W6/7/8

Question 1

MacConkey plates:
1) what they contain
2) how they work
3) what bacteria do on them hint - what’s the process
4) how they are interpreted. think neutral red
5) what bacteria they are useful for identifying

Answer 1

1) Bile salts + crystal violet (inhibit Gram+), lactose, neutral red (pH indicator)

2) Function: Selective for Gram-bac; differentiates between non/lactose fermenters.

3) Bacterial Growth
a. Lactose fermenters → Pink/red (E. coli, Klebsiella).
b. Non-lactose fermenters → Colorless (Salmonella, Shigella)

4) Interpretation: Pink = acid from lactose fermentation; colorless = no fermentation

5) Use: Identifies Enterobacteriaceae, differentiates enteric pathogens (gut organisms)

Question 2

How would you isolate one bacterial strain out of a community of bacteria?

Answer 2

T-streaking onto selective + differential media

Selective media - inhibits unwanted bacteria, allowing only your target group to grow (e.g., MacConkey agar for Gram-negative bacteria).

Differential media - allows you to distinguish between different types of bacteria based on their metabolic activity (e.g., lactose fermentation on MacConkey agar).

Question 3

Purpose of general purpose media.

What does this type of media by composition is it equivalent to?

How would you know the media is a general purpose media?

Answer 3

Supports growth of a wide variety of organisms

rich, complex media

If it has poorly defined source of organic material, then its general
− yeast cells
− milk
− animal tissue (e.g. meat, gelatin)
− soy beans

Question 4

can any growth medium support the growth of all microbes?

Answer 4

NO

Question 5

Purpose of Defined/minimal media

How would you know the media is a defined/minimal media?

Answer 5

To grow microorganisms w/ known nutritional requirements by providing just enough nutrients to support basic metabolic functions of organisms, w/o any excess components

if every ingredient is precisely known and measured (synthetically produced) + is composed of pure biochemicals, then its defined/minimal

Question 6

True or false: All minimal media are defined, but defined media are not necessarily minimal Growth media

Answer 6

TRUE

Question 7

Purpose of Selective media

How would you know the media is a selective media?

what are the approaches of a selective media? (2 total)

Answer 7

To isolate rare or slow-growing species in a complex bac pop by killing organisms you don’t want, so only organism you want survives

If there are specific Inhibitory Agents, then its selective media

• two approaches
  − minimal medium
  − add a toxin to a general, all-purpose medium Growth media - to selectively kill/inhibit unwanted organisms while allowing certain ones to grow

Question 8

purpose of differential media

How would you know the media is a differential media?

This media can be differential AND what else at the same time?

Answer 8

to allow you to distinguish between two or more organisms (that all grow on the medium)

If it has pH indicators or dyes, then its differential media

A medium may be selective AND differential at the same time

Question 9

Purpose of enriched media?

How would you know the media is an enriched media?

what type of media do you start w/? what do you do after? why?

This media can be enriched AND what else at the same time?

Answer 9

To enhances the growth of fastidious
organisms

If it has extra nutrients, then its enriched

start with all purpose medium, then add MORE nutrients to make it even
RICHER

A medium may be selective AND enriched at the same time

Question 10

What is the purpose of MRS agar?

How does this plate work?

They are in which category(ies) of media?

Answer 10

promotes growth of Lactobacillus and streptococcus

provides a nutrient-rich, slightly acidic environment that favors the growth of Lactobacillus species while inhibiting many other bacteria.

enriched bc Lactobacillus Is fastidious; selective bc pH is lowered, tries to inhibit streptococcus

Question 11

What is the purpose of Streptococcus agar?

How does this plate work? They are in which category(ies) of media?

Answer 11

promotes growth of
Streptococcus

enriched bc streptococcus Is fastidious; somewhat selective bc sucrose is fermented by S but not by L and higher pH to inhibit lactobacillus

Question 12

Why do cells sporulate? How does it help the bacteria?

What are the characteristics of endospores?

How can one observe spores?

What is a vegetative cell? a sporangium?

