Friday, October 19, 2012

MicroBiol Coredump E2

3 topics covered in exam
Microbial Nutrition and Growth

essential nutrients are needed for survival

general essential nutrients (GEN)
SPONCH = [Sulfur, Phosphorus, Oxygen, Nitrogen, Carbon, Hydrogen] are needed at an absolute minimum (at the very least, not amt)

growth factor = organic molecule the microbe cannot make itself. Might need preassembled aminic acid. Includes amino acids, vitamins, nucleotides

S by proteins; P from ATP, nucleic acids, phopolipids (easy)
N: via proteins, nucleic acids (considered difficult)

Generally parasites use organic form for S

specific essential nutrients (SEN)
individual needs of microbes. Diatons need silicon, bacteria does not need Si, but needs Fe
Difference between (GEN) and (SEN)
GEN are universal, while SEN are specific? Not All SEN's are needed at an absolute minimum like GEN.

  • energy source types
    • phototroph
      • Get energy from light → photons
    • chemotroph
      • Get chemicals from inorganic chemicals
      • lithotrophs – Get E from inorganic chemicals
      • organotrophs – Get E from organic chems (C,H) at a min

  • Carbon source types
    • autotroph
      • uses inorganic CO2
      • mentions methanogens
        • 4H2+CO2 → CH4+2H2O
    • heterotroph
      • Obtain C from organic form
      • CHO's, proteins, lipids, etc
  • Some combinations
    • Photoautroph – E from light, C form CO2
      • eg. Thermus aqnaticus
    • Chemoheterotroph – energy from chem, carbon from organic source
//These are the 4 main arrangements, 16 possible combinations?

Know the various oxygen requirements/tolerances for the categories we went over in class

Oxygen is used in macromolecules and sometimes in respiration
organisms that use O2 as their final e- acceptors in respiration are called obligate aerobes. That acceptor needs to be there waiting at the end or you won't make ATP → dead.

Aerobic respiration
Glucose [(CH2O)n] + O2 → CO2 + H2O + Energy (ATP)

obligate anaerobes are killed by O2. There might be intermediate steps.. e- + O2 → radicals. They won't have the enzymes to break down these and they'll damage via free radicals.
facultative anaerobe → don't need O2 , but can use it
aerotolerant anerobe → don't need O2, aren't killed by it, don't use?
catalase : if bubbles are seen, it's not an obligate anaerobe since we can see that it can properly deal with O2, or at least is trying to

concerning hydrogen, don't worry about with with SPONCH. Too much of it is around already.



cardinal temperatures: set of temps unique to each type/species
minimum : lowest temperature for survival, they aren't dead yet but close. Just surviving, not much growth
optimum: best temperature for growth/enzymatic reactions, lots of growth
maximum: highest temperature organisms can survive at, not much growth

psychrophiles : 0(or below)-20C, opt@15
psychotrophs
facilitative psychorophiles
mesophiles: 20-45C, opt@20-40
thermophiles: by plot 40-80C. Opt @~65C
hyperthermophiles
extremethermophiles

//do not relie on freezing to kill a microbe unless if hyperthermohile, they can exist at low temps. Boiling is preferred
//Thermus aqnaticus



acidophiles 0-5.5 ph
alkaliphiles – 5.5 – 8.0 ph
halophiles - 9 – 30% salinity (exist in pink crystals)?
Halotolerant 5-6% salinity
osmophiles
osmotolerant

Q: If you pull out infested Jam from fridge, wha tis it?
Osmotolerant and pschrotroph (possible)


    binary fission / transverse fission
    • Replication/Partitioning
      • 1 mother cell --> 2 daughter cells
      • origin of replication → start copying DNA
    • Septation/cytokenesis
      • forms a ring in the center of the cell, attached to cell membrane
      • add peptidoglycan
      • Z-ring contracts → splits cell apart

//cycle time = generation or doubling time
what happens during each of those two main stages to create two cells

Know the stages of the microbial growth curve and what is happening with the cells at those various stages

  • Lag phase
    • adjusting to environment, gathering nutrients getting ready to divide
  • Log/exponential phase
    • rapid cell division, more cells being produced than dying
    • Nf = (Ni)2n
  • stationary
    • net = 0, nutrients get used, waste products get made and kill off population
  • death
    • more cells dying than produced
there is a sample problem with this.
Q: to get n, n = (duration/standard doubling time)
thus, if sdt = 20, and duration = 240, n = 240 / 20, then Nf = (Ni)2n
//she used the book example*


Know the difference between symbiosis and non-symbiosis; know the various categories in those types of interactions

