Microbiology midterm

Which of the following are components of the anaerobic jar? (select all that apply).

Gas Pack Indicator strip Palladium Pellets

Briefly explain how different temperatures produce different growth rates. (Hint: how do enzymes play a role in this process?)

Higher temperature will produce higher growth rates. An increase in temperature will increase enzyme activity and increase the growth rate. Likewise a decrease in tempeture will decrease enzyme activty and decrease growth.

Animal cells

Huge assortment of media and they can often contain both complex and defined ingredients depending on what type of cell you are working on. (Fetal calf serum, antibiotics, antifungal and specialized proteins) since the cells lack and immune system Unlike bacterial culture much more care needs to be taken care of to make sure everything is sterile and the growth environment usually needs to be carefully controlled using things like specialized co2 incubators.

Select the answer option that best describes the effect of UV exposure on microbial growth.

UV radiation affects thymine in the DNA, creating abnormal bonds between pairs of thymine called thymine dimers that lead to mutations in cellular function and ultimately death of the organism.

Plant and animal cell culture and why it's different:

Unlike bacteria where a single cell is the whole organism, animals and plants are multicellular. This means individual cells can't do all the things that the full organism can do. Extra care needs to be taken to keep these cells alive outside their host organism.

List the minimal nutritional components to grow E.coli cells in the laboratory.

energy source carbon source water vitamins minerals nitrogen

You are trying to determine the oxygen requirements of 3 organisms. You instructor has inoculated the organisms in different oxygen environments and asks you to observe the results of the organisms grown in the anaerobic jar. Based on the image above, select the organism that requires oxygen in order to survive.

farcalis

Broth:

good for growing lots of organisms but gives very little control over the individual organisms

Lb:

isn't the only complex media and often isn't the best choice Example: If you are growing fungi you'll want something like potato dextrose broth or sawdust or brown rice depending of the species Complex media just means it comes from natural source and you aren't 100% sure whats in it. This can mean dirt, extract, fruit, blood serum or almost anything else

(2-9 results) Absorbance @ 600nm Organism: 10°C 25°C 35°C 45°C 60°C Orphan 1: 0.394 0.699 0.768 0.650 0.050 (Incubated at 20°C) (Incubated at 37°C) The temperature classification of Orphan 1 is:

mesophile

(2-10 results) The pH classification of Orphan 1 is a(n):

neutrophil

Why is the mouth of a culture tube passed through the Bunsen burner flame or bacteriological incinerator after removing and before replacing the cap?

prevent the entry air which might introduce contaminants.

Pick the location on the plate where is the highest probability of picking an isolated colony?

quadrant 1

When you are about to use new media, such a TSA plate or a phenol red broth tube, after the preparation process. Which of the following would be a valid reason for NOT using the media. (Pick all that applies)

the broth tube is cloudy the perti dish lid was left off before I used it the media is at room temperature there are colonies that appear of the solid plate

Which of the following best describes a bacterial colony?

visible mass of daughter cells on a piece of solid media all originating from a single parental cell

How does the antibiotic get from the disk to the agar?

The antibiotic diffuses from the disk onto the agar plate

Does the agar have an antibiotic beyond the zone of inhibition? How does your answer tie with (MIC) minimum inhibitor concentration?

The antibiotic is diffused on the plate but there's a MIC where only some growth is stopped

What would have been the result of keeping the Petri dish lids on the plate during UV light exposure?

The lid would have filtered out some of the UV light, decreasing the organism's UV exposure.

Complex/Basal Media: (Culture media):

The most common version of this is called LB or Lysogenic broth sometimes also called Lenox broth. -made by taking cells and breaking or licen them open releasing their contents -Manufactures usually use yeast for this as it is plentiful and easy to grow in large quantities and they contain almost all of the nutrients that's many microorganisms need to survive. (Also, why it is called complex since the exact concentration will vary from batch to batch and contain sorts of metabolites and molecules that you may not know are present.)

Germicide is most effective when:

There is no growth

Disinfecting your bench top with EtOH or bleach is common BEFORE AND AFTER each laboratory session. Why is this a standard practice in a microbiology laboratory?

To ensures any leftover microbes do not contaminate your project and to do not contaminate the researcher.

Which of the following statements correctly identifies the goal of a quadrant streak?

To isolate bacterial colonies on the surface of agar plate.

Applying betadine on a patient's skin before surgery.

Antiseptic

Rate the amount of growth using a scale of 0-3 (0 indicates no growth, 3 indicates a lawn of growth).

