Lab Practical Study Questions
Which of the following are the spore-forming bodies of fungi? Choose all that apply
- Basidium - Sporangium
Which of the following are eukaryotes? Choose all that apply
- Ciliates - Chromists - Fungi - Yeasts
Which of the following is (are) required PPE for a BSL2 lab. Choose all that apply
- Long, fabric pants -Gloves -Safety glasses, when working with aerosols -Lab coat
Euglena gracilis is a member of the Domain Eukaryota and is known for its flagella. What use does the flagellum have to the organism? Choose all that apply
- Movement - Feeding
Nostoc is a ____________?
- Prokaryote - Cyanobacteria
Algae are able to reproduce by which of the following methods? Choose all that apply
-Oogamy -Fragmentation -Fusion -Meiosis -Zoospores -Binary Fission
Which of the following would you expect to be Acid-Fast negative? Choose all that apply
-Streptococcus mutans -Bacillus subtilis -Haemophilus hemolyticus -Staphylococcus aureus -Bacillus tetani
You spill a small amount (~2mL) of culture broth on the lab bench. You do not have any on you or your lab partner. Who should be informed? Choose all that apply
-The Ta -The Laboratory Coordinator
What is (are) the main difference(s) of the Quadrant and T-Streak methods of isolation streaks
-The main difference is personal preferance -The use of either three sections or four sections of a petri dish
Choose all that apply: What information does simple staining provide and what information can we derive from simple staining?
-The shape of the bacteria -Arrangement of the bacteria -The size of the bacteria
Why do we cool the molten agar after autoclaving? choose all that apply
-To prevent the destruction of temperature - sensitive organisms -To increase safety when pouring
You spill a large (~10mL) culture of bacteria. Who should be notified, either by you or the TA? Select all who apply
-University Police -Environmental Health and Safety -The Laboratory Coordinator -The TA
When should a Biological Safety Cabinet be used? Choose all that apply
-When working with Biosafety Level Two organisms -When working with a high-risk BSL1 organism whose genus is the same as a BSL 2 organism
Choose each unsafe practice
-Your lab partner has spilled culture on themselves and not informed the TA -You are taking pictures with your cellphone in lab -You see a lit, unattended bunsen burner -The person behind you has their long hair down and are working with a lit bunsen burner -You didn't clean your work area before you started
Which of the following is (are) NOT a part of a microscope. Select all that apply
-leg
Which of the following should be used to clean your microscope?
-lens paper -lens cleaner
Which of these are states of media? Choose all that apply
-solid -semisolid -liquid
A student transfers one milliliter of a sample with a concentration of 0.1 and transfers it into an autoclaved tube of 10mL sterile water. What is the ending concientration?
.01
A student places 10g of soil into 90mL of water. What is the concentration ?
0.1
Order the steps in simple staining, from the start to the end. Observe under the scope Blot dry with bibulous paper Heat fixed, labeled smear prep Add stain let rest for the required time DI rinse
1. Heat fixed, labeled smear prep 2. Add stain 3. Let rest for the required time 4. DI rinse 5. Blot dry with bibulous paper 6. Observe under the scope
Order the following steps in an aseptic transfer. Assume all proper disinfection protocols have been performed. Assume you are wearing proper PPE. Assume your burner is already attached to the gas valve, and is working Go back to the fire, holding your tool into the fire at a 45 degree angle Perform your transfer Light the Bunsen burner Hold inoculating loop at a 45 degree angle Put the loop into the fire until it turns orange Let the loop cool
1. Light the Bunsen burner 2. Hold inoculating loop at a 45 degree angle 3. Put the loop into the fire until it turns orange 4. let the loop cool 5. perform your transfer 6. Go back to the fire, holding your tool into the fire at a 45 degree angle
As a budding microbiologist, you have been selected by the Nueces County Public Health District to run a toxicological analysis of the soil around Oso Bay. You know that there are Bacteria and Fungi growing in your collected soil samples. How do you seperate out the bacteria from the fungi? What do you do to prevent growth of non-target organisms?