Answer 12

Sporulation is a survival mechanism to withstand harsh environmental conditions

Highly Resistant, desiccated (10-20% water), metabolically inactive

malachite green stain - a structural stain, need heat and solvent to penetrate spore, decolorize and stain is removed

Vegetative cell - have no spores inside (80-90% water); sporangium - mother cell or cell around spore

Question 13

What is negative staining? How do you do it?

Answer 13

Negative staining (acidic dyes): The (-) charged dye is repelled by the (-) charged cell surface, so it stains the background instead, leaving the cells as clear, unstained shapes

Question 14

What is dark-field optics? When would you use this? Are the cells stained?

Answer 14

a form of microscopy where cells become visible due to the light refraction of light

when negative staining

cells aren’t stained

Question 15

When would you use a basic dye? When would you use an acidic dye?

Answer 15

basic dye if you want to satin cell

acidic dye if you want to stain background

Question 16

What is a simple stain? differential stain? structural stain? Why would you use each?

Answer 16

Simple - one stain to visualize shape and size

differential - two stains to distinguish between cell wall structures

Structural - two stains to identify the presence or absence of a particular cell structure(flagella, endospores, capsules)

Question 17

What is the acid fast stain?
What type of bacteria are characterized as being acid fast? Why? What diseases are caused by these bacteria?

Answer 17

staining used on gram indeterminate bac

bac of genus Mycobacterium characterized as acid-fast due to the [high] of mycolic acid that makes them resistant to decolorization by acid-alcohol.

Mycobacterium tuberculosis: Causes tuberculosis (TB).
Mycobacterium leprae: Causes leprosy.

Question 18

What are viruses? What is their structure?

Answer 18

infectious agents composed of genetic material (DNA or RNA) enclosed in a protein coat, sometimes with an additional lipid envelope.

Question 19

Why might you think they are alive?

Answer 19

Viruses evolve, replicate, and interact with host cells, resembling characteristics of living organisms.

Question 20

Why might you think they are not alive?

Answer 20

They lack cellular structures and metabolism and cannot reproduce without a host.

Question 21

Describe the viral life cycle.

Answer 21

The viral life cycle involves attachment, entry, replication, assembly, and release, often through host cell lysis or budding.

Question 22

What type of viruses infect bacteria?

Answer 22

Bacteriophages (or phages)

Question 23

What are the implications for human health and economics?

Answer 23

Bacteriophages can be used to treat antibiotic-resistant infections

can also infecting starter cultures, affecting yogurt production

Question 24

How might strains of lactic acid bacteria be rendered immune to viruses? why does this work?

Answer 24

They can acquire resistance by integrating viral DNA into their CRISPR arrays, enabling bacteria to recognize + destroy viral DNA upon reinfection

acts as an adaptive immune system.

Question 25

What are the steps of the scientific method?

Answer 25

Observation, question, hypothesis, experiment, data analysis, conclusion, and communication.

Question 26

What is a hypothesis?

Answer 26

A testable statement predicting the outcome of an experiment.

Question 27

What is a control? What types of controls might you use?

Answer 27

a baseline for comparison in an experiment; types include positive controls (expected response) and negative controls (no response). baseline - dont know what is gonna happen

Question 28

What are independent & dependent variables? What are controlled variables?

Answer 28

independent variable - manipulated, dependent variable - measured in response. Factors kept constant to ensure a fair test.

Question 29

What features are necessary in a well-designed experiment? What features are necessary in a clinical trial? Why?

Answer 29

Clear hypothesis, reproducibility, controls, randomization, and sufficient sample size. Randomization, blinding, placebo controls, and large sample size to reduce bias and ensure reliable, generalizable results.

Question 30

How long do spores last? How can we kill them?

Answer 30

can last for decades or even centuries under harsh conditions. can be killed using autoclaving, strong disinfectants (e.g., bleach), or specialized sterilization techniques (e.g., ethylene oxide gas).

Question 31

Recognizing and identifying organisms: Volvox, Paramecium, Amoeba, tardigrades, rotifers

Answer 31

Volvox – Green, spherical colonies of flagellated cells. Paramecium – Oval-shaped, covered in cilia, moves by cilia beating. Amoeba – Irregular, shapeless, moves via pseudopodia. Tardigrades – Microscopic, segmented, eight-legged “water bears.” Rotifers – Tiny, wheel-like cilia at the head, elongated body.