  • symbiotic
    • @least needs the other to survive
    • Mutualism
      • both benefit
    • Commensalism
      • one benefits, the other is unaffected
    • Parasitism
      • One benefits, one is harmed
      • //anytime we get sick..
    • //Termite example:
      • They don't have the enzymes needed to digest wood cellulose, but they have the enzyme cellulase
      • protozoa have bacteria on/in it
      • bacteria live on/in protozoa, helps them produce cellulase
      • termite gets glucose
      • err.. cellulose ---(with cellulase)--> glucose
      • orgs xfered through silava
    • another ex. Us e.coli, et other one
      • e.coli will take O2 in our intestents, and little pocktes inbetween cells won't have O2
      • now, there's bacteriodes → obligate anaerobes that live there. If they get hit with O2, they die


  • non-symbiotic
    • Synergism
      • both benefit
    • Antagonism
      • one benefits, one is harmed
        • ex. penicillum colony vs bacteria
        • fungi produces toxin...
    • interacts /w one another, does not need other to survive
    • Some are beneficial, some are harmful



-Know the stages of biofilm formation and what is happening in those various stages

  • Van Leeuwenboek via teeth scraping
  • microbes grow together in close assocation
    • symbiotic
    • non-symbiotic?
  • Bio-Films
    • attachment
      • Planktonic bacteria attachment
      • inducer molecules releases
        • these inducer molecules signal others to come over to initial attachment sites
        • /w enough they'll eventually reach a quorum
    • quorum
      • number of individuals needed to start forming matrix/biofilm
    • matrix development/maturation
      • cells act as a single unit, not individuals
    • matrix composition
      • polysaccharides, Proteins,Fimbriae, Pili
    • matrix function
      • Protection
        • film can block antibodies, keep antibodies from reaching cells
      • Genetic exchange
        • DNA binding proteins → help them hold onto DNA and maybe use it. One cell would spill its contents, another can pick I tup
    • matrix structure
      • differences in nutrients, temperatures, and oxygen levels
  • There are a number of quorum-dependent proteins (qdp)
    • inducer molecule → chromosome → qdp
  • //nitch concentration differences?
    • Top may offer most? These changes allow cells to behave differentially, than each other. They'll function as a unit and divide up tasks.
    • //@ top, lots of O2 and it's used for respiration → ATP
    • //at bottom, cells have acccess to a lot of nutrients
    • //cells at top will produce a lot of energy that they'll share with cells at the bottom, while bottom cells share nutrients with them. Thus, a biofilm can act as a multicellular organism.


===
TBC 

Saturday, October 13, 2012

Core Dump: Microbiol Lab Notes

Just posting some of my notes in case anyone from UNLV or CSN (or anywhere) wants  something to reference to.We had our first lab practicum mid-this-week. Some items aren't complete - differential mediums - some basic stains are explained well though.
---

Practical Part:

1: Make GS and AF stains.

GS algorithm
  • Saline+transferred bacteria ---> prepare smear, let air dry, then heat fix ~15s
  • Add Crystal Violet, 1min, rinse with water
  • Add Gram's iodine, 1min, rinse with water
  • Add Gram's Alcohol, slant it**, it'll decolorize, then immediately rinse with water
  • Counter stain safainin,1min, rinse with water
  • Let dry with bibulous paper

AF Algorithm

  • Saline+xfer bacteria ---> prepare smear, let air dry, then heat fix~15s
  • Add carbolfuchsin, let stand 5min
  • Add Acid-fast Alcohol, wash off last thing, then immediately rinse with water
  • Add methylene blue, 1 min rinse with water
  • let dry with bibulous paper

Remarks on interpretation: know result, and shape/arrangement, for an unk org.

  • if Gram stain +, we should see blue-purple stains on our organisms
  • if Gram stain -, we should see red-pink stains on our organisms
  • G variable is??? both, dependent on age, culture mix, etc.
  • if acid-fast +, reddish-purple
  • If acid-fast -, (methylene blue)

//ahh, streptoooooo, ooo as in “oo” not grapes. Don't see any grape bacillus.
Cocci (little circle dots)

Coccus: o, single
diplococci: oo, paired together
streptococci: oooooooo, chains
tetrad: o^2, 4 square formation
sarcina: o^3
staphylococci: grapes*
coccobacillus: meso between cocci and bacilli
Bacilli (rod shaped bacterium)
Bacillus
diplobacilli
streptobacilli
palisades: zig-zag /|/\/\/\|/\/\/
Spirillum (spiral)

Some remarks: Basically the arrangements tend to occur when a daughter cell sticks with its parent after division. Concerning the cocci, everything is the same. Just, if the plane become irregular it becomes a “staphylo-” Nothing too special. Also note that pleomorphic may exist. Report what you see and emphasize on the most complex arrangement, many will probably exist.

