.0. When methyl red is added to the broth, cultures demonstrating mixed-acid fermentation will turn red because the mixed-acid fermentation creates acidic bi-products that do not neutralize in the buffered solution.

Pickling has been used to preserve foods for over 4000 years. Research how foods are pickled and relate your experimental results to this preservation technique.

Pickling is a the process of preserving foods by either anaerobic fermentation in brine or immersion in vinegar. The acetic acid contained in the vinegar increases the acidity of the food being pickle, killing microorganisms and effectively preserving the food by preventing spoilage. In the experiment both the S. epidermis and S. ceresiviae were found to be neutrophiles, although they could potentially live in an acidic environment they would not prefer it and would likely not grow/survive in the acidic environment of pickled foods.

Why are plate counts of 30-300 considered viable?

Plates with counts of 30-300 are considered viable because plates with less than 30 colony forming units reduce the statistical accuracy of the sample while plates with more than 300 colony forming units , the cells begin to compete with each other for nutrients and colonies may merge.

Compare the growth of E. coli and S. epidermidis on each of the media. What can you conclude about each microbe based on these tests?

S. epidermis only grew on the TSA media and E. coli grew on all three of the medias. I can conclude that E. coli is a gram-negative bacteria and S. epidermis is a gram-positive bacteria since MAC and EMB media's both inhibit the growth of gram-positive bacteria and both select for gram-negative bacteria.

Based on your experimental results, categorize each microbe as preferring isotonic environments, being halotolerant, or as a halophile.

Since S. cerevisiae grew in the 1% solution but not the 7% or 15%. I can conclude that S. cerevisiae prefers an isotonic environment. S. epidermis grew at 1% and 7% but not at 15%. I can conclude that S. epidermis is halotolerant, meaning it will grow in a more hypertonic (salty) solution. Since both S. epidermis and S. cerevisiae grew at the 1% neither are halophilic. Meaning they are not requiring a more hypertonic solution. They can grow without the high salt environment

Was the bacterial growth on the "pre" side of the "water only" and "water + soap" plates the same or different? Create a hypothesis to explain the results.

The bacterial growth on the "pre" side of the "water only" and the "water + soap" plates was different. My hypothesis for this result is, I had recently washed my hand prior to beginning the experiment and the first plate I touched with my index finger was "water only" plate. The instructions of the experiment then explicitly stated to,"go about my day and collect transient flora on my hands and create the next sample (water + soap) before washing my hands". I was much more mindful about collecting bacteria on my hands for the water + soap pre-portion of the experiment than I was for the water only portion.

Which of the two agents were most effective at inhibiting microbial growth? Use your MIC results and observations to support your answer.

The bleach is the most effective agent at inhibiting microbial growth. None of the bleach tubes appear turbid and the MIC for bleach was test tube M4 with a dilution of 1:14,641. For mouthwash the MIC was test tube M1 with a dilution of 1:11. Also, bleach contains chlorine. Chlorine breaks organic molecules and disrupts their enzyme function permanently. Chlorine kills most bacteria, viruses and fungi in about 30 minutes.

The oxidase test is another biochemical test used to distinguish aerobic versus anaerobic metabolism in microbes. Research the oxidase test and describe how it is performed. Include examples of oxidase-positive and oxidase-negative microbes in your answer.

The oxidase test is performed often by using a reagent, tetramethyl-p-phenylene-diamine (although others can be used). When the reagent is oxidized by cytochrome c, it changes from colorless to a dark-blue or purple compound (positive-oxidation result). The test can be used to distinguish Neisseria Gonorrheae (oxidase-positive) from Staphylococcus spp. and Streptococcus spp. (oxidase-negative).

What is the purpose of heat fixing? Why did the slides need to be heat fixed during the direct stain but not during the negative stain?

The purpose of heat fixing is to ensure bacteria is kille. It is not necessary to heat fix the slides during the negative stain because the goal of the experiment is to view bacteria that has not been distorted.

What was the purpose of placing the drop of distilled water onto the slide before smearing the bacterial colony onto the slide? What do you think would have resulted if the distilled water was not used?

The purpose of placing the drop of distilled water onto the slide before smearing the bacterial colony onto the slide was to fix the bacteria to the slide so it would not be lost during the staining process. If the distilled water was not used the bacteria may not have been fixed to the slide, the stain would not have worked and the bacteria would not have been visible under the microscope.

What was the purpose of the control tube in this experiment?

The purpose of the control tube in this experiment was to serve as a comparison to the mouthwash, bleach and stock tubes to help observe and determine the MIC of the mouthwash and bleach tubes.

What is the purpose of wiping down your work area with bleach before and after an experiment?

The purpose of wiping down your work area with bleach before and after an experiment is to decontaminate the work area. Wiping down the work area before the experiment ensures you do not introduce unwanted microbes to your culture. Wiping down the work surface after your experiment ensures you disinfect any microbes you have on your workspace to prevent spreading them.