micrb lab analysis Q
What happened if you got a large lawn of growth in only one third of the streak plate and the rest of the plate was empty?
You likely did not drag enough cells from the first streak into the second and third streaks or you forgot to cross into the previous section, so all the bacteria stayed concentrated in the first area instead of being diluted across the plate.
If two different bacterial cells were clumped together in the third streak, how would the colony appear after incubation?
They could grow into one mixed colony that looks like a single colony to the eye because both cell types multiplied in the same spot.
Could you distinguish two species if they started as a clump and formed one colony?
Not reliably, because a mixed colony can look like one colony even though it contains two species.
How could a mixed colony affect your supposedly pure re-streaked plate?
If you pick that colony, you may carry both species onto the next plate and your new plate will not actually be pure.
What is the purpose of crystal violet in the Gram stain?
Crystal violet is the primary stain and colors all cells purple at the start of the procedure.
What is the purpose of iodine in the Gram stain?
Iodine is the mordant that complexes with crystal violet and helps trap it more strongly in the cell wall.
What is the purpose of 95% ethanol in the Gram stain?
Ethanol is the decolorizer that removes the crystal violet-iodine complex from Gram-negative cells but not from Gram-positive cells when the stain is done correctly.
What is the purpose of safranin in the Gram stain?
Safranin is the counterstain that colors decolorized Gram-negative cells pink so they can be seen.
Why do only Gram-negative cells decolorize during Gram staining?
Their thin peptidoglycan layer and outer membrane do not retain the crystal violet-iodine complex after ethanol treatment, so the dye is washed out, whereas Gram-positive cells trap the complex in their thick peptidoglycan.
Why would a Gram stain look very dark or almost black with cells covering the whole field of view?
The smear was too thick because too much culture was added, so the cells overlapped heavily and held too much stain.
How would you fix a Gram stain that looks too dark and overcrowded?
Make a new smear using much less inoculum and spread it into a thin even film before drying and staining.
Why might a well-isolated colony still give a mixture of pink and purple cells on a Gram stain?
The most likely problem is improper decolorization, usually under-decolorizing or uneven decolorizing, which makes some cells keep crystal violet while others lose it.
How would you correct a mixed pink and purple Gram stain caused by staining error?
Repeat the stain with a thin smear and use ethanol carefully and evenly for the correct time.
Why would small pink rods be hard to distinguish if the background is also pink?
The slide likely still had too much safranin or stain residue left on it because it was not rinsed well enough, so the background stayed pink.
How do you fix a Gram stain where the background is pink?
Rinse more gently but more thoroughly after safranin and blot dry instead of wiping.
What kinds of information can you learn from an organism’s genome sequence?
You can learn its identity or closest relatives, what genes it carries, possible proteins and pathways it can make, possible resistance genes, possible virulence genes, and evolutionary relationships.
Can genome sequence alone prove an organism’s metabolic function or traits like pathogenicity motility or temperature range?
Not with certainty, because a genome shows what the organism could potentially do, but it does not prove the genes are expressed or functional under the conditions you care about.
What are the main limitations of using bioinformatics alone?
It depends on database quality and existing annotations, predicts potential rather than actual behavior, can miss regulation and expression, and still needs experimental testing to confirm real phenotype.
What is the main difference between FASTA and GenBank formats?
FASTA is a simple sequence format with a header and raw sequence, while GenBank is a richer annotated record that includes sequence plus detailed metadata and feature information.
What is the main similarity between FASTA and GenBank formats?
Both can store the same biological sequence information and identify the sequence entry.
Why are Lactobacillus and Bifidobacterium grown in different oxygen conditions?
Lactobacillus is an aerotolerant anaerobe that can tolerate oxygen, while Bifidobacterium is an obligate anaerobe that grows poorly or not at all in oxygen.
What are examples of natural probiotics?
They are found in foods such as yogurt, kombucha, pickles, and other fermented foods.
What are probiotic supplements?
They are capsules or powders containing specified numbers of live probiotic strains.
How would you dilute a bacterial culture to 10^-7?
Make seven sequential 1 to 10 dilutions by transferring one part culture into nine parts sterile diluent each time, mixing at every step, so each tube is ten times more dilute than the one before it.
Would Lactobacillus grow on MRS agar?
Yes, MRS is designed to culture Lactobacillus.
Would Bifidobacterium grow on MRS agar?
Yes, it can grow on MRS, but MRS is not ideal for mixed samples because it does not exclude Lactobacillus well enough for accurate Bifidobacterium counts.
Would Lactobacillus grow on BSM agar?
No or very poorly, because BSM is highly selective against non-Bifidobacterial microbiota.
Would Bifidobacterium grow on BSM agar?
Yes, BSM is designed to culture Bifidobacterium.
