Microbiology Final- Labs

At what temperature should you refrigerate bacterial samples? Explain why this is beneficial.

4°C. This temperature slows bacterial growth and prolongs the life of the sample.

How many milliliters of water were added to the tray prior to incubating the strip at 37C overnight?

5 mL

You arrive to your first day of work at a new lab. You are taking over for someone who took a new job at another lab. Your boss informs you that because of time restraints, this person did not exactly follow the experimental protocol. In order to proceed, you must know what he did differently. (1) According to the lab lecture, under what section of his notebook would you look to find the experimental steps? (2) As changes to the experimental steps were made, what are these differences called, and how should it appear in the lab notebook?

(1) Procedure—this is where the steps for the experimental protocol are recorded.(2) Deviations. All deviations should be written in red to immediately bring attention to the changes in the protocol.

List the four main steps used to prepare a WET mount, and indicate which (if any) step is optional.

1 - Clean slide 2 - Circle area on slide with wax/hydrophobic pen 3 - Apply organism to slide 4 - View under microscope *Note: there are no optional steps in the wet mount.

What THREE rules were discussed in regards to lab safety that would protect you and others from contamination?

1. Never eat or drink in the lab2. Always wear appropriate PPE3. Never leave the lab wearing PPE

Once you inoculate the rabbit plasma containing media for the coagulase test, how long do you wait to read the sample results?

12-14 hours 12-14 hours which is the equivalent of an overnight incubation.

What is the minimum temperature an autoclave must be set at to achieve sterile condition?

125°C

You are viewing a bacterial sample that is 7 micrometers (µm) in diameter through your oil objective lens which has a magnification of 100x. The eyepiece has a magnification power of 5x. What size will the sample appear through the eyepiece?

3500 um or 3.5 mm in diameter (or 500x's larger)

At what temperature is the fixed incubator set to, as presented in the lab video?

37°C

Identify the plating method (below) as demonstrated in the lab.

4-phase dilution streak

What objective power is best suited if you are uncertain what the sample is and where to begin?

4x (or lowest power objective)

Based on the microscope shown in the lab lecture, which objectives would NOT require placing oil on the slide?

4x, 10x and 40x

When might you want to utilize a negative stain technique in the lab? What is the name of the primary dye used in this technique?

A negative stain is used to identify organisms with an opaque structure. Nigrosin dye.

You wish to study the motility of a particular bacterium. What type of mounting technique would you use? Explain your answer.

A wet mount technique is the best technique to use when studying motility of an organism because the sample remains viable (heat fixing kills the sample).

What is the proper way to dispose of all materials used during the lab?

All materials must be place in a biohazardous waste bag and placed into an autoclave for sterilization.

What would happen in terms of the colorimetric readout if you forgot to do the wash step after Step D (above)?

All of the wells would have a dark color. The intensity of the readout is dependent upon the amount of the labeled secondary antibody present in the well. Since the unbound secondary antibody was not washed away, all wells would react equally.

Name one advantage that the ability to breakdown tryptophan brings to a microorganism.

An increased resistance to drugs/medications OR an increased ability to survive harsh environments—often seen via spore formation.

The word 'antigen' is actually a combination for what two words?

Antibody-generating

Enteric bacteria are often found where? Give an example of one.

Enteric bacteria around found in either the intestines or gut. Examples given in the video were either Salmonella or Shigella.

Given the following image, determine whether the bacterial sample is resistant or susceptible to the following antibiotics.

A—24mm (because radius is given) SusceptibleB—9mm Susceptible, despite small zone of inhibition.C—14mm Resistant

Identify the stain and shape of the bacteria pictured below.

Gram-positive; Cocci (spherical)

As stated in the lab video, an example of a lipase-positive bacterium is:

Bacillus subtilis

When setting up a western blot, what is the purpose of the blocking step post-transfer?

Blocking prevents non-specific binding of the antibody—you only want the antibody to bind to its specific target.

When developing a western blot what two factors influence the intensity of the band?

