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Biod 151 Lab exams 1 to 9 / Portage
College study guide 2023
LAB 1
Answer the following questions
- What three elements are used in an autoclave to sterilize equipment? heat, pressure, and steam
- What is the minimum temperature an autoclave must be set at to achieve sterile condition? 125°C
- If you are working in a lab in which an autoclave is not available, and you are pressed for time, which would you chose 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.
- What type of incubator is pictured below? Fixed incubator
Answer Key
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.
LAB 2
- Identify the part of the microscope indicated by the arrow. Oculars (or eyepiece)
- Identify the part of the microscope indicated by the arrow. Objectives (or objective lens)
- Identify the part of the microscope indicated by the arrow. Course focus
- 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.
- What 2 parts of the microscope contributes to the total magnification to your sample? Objective and oculars (eyepiece)
Creating a hydrophobic barrier (the circle) helps to keep the water within the circle so it does not spill off of the slide.
- What dye do Gram-positive bacteria primarily retain? Crystal violet
- 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.
- Identify the Gram status (positive or negative) and shape of the bacteria pictured below. Gram-Negative; Bacillus (rod)
- An acid-fast stain is most commonly used to identify what type of bacterium? What is the name of the primary dye used in this technique? Mycobacterium; carbolfuchsin dye
- 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 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 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 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 #5 from the Gram stain procedure? Gram-positive; Cocci (spherical) chain; Streptococcus
LAB 4
- What type of media is best used to eliminate certain bacteria from within a mixed culture? Selective media
- According the lab module, what type of agar plate is the most commonly used nutrient agar? What color is it? LB agar; Light (or pale) yellow
- What was the name of the selective agar plate (shown below) that is similar to a blood agar plate? MacConkey agar
Individual colonies were observed within Phase 3.
- Identify the plating method (below) as demonstrated in the lab. Quadrant growth
- Identify the organism growing on the TOP half of the agar and describe the observed hemolytic properties. Staph Aureus; Beta hemolysis is observed based on the zones of clearing within the red agar.
- Would you expect to see a color change when pseudomonas is streaked onto an EMB agar plate? Explain your answer. No. There would not be a color change because pseudomonas does not ferment lactose.
Lab 5
- The Kirby-Bauer method for examining antibiotic sensitivity is also known as what? The Standardized Disc Susceptibility Test
- 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.
- 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.
- 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 unit of measurement is used when determining the size of the zones of inhibition? A. Centimeters B. Micrometer C. Millimeters D. Meters C
- True or False. In order to maintain proper spacing the antibiotic discs should be place around the edge of the plate. False. 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.
True
- Which STD is most often identified using the Oxidase test? Gonorrhea
- Using the catalase test, a Staphylococcus sample would be: A. Gram (+), Catalase (+) B. Gram (-) Catalase (+) C. Gram (-), Catalase (-) D. Gram (+), Catalase (-) A. Gram-positive, Catalase-positive.
- True or False. The catalase test is a qualitative and selective assay. False. The catalase test is a qualitative and differential assay.
- Fibrin is another term for? blood clots
- Once you inoculate the rabbit plasma containing media for the coagulase test, how long do you wait to read the sample results? A. 1-2 hours B. 3-6 hours C. 8-10 hours D. 12-14 hours D. 12-14 hours which is the equivalent of an overnight incubation.
- What specific bacterium was mentioned to be coagulase positive and resistant to antibiotics? Staph aureus.
- The hydrolysis of triglycerides on a spirit blue agar plate most closely resembles that of hemolysis on a blood agar plate. A. Gamma
B. Beta C. Alpha D. None of the above. B. Beta.
- As stated in the lab video, an example of a lipase-positive bacterium is: A. Staph aureus B. Pseudomonas C. Bacillus subtilis D. Streptococcus C. Bacillus subtilis An unknown bacterium (Sample A) 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.
ADH, LDC, ODC, H2S and URE
- How many milliliters of water were added to the tray prior to incubating the strip at 37C overnight? 5 mL
- 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.
- Enter the API Code for S1 and S2 from the lab here: S1: 5044550 S2: 2206002 API ANSWER KEY
LAB 8
- The word ‘antigen’ is actually a combination for what two words? Antibody-generating
- For the ELISA assay depicted below, what do the arrows indicate? The arrows indicate the wash steps.
- 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.
- In a Western blot are separated based on its size. proteins
- 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.
- Identify (specific name) given to the region of the western blot indicated by the arrow. The arrow is pointing to the dye front.
- What is the common name for agglutination? Clumping
LAB 9
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.
- 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.
- 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.