(2-13 lab results)

Select all that apply regarding the relationship between absorbance (as measured by a spectrophotometer) and growth.

- The higher the reading on the spectrophotometer, the more microbial growth has occurred. - The more cloudy (turbid) the sample, the more microbial growth has occurred. - There is a direct relationship between turbidity and growth.

Select all that apply regarding pH and microbial growth:

- Though the organism Vibrio cholerae is classified as an alkaliphile, it is still able to withstand the environment of the human stomach and produce disease when ingested in large quantities -The pH scale tells us how acidic or basic a substance is -Organisms of the Lactobacillus genus are examples of acidophilic organisms, and create their own lactic acid -pH is a measure of hydrogen ion concentration

Which of the following would violate the guidelines of aseptic technique? (Pick all that apply)

- waving the loop in the air after it was placed in a bacteriological incinerator to ensure that it is cool enough to use -allowing the sterile loop to cool on the bench top -touching the agar surface with you gloved finger -Placing a closed water bottle on the lab bench -bring your cell phone in the lab -touching the loop after it was placed in a bacteriological incineratorto ensure that it is cool enough to use -touching the loop after it was placed in a bacteriological incineratorto ensure that it is cool enough to use

Select all that apply regarding the effects of osmotic pressure on microbial growth:

-In an isotonic environment, there is equal movement of solute and water in and out of the cell. -Halophiles are examples of organisms that thrive in high salt environments -In a hypertonic environment, water moves out of the cell resulting in cell shrinking (plasmolysis) and death. -In a hypotonic environment, water moves into the cell resulting in cell swelling

Select all that apply about the organism represented by curve C. (mesophile)

-The organism is a Mesophile -The organism's optimum growth occurs at approximately 37 degrees Celsius -Outside of 15 to 45 degrees Celsius the enzymes of this organism misfold, leading to microbial death -This organism is able to reproduce in the human body

What is lab safety and why is it important when working in a microbiology laboratory?

-When working with microorganisms and possibly some chemicals can be harmful, therefore to prevent infection or accidents its important to practice lab safety and be aware of hazards.

For plate "D" shown in the results section is a swab from a water fountain. Based on colony morphology, estimate the number of different microbes that could be present on that media.

0

(2-11 results) Organism: Orphan 1 NaCl 0% : 0.893 NaCl 5%: 0.675 NaCl 10%: 0.463 NaCl 15%: 0.100 NaCl 20%: 0.067 NaCl 25%: 0.009 The optimum growth of Orphan 1 occurs at:

0 %

For plate "C" shown in the results section is a swab from a keyboard. Based on colony morphology, estimate the number of different microbes that could be present on that media.

1

Using the terms in Exercise 2-2 describe the representative organisms in the results section.

1. Micrococcus luteus: yellow, smooth and convex with an entire margrin 2. Klebsiella pneumonia: Mucoid, raised and shinny. 3. Bacillus subtilis: large, grey, granular and irregular colony 4. Pseudomonas aeruginosa: green, mucoid 5. E.coli: round, convex, off white and shinny, and raised 6. Orphan 1: circular entire and pulvinate

For plate "A" shown in the results section is a swab from a persons hand. Based on colony morphology, estimate the number of different microbes that could be present on that media.

10

(2-9 results) Use the table feature to create a table like the one below to record the growth characteristics of the Serratia marcescens results. (Hint: your table should be 2x3)

20 C: growth is reaching optimal however a higher temperature will produce optimal growth 37 C: Growth is produced at optimal

Determine the original cell concentration of based on the data provided in module 6-1. (Do not forget your units!)

30 CFU/1mL x 1/10-8 = 3.0 x 109 CFU/mL

What is the accept range of plaques to count in this assay?

30-300

Growth media

5 different types of media and you choose depending on what you're trying to do

For plate "B" shown in the results section is a swab from a cell phone screen. Based on colony morphology, estimate the number of different microbes that could be present on that media.

7

Which answer option best explains the mechanism of action of the candle jar?

As the candle burns it utilizes the oxygen in the jar and produces carbon dioxide, creating an environment with reduced oxygen levels that is conducive to the growth of microaerophiles.

Which of the following best explains the definition of aseptic technique.

A technique that utilizes specific procedures performed under controlled conditions to avoid contamination and obtain a pure culture.