Question 32

Bacterial shapes and arrangements: * Bacillus * Staphylococcus * Streptococcus * Lactobacillus * Lactococcus * Pseudomonas * Rhodospirillum * e.coli

Answer 32

Bacillus – Rod-shaped, can be single or in chains. Staphylococcus – Spherical, clusters (grape-like). Streptococcus – Spherical, chains. Lactobacillus – Rod-shaped, often in chains. Lactococcus – Spherical, pairs or short chains. Pseudomonas – Rod-shaped, single or pairs, motile. Rhodospirillum – Spiral-shaped, single cells. E. coli – Rod-shaped, single or short chains, motile.

Question 33

If bacteria are moving on the slide, what might they be doing? Does that indicate they are motile? If bacteria are not moving on the slide, what does that mean?

Answer 33

could be exhibiting true motility or Brownian motion due to collisions with water molecules. nope, true motility involves directed movement. Brownian motion is random They may be non-motile, dead

Question 34

What is swarming?

Answer 34

coordinated, rapid movement of bacteria cells across a solid surface, often involving flagella.

Question 35

Do eukaryotes or bacteria have flagella? Can you see bacterial flagella on a light microscope? Can you see eukaryotic flagella on a light microscope?

Answer 35

both have flagella structures differ—bacterial flagella made of flagellin, eukaryotic flagella composed of microtubules. No, bacterial flagella are too thin but can be seen after structural staining Yes, bc eukaryotic flagella are larger

Question 36

What are the general steps of Illumina sequencing? What happens during each of the major steps?

Answer 36

Library preparation: DNA is fragmented, adapters are attached, and fragments are PCR amplified. Cluster generation: Fragments bind to the flow cell and undergo bridge amplification to form clusters. Sequencing by synthesis: Fluorescently labeled nucleotides are incorporated, and the emitted signals are recorded. Data analysis: The sequence reads are aligned and analyzed using bioinformatics tools.

Question 37

How is the library made? Why is this necessary?

Answer 37

DNA is fragmented and adapters are ligated via PCR to modify DNA samples, making them suitable for sequencing with a specific company

Question 38

What is the purpose of the “tape station” (bioanalyzer)?

Answer 38

checks DNA fragment size and quality before sequencing.

Question 39

What is a flow cell? How do our DNA fragments stick to it? Why is clonal amplification performed? How is the sequence of the new strand detected on the flow cell?

Answer 39

flow cell - a glass slide coated with oligonucleotides that capture adapter-ligated DNA fragments via complementary bp To generate strong fluorescent signals for sequencing by making millions of copies of each DNA fragment. Fluorescently labeled nucleotides added one at a time, and the emitted light is recorded to determine the sequence.

Question 40

Advantages? Disadvantages? of Sanger seq

Answer 40

Advantages: High accuracy, good for short sequences. Disadvantages: Low throughput, expensive for large-scale sequencing.

Question 41

How do you do Sanger seq?

Answer 41

DNA is amplified, mixed with primers, DNA poly, dNTPs, and fluorescently labeled ddNTPs, then run through capillary electrophoresis.

Question 42

What reagents are included in Sanger seq? Why?

Answer 42

Template DNA: To be sequenced. Primer: Provides a starting point for DNA polymerase. DNA polymerase: Synthesizes new strands. dNTPs: Regular nucleotides for strand elongation. ddNTPs: Terminator nucleotides that stop synthesis at random points to generate fragments of different lengths.

Question 43

What is a chromatogram (electropherogram)? How do you read it ? basic DNA replication steps?

Answer 43

A chromatogram is a graph showing fluorescence peaks that correspond to the DNA sequence; it is read from left to right in the 5’ to 3’ direction. DNA replication involves helicase unwinding the DNA, primase adding primers, DNA polymerase synthesizing new strands, and ligase sealing gaps.

Question 44

What is breseq? What does it tell you? What did we use it for in our class? What were the goals of the second (genotyping) part of the microcosm experiment?

Answer 44

a tool to identify mutations tells me genetic changes that impact gene function between a reference genome + sequenced samples to identify muts that occurred in our microcosms, possibly contributing to the observed pheno

Question 45

Explain how bacteriophages may be helpful or harmful for humans.

