MICROBIO WEEK 3 LABS

If you stopped the procedure here and analyzed your quadrant streak plate after incubation, which of the following would correctly describe the outcome?

Heavy growth visible, appears only in one quadrant, no isolated colonies present

You will be given 3 bacterial cultures in this lab. Your task is to evaluate their relative growth rates. Choose a strategy you predict will best fit this upcoming experiment.

I will frequently measure the absorbance of each bacterial culture over several hours and then plot the absorbance against time

Why is it important to first moisten the applicator swab with sterile broth media?

It aids in picking up bacteria on objects and spreading them evenly over the agar plate

What phase of bacterial growth likely describes the culture at 2 hours of growth?

Lag phase

A new co-worker in the lab is practicing their technique of quadrant streak plating. They ask you to review their work. Did their quadrant streak plate successfully isolate single bacterial colonies compared to your quadrant streak plate?

No

Where is the label placed on a petri plate?

On the bottom of the plate = Plates are incubated upside down to keep lid condensation from falling onto the agar surface. This keeps the label with the bacterial culture if the lid were to be separated from the agar plate.

How many types of bacteria appear on a successful subculture to a slant?

One

Which of the three plates seen in the image would represent a successful quadrant streak method?

Plate C

Surface colony = grows on the top of agar Subsurface colony = grows inside agar

Scanty growth = low number Heavy growth = high number

At this phase, the growth has slowed, what phase of bacterial growth likely describes the culture at 8 hours of growth?

Stationary phase

The tubes are already inoculated, and we do not need the loop for this part of the protocol. However, some in-person microbiology lab procedures may recommend keeping your Bunsen flame on and working nearby it on the benchtop as you complete this experiment. Why might this recommendation be acceptable during an isolation methods protocol? The Bunsen flame remains on to provide extra light for the scientist as plates are poured.The Bunsen flame will need to be passed over the top of the melted agar, so as to sterilize the surface of the pour plate.The Bunsen flame augments our aseptic technique, as working near the flame reduces the chances that airborne microorganisms land on the open petri dishes when pour plates are being created. The Bunsen flame will be used as the heat source for incubation. The Bunsen flame could be used to sterilize hands before creating pour plates.

The Bunsen flame augments our aseptic technique as working near the flame reduces the chances that airborne microorganisms land on the open petri dishes when pour plates are being created

Why was the spectrophotometer absorbance reset to "zero" without bacteria in the broth?

The goal of the experiment is to record absorbance by bacteria as the measurement of bacterial growth, and this sets the instrument to detect how "zero" bacteria would appear in broth

What would you expect if the outside of the can was sampled instead?

The outside of the can would be covered in microbes.

What caution(s) should be taken when pour plates are placed in the incubator?

The plates should be placed in the incubator upside-down to prevent contamination from condensation The plates should be accurately labeled, with writing on the agar side of the petri dish

It is important to wet your hands before using soap to ensure a good lather and to prevent the soap from washing off too quickly.

True

Which of the following statements is correct regarding the samples you created?

Tube I contains the most bacteria in the series The original, undiluted E. coli culture could be used in pour plating but would likely grow too many colonies to be analyzed Tube III will be expected to contain the fewest number of CFUs (colony forming units)

Why were bacteria present on the hand after washing with soap and water?

Washing with soap and water does not make the hand sterile; it merely reduces the number of bacteria present.

Did your pour plates show isolated colonies, both surface and subsurface, from E. coli sample and dilutions?

Yes

Was the use of ethanol on the lab bench useful in reducing the number of bacteria present?

Yes

If your completed quadrant streak plate showed two different and distinct colony appearances, what could you conclude?

Your plate was inoculated with a single species, but break in aseptic technique could have allowed contaminant bacteria to also grow Your initial bacterial culture inoculum contained two unique bacteria species

In your experiment, the spectrophotometer will pass light through the culture. The light will be __________ by bacterial cells.

absorbed

Specialized materials

agar in a liquid state / At ~50°C, liquid agar can be inoculated with bacterial culture without harming the microorganisms. When agar reaches room temperature, it forms into solid state + bacteria will grow where they settled in media and form visible colonies. Most bacteria grow well between 20-40°C and are commonly incubated at 37°C (human body temperature)

Isolation techniques

allow you to select out a specific bacterial organism from a source containing complex mixtures or combinations of different microorganisms for bacterial identification process and help us understand bacterial growth patterns

In your experiment, as light moves through the culture, it is detected on the opposite side of the sample. As the bacteria replicate to higher numbers, the amount of the light passing through the culture will __________ as bacterial cell numbers increase.

