Lab Quiz #2 (Gram Stain through pGLO Lab)

It uses alcohol as a decolorizing agent which extracts the carbolfuchsin from the acid-fast (-) cells while ineffective on the acid-fast (+) cells. The acid-fast (-) cells are then counterstained with methylene blue.

Are acid-fast negative cells stained by carbolfuchsin? If so, how can this be a differential stain?

b, g, a, f, a, d, a, e, a, c

Arrange the following steps in order of completing a successful Gram Stain of an unknown bacteria. (NOTE: A choice may be used more than once) a) Rinse slide with distilled water until it runs clear b) Heat fix specimen to slide c) Gently blot with bibulous paper d) Decolorize with decolorizer until it runs clear e) Flood slide with safranin for 1 minute f) Flood slide with Gram iodine for 1 minute g) Flood slide with crystal violet for 1 minute

b, a, e, d, e, f, e, c

Arrange the following steps in order of completing a successful Kinyoun Method acid-fast stain of an unknown bacteria. (NOTE: A choice may be used more than once) a) Flood with Kinyoun carbolfuschin for 5-10 minutes. b) Heat fix specimen to slide c) Gently blot with bibulous paper d) Decolorize with decolorizer until it runs clear e) Rinse slide with distilled water until it runs clear f) Flood slide with methylene blue for 1 minute

b, e, a, d, a, f, a, c

Arrange the following steps in order of completing a successful ZN Method acid-fast stain of an unknown bacteria. (NOTE: A choice may be used more than once) a) Rinse slide with distilled water until it runs clear b) Heat fix specimen to slide c) Gently blot with bibulous paper d) Decolorize with decolorizer until it runs clear e) Place slide over a steam bath, place a small piece of bibulous paper over the slide, and flood with carbolfuschin for 8 minutes. After the 8 minutes, remove the paper. f) Flood slide with methylene blue for 1 minute

b, a, d, e, d, c

Arrange the following steps in order of completing a successful endospore stain of an unknown bacteria. (NOTE: A choice may be used more than once) a) Place slide over a steam bath, place a small piece of bibulous paper over the slide, and flood with malachite green for 8 minutes. After the 8 minutes, remove the paper. b) Heat fix specimen to slide c) Gently blot with bibulous paper d) Rinse slide with distilled water until it runs clear e) Flood slide with safranin for 3 minutes

d.

Multiple Choice (Choose the BEST answer) When using an EMB agar, growth that is "dark" (purple to black, with or without green metallic sheen) indicates what type of growth? a) Gram positive b) Gram negative c) Possible coliform d) Probable coliform e) Noncoliform

e.

Multiple Choice (Choose the BEST answer) When using an HE agar, growth that is blue-green growth with black precipitate (Bppt) indicates what type of growth? a) Gram negative b) Gram positive c) Not Shigella or Salmonella d) Probable Shigella or rarely Salmonella e) Probable Salmonella

d.

Multiple Choice (Choose the BEST answer) Which is NOT part of a pGLO Plasmid a) Replication origin b) Antibiotic resistance gene c) DNA binding protein d) Glucose promoter e) Green fluorescent protein f) None of the above

b

Multiple Choice (Choose the BEST answer) Which of the following stains is NOT categorized as a structural stain? a) flagella stain b) acid-fast stain c) capsule stain d) spore stain e) none of the above

a, d, e

Multiple Choice (Choose the BEST answers. There is more than one answer for this question) Choose the antibiotics that are protein synthesis inhibitors. a) Chloramphenicol b) Ciprofloxacin c) Penicillin d) Streptomycin e) Tetracycline f) Trimethoprim

Cocci and Gram positive (Streptococcus)

Name the shape and Gram stain result for the following image:

Bacillus and Clostridium

Name two genus of bacteria that can be identified by using an endospore stain?

+pGLO/LB/AMP/ARA Only this plate should contain the fluorescent bacterial cells as it is the only medium that contains the arabinose sugar that "turns on" the arabinose operon that transcribes for the GFP genes that allow the cell to glow.

On which plate should you expect to find glowing fluorescent bacterial cells? Explain your prediction.

-pGLO/LB Since there is no antibiotic on the agar, no antibiotic resistant gene is in need of expression. Therefore E. coli can grow freely.

On which plate would you expect to find untransformed bacterial cells? Explain your prediction

-pGLO/LB/AMP No E. Coli Growth because the plasmid does not contain the antibiotic resistance gene, therefore E. coli's growth is inhibited.

On which plate would you expect to not find any bacterial cells? Explain your prediction.

+pGLO/LB/AMP/ARA & +pGLO/LB/AMP Since both of these plasmids contain the antibiotic resistance gene, they can both be expressed.

