microbio quiz 8 Flashcards
What is the first step in determining the number of organisms in one liter of water from a local water source
The first step is to conduct a direct count of the number of bacteria present in a sample using the direct counting method
What is the second step in the experiments for estimating the number of organisms in the water?
The second step is to plate out an aliquot to estimate the number of viable bacteria in the sample.
What is the third experiment conducted to estimate the number of organisms in the water?
The third experiment estimates the number of potential coliform bacteria (indicators of fecal contamination) in the sample.
How are coliform bacteria defined?
Coliform bacteria are aerobic or facultatively anaerobic, gram-negative, non-spore-forming, rod-shaped bacteria that ferment lactose with acid and gas formation
What does the direct count method reveal about the bacteria in the sample?
The direct count method tells us the total number of bacteria, both dead and viable.
What should you do if too many bacteria are present in the water sample during direct counting?
If too many bacteria are present, you may need to dilute the sample
What should you do if no bacteria are visible in the sample during direct counting?
If no bacteria are visible, you will concentrate the sample by centrifugation
What happens during centrifugation in the direct counting process?
Centrifugation concentrates the bacteria into a pellet that must be resuspended before counting.
What additional sample is provided to compare with the water sample?
A broth culture is provided to compare with the water sample.
What is the first step in the direct count procedure?
The first step is to gently mix the samples to ensure that the bacteria are in suspension.
After transferring a drop of culture to the counting chamber, what should you do next?
Place the coverslip on the calibrated surface of the counting chamber and gently push down to force excess solution out of the counting area.
How do you find the large squares on the counting chamber using a microscope?
Place the chamber on the microscope stage, scan under low magnification, and locate the large squares. Then, move the slide to identify the small squares, which subdivide the large squares.
How many small squares are there per large square on the counting chamber grid?
There are 16 small squares per large square.
What magnification should you use to count the number of bacteria in the small squares?
Switch to high-dry magnification and count the number of bacteria in 20 small squares.
What is the ideal number of bacteria to see per small square during the direct count?
Ideally, there should be between 5 and 15 bacteria per small square.
How do you count bacteria that fall on the lines of the small squares?
Count the bacteria that fall on the top and left-hand lines, but not the bottom or right-hand lines.
What should you do if the number of cells in the sample is too great to count?
Dilute the sample and start the counting process again.
How do you calculate the number of bacteria in the original sample?
The conversion factor accounts for the area of the square and the volume of liquid.
What is the final step after completing the direct count procedure?
Wash the slides with 70% alcohol before returning them to the box.
What does the viable count method determine?
The viable count method provides an indication of the total number of viable bacteria present in the water sample.
How is the water sample prepared for the viable count method?
he sample is diluted to 10^-3.
How are the diluted samples plated for the viable count method?
Aliquots of the diluted sample are plated out in triplicate
What is the purpose of plating aliquots in triplicate during the viable count method?
Plating in triplicate helps to ensure accuracy and consistency in the results.
What is one use of the organisms isolated in the viable count method?
The isolated organisms are used in the characterization of gram-negative organisms.
What should be labeled on the MacConkey agar plates before beginning the viable count spread plate procedure
The plates should be labeled with the dilution factor (100, 10^-1, 10^-2, and 10^-3).
How many tubes of diluent are needed in the procedure, and what dilution factors should they have?
hree tubes are needed, labeled with the dilution factors 10^-1, 10^-2, and 10^-3.
What is the first step in creating serial ten-fold dilutions of the bacterial culture?
Mix the water sample to ensure the cells are evenly dispersed.
What should you avoid doing when transferring 0.5 ml of water into the 10^-1 dilution blank?
Do not put the pipette into the liquid, as it could introduce more bacteria from the outside of the pipette.
What is the dilution factor after transferring 0.5 ml of the sample into the dilution blank labeled 10^-1?
The dilution factor will be 10^-1, a ten-fold dilution.
What should you do after transferring the bacterial culture into each dilution blank?
Mix the diluted water thoroughly.
How should you transfer the bacterial samples onto the agar plates?
transfer 0.1 ml of the 10^-3 dilution to the surface of an agar plate and repeat this for 3 plates. Then, repeat for the 10^-2, 10^-1, and undiluted samples, using the same pipette.
How do you spread the bacterial sample evenly over the agar surface?
Use a sterilized glass spreader, dip it in alcohol, and pass it through the Bunsen flame to sterilize. Then, spread the sample over the surface by rotating the plate on a turntable.