1. Serial Dilution 2. Pour plate of NA (for bacteria) and PDA w/ Tartaric Acid (for Fungi) 3. Grow and islolate colonies Tartaric acid prevents the growth of bacteria for the PDA plates
You are told to make a 1.5% solution of Agar in 1L of water. How many grams of Agar do you need?
1.5% = 0.015 1L = 1000mL 1000 * 0.015 = 15 15g/1000mL = 15g/L
Match the media type to the technique used to inoculate it Deeps: Slants: Plates: Broths:
1.Stab 2.zig-zag streak 3.Clamshell 4.Dip and Shake
Under which objective should oil immersion microscopy take place?
100X
How long would Methylene Blue be left to sit when simple staining?
30 seconds
A student autoclaves media for use in culturing. They add glycerol before the autoclaving. After autoclaving, the student lets the molten agar cool in a water bath. To what temperature should the agar be cooled so that it remains molten, but can be used for a pour plate
45-50 degrees
How many grams to TSB are needed to prepare 20 tubes of TSB? Assume the following: The bottle has a range of 30g powder per 1 Liter of DI water Each tube holds 9mL broth 10mL allowance for the total volume
5.7
You are asked to prepare a 2 Litre batch of Tryptic Soy Broth. How much powder do you need? assume 30g/L
60
How long would safranin be left on the bacteria when staining?
60 seconds
You are asked to prepare 100 tubes of Nutrient Agar (NA). How much NA powder is needed? Assume the following: 1 Tube holds 8mL of agar 20mL allowance formulation is 50g powder per liter
Amount required = (8mL*100tubes) + 20mL = 820mL Bottle = 50g/1L Math Xg/820mL = 50g/1000mL Math (820mL)(Xg/820mL) = (50g/1000mL)(820mL) Math X = (50g*820mL)/1000mL Math X = 41
Analyze the growth of the following organism on Simmons Citrate Agar. What are the results? Why? (the slant is royal blue with a yellow zigzag growth)
Bacteria is + for using citrate as main carbon source and is Acid +
Which of the following is the correct entry procedure for a BSL2 lab? Choose the best answer
Before entering, you put up your long hair into a bun. Upon entering, you wash your hands. then put on your lab coat. Then put on gloves and clean your work area
On Hektoen Enteric Agar (HEK), Salmonella enterica is grown. Individual colonies appear _____________, while the agar appears ______________.
Black, Blue
Citrate postitive orgainisms (organisms that can utilize citrate) will change the color of Simmons Citrate Agar to what color?
Blue
Microcystis aeruginosa is a blue-green alga commonly found in the Great Lakes. It is a Harmful Alga, capable of producing neurotoxins, hepatotoxins, and other cyanotoxins. In what domain of life would you put Microcystis? Why? (Hint: What is the specific anatomical difference between domains of life)
Blue-green algae are cyanobacteria, and are in the Domain Bacteria, not Domain Eukaryota. They are in that domain because they don't have a true nuclear envelope, and are thus a Prokaryote, not a Eukaryote
Voges-Proskaur positive organisms use the _____________________ metabolic pathway
Butylene Glycol
A student performs a serial dilution, starting with 10g of loamy soil. They then dilute the the sample to 10^-4 and plate 0.5mL of sample on to a plate of of Potato Dextrose Agar. This process is repeated for 5 replicates. After one week of growth, the student counts an average of 523 colonies across all their replicates. Calculate the colony forming uints per gram of soil. Give your answer as a number, not in scientific notation
CFUs = (523 * 1.0e4)/0.5 =10,460,000
A large spill (~10mL) has spilled on you. It has soaked through your lab coat and has made contact with your skin. Which of the following is the best response
Calmly inform your TA. The TA will then have you remove any contaminated PPE or cloths to be autoclaved. You will then wash in a safety shower. The BIMS Coordinator, EH&S, and UPD will be informed, and a police report filed
An unknown bacteria is grown on MacConkey's Agar. The bacteria in question produces clear colonies meaning it __________________ ferment lactose. The growth of the bacteria implies it is a gram ____________ organism.