On which plate(s) would you expect to find genetically transformed bacterial cells? Explain your prediction

Answers may vary but may include the following: 1. Under-decolorize 2. Over-decolorize 3. Not rinsing stain off therefore viewing dry stain on slide that can be confused as a bacterial cell 4. Some specimens (e.g. Bacillius & Staphylococcus) can only retain their stain for a 24 hr period 5. Failure to add the iodine 6. Failure to use both stains or reversing their order.

What are ways in which an individual could receive poor/bad results for a Gram stain?

Gram (-) bacteria have a cell wall that is composed of a thinner peptidoglycan layer and replaced with an outer membrane composed of lipid. Therefore the crystal violet forms a complex with the iodine and can get trapped in the cell wall of the Gram (+) bacteria while in Gram (-) bacteria the more lipid based outer membrane layer can be broken down by the decolorizer, therefore not trapping the crystal violet iodine complexes.

Structurally, what causes a bacterial sample to result in a Gram (+) or Gram (-) result?

Chlorampenicol is the best choice w/ a zone diameter 25 mm, if falls to the right suceptible zone (>18) Penicillin w/ zone diamter of 25mm, falls to the left of the resistant zone (<28). It lands in the intermediate category.

Suppose you do this test on a hypothetical staphylococcus species w/ the antibiotics pencilllin (p-10) and chloramphenicol (C30). You record zone identical diameters of 25 mm for the chloramphenicol and penicillin disks. Which antibiotic would be more effective against this organism? What does this tell you about comparing zone diameters to each other and the importance of the zone diameter interpretive chart?

Answers may vary but may include the following: 1. Time of exposure 2. Lamp intensity 3. Distance to the target (e.g. 1/distance squared) 4. No barriers (e.g. petri dish lid) to penetrate 5. Dust, organic materials, or other objects to block its path. 6. Must have "line of sight" to the surface being decontaminated.

What are ways in which you can alter the effectiveness of UV light on controlling microbial growth?

It is a process where foreign DNA is introduced into a cell, it assists in identification of transformed cells, and regulation of an introduced gene's expression that are used in genetic engineering.

What is the application (purpose) of Bacterial Transformation: The pGLOW System?

EMB agar favors growth for Gram (-) bacteria. More specifically califorms, who either ferment lactose vigorously (e.g. E. coli) or less-aggressively (e.g. Enterobacter or Klebsiella).

What is the application (purpose) of EMB agar?

HE agar is selective for Gram (-) bacteria and by containing bile salts it inhibits the growth of Gram (+) bacteria. HE is a differential medium designed to isolate Salmonella (which doesn't ferment lactose but reduces sulfur to H2S) or Shigella (doesn't ferment sugars and doesn't reduce sulfur) from other Gram (-) enteric organisms in a patient's stool sample.

What is the application (purpose) of HE agar?

UV light is commonly used to disinfect laboratory and health-care environment work surfaces and surrounding air.

What is the application (purpose) of UV radiation on microbial growth?

The Kirby-Bauer test is a standardized method used to measure the effectiveness of antibiotics and other chemotherapeutic agents on pathogenic microorganisms. It can also be an essential tool in prescribing the appropriate treatment for a patient.

What is the application (purpose) of the Kirby-Bauer Method?

There seems to generally be a direct relationship. Increase the exposure time and increase the amount of bacterial death for each species of bacteria.

What is the general trend between bacterial death and UV exposure time?

between 30-300 colonies

What is the range in which you would have a "countable plate" ?

Zone of Inhibition

What is this "ring" around the antibiotic disc called?

This depicts a serial dilution experiment and it is important because it allows individuals to estimate the population density in a liquid sample by plating a very dilute portion of the sample and counting the number of colonies it produces.

What is this visual depicting and what is its significance?

1. Aerobe 2. Anaerobe 3. Facultative anaerobe 4. Microaerophile 5. Aerotolerant anaerobe

What type of microbe would exhibit these types of growth? (Hint: There is a total of 5 answers)

OCD = CFU / D x V

When participating in a Standard Plate Count what is the equation you'd use to calculate original cell density?

Hydrogen peroxide was the least effective as 24/27 samples had growth. NOTE: The professor thought this was slightly strange and hypothesized that the samples may have been expired/old.

Which germicide was least effective? Defend your choice.

Bleach was the best germicide at 1% concentration as out of 9 trials from all classes, no growth existed.

Which germicide was most effective and at what concentration? Defend your choice.

P. aeroguinosa was the most resistant as 21/36 samples had growth in comparison to E. coli with 17/36 and B. subtilis with 19/36

Which organism seemed to be more resistant to the germicides?

There were some examples were Bacillus was able to grow in harsher conditions versus E. coli. E. coli can be more effected by UV because it hasn't had to evolve to be resistant and because Bacillus can produce endospores to make it more resilient.

Which organism survived the longest exposure: Why do you suppose it did?

Bacteria would be the best choice as they are single celled, reproduce quickly, and are cheap!

Which would be the best choice for a genetic transformation: a bacterium, fish, or mouse? Describe your reasoning.