What should you do after using the glass spreader to spread the bacteria on the agar plate?
Flame the spreader to ensure it is sterile before placing it down on the bench.
At what temperature should the agar plates be incubatee
the plates should be incubated at 37°C.
What should be done after the plates are incubated?
Count the number of colony-forming units (cfu/ml) to determine the number of bacteria in the original sample.
Why is it important to know both the type and the number of bacteria present in a sample?
It’s important to know both the type and the number of bacteria to properly assess the sample and understand the microbial population.
What are some direct methods for determining bacterial count
Direct methods include direct microscopy, counting chambers, and electronic cell counters (Coulter counter).
How does an electronic cell counter (Coulter counter) work?
It counts cells that pass through an orifice with an electric current passing through it, measuring the disruption caused by each cell.
What is the principle behind using a spectrophotometer for bacterial count?
A spectrophotometer measures the turbidity of a culture, with increases in turbidity being directly proportional to the number of cells in the culture.
What is a chemical method for estimating bacterial count?
Chemical methods measure the amount of cell components such as DNA or ATP to estimate bacterial numbers.
What does an increase in turbidity indicate in spectrophotometer analysis?
An increase in turbidity indicates a higher concentration of bacterial cells in the culture.
How is the Petroff-Hauser Chamber used to measure bacterial count?
A measured volume of suspension is dispersed over a known area of the counting chamber, and the number of bacterial cells in a defined area is counted.
What is the advantage of using the Petroff-Hauser Chamber?
It allows for a direct conversion of the number of bacteria in a defined area to the total number of microorganisms (m/o) in a sample.
What are some of the limitations of the Petroff-Hauser Chamber?
The method is prone to errors due to difficulties in getting reproducible numbers, absorption of bacterial cells to glass surfaces (like pipettes), and human errors in counting.
What is a major drawback of the Petroff-Hauser Chamber in terms of bacterial count?
The method counts all cells, including dead cells, which may not accurately represent viable bacteria in a sample.
What magnification should you use to find the large squares on the counting chamber grid?
Use low power (10x) magnification to find the large squares.
After finding the large squares under low power, what should you do next?
Scan until you can find the small gridded squares that subdivide the large squares.
What magnification should you use to count the bacteria in the small squares of the counting chamber?
Use high dry (40x) magnification to count the bacteria in 10-20 small squares.
How should you calculate the average number of bacteria when counting them in the small squares?
Take the average number of bacteria from 10-20 small squares.
What should you do if the number of bacteria in a small square is too high to count?
Dilute the sample to bring the count down to approximately 0-14 cells per square.
How do you calculate the number of bacteria per milliliter from the count?
Use the formule
What is the most common method for determining bacterial count?
The most common method is the viable count, which measures living cells
How is a bacterial suspension prepared for a viable count?
The bacterial suspension is spread across the surface of an agar plate, incubated, and then colonies are counted to determine the colony-forming units (cfu) per milliliter.
What is the ideal number of colony-forming units (cfu) to count on a plate?
The ideal range is between 30 to 300 cfu per plate.
What is a disadvantage of the viable count method?
A disadvantage is that the plates are selective and biased for particular conditions, such as the type of media, temperature, and aeration, which may affect the types of bacteria that grow.
What is the purpose of MacConkey Agar?
The purpose is to isolate and differentiate members of Enterobacteriaceae based on their ability to grow on bile salts and crystal violet, and ferment lactose.
What type of media is MacConkey Agar?
MacConkey Agar is both selective and differentia
What ingredients in MacConkey Agar make it selective for Gram-negative bacteria?
Bile salts and crystal violet inhibit the growth of Gram-positive bacteria.
What is the role of lactose in MacConkey Agar?
Lactose is a sugar/carbohydrate that allows for the differentiation of bacteria based on their ability to ferment it.
What does the neutral red pH indicator do in MacConkey Agar?
The neutral red pH indicator turns red at pH < 6.8, indicating lactose fermentation and acid production.
What do coliform bacteria indicate in water?
Coliform bacteria are indicators of fecal contamination in water.
What is a key characteristic of coliform bacteria in terms of spore formation
Coliform bacteria are non-spore forming.
What type of bacteria are coliforms (gram stain and shape)?
Coliform bacteria are Gram-negative bacilli (rod-shaped).
What type of sugar do coliform bacteria ferment?
Coliform bacteria ferment lactose.
What do coliform bacteria produce during fermentation?
Coliform bacteria produce gas during lactose fermentation.
Can you name some examples of coliform bacteria?