Both the amount of protein loaded as well as the duration (time) of development is directly proportional to band intensity.

According to the lab module, which 3 wells of the API strip should be filled entirely?

CIT, VP and GEL wells.

What is the common name for agglutination?

Clumping

You are about to study a bacterial sample under a light microscope. You look into the oculars and see two circles. What adjustments need to be made?

Compress or expand the oculars until a single circle can be seen while using both eyes simultaneously.

Using the observations made and recorded from PL09 Unknown Pathogen, complete the results sections accordingly. Culture A and Culture B were assessed via the catalase test as shown below. State your observations for each culture.

Culture A and B were both catalase positive meaning both are able to breakdown hydrogen peroxide (damages cellular integrity) into harmless hydrogen and water molecules.

Using the observations made and recorded from PL09 Unknown Pathogen, complete the results sections accordingly. Culture A and Culture B were assessed via the oxidase test as shown below. State your observations for each culture.

Culture A and B were both oxidase negative. This suggests both bacteria are anaerobic (or at the very least facultative). The top left panel is simply the negative control, while the top right quadrant is the positive control.

Using the observations made and recorded from PL09 Unknown Pathogen, complete the results sections accordingly. The hemolytic properties of both cultures were next examined by streaking each culture on a blood agar plate (shown below). What is the hemolytic status of Culture A? Culture B?

Culture A exhibits beta hemolysis as defined by the region of clearing around the growing bacterial streak. Culture B exhibits gamma-hemolysis (non-hemolytic) as indicated by the white colonies growing on the still red agar.

Using the observations made and recorded from PL09 Unknown Pathogen, complete the results sections accordingly. Culture A and Culture B were assessed via the coagulase test as shown below. State your observations for each culture.

Culture A is coagulase positive. The inverted tube showcases the clotting/hardening effect. This strongly suggests Culture A is Staph aureus as shown in the Lab#6 coagulase test. Culture B is coagulase negative. The tube could not be inverted as the culture was entirely in liquid form.

Using the observations made and recorded from PL09 Unknown Pathogen, complete the results sections accordingly. State your conclusions on the identifications of Culture A and Culture B based on your summary of results.

Culture A: Staph aureusGram-positive cocciBeta hemolyticCatalase positiveOxidase negativeCoagulase positive Culture B: E. coliGram-negative bacillusRed colonies on Mac; lac+Gamma hemolyticMetallic green on EMBCatalase positiveOxidase negativeCoagulase negative

You are looking for media that will help you distinguish between two gram positive bacteria. What type of media would be best for this?

Differential media

What pathogen is best identified using a SMAC agar?

E coli O157:H7. SMAC agar specifically differentiates between non-pathogenic E coli and the pathogenic E coli strain O157:H7.

What color will E.coli exhibit when grown on an EMB agar plate? Explain your answer.

E coli would exhibit a green color because it ferments lactose.

https://portagelearning.instructure.com/courses/906/quizzes/23195?module_item_id=85788#:~:text=Identify%20the%20organism%20growing%20on%20the%20BOTTOM%20half%20of%20the%20agar%2C%20and%20describe%20the%20observed%20hemolytic%20properties.

E. coli; Non-hemolytic activity (gamma) is observed as E coli does not lyse red blood cells.

Using the observations made and recorded from PL09 Unknown Pathogen, complete the results sections accordingly. 1. Culture A and Culture B were streaked onto EMB plates. What does this tell you about each of the cultures? Describe your observations.

EMB agar is both selective and differential media. EMB restricts Gram-positive and as such this again confirms Culture A is Gram-positive. Culture B clearly grew on the EMB plate, further confirming it is Gram-negative. A green metallic sheen was also observed which immediately suggests Culture B is E. coli.

True or False. The antibiotic discs are placed onto the LB agar plate before spreading the bacterial culture on the plate.

False The antibiotic discs are place onto the plate AFTER the culture has been spread.