Answer 45

Helpful: used to treat bacterial infections, resistant to antibiotics Harmful: can also infecting starter cultures, affecting yogurt production

Question 46

How does CRISPR protect bacteria?

Answer 46

CRISPR stores viral DNA sequences (spacers) from past infections, allowing bacteria to recognize and cut invading viral DNA upon reinfection.

Question 47

What can we learn from examining the size and sequence of the CRISPR array?

Answer 47

size indicates # of past infections seq reveals which viruses bac have encountered + possibly developed immunity against.

Question 48

Did we sequence the newest or oldest spacers in the CRISPR array? How do you know?

Answer 48

sequenced the newest spacers bc they are closest to 5'. CRISPR arrays grow by adding new viral DNA at 5'

Question 49

What is horizontal gene transfer? Briefly explain each type.

Answer 49

the transfer of genetic material between bacteria, not from parent to offspring. Types include Transformation: Uptake of free DNA from the environment. Transduction: DNA transfer via bacteriophages. Conjugation: Direct transfer of DNA through a pilus between bacterial cells.

Question 50

What characteristics may be used to identify bacteria? What is Bergey’s Manual?

Answer 50

Morphology (shape, arrangement), staining (Gram stain), metabolic properties, genetic sequencing, and biochemical tests. A reference book that classifies and identifies bacteria based on their characteristics, including morphology, metabolism, and genetics

Question 51

How do you do a Gram stain? WHY does it work?

Answer 51

The primary stain Gram iodine (moderant) Decolorizer Counter stain

Question 52

What should it look like? What optics should you use?

Answer 52

Gram (+) = purple; Gram (-) = pink Brightfield optics

Question 53

What would happen if you accidentally mixed up the dyes?

Answer 53

If Crystal violet and safranin swapped, then all bac would appear purple, masking Gram-

Question 54

What are common sources of error in gram staining?

Answer 54

Incorrect smear preparation = inconsistent decolorization Inadequate fixing of the slide = cells to wash off during staining Decolorizing gram (-) too little = purple cells instead of pink bc retention of primary dye Decolorizing gram (+) too much = pink cells bc they lose primary stain

Question 55

How could you tell if you made a mistake? How would you make a correction?

Answer 55

use controls with know gram characteristics - Any result that does not lead to Gram+ bac as purple and Gram- bac as pink try making thinner smears, ensuring slide is heat fixed, adjust decolorizer time

Question 56

Name one bacterial strain that you could use as a positive control. Name one bacterial strain that you could use as a negative control.

Answer 56

Staphylococcus epidermidis E. coli

Question 57

What is Gram indeterminate? WHY might a culture be Gram indeterminate? What is Gram variable? WHY might a culture be Gram variable?

Answer 57

It cannot be stained by gram staining Bc culture is old, meaning cell wall is compromised or its waxy, full of mycolic acid Where pure culture doesn’t consistently stain as either clearly Gram+ or Gram- ; Gram + culture that is too old bc cell wall is deteriorating

Question 58

Why is it helpful to turn milk into yogurt? Who is lactose intolerant? Is this “normal?”

Answer 58

to preserve milk; People without lactose tolerant mutated gene

Question 59

what is in the milk?

Answer 59

87% H20 proteins 2.5% of milk is casein lactalbumin lactoglobulin 5% of milk is lactose fat vitamins minerals

Question 60

What bacteria are in yogurt? What are their characteristics?

Answer 60

Streptococcus thermophilus Gm (+) coccus, aciduric, thermophiles Lactobacillus bulgaricus Gm (+) rod, acidophilic, thermophiles Lactobacillus acidophilus Gm (+) rod, acidophilic Lactococcus lactis Gm (+) coccus, mesophile Bifidobacterium Gram(+), branching rods

Question 61

What are bad doing in yogurt do? How do they cause the changes that result in yogurt?