Examples include Enterobacter, Escherichia, Hafnia, Klebsiella, Serratia, and Yersinia.
Why is testing for coliform bacteria preferred over testing for specific organisms and pathogens in water?
Testing for coliform bacteria is faster and cheaper than testing for specific organisms and pathogens.
What are fecal coliforms?
ecal coliforms are coliform bacteria that specifically originate from the intestinal tract of warm-blooded animals, such as humans, beavers, and raccoons.
How are laws regarding acceptable levels of coliform bacteria in water determine
aws determining the “standard” for coliform bacteria in water vary from state to state and are based on the intended use of the water (e.g., drinking, recreation, irrigation).
Why is Escherichia coli (E. coli) commonly used as an indicator bacterium in water testing?
E. coli is commonly used because it is one of the easiest microorganisms to grow, has a fast reproductive rate, and is easier to detect than most other organisms.
What does the presence of E. coli in water suggest?
The presence of E. coli suggests the presence of fecal matter and the possibility of enteropathogenic organisms, but does not confirm the presence of harmful bacteria.
Does the presence of E. coli guarantee harmful bacteria in a water sample?
No, the presence of E. coli suggests the possibility of harmful bacteria but does not prove that harmful bacteria are present.
What are some of the key reasons why E. coli is used as an indicator organism?
E. coli is easy to grow, has a fast reproductive rate, is easily detectable, and provides a safety factor in assessing water quality.
What are the six well-described pathogenic strains of Escherichia coli?
The six pathogenic strains are:
Enteropathogenic E. coli (EPEC)
Enterohaemorrhagic E. coli (EHEC)
Enterotoxigenic E. coli (ETEC)
Enteroaggregative E. coli (EAEC)
Enteroinvasive E. coli (EIEC)
Diffusely adherent E. coli (DAEC)
What does the Most Probable Number (MPN) measure?
MPN measures the most probable number of coliforms, including E. coli, present in 100 mL of a water sample.
How is the MPN method used to measure coliform density?
The MPN method involves replicate dilutions of a bacterial sample in a growth medium, and the number of tubes showing growth is recorded
What is done with the number of tubes showing growth in the MPN method
The number of tubes showing growth is used with statistical probability tables to estimate the coliform density in the sample.
What types of statistical probability tables are available for MPN?
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A: Tables are available for a replica of 3, 5, and 10 tubes to calculate the MPN.
What types of bacteria are measured using the MPN method?
The MPN method measures all coliforms, with a specific focus on E. coli.
What is the purpose of the Presumptive Phase in the MPN method?
The Presumptive Phase detects coliform bacteria by adding aliquots of water to fermentation medium (lauryl tryptose) with lactose and a Durham tube. Growth and gas production are considered positive results.
What does a positive result in the Presumptive Phase indicate?
A positive result indicates the presence of growth and gas production, but it could be from non-coliform bacteria, which is why the Confirmed Phase is necessary.
What is the purpose of the Confirmed Phase in the MPN method?
The Confirmed Phase confirms that the organisms producing gas are enteric organisms by inoculating the positive samples into a lactose medium containing bile salts (brilliant green lactose broth - BGLB).
Why is Brilliant Green Lactose Broth (BGLB) used in the Confirmed Phase?
BGLB is selective media that inhibits the growth of non-coliforms, confirming that gas-producing organisms are coliforms
What is the purpose of the Completed Phase in the MPN method
the Completed Phase definitively confirms the presence of coliform bacteria by streaking positive BGLB tubes onto Eosin Methylene Blue (EMB) agar.
What characteristic indicates coliform growth on Eosin Methylene Blue (EMB) agar?
Coliform bacteria produce green metallic colonies on EMB agar, indicating vigorous fermentation of carbohydrates, such as lactose.
What is the purpose of Eosin Methylene Blue (EMB) agar?
EMB agar is used for the isolation of fecal coliforms and differentiates coliform bacteria based on their ability to ferment lactose.
What ingredients in EMB agar contribute to its selective properties?
EMB agar contains peptone to promote microorganism growth, and the dyes eosin Y and methylene blue inhibit the growth of Gram-positive bacteria
How do the dyes in EMB agar affect bacterial growth?
The dyes (eosin Y and methylene blue) inhibit the growth of Gram-positive bacteria and create dark purple complexes that turn green in acidic conditions, indicating vigorous fermentation of carbohydrates.
What sugars are included in EMB agar to promote growth of fecal coliforms?
EMB agar contains lactose and sucrose to encourage the growth of fecal coliforms.