Visuals:

Gram positive staphylococcus
Gram negative cocci
streptococci









Gram positive bacillus
diplobaciili










palisades
Gram negative bacillus


2: identify endospore, flagellar, and capsule stains. Structure/function
    Endospore Stain:
  • Impossible to see. In a strepto arrangement they were the little dots between dead cells. Stain is forced within cell, and declorization removes stain in cells except spores. A counter stain will stain the cell but not spore. So spore can be seen as a different color
  • an endospore is a dormant form of a bacterium that allows it to survive poor environment conditions. Some papers also attribute it to as a form of primitive egg laying since multiple copies can be saved. Regardless, RNA is conserved. In slide you see vegetative cells and spore mother cells.
Capsule Stain
  • The capsule was not stained in our slides. The stained cells were covered by nothingness, blank, nada.
  • remarks: capsules are composed to mucoid polysaccharides/polypeptides that repel most stains. So they stain around the cell by the technique. Acidic stain (negative***) stains background. And the basic stain colourizes the bacteria. Thus, the capsule remains unstained and we see a white halo (the nothingness)
  • Also, the function is that a capsule increases virulence in microbes. They make them less vulnerable to phagocytosis. Helps in harsh environments, can live longer on doorknobs and stuff
Endospore Stain
  • stain is forced within cell, and declorization removes stain in cells except spores. A counter stain will stain the cell but not spore. So spore can be seen as a different color
  • an endospore is a dormant form of a bacterium that allows it to survive poor environment conditions. Some papers also attribute it to as a form of primitive egg laying since multiple copies can be saved. Regardless, RNA is conserved. In slide you see vegetative cells and spore mother cells.
Flagella Stain
inject Ryu stain with needle underneath cover.
Flagella functions.. creates motility in cells.
1 tail = polar, monotrichous arrangement, amphitrichous (both ends), lophotrichous (one end ,many) peritrichous (all over, like ex)

  1. differential, enriched, selective media. G+/-, for HEA, EMB, MacConkey, BAPcoliform bacteria produces gas from lactose fermentation
    selective: encourages specific, discourages other growth
    differential: allows to distinguish between diff microbes
    defined: chemicals are known and so is their composition
  • undefined: composition is unknown; however ingredients are known
Below is a list of common agars: exact details are in text
  • NA is w/eb
  • Phenylethyl Alcohol Agar (PEA) is an undefined, selective medium that encourages G+ and inhibits growth of most G- orgs. Not considered a differential medium since it doesn't distinguish between different orgs (I question this) it's considered selective**
  • columbia CNA /w 5% SBA: undefined, differential, selective medium. Allows growth of G+and stops/inhibits G-.
    straphylococci, streptococci, enterococci love it
  • concerning blood agar. Alpha hemolytic → (eats away, leaves a green partial clearing) gamma is nonhemolytic, beta is a complete hemolytic (all RBCs are hemolyzied, but not all)
  • HEA Hektoen Enteric Agar
    • selective differetial
    • selective: choose between G+, G- (allows G- grow,not so much G+)
    • differential based on G- (what species. Black color → salmonila)
  • Eosin Methylene Blue (EMB)
    • complex(chem undef), slective, differential medium. Has peptone, lactose, sucrose, and dyes eosin Y /w methlyene blue.
    • Dyes inhibit G+ orgs, rxn with lactose fermenter in acidc envoinrments.


result interpretation “presumptive ID”
No growth (P) inhibited G+
Good growth (G) Not inhibited G-
Pink and mucoid (Pi) Lactose ferment, lil acid Maybe coliform
(purple, black wo green) (D) Ferment lactose/sucrose lots of acid X2Maybe coliform
Colorless (C) No rxn Not coliform

  • MacConkey Agar
    • selective+differentiam medium /w lactose, bile salts, neutral red, and CY.
    • Inhibits growth of G+ orgs. Neutral red dye is a pH indicator. Red < 6.8 colorless otherwise
    • Used to isolate and differentiate members of the Enterobacteriaceae
    • remarks: if org. is a lacctose fermenter, it turns red/pink



over all remark: if it grows on Macconkey, EMB, or HEA it's G-.