decrease proportionally

Quadrant streak plate

isolate one bacterial strain from a mixture of bacteria and are helpful in determining whether a bacterial culture may be pure

Light absorbance by bacterial cells

light passing through the culture is decreased proportionally by the number of cells present in the culture = increases the absorbance registered by the spectrophotometer (both living and dead cells contribute to the turbidity)

A spectrophotometer is an important microbiology instrument used to measure optical density and bacterial growth curve turbidity by

measuring light absorbance

Subculturing of a bacterial sample

method for obtaining pure cultures of bacterial species from mixed microbial populations

Pour plate

obtaining isolated colonies of bacteria, determining estimated bacterial numbers in a sample, and could be used to make further pure cultures / organisms causing disease states, determining infectious doses, or assessing purity of a sample

During the steps of the streak plate method, the bacterial culture is

only added directly to the agar plate one time

Successful streak plate

provides isolated colonies on the top of the agar by drag-diluting the colony forming units (CFUs) out across 4 sections of the plate = bacterial culture is only added to the plate one time. Each subsequent quadrant is a dilution of the initial inoculum and shows less bacterial growth than the previous quadrant area. Typically, the last dilution quadrant shows the isolated colonies.

Series of dilutions

reduce numbers of bacteria grown in/on the pour plate, allowing colonies to be easily counted. The goal is to obtain an agar plate with isolated pure colonies = transfer bacteria into a series of melted agar pours and make plates from the inoculated media.

Isolation methods

separate microorganisms on a solid agar medium, how to estimate the number of bacteria in a sample, and how to obtain pure cultures

Growth rates

some replicate within 20 minutes, other bacteria take much longer. But as bacteria grow, turbidity increases and can help determine growth rates

Bacteria live in and on our bodies, outnumbering our own human body cells 10 to 1. Microbes can exist in harsh environments:

temperatures between 20 to 40°C moist conditions rather than very dry conditions an oxygen-rich environment an environment close to neutral pH

Quantitation by optical density

useful when quantification (counting) over time is necessary. This data is commonly plotted on a graph to form a bacterial growth curve

Which of the following streak plate diagrams shows the correct pattern for a quadrant streak plate method?

Diagram C

According to this data, which organism has the fastest growth curve under the conditions of this simulation?

E. coli

What phase of bacterial growth likely describes the culture at 6 hours of growth?

Exponential phase

3 steps of subculturing technique:

1. Choose carefully when picking which colony/colonies to subculture. Pick an isolated colony (not mixed with or directly adjacent to another colony) to avoid mixing bacterial types and contaminating the pure culture. 2. Use a small amount of colony material, rather than a large glob of bacteria, to reduce risk of cross contamination during sample acquisition. 3. Always use aseptic technique to transfer bacteria between growth media.

4 steps of optical density:

1. Prepare spectrophotometer and set to proper wavelength 2. Calibrate the instrument by setting zero as the absorbance of uninoculated sterile medium 3. Insert a cuvette containing the bacterial sample of interest and read absorbance 4. Remove cuvette, pour sample into waste, and clean cuvette for next sample

Pour plating steps:

1. Transfer a bacterial culture sample into melted agar tube 2. Create a dilution series into several melted agar tubes 3. Pour inoculated, melted agar into petri dishes and allow to solidify 4. Incubate plates to grow colonies and subsurface colonies

4 steps in pour plating technique:

1. Transfer of a bacterial culture into a tube of warmed, melted nutrient agar. 2. Reduce numbers of bacteria in the culture by making serial dilutions into similar nutrient agar tubes. 3. Pour melted, inoculated nutrient agar into a petri dish, distributing evenly across the surface, and allowing media to solidify. 4. Incubate pour plate, allowing for growth of colonies.

4 steps quadrant streak plate technique:

1. Use aseptic technique to transfer a bacterial sample to an agar plate. 2. Streak the bacterial sample across one quadrant (approximately ¼ of the agar surface) in a back-and-forth motion. 3. Re-sterilize the loop and then drag the loop through the previously inoculated quadrant to pick up a small fraction of sample to the next quadrant surface. Repeat until four quadrants are completed in the same manner. 4. Incubate the agar plate for 24 hours to grow isolated bacterial colonies.

Why is 550 nm a correct wavelength setting for the spectrophotometer?

Bacterial cells absorb light well in the wavelength range of 550-600 nm, allowing for measurement of growth as more bacteria are present

Why was the liquid in the can found to be free of microbes?

Canned liquids like juice or soda are prepared to be free of microbial contamination.

If you are attempting to obtain a pure culture, what must you avoid when subculturing bacteria from a mixed plate to a sterile agar slant?

Contamination of the transfer tool or media containers