Answer 61

fermentation (lactic acid) 1) lacotse hydrolysis: bacteria break lactose into glucose + galactose 2) Glycolysis to produce pyruvate 3) lactic acid fermentation where Pyruvate reduced to lactic acid 3) lactic acid lowers pH, leading to protein denaturatoin 4) denaturation leads to protein precipitation out of the whey

Question 62

explain glycolysis, Krebs cycle, electron transport chain

Answer 62

Glycolysis: Occurs in the cytoplasm. - Glucose (C₆H₁₂O₆) is broken down into two molecules of pyruvate. - Produces 2 ATP and 2 NADH. - Occurs in both aerobic and anaerobic respiration. Krebs Cycle (Citric Acid Cycle): Occurs in mito (eukaryotes) or cytoplasm (prokaryotes). - Acetyl-CoA (pyruvate that underwent oxidative decarboxylation) enters, and a series of reactions release CO₂, generate NADH and FADH₂, and produce a small amount of ATP. - Generates high-energy electron carriers (NADH, FADH₂) for the next step. Electron Transport Chain (ETC): - Located in the inner mitochondrial membrane (eukaryotes) or plasma membrane (prokaryotes). - NADH and FADH₂ donate electrons, which pass through protein complexes, creating a proton gradient. - ATP synthase uses this gradient to generate ATP (oxidative phosphorylation). -O2 final electron acceptor, forming water.

Question 62

Generally, from where do microorganisms get their energy?

Answer 62

Glycolysis - resulting in production of 2 ATP → cell resp Pyruvate produced then undergoes cell resp

Question 63

Explain fermentation. Why do bacteria do this?

Answer 63

Fermentation: glycolysis --> Pyruvate + NADH + ATP generated→ Pyruvate + NADH --> Lactic Acid → regenerating NAD⁺ allows glycolysis to continue in absence of oxygen/ETC by recycling NAD⁺, making it a survival mechanism in anaerobic conditions.

Question 64

Describe the relationship between Lactobacillus bulgaricus and Streptococcus thermophilus.

Answer 64

Mutualistic relationship: Bulgaricus produces aa → thermophilus can grow, producing lactic acid and lowering pH → bulgaricus comes back and produces majority of lactic acid

Question 65

What compound is produce by lactic acid bacteria that gives yogurt its distinctive taste/aroma?

Answer 65

Acetaldehyde

Question 66

What is the difference between how heirloom yogurt is made and commercial yogurt?

Answer 66

Heirloom starter - undefined + self perpetuating cultures commercial - standardized strains + not self perpetuating (L. bulgaricus + S. thermophilus)

Question 67

How would you make yogurt at home?

Answer 67

1) Obtain Commercial yogurt w/ live + active cultures 2) warm milk to 115C 3) Whisk store bought yogurt, starting fermentation process 4) Submerge jars in water at 120C, place in an off oven alongside a second pot of boiling water 5) Check after 5 hrs, 6) Tarter+thicker, leave it fermenting

Question 68

What is PCR? What is an amplicon? How does it work?

Answer 68

PCR = Technique to amplify target DNA region Amplicon = PCR products Denaturation (~94-98°C) - dsDNA heated, breaking H bonds, resulting in ssDNA Annealing (~50-65°C) - Short DNA primers (specific to target seq) bind to complementary regions on each strand Extension (~72°C) - Taq polymerase (a heat-stable DNA polymerase) adds nucleotides (dNTPs) to the primers, synthesizing a new complementary strand.

Question 69

How does PCR assist in the preparation of the DNA library for Illumina sequencing?

Answer 69

Amplifies DNA template for Higher throughput sequencing (More DNA means stronger signals, allowing for more data.) Adds adapters: Essential for binding to the flow cell. Adds indexes: Allowing for multiplexing and sample identification.

Question 70

How does PCR assist our preparation of DNA from yogurt bacteria?

Answer 70

PCR acts as a screen, selectively amplifying the S. thermophilus DNA while excluding other DNA

Question 71

What are the reagents used in PCR?

Answer 71

template DNA 2 primers nucleotides (A, G, T, C), specifically dNTPs (deoxyribonucleotide triphosphates) polymerase enzyme buffer well suited to the enzyme (pH, salts,cofactors, etc)

Question 72

How do the primers define the region that will be amplified (copied)?

Answer 72

Primers guide Taq polymerase to amplify the specific DNA segment.