Prefer pH 5.5 and below

Acidophile

Prefer pH 8 and above

Alkaliphile

Plates

Allow you to isolate single cells and also do analysis, but isn't great for producing large quantities of organism

Solid:

Also, similar as broth but gelling agent extracted from seaweed is added called Agar which allows the material to be cast into petri dishes and hold its shape once it sets. Agar is used instead of gelatin because most organisms can break down gelatin but can't break down agar

Administering doxycycline to your dog to treat an infection.

Antibiotic

According to the above results, which organism survived the longest exposure to UV light? Identify the correct organism, and generate a hypothesis as to why this organism was able to survive the longest. (Hint: think about cellular structure(s) that help protect a microbe in hostile environments).

B subtilis is gram positive and survived the longest when exposed to UV light. The longer survival rate can be because B subtillis can produce endospores which are more resistant to UV light.

Liquid:

Broth because culture media is very similar to soup broth. (If you can't use lb than chicken broth will work just as well)

When Professor Daydif demonstrates how to inoculate an FTM tube, he stresses the importance of not shaking the media. Imagine you are in lab and you see your partner aggressively stirring the media at your table. How do you explain to him the reasoning behind Daydif's instructions?

By shaking the tube, your partner is disrupting the oxygen gradient present in the media and confounding the results

What is the role of carbon in the media?

Carbon is an atom found in all biological molecules

Colony Morphology provides important pieces of information for characterizing a microorganism, what other information should be considered for characterizing/ classifing a microorganism such as your orphan? (Pick all that apply)

Cell shape Type of energy source need for growth Growth Temperature The genetics of the microbe Type of carbon source need for growth Incubation time pH of the media Salt concentration of the environment

place the following procedural steps for the UV light exercise in the correct order.

Collect your materials: cotton applicator, TSA plates, the organism(s) to be utilized, a sharpie, and a shield. Label the agar plates appropriately, including: organism, time, area of exposure. Remove the lid and spread the organism over the entire agar plate. Cover half of the agar plate with a shield to protect against UV light exposure. Carefully place the plate under the UV light for the appropriate amount of time. Remove the shield and replace the lid to the agar plate. Incubate the plates overnight. Read and record your results!

Determine the original virus concentration of based on the data provided in module 6-5. (Do not forget your units!)

Correct 170 PFU/mL x 1/10-7 = 1.70 x 109 PFU/mL

Using EtOH on your MIC 206 lab bench before class.

Disinfectant

Can grow in the presence or absence of oxygen.

Facultative anaerobe

For each of the plates in that results section they were incubated at 30 degree Celsius, at pH 7, for 24 hours. If these environmental parameters were to change, what would be the change in the pattern of growth and why?

If the teperature is increased growth will increase as well. Likewise if the temperature is lowered growth will decrease because growth only occurs in specific environments that they can live and survive in. Most bacteria are in neutral pH however will increase or decrease the pH can allow some bacteria to grow.

Which of the follow BEST describes the role of the top agar overlay in this procedure.

It increases the interaction of the virus attaching to the host cell.

Imagine there was only faint growth observed for the anaerobic organism Clostridium sporogenes when incubated in an anaerobic jar.

Jar was may have been opened and to much oxygen was introduced. Clostridium sporogenes is a Gram positive anaerobic spore-forming bacterium. (requires little to no oxygen) significant cause of food spoilage highly resistant dormant state permits survival in hostile conditions (e.g. oxygen, absence of nutrients, heat treatment, desiccation, high pressure, toxic chemicals)

MIC definition

Lowest concentration of the chemical that prevents bacteria from growing

Explain the importance of fresh media (one day old vs. 3 month old) in regard to the FTM tube. Which two organisms would be negatively affected by the use of old media? (Think about the laws of chemistry and equilibrium.)

Media should be fresh so there is less time for oxygen to diffuse to the bottom, old media would negatively affect the growth of anaerobic organisms and microaerophilic because of the presence of oxygen.

growth quiz

Mesophiles: microbial growth from +15 to +45 c Strict aerobes: microbes that require oxygen for growth Facultative anaerobic: Microbes that can grow in the presence of oxygen (O2) or without O2 Acidophiles: Microbial growth in a PH of 3 Thermophiles: Microbial growth from +40 to +80 C Strict anaerobes: Microbes that cannot grow in the presence of oxygen Phychrophiles: Microbial growth from -5 to +20 C Microaerophiles: Microbes that grow in 2% to 10% in oxygen Alkaliphile: Microbial growth in pH of 10 Autotrophs: Microbes that utilize CO2 as a carbon source

Requires less than atmospheric levels of oxygen.