Answers may vary, here are some possible answers: 1. Useful to identify bacteria in the genus Mycobacterium, or some other pathogens 2. Useful in identifying acid-fast bacilli (AFB) 3. Rapid, preliminary diagnosis of tuberculosis and leprosy 4. Performed on patient samples to track the progress of antibiotic therapy

Describe a couple benefits/applications for using the acid-fast stain in a clinical setting.

Answers may vary, here are some possible answers: 1. The location of the spore within the mother cell 2. Shape: spherical or elliptical (oval) 3. Size relative to the cell - whether they cause the cell to look swollen or not

Describe a couple benefits/applications for using the endospore staining in a clinical setting.

green, red, and red

Fill in the blanks A spore producer result can be observed as the bacterial cells having a spore colored __________ while the mother cell surrounding the spore will be a _______________ color. A spore nonproducer result can be observed as the bacterial cells having no spore color, while the mother cell will be a _______________ color.

bactericidal, bacteriostatic

Fill in the blanks Drugs that kill the organism are said to be ___________________, but other drugs are____________________; that is, they stop the bacteria from dividing, but do not kill them.

red, purple

Fill in the blanks Gram (-) results can be observed as the bacterial cells being a _______________ color. Gram (+) results can be observed as the bacterial cells being a _______________ color.

Yes drugs (e.g. Bacitracin) that target cell wall are less effective on gram (-) cells; cannot get past outer membrane

For a given antibiotic, is there a difference in susceptibility between the Gram (+) and Gram (-) bacteria? If so, what difference(s) do you see?

Heating melts the mycolic acid and allows the stain to penetrate the cell walls.

How does heating the bacterial smear during a ZN stain promote entry of carbolfuchsin into the acid-fast cell wall?

Simple diffusion

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

In presence of arabinose, the regulatory gene turns the operon on. Also, instead of producing enzymes to break down arabinose, the operon codes for green fluorescent protein

How does the pGLO recombinant plasmid work?

d.

Multiple Choice (Choose the BEST answer) The _______ - _____________ Test is a standard procedure used to measure the effectiveness of disinfectants specifically against Staphylococcus aureus, Salmonella enterica server Choleraesuis, and Psuedomonas aeruginosa. a) Kirby Bauer b) Gram Negative c) Antibiotic Efficacy d) Use Dilution e) None of the above

acid-fast (+)

Is this an acid-fast (-) or acid-fast (+) result?

ae, bf, cd

Matching Match the type of electromagnetic energy with its corresponding wavelength range: a) UV-A b) UV-B c) UV-C d) 100 nm - 280 nm e) 315 nm - 400 nm f) 280 nm - 315 nm

b

Multiple Choice (Choose the BEST answer) A Gram Stain is considered a _____________ stain a) sensitive b) differential c) distance d) messy e) none of the above

d.

Multiple Choice (Choose the BEST answer) C. sporogenes is classified as: a) Aerobe b) Microaerophile c) Facultative anaerobe d) Anaerobe e) Aerotolerant f) None of the above

c.

Multiple Choice (Choose the BEST answer) E. coli and S. aureus are typically classified as: a) Aerobes b) Microaerophiles c) Facultative anaerobes d) Anaerobes e) Aerotolerants f) None of the above

e.

Multiple Choice (Choose the BEST answer) L. lactis & E. faecalis are typically classified as: a) Aerobe b) Microaerophile c) Facultative anaerobe d) Anaerobe e) Aerotolerant f) None of the above

a.

Multiple Choice (Choose the BEST answer) P. aeruginosa & B. subtilis are typically classified as: a) Aerobes b) Microaerophiles c) Facultative anaerobes d) Anaerobes e) Aerotolerants f) None of the above

True

True or False (NOTE: Select true or false and if you select false, correct the statement to make it a true statement) Bacterial transformation is the process by which competent bacterial cells pick up DNA from the environment and make use of the genes it carries

False. Because so few organisms are acid-fast, the acid-fast stain is run only an infection by an acid-fast organism is suspected.

True or False (NOTE: Select true or false and if you select false, correct the statement to make it a true statement) Because so many organisms are acid-fast, the acid-fast stain is run most times an infection by an acid-fast organism is suspected.

False, Eosin methylene blue (EMB) agar is a complex, selective, and differential medium.

True or False (NOTE: Select true or false and if you select false, correct the statement to make it a true statement) Eosin methylene blue (EMB) agar is a complex, non-selective, and differential medium

True

True or False (NOTE: Select true or false and if you select false, correct the statement to make it a true statement) Germicides designed for use on or in livings tissues are called antiseptics.

False. Hektoen enteric (HE) agar is a complex, moderately selective, and differential medium.

True or False (NOTE: Select true or false and if you select false, correct the statement to make it a true statement) Hektoen enteric (HE) agar is a simple, moderately selective, and differential medium