True or False. The presence of cytochrome C is often associated with anaerobic bacteria.

False Cytochrome C is a component of the ETC and is associated with aerobic bacteria.

What type of incubator is pictured below?

Fixed incubator

Using the catalase test, a Streptococcus sample would be:

Gram (+), Catalase (-)

What type of bacteria does MacConkey agar select for?

Gram-negative bacteria

What are the Gram status, shape and identification of organism #2 from the Gram stain procedure?

Gram-negative; Bacillus (rod); E. Coli

What are the Gram status, shape, and identification of organism #4 from the Gram stain procedure?

Gram-negative; Bacillus (rod); Pseudomonas

Using the observations made and recorded from PL09 Unknown Pathogen, complete the results sections accordingly. Given Culture A and Culture B were accidentally mixed, what observations were made from the Gram stain (shown below)?

Gram-positive cocci (spherical) clusters and Gram-negative bacillus (rods) were observed.

If you are working in a lab in which an autoclave is not available, and you are pressed for time, which would you choose to best sterilize your equipment? Hot steam or hot air? Explain why you chose your answer.

Hot steam is the best choice as you can achieve a sterile environment in a matter of minutes whereas hot air will take several hours to achieve the same effect.

Why was an LB agar plate used to test the Staph culture as opposed to a selective/differential agar that only grows Staph?

LB agar is used as it simply provides the nutritional requirements to encourage bacterial growth. Since the results of the Kirby Bauer method is directly based on bacterial growth patterns, no other selective or differential additives should be present that may hinder or inhibit the samples growth.

What are the FOUR types of gloves presented in the lab video?

Latex, Nitrile, Thermal cold, Thermal heat

Based on the 4-phase dilution streaking experiment, in what phase were bacterial isolates (colonies) observed?

Individual colonies were observed within Phase 3.

The Standardized Disc Susceptibility test for examining antibiotic sensitivity is also known as what?

Kirby-Bauer Method

In order to assess a liquid bacterial culture for tryptophan breakdown, ~5 drops of _______ reagent is added

Kovac's

What was the name of the selective agar plate (shown below) that is similar to a blood agar plate?

MacConkey agar

Using the observations made and recorded from PL09 Unknown Pathogen, complete the results sections accordingly. Given the results from a MacConkey agar plate shown below, what is the shape of Culture A? Culture B?

MacConkey agar is selective for Gram-negative bacteria. Thus, Culture A must be Gram-positive as no growth was observed. Coupled to the results of the gram stain, Culture A (Gram-positive) must be the cocci. Culture B must be Gram-negative (growth observed) and this correlates to the bacillus or rod-shaped bacteria.

How would a microorganism be classified (positive or negative) if a pink/red band was observed at the top of a liquid culture containing tryptophan?

Positive. The microorganism is capable of breaking down tryptophan into indole and would thus be classified as indole positive.

Why do bacteria repel the dye nigrosin?

Nigrosin is a negatively charged dye. The membranes of most cells are also negatively charged. The membrane will repel the dye not allowing it to be absorbed.

What 2 parts of the microscope contributes to the total magnification to your sample?

Objective and oculars (eyepiece)

What are the main sections that should be found in a lab notebook? Name at least 4.

Objective, Procedure, Notes, Results and Deviations

1. Identify the part of the microscope indicated by the arrow.

Oculars/eyepiece

Eosin Methylene Blue (EMB) agar is:

Selective and differential media

A patient with type O blood is sent to the lab for a blood-clotting assessment. Since type O blood contains no antigens, which quadrant (#1-6) best depicts the result you would expect to see once the assay is complete?

Quadrant 6 shows no signs of agglutination, which is only possible in the presence of antigens. Since Type O blood has no antigens, no clumping will occur during the test. Note: Quadrant 2 was stated to have clumping (albeit minimal) during the wet lab and is thus not an acceptable answer.

If you were required to grow 4 types of bacteria on a single agar plate without cross contaminating the samples, what method would you use?