Would Lactobacillus grow under aerobic incubation?
Yes, because it is aerotolerant.
Would Bifidobacterium grow under aerobic incubation?
No or very poorly, because it is an obligate anaerobe.
Would Lactobacillus grow under anaerobic incubation?
Yes, because it does not require oxygen.
Would Bifidobacterium grow under anaerobic incubation?
Yes, that is the condition it needs.
What would happen if MRS plates were accidentally incubated anaerobically and BSM plates aerobically?
Lactobacillus would still grow on MRS, but Bifidobacterium on BSM would be greatly reduced or absent, so your counts would be distorted.
How would that incubation mix-up affect your ability to assess the probiotic contents?
You would likely undercount Bifidobacterium and possibly misjudge the balance of genera in the probiotic, making the results unreliable.
If Jamieson Probiotic 30 Billion Extra Strength contains the advertised number of cells, what Lactobacillus CFU total should be present per capsule?
About 18.3 billion CFU per capsule from the advertised Lactobacillus strains combined.
If Jamieson Probiotic 30 Billion Extra Strength contains the advertised number of cells, what Bifidobacterium CFU total should be present per capsule?
About 11.1 billion CFU per capsule from the advertised Bifidobacterium strains combined.
If 0.25 mL of a 10^-7 dilution is plated on MRS and the advertised Lactobacillus amount is present, what count would you expect?
About 458 colonies because 18.3 × 10^9 × 10^-7 × 0.25 = about 457.5.
If 0.25 mL of a 10^-8 dilution is plated on MRS and the advertised Lactobacillus amount is present, what count would you expect?
About 46 colonies because 18.3 × 10^9 × 10^-8 × 0.25 = about 45.75.
If 0.25 mL of a 10^-7 dilution is plated on BSM and the advertised Bifidobacterium amount is present, what count would you expect?
About 278 colonies because 11.1 × 10^9 × 10^-7 × 0.25 = about 277.5.
If 0.25 mL of a 10^-8 dilution is plated on BSM and the advertised Bifidobacterium amount is present, what count would you expect?
About 28 colonies because 11.1 × 10^9 × 10^-8 × 0.25 = about 27.75.
How do Lactobacillus and Bifidobacterium cell morphologies compare with many other bacteria from the semester?
Both are Gram-positive rods, so they differ from Gram-negative pink rods and from Gram-positive cocci such as staphylococci or streptococci.
How is Bifidobacterium cell morphology often especially recognizable?
It often appears as irregular branched or bifid rod forms rather than simple straight rods.
How does colony morphology of Lactobacillus and Bifidobacterium compare with many earlier lab organisms?
Their colonies are often smaller and less dramatic than some earlier selective-media colonies, and the media background and growth conditions are very important for interpretation.
What do catalase and oxidase test results tell you about Lactobacillus and Bifidobacterium?
Both genera are generally catalase negative and oxidase negative, which fits their fermentative lifestyle and lack of reliance on cytochrome c oxidase-based aerobic respiration.
What is one way to unequivocally determine the identity of each probiotic species?
Sequence-based identification such as 16S rRNA gene sequencing or whole-genome sequencing.
In the Week 10 quality control question, which MRS dilution should be used for calculation?
Use the 10^-9 MRS plates because 10^-8 is too high and 10^-10 is too low, and 10^-9 is the closest countable dilution.
In the Week 10 quality control question, which BSM dilution should be used for calculation?
Use the 10^-8 BSM plates because that dilution gives the countable range closest to the 30 to 300 rule.
What MRS CFU per capsule is estimated from 35 colonies at 10^-9 with 1 mL plated?
3.5 × 10^10 CFU per capsule.
What BSM CFU per capsule is estimated from 220 colonies at 10^-8 with 1 mL plated?
2.2 × 10^10 CFU per capsule.
What is the total probiotic CFU per capsule in the Week 10 quality control problem using those valid plates?
5.7 × 10^10 CFU per capsule.
Does that probiotic pass the Week 10 quality control test if at least 6.0 × 10^10 CFU per capsule is required?
No, because 5.7 × 10^10 CFU per capsule is below 6.0 × 10^10.
Why do we use plates with 30 to 300 colonies for CFU calculations?
Because fewer than 30 gives poor statistical reliability and more than 300 increases crowding and counting error.
Does the probiotic contain the advertised amount of live culture if your measured total CFU per capsule is at or above the label claim?
Yes, if the measured total is equal to or greater than the advertised amount, it supports the claim.
Do results support the hypothesis that the advertised concentration is accurate if your measured CFU is close to the label claim?
Yes, because the observed viable count agrees with the expected amount.
How many different species can you prove are present from colony appearance alone on Lactobacillus and Bifidobacterium plates?
You cannot prove exact species from colony appearance alone; you can only say there may be multiple colony types or morphotypes.