Cannot, negative
Analyze the following Capsule Stain. Give the following, if applicable: Capsule status, primary stain, counterstain, reagent
Capsule: + Primary Stain: Crystal Violet Countertain: None Reagent: Copper sulfate 20%
Bacillus subtilis is grown on Casien agar. The areas around the bacteria are clear. This means that B. subitlis produces _____________________ an enzyme that breaks down _________________.
Casease, Casien
The pyrenoid is a body in what organelle?
Chloroplast
__________ are used to look at specific colonies
Isolation Streaks
What is the purpose of adding glycerol to Actinomycete Isolation Agar?
It is a carbon source
Describe the shape below, using scientific terminology (circular shaped bacteria)
Coccus
Explain the rationale for Capsule Staining. Explain why it works as it does. Is the wash water? why or why not?
Copper sulfate is used as a wash because a capsule is water soluable. Primary stain stains the bacterium, wash clears out the capsule. you look for halos
After taking a swab of a bacteria and adding the Oxidase reagent, the swab turned blue after five seconds. What does this mean?
Cytochrome c oxidase was produced
Explain the rationale for Endospore Staining. Explain why it works as it does
Endospores are resistant to staining. Heat drives the MG into the cell and spore. MG is a water soluble stain, thus the MG is washed away from any aspect that is not hydrophobic (ie, not the endospore). Counterstain for contrast
All algae are photosynthetic
False
All photosynthetic live is either an alga or a plant (Verdeplantae)
False
You are called over by a fellow lab mate to check their slide. Is the following statement True or False You do not have to adjust their settings
False
Analyze the following surcrose fermentation tube. What can we tell from this photo, and why? (The tube has yellow broth with turbidity at the bottom and a bubble in the Durham tube)
Fermentation + , gas produced
________are used to create stock cultures, not isolate colonies
Lawn
When autoclaving media, which cycle should be used?
Liquid cycle
Mycobacterium nonchromogenicum is grown on Casien agar. The colonies are a white color without an area of clearing. What does this tell us?
M. nonchromogenicum does not produce casease
A student grows an unknown bacteria in MRVP broth. After adding the reagents, their MR results are shown below. Analyze their results. What are the reagent(s)? What does this tell us? Why do you know? (The tube is red and almost looks like jelly)
MR + (red color) Mixed Acid Fermenter Methyl Red (Reagent)
When performing a pour plate, what is the most critical step?
Maintaining proper temperature
After adding in reagents and not noticing a change in color, a student adds powdered zinc to a culture grown in nitrate broth. The culture then changes to a red color. How would you analyze this?
Nitrate was never reduced
Intemperate the Hektoen Enteric Agar plate below. Describe the bacteria using proper, scientific language. What is the gram status of the organism? Does it ferment? Can you determine the the species? Why do you say that? (its a zigzag line of bubbles on a blue petri dish)
Non fermenting gram negative organism. A case can be made for a Shingella sp - Blue agar, with blue colonies
The lab coordinator considers this the most dangerous lab. Why do you think that is? do you agree with their assessment? Why or why not?
OPINION BASED
Where does labeling occur on a petri dish?
On the bottom edge of the plate
When looking through a scope, what is changing? Choose the best answer
Our perception of what is on the stage
Which of the answers below best describes the picture? (picture of obscure bacteria shape)
Pleomorphic
Pseudo-nitzschia australis is a harmful diatom known for its large blooms off the coasts of California, Washington, and Oregon. One of the defining aspects of its anatomy is an outer shell (or frustrule) composed of what?