Microaerophile

Selective Media:

Most diverse types and varies wildly The simplest is to add an antibiotic that one kind of bacteria is resistant to. Technique for isolating genetically modified bacteria

Prefer pH 5.5-8

Neutrophile

Can only grow in the presence of oxygen.

Obligate aerobe

Oxygen is lethal to this organism. It can only grow in the absence of oxygen.

Obligate anaerobe

Transport media:

Only for a short term. Meant to keep your organism happy while its being moved between locations. This can mean adding things that allow the organism to be frozen or slow down their metabolism and make them easier to move

Briefly explain how pH affects microbial growth rates. (Hint: How do enzymes play a role in this process? This question should feel familiar!)

Optimal growth is the most favorable for the growth of an organism. If the pH is to low for the enzyme the concentration of hydrogen ions will be greater than normal, there will be a change in shape of active site and reduce the rate of reaction. Maximum growth is at pH of 8 or greater, Minimum growth is at a pH of 5.5 of less and optimum growth is between pH of 5.5-8.

What are the correct units to use when titering a virus sample?

PFU/ml

Minimal media:

People figured out the exact minimum of every nutrient that a growing organism will need. Individual chemicals and the carbon source of your choice, weight them out, combine them and add water (this way you can change the concentration of any individual component and see how it effects the growing organism) Use this idea to allow the growth of one sort of organism over another

Examine the quadrant streak results labeled A-D in the in the results for module 1-4. For each of the results, write a brief critique addressing the following: Was isolation produced? What could be done to improve results?

Plate A: No isolation was produced to improve the results loop must be heated over flame between plate B: Isolation was produced Plate C: No isolation produced to improve these results avoid taking off the lid completely to avoid contamination from environment Plate D: No isolation produced to improve these results use proper sterilization and avoid contamination

Order the following steps for an inoculating a test tube from starter culture. (Think about the steps of aseptic technique)

Put on all required PPE Wipe the bench down with EtoH Label all new media to be used. Hold the inoculating loop in the bacteriological incinerator until it glows red After exposure to the bacteriological incinerator allow the loop to cool Remove the test tube cap from the starter culture and place the mouth of the tube in front of the bacteriological incinerator for at least 3 seconds. Stick the sterile loop into the starter culture broth Remove the loop from the starter culture Place the mouth of the starter culture in front of the bacteriological incinerator for at least 3 seconds and replace the cap Place the mouth of the new media front of the bacteriological incinerator for at least 3 seconds and replace the cap Stick the loop into the new media broth Remove the test tube cap from the new media and place the mouth of the tube in front of the bacteriological incinerator for at least 3 seconds.

Cleaning a public bathroom with toilet bleach to lower the amount of bacteria present.

Sanitizer

Plasmid:

Small circle of DNA. There is usually a gene for antibiotic resistance (usually ampicillin)

Plant cells:

Use of special media like Murashige and skood media which contain all the nutrients and growth hormones that a plant needs.

Differential Media:

Used to learn what the organism is doing and figure out what organism it is. Example: Mannitol plates contain mannitol's, their carbon source and allow you to test if the organism can break it down. Excal plates allow you to check if the bacteria have the lac operon in their DNA. Many contain dyes to known which are doing what.

Imagine you are in charge of disinfecting a room in a hospital that is known to be contaminated with your orphan. Based on the above results, describe a method to remove the bacteria. Include which disinfectant(s) you would use and how to appropriately clean different aspects of the room, such as: table tops, bed sheets, floors, and medical instruments (stethoscopes, blood pressure cuffs, etc). (Hint: if a certain chemical was not very effective, what actions would increase effectiveness?)

When disinfectin a room in a hosiptal I would use bleach to kill any bacteria on the table tops and medical equipment. Lysol will also be effective to disinfectant to use in the hospital to eliminate the growth of the orphan.

M9 is

a minimal media

MSA is

both selective and differential media

Mac conkey agar is

both selective and differential media

blood agar is

differential media, but it is also an enriched media because it helps in the growth of organisms requiring lots of nutrients which are present in blood

TSA is a

complex media because any inclusion of proteins like BSA or TS does not give us an exact percent of concentration of nutrients in the media and hence it is complex

Enriched media

contain the nutrients required to support the growth of a wide variety of organisms, including some fastidious ones. They are commonly used to grow as many different types of microbes as are present in the specimen.

m-Endo agar is

differential because it helps in the growth of coliforms and Salmonella to produce pink color

phenol red is

differential media because it differentiates between gram negative bacteria from coliforms due to the production of gas in the Durham's tube'