Quadrant growth method

Based on the image below, which row (A or B) is positive for agglutination?

Row B

You are a lab instructor, and Sally Miller has turned in her lab notebook for you to grade the lab experiment #1 on lab safety. Based upon what was covered in the lab video, how should Sally have titled her experiment?

SM01 Lab Safety

What dye do Gram-negative bacteria primarily retain?

Safranin

For the ELISA assay depicted below, which step (A-E) first contains only specifically bound antigens (aka a 'pure' population)?

Specifically bound antigens are present in step C. Specific and non-specific antigens are present in B but then the wash step removes the non-specific (unbound) antigens, leaving only the specific population seen in step C.

As you looking through the microscope you notice your sample is slightly off-center—what component of the microscope would be used to bring the sample into the center of the image?

Stage adjustment knobs

In the wet lab portion of the video, what pathogen was the agglutination assay specifically designed to test for? Choose all that apply.

Staph

What specific bacterium was mentioned to be coagulase positive and resistant to antibiotics?

Staph aureus

If you were to remove the red bloods cells from at blood agar plate (BAP), what kind of agar plate derivative would you have?

TSAYE (Tryptic soy agar yeast extract)

An enzymatic test that assesses the ability of a microorganism to produce hydrogen sulfide gas and/or ferment sugar is known as the __________ test.

TSI (triple sugar iron) test.

What is the main advantage of using the API test?

The API test allows for rapid characterization by simultaneously assessing 20 different enzymatic properties of a given microbe.

Identify (specific name) given to the region of the western blot indicated by the arrow.

The arrow is pointing to the dye front.

What was the purpose of the first quadrant of the agglutination assay containing only the reagent?

The first quadrant served as the reagent only negative control—you want to ensure the reagent by itself does not clump but rather requires the presence of Staph.

What is one disadvantage of heat fixing a sample?

The heat fixing procedure kills the specimen. This also prevents any observations on motility and enzymatic properties.

What is the purpose of doing a 4-phase dilution streak?

The purpose of a 4-phase dilution streak is to isolate individual bacterial colonies.

Answer the following. An unknown bacterium (Sample B) was tested using several of the rapid, qualitative tests described within this lab lecture. Using the images of the results below indicate the lab results for each test (1-3) as either positive or negative, then using the decision tree identify the unknown bacterium by the corresponding letter.

The unknown sample is: Oxidase-negative (anaerobic), catalase positive and coagulase-positive, which on the decision tree, corresponds with the letter O.

Why are Gram-positive bacteria able to retain the crystal violet dye?

They contain a thick peptidoglycan layer in their cell wall that readily retains the dye.

True or False. In order to maintain proper spacing the antibiotic discs should be placed approximately a finger's width from the edge of the plate.

True The disc should be placed approximately a fingers width from the edge so that a uniform zone of inhibition can be seen around the entire disc.

What color(s) was the TSI agar slant containing Salmonella?

Yellow and Black. The agar slant was yellow at the bottom while black on the sloped gradient portion of the agar.

Why is it important to first clean your slide before applying your sample?

You must first remove any unwanted contaminants from the slide otherwise it may be difficult to distinguish between the pathogen of interest and a contaminant.

When performing the Kirby-Bauer method the areas of clearing surrounding an antibiotic disc after an overnight incubation are known as what?

Zones of Inhibition

The hydrolysis of triglycerides on a spirit blue agar plate most closely resembles that of __________ hemolysis on a blood agar plate.

beta

Fibrin is another term for _______________?

blood clots

1. Identify the part of the microscope indicated by the arrow.

coarse focus

What three elements are used in an autoclave to sterilize equipment?

heat, pressure, and steam

What unit of measurement is used when determining the size of the zones of inhibition?

millimeters

1. Identify the part of the microscope indicated by the arrow.

objectives/objective lens

In a western blot ____________ are separated based on its size.

proteins

An oxidase positive test result will appear as ______ in color

purple

True or False. The catalase test is a qualitative and differential assay.

true