Silica
Which of the answers below best describes the picture? (its a purple rod shaped bacteria)
bacillus
What are actinomyces?
bacteria found in soil with a hard shell made of sugars and have filaments call hyphae
After the addition of Barret's reagents, a mixed acid fermenter will appear what color in the Voges-Proskauer test?
brown
Often times when doing an environmental isolation, scientists wrap the plate in parafilm. Why do you think this is?
create anoxic conditions, prevent contaminants from leaving the plate, etc
________________ media contain known quantities of chemical components
defined
Which of the answers below best describes the picture? (Its a picture of what looks like two bacteria stuck together but its just one bacteria)
diplococcus
Escherichia coli K-12 is grown on Hektoen Enteric Agar (HEK). Individual colonies appear _______, while the media appears __________
orange, orange
You should respect the organism you are working with, not fear it
true
_________________ media do not have a known, exact, chemical composition
undefined
Sulfur Reduction can be tested by which of the following? Choose all that apply
- TSI - HEK - SIM
What are the benefits of knowing your microscope's calibration factor? Choose all that apply
-Ability to measure a single organism across magnifications -Ability to gain an accurate measurement of an organism on a microscope
Where are broths or slants labeled?
-Around the test tube, just below the cap
Select all the tools necessary for basic aseptic transfers
-Disposable inoculating loops - Bunsen burner -metal needle -metal loop
Which of the following tests limit the growth of non-enteric bacteria? Choose all that apply
-HEK -MAC -EMB
Choose all that go into a properly labeled plate
-Media type -Genus species of the inoculant -Date of incoulation -Initial(s) of the one(s) performing the inoculation -Growth temperature
Analyze the following Acid-Fast stain. Give (if applicable) AF status, primary stain, counterstain, regaent
AF: - Primary: Carbol fuchsin Counter: Methylene Blue Reagent: Acid-Alcohol
Why are actinomycetes clinically important?
Actinomycetes are useful because of the componentsthey are able to produce. Specifically, several types of antibiodics, chemotherapeutics, and immunosuppressants are derived from Actinomycetes While there are fungicides and herbicides that can be derived, clinically refers to medical treatment, where the antibiodics, chemotherrapeutics and immunosupperessants are more important
Why is the spread plate technique unusually dangerous?
As a part of the sterilization process, we ignite EtOH
What does "parfocal" mean?
As power increases, you don't need to adjust the coarse adjustment. Just the fine
Escherichia coli K-12 is grown on Eosin Methylene Blue Agar. The colonies are ______________ with a ___________ sheen. The agar is _______________.
Green, metallic, Black
Which of the following is not a part of the protocol for capsule staining?
Heat fix
Describe the biochemical rationale for Acid-Fast staining. Explain why an AF stain works like it does
Members of the genus Mycobacterium are resistant to staining due to the Mycolic acid in the cell wall. AF staining uses heat to drive the primary stain (carbolfuchsin) into the cell. The acid alcohol then decolorizes cells that don't have mycolic acid, and the counterstain stains them blue
Which of the answers below best describes the picture? (picture of four circular bacteria stuck together)
Micrococcus
A methyl red positive organism uses the _________________ metabolic pathway
Mixed Acid
Which of the answers below best describes the picture? (Cube shaped bacterie)
Sarcina
Which of the answers below best describes the picture? (its a picture of a yellow zigzag bacteria)
Spirillum
Which of the answers below best describes the picture? (three purple bacteria linked together that are all rod shaped)
Streptobacillus
Analyze the TSI slant below. What is it positive for? Why? (its yellow at the top and dark blue-black at the butt)
Sugar Fermentation + -> Yellow media throughout Sulfur Reduction + -> Black butt
You spill a small amount of culture broth on the bench. Who should clean it up?
The TA or Instructor
What is the practical application of using a calibration factor? choose the best answer
The ability to measure the same organism on multiple magnification powers
An unknown bacterium is grown on starch agar. after twenty four hours of growth, the student adds iodine to the plate. The student then notices a halo of clearing around the bacterial colonies. What does this imply?
The bacteria has enzymes that can hydrolyze starch
A smear of an unknown bacteria is placed on a slide. Hydrogen peroxide is added, and bubbles are formed. What does this tell us about the bacteria?
The bacteria produces catalase
Why do we plate the L rod in ethanol as a part of sterilization?
