Kohlering/Serial Dilution/Streak/Enumeration
When isolating a microbe by streaking on an agar plate, what two elements of good streaking practice will ensure that you get isolated colonies by your fourth quadrant?
If you focus your given bacterial sample on the slide after Gram staining and each cell is stained dark purple, is the organism Gram positive or Gram negative?
Gram-positive.
Your sample did NOT grow on MacConkey agar. Is your sample most likely Gram-positive or Gram-negative?
Gram-positive.
You got your motility broth back and you can only see growth down a very narrow tunnel in the center of your broth. Your partner's motility broth exhibits dark red growth in the shape of a wide funnel. Was either organism motile and if so, which one?
Neither are motile.
What does a faint but intact and visible band on your gel indicate? Should you just ignore this?
It simply indicates that your sample had a low concentration. Do NOT just ignore it, as an intact band means that you got your desired result. The intensity/brightness/thickness should not scare you - it's okay if it's faint. As long as you can still see it, that counts as a result.
If each Eppendorf tube in a serial dilution has 1 mL of fluid TOTAL, how many microliters of the INITIAL, undiluted sample should be in the 10^-3 dilution, theoretically?
1 uL.
Staphylococcus can NEVER be Gram...?
Gram-negative.
Your microbe GROWS on Mannitol Salt Agar and its colonies have turned the medium BRIGHT YELLOW. What two things can you deduce about your organism?
Your organism is likely Staphylococcus because of its ability to grow in a medium containing such a high salt concentration. Your organism is also capable of fermenting mannitol.
What color should the cotton swab turn after the oxidase test IF your organism produces cytochrome c oxidase?
Violet to purple (DARK) in seconds. If it's light pink or yellow, it's NEGATIVE.
You are given an image of a gel to analyze. You can see four wells: one ladder and three unknown samples. The bands on the ladder have their sizes clearly indicated and are as follows: 1000, 960, 840, 750, 600, 500, 400, 310, 220, 100. You are told that these are the results of a gel after loading the PCR product of a sample that had a target length of 450 base pairs into the unknown wells. What are you looking for on the gel to determine if the PCR worked for each of the samples in the unknown wells?
Look for an intact band between the bands on the ladder that are labeled 400 and 500 base pairs in length. Faint lines are okay and are still indicative of the PCR reaction working. If the lines are heavily smeared or nonexistent, then you can conclude that the PCR did not work for the sample in that well.
You are given the Petrifilm of a 10^-5 dilution sample. You count 62 colonies. Calculate the number of colony forming units (CFU) per milliliter of undiluted sample. Give your answer in scientific notation and use correct units.
6.2 x 10^6 CFU/mL
If the majority of the cells in your sample are rod-shaped and arranged in a single-file chain, what would the technical term for that arrangement be?
Streptobacillus.
What is the difference in appearance on blood agar between organisms capable of beta hemolysis and those capable of alpha hemolysis?
Beta hemolysis: media around colony is fully transparent, lost its color.
Alpha hemolysis: Green or brown discoloration around colony (especially visible on bottom of plate, looks very dark).
Gamma hemolysis: NO hemolysis occurred (no color change at all, just see the colony growing as normal on the agar).
Your organism appears pale/yellow on MacConkey agar. What can you conclude about its ability to ferment ... (name the sugar)?
Your organism cannot ferment lactose.
You got the results of your Sanger sequencing back after performing a PCR. What online resource would you use to find the species that your nucleotide sequence belongs to?
BLAST (either BLASTn or BLASTx).
Name the steps involved in properly Kohlering a microscope.
Focus on calibration slide using 4x or 10x lens, close field diaphragm completely, adjust condenser height to get the polygon with crisp edges (condenser all the way UP then slightly down - orange to blue on the edges), center circle, open field diaphragm to enlighten whole field of view.
NEVER touch WHICH lens with the immersion oil?
40x. ONLY use immersion oil on the 100x lens.
You inoculated your organism on DNase agar last week and you are now reviewing its growth. You conclude that your organism does produce deoxyribonuclease and can therefore cleave DNA. What visual cues must you have seen in order to reach this conclusion?
A clear zone of discoloration is present around your colony's growth. The agar is clearly losing color around the perimeter of your colony.
How will you know if your organism tested positive on the catalase test?
Bubbles formed on your microscope slide within seconds after adding a drop of hydrogen peroxide (H2O2) onto your cells.
You are given a nucleotide sequence and you want to find the protein sequence that it likely corresponds to. You also want to be able to easily check the structure and function of the protein. Which resource would you use on BLAST and which database is best?
Blastx, UniProt/Swissprot database.
What function in Excel will calculate the standard deviation for your chosen data set?
=STDEV
Name the 5 crucial steps involved in Gram-staining. Go by the substance used in each AND the duration for which the sample should be exposed to the substance before letting it run off or rinsing it (in seconds). Then, name the TWO steps you must take AFTER step 5.
1) Water + cells, dry FULLY, add decolorizer to fix/glue cells (30 s). 2) Crystal violet (60 s). 3) Iodine/mordant (60s). 4) Decolorizer (drop by drop until it runs clear). 5) Safranin/counterstain (60 s). (After) Rinse with stream of water (2 s), blot gently with bibulous paper.
You are looking at the results of your phenol red broth test and you noticed that your sample turned bright yellow. There is also a clear/transparent bubble in the top of the inverted Durham tube in the vial. What do you know about your sample?
Your organism was CAPABLE of fermenting the glucose in the medium AND it is heterofermentative (CO2 gas was produced as a byproduct along with acid).
If your organism is capable of utilizing citrate as the SOLE carbon source, what will your citrate agar look like? If incapable, what will your citrate agar look like then?
DEEP, dark blue if positive. Greenish if negative.
Which formula is used to manually calculate the primer's melting temperature (when the primer melts off the DNA)?
(G and C x 4) + (A and T x 2)