How can you tell colonies on the same plate may be different types?
They can differ in size color shape edge texture opacity elevation or presence of halos.
How would colony differences support or refute the advertised genera in the probiotic?
If colony types are consistent with expected growth on the correct selective media, that supports the presence of the advertised genera, but it still does not prove species identity.
How well did the probiotic experimental design address the project research questions?
It addressed them reasonably well by combining selective media serial dilution CFU counting and preliminary identification tests, so it could estimate viable counts and assess likely genera present.
What were the main flaws in the probiotic experimental design?
Media were selective but not absolutely perfect, some cells may have been nonculturable or stressed, colony morphology is not definitive, and the workflow only identified bacteria confidently to about the genus level.
What additional experiments would improve the probiotic study?
Use more replicates, species-specific molecular tests, sequencing, stricter controls, and additional confirmatory biochemical or molecular identification methods.
What kinds of information can you learn from an organism from its genome sequence?
Its likely identity relatedness gene content potential proteins pathways possible resistance genes possible virulence genes and evolutionary relationships.
Could you determine metabolic function or traits such as pathogenicity motility or temperature limits from genome sequence alone?
You can often predict them, but you cannot confirm them without experimental evidence because genes may not be expressed or functional.
What are the limitations of bioinformatics approaches alone?
They predict potential rather than confirmed phenotype, rely on database quality, and cannot replace functional testing of the actual organism.
Gram stain is the first step in bacterial identification. What does it tell you?
It tells you whether the bacteria are Gram-positive or Gram-negative and their shape, narrowing down possible organisms.
What are the two main groups after Gram staining?
Gram-positive and Gram-negative bacteria.
After Gram staining, what is the next step?
Determine the cell shape (cocci or bacilli).
What are the two shapes of Gram-positive bacteria in this scheme?
Cocci and bacilli.
If bacteria are Gram-positive cocci, what is the next test?
Catalase test.
What does a catalase-positive result indicate in Gram-positive cocci?
It indicates Staphylococcus.
What does a catalase-negative result indicate in Gram-positive cocci?
It indicates Streptococcus or Enterococcus.
If Gram-positive cocci are catalase positive, what test is next?
Blood agar hemolysis test.
If Gram-positive cocci are catalase positive and beta hemolytic, what organism is it?
Staphylococcus aureus.
If Gram-positive cocci are catalase positive and gamma hemolytic, what organism is it?
Staphylococcus epidermidis.
If Gram-positive cocci are catalase negative, what test is next?
Bile esculin test.
If Gram-positive cocci are catalase negative and bile esculin positive, what organism is it?
Enterococcus faecalis.
If Gram-positive cocci are catalase negative and bile esculin negative, what is the next test?
Blood agar hemolysis test.
If Gram-positive cocci are catalase negative, bile esculin negative, and alpha hemolytic, what organism is it?
Streptococcus mutans.
If Gram-positive cocci are catalase negative, bile esculin negative, and beta hemolytic, what organism is it?
Streptococcus thermophilus.
If bacteria are Gram-positive bacilli, what is the next test?
Catalase test followed by starch hydrolysis.
If Gram-positive bacilli are catalase positive and starch hydrolysis positive, what determines final identification?
Motility.
If Gram-positive bacilli are motile, what organism is it?
Listeria innocua.
If Gram-positive bacilli are non-motile, what organism is it?
Bacillus subtilis.
If bacteria are Gram-negative bacilli, what is the first test?
Oxidase test.
What does an oxidase-positive result indicate?
The organism uses cytochrome c oxidase (aerobic respiration).
If Gram-negative bacilli are oxidase positive, what test is next?
ONPG test.
If Gram-negative bacilli are oxidase positive and ONPG negative, what organism is it?
Pseudomonas fluorescens.
If Gram-negative bacilli are oxidase negative, what is the next test?
ONPG test.
What does ONPG test determine?
Ability to ferment lactose (β-galactosidase activity).
If Gram-negative bacilli are oxidase negative and ONPG positive, what is the next test?
Indole test.
If Gram-negative bacilli are oxidase negative, ONPG positive, and indole positive, what organism is it?
Escherichia coli.
If Gram-negative bacilli are oxidase negative, ONPG positive, and indole negative, what organism is it?
Klebsiella aerogenes.
If Gram-negative bacilli are oxidase negative and ONPG negative, what is the next test?
Urease test.
If Gram-negative bacilli are oxidase negative, ONPG negative, and urease positive, what is the next test?
Indole test.
If Gram-negative bacilli are oxidase negative, ONPG negative, urease positive, and indole negative, what organism is it?
Proteus myxofaciens.
What is the overall order of identification in this flowchart?
Gram stain → shape → catalase/oxidase → fermentation tests → confirmatory test.