The burning of the ethanol sterilizes the rod
Analyze the photo of a urea slant. What can we tell about this organism, and why? (Its bright pink with a zigzag line with a slight bit of growth)
Urea +
Explain the rationale for Negative Staining. Explain why it works as it does
Uses a negatively charged stain to stain the background rather than the bacterium
After growing an unknown bacteria in MRVP broth, a student performed a VP test. Their results are shown in the picture below. Analyze the results. Is this a positive or negative? What does this tell us? What reagents were used? (The tube has dark purple at the top and yellow bellow)
VP +; due to the red color Organism uses the Butylene Glycol pathway Used Barrett's Reagents (A + B)
Isolation streaks still contain human error, thus may not be exact from person to person or bacteria to bacteria
true
Where do the proportions for making media come from ?
The media bottle
You grow an unknown bacteria in nutrient gel. After incubation, you cool the gel in the culture fridge. Upon pulling the gel deeps out, you notice that the upper layer of gel has not resolidified. What does this tell you?
The organism produces gelatinase
Which of the following is the proper technique for transporting a microscope?
The scope is supported on the base by your dominant hand, with your non dominant hand controlling the scope from the arm
Why do we care about isolating actinomycetes?
They have antimicrobial properties, leading them to be major sources of antibiotics
What is the purpose of a serial dilution?
To gain individual colonies, or isolates, by reducing the the concentration of the inocculum
How many grams of agar are needed to make 10 TSA slants and 10 TSA plates? Round your answer to the nearest tenth. Assume the following 1 TSA Slant holds 8mL of agar 1 TSA Plate holds 15mL of agar 1.5% agar concintration 15mL allowance for the entire batch
Total mL = 80 (Slants) + 150 (Plates) + 15 (Allowance) = 245mL 1.5% agar - 15g / 1000mL Setup: Xg/245mL = 15g/1000mL Math (245mL)(X/245mL) = (15g/1000gL)(245mL) Math X=(15g*245mL)/1000mL Math X= 3675g*mL/1000mL Math X = 3.675; Rounded 3.7
You are asked to make 30 tubes of Nutrient Broth (NB). How many grams of dry powder are needed? Give the full answer without rounding. Assume the following: 6mL of broth per tube 15mL allowance Bottle formulation is 10g of powder per liter
Total volume = (6mL * 30 Tubes) +15mL allowance = 195mL Bottle Formula = 10g/1000mL Setup Xg/195mL = 10g/1000mL Math (195mL)(Xg/195mL) = (10g/1000mL)(195mL) Math Xg = (10g*195mL)/1000mL Math Xg = 1.95g
Microscopy is more of an art than a science
True
The term "Algae" is not a formal taxon
True
Laboratory Acquired Infections (LAIs) are a serious problem, for all who work in the lab
true
A defined media is a complex of media
false
All media are created equally, thus proper monitoring is not necessary
false
All members of a single genus (like Mycobacterium or Bacillus) are always harmful
false
All modern microscopes are made to such high standards, that there is not any physical error between scopes
false
Bunsen burners are used with Biological Safety Cabinents (BSCs)
false
Nothing in this lab can hurt you
false
Sterile technique when using a disposable loop includes the use of a bunsen burner or bacti-incinerator
false
The Olympus BX41 and Olympus CX41 scopes are so similar, there will not be any differences between them
false
The phase contrast objective can be used to replace the bright field objective of the same power
false
Fill in the blanks. When pouring agar plates, the__________ is autoclaved, but the __________ is not
media, dish
Bacteria have a __________ charge
negative
When staining a bacteria, we use a stain with a ________ charge
positive
Complete the equation: CF @ Low Power = # of spaces on the ___________ micrometer __________________________________________x10 # of spaces on the _________ micrometer
stage, ocular
Fill in the blanks: When calibrating a microscope using a stage and ocular micrometer, the perceived size of _________________ micrometer changes, but the perceived size of the micrometer does not ______________
stage, ocular
Which of the answers below best describes the picture? (its a long chain of coccus bacteria)
streptococcus
Since each of us are different (different eyes, different technique) there will be subtle differences in the exact calculation we make for each microscope
true
The calibration factor must be calculated for each scope
true
A dust cover should always be used when storing your scope
true
A nonsynthetic media is also called a complex
true