Question 73

What will you include in the mix?

Answer 73

Everything but DNA template DNA polymerase: The enzyme for DNA synthesis. dNTPs: The building blocks of DNA. Primers: Both forward and reverse primers. Buffer solution: To maintain optimal pH and conditions. Magnesium chloride (MgCl2): A cofactor for the polymerase.

Question 74

What will you use as controls in PCR? Why?

Answer 74

Positive control - pure bacterial culture to ensure PCR successfully amplified DNA Negative control - no bacterial DNA to ensure no contamination

Question 75

What do we add to the gel to allow us to see the DNA? How does this work? How do you decide what concentration of agarose to use? The DNA runs toward which electrode? Why?

Answer 75

Add SYBR safe, binds to DNA, and when excited by blue light, DNA becomes visible Lower % gels will better resolve larger fragments, higher % gels will make smaller fragments easier to identify anode bc its positive and DNA is negative

Question 76

What is in the loading dye (loading buffer)? What is its purpose?

Answer 76

Contains two dyes + glycerol to make sol heavy. Purpose is to help you see sol as you load it into wells, watch dyes travel through the gel for tracking purposes, all while NOT binding directly to DNA

Question 77

What are DNA ladders? What is their purpose?

Answer 77

a set of DNA fragments of known sizes. to provide a reference for estimating the size of DNA fragments in our samples

Question 78

What is in the running buffer? What is its purpose?

Answer 78

contains Tris base to maintain a stable pH, preventing damage to DNA acetic or boric acid to provide ions for conductivity EDTA protects DNA from degradation by DNases during gel electrophoresis.

Question 79

what is CRISPR? What organisms have it?

Answer 79

bacterial adaptive immune system that defends against viruses by storing and using viral DNA sequences to target and destroy future infections. bacteria + archaea

Question 80

What is the structure of the CRISPR locus? What is encoded? How are new spacers added?

Answer 80

CRISPR locus consists of a 5' seq, palindromic repeats, viral-derived spacers has genes encoding Cas proteins, which process RNA and cut DNA. Cas1 + Cas2 proteins capture fragments of invading viral DNA and integrate them at 5' end of the CRISPR array

Question 81

How is the CRISPR system deployed? What is the goal?

Answer 81

CRISPR array is transcribed into pre-crRNA, processed into individual crRNAs, guiding Cas proteins to target and destroy viral DNA. To provide adaptive immunity against repeated viral infections.

Question 82

how does crispr work?

Answer 82

When bacterium is infected by a virus, it captures a small piece of viral DNA (a spacer) and stores it in its CRISPR array; upon reinfection, CRISPR RNA guides Cas proteins to recognize and cut the matching viral DNA.

Question 83

How may CRISPR be used for genetic engineering? What are the ramifications for human health? What type of ethical considerations are there?

Answer 83

CRISPR-Cas9 programmed w/ a guide RNA to target + edit specific genes in various organisms, enabling gene knockout, correction, or insertion. CRISPR holds potential for treating genetic diseases, creating personalized medicine, and improving disease resistance, but it also raises safety and regulatory concerns Issues include unintended genetic modifications, germline editing risks, consent for gene therapy, and the potential for misuse (e.g., designer babies, bioweapons).

Question 84

How would you determine whether your yogurt bacteria are resistant to infection by particular viruses? Why is the BLAST tool useful? What can you do with it?

Answer 84

By sequencing CRISPR array to check for spacers matching known viral sequences BLAST is a database used to identify organisms based on sequences and infer evolutionary relationships

Question 85

How would you use the data from our experiment to compare the lengths of CRISPR loci in heirloom and commercial yogurt? What would a difference indicate? What might you expect? How did we sequence our CRISPR loci? How many primers did we use? Did we sequence the entire array? How do we figure out the locations/sequences of the spacers and repeats?

Answer 85

1)Longer loci suggest a greater history of bacteriophage exposure 2)difference indicative of differences in viral infections 3) heirloom - longer loci bc of uncorntolled fermentation vs commercial - shorter loci bc of controlled environment 4) sanger seq, one primer 5) no 6) spacers + repeats found by CRISPRCasfinder website; spacer matches found by blast