Written Portion1.Magnification and Resolution. There is also contrast. We stain for this some. Impossible to get 10Kx with a light microscope (text) blue light?
2.
3.
4.
5. procedure/purpose of smear prep.
  • Algorithm: emulsion
    • set-up includes the staining tray
    • place drop of water /saline on clean slide if not staining from a broth culture
    • aseptically add bacteria to water/saline. Mix and spread out.
      • Adding it to saline causes a dilute solution. smear concentration/cell densities affects stain
    • Flame it to heat fix.
      • heat fixing kills the bacteria, makes adhere to slide, and coagulates their protein for a better staining. This could distort cells, and create aerosols.
    • Let air dry, if done correctly should be cloudy
6. Describe G and AF stains. Justify, principles, explain in detail, step-by-step
Negative stains not discussed, but they have neg ch­arged chromogen, repelled by bacteria, and … heat fixing need not be invloved.
  • gram stain is a differential stain because it divides bacteria into two classes, gram + (purple) and gram – (pink)
  • cells from fresh culture are xfer to a clean slide and allowed to dry. If from agar plate, first xfer to a liquid medium for dilution. create thin, barely visible film. You must smear. fresh culture should be used since as cells age, they'll lose their ability to retain the stain. Fix cells on slide by heating (Bunsen burner, slide should feel warm afterwards)
  • Stain with basic dye – crystal violet for 30-40s, then rinse to remove excess stain. Cells appear purple under microscope
  • Add Gram's Iodine solution and retain on slide for ~1min. Iodine combines with crystal violet to form a dye-iodine complex. This decreases its solubility within the cell. Cell still appears purple. This means it prevents it from being dissolved?
  • Decolorated by washing with ethanol or acetone. This is the differential step. G+ bacterial will retain the crystal violet, but gram negative bacteria do not. Add ethanol/acetone drop-wise, /w slide tilted at an angle. Do this until drops coming off edge start to become colorless
  • WARNING: even gram + cells can lose the crystal violet-iodine complex if decloration is excessive. wash excessive ethanol with water. At this point G+ are purple and G- are colorless.
  • The rinse cells are covered with a counter-stain called “safrainin” for 20-30s. This will make G- bacteria pink. Rinse with water and dry with filter paper

Theory behind G stains correlates with cell wall structure among bacterial. Ethanol is thought to shrink the thick peptidoglycan in G+ bacteria, forcing it to retain the dye. So, this thick, dehydrated peptidoglycan layer of G+ bacteria is now like “permeability barrier” this prevents the loss of the crystal violet-iodine complex.

In contrast. Since the peptidoglycan in G- bacteria is very thin and has large pores, ethanol may extract lipids and increase the cell's porosity. This will remove the crystal violet-iodine complex

again, G- cells have a higher lipid content -because of the outer layer - and thinner peptidoglycan layer than G+ cells . Also G+ cells with their thicker peptidoglycan layer have a greater degree of cross links due to teichoic acids .
Note: Iodine is not a mordant agent, it's more proper to consider it a trapping agent? Why does text continue to use “mordant”?

Acid-Fast Stain

Mycolic acid is a waxy substance that gives acid-fast cells a higher affinity for the primary stain and resistance to decolorization by an acid alcohol solution.
  • two methods. Ziehl-Neelsen (ZN) and Kinyoun (K) methods. K is coldstain*. We do coldstain
  • carbolfuchsin is a phenolic compound

basic stains have a negatively charged chromogen, which forms an iconic bond with a negatively charged bacterial cell.
Differential and structural stains, allow to see compartmentalizations and stuff
the acid-alcohol is (95% ethanol + 3% HCL), the carbolfuchsin would differ based on method

For Kinyoung Method
  • prep smear. they recommend sheep serum vs water, but I always use saline solution. But continue on with heat-fix.
  • Apply Kinyoung carbolfuchsin stain for 5min. Don't remember if it smells, but don't breath this in. rinse with water
  • declorize with acid-alcohol, not to excess until run off is clear
  • counterstain with something, (methylene blue) for a min. then rinse with water
  • blot dry with bibulous paper or paper towels
more remarks. Do not rub with the bibulous paper - this is bad.


Wednesday, October 10, 2012

ME402 Some Overview Notes ch 8,9,10

core dump


Cramer's rule:
Use on small systems, finding dets gets more and more time consuming
, det/a
Naive GE.
Forward elemination, backward substitution
Operation counting
er...
most of the effort is incurred in the elimination portion. as opposed to the back sub.
pitfalls
dividing by 0 is bad. also dividing by numbers close to 0. pivoting trys to work around this
round off errors : every result is based on previous result, thus errors tend to propagate
RoT: very imp. when dealing with 100+ eqs
ill-conditioned systems
  • well conditioned systems are ones where a small change in one or more of the coefficients results in a small change in the solutoin
  • Ill-conditioned systems are those where small changes in coefficients result in large changes in the solution.
  • an ill conditioned system is one in which the determinant is close to 0
    if slopes = 0, then they're the same line or there is no soln...
    But, determinant is a relative factor affected by the scales of its cofactors?
    • Slopes are same... x1x2 - y1y2 = 0, etc etc
/determinate of a diagonal matrix is just the product of its diagonal elements
the determinate of a singular system IS 0
GE will recognize this at the end the elemination step, but you can check the determinate once you have set up the triangular matrix

Techniques for improving solns.
use more sig.figs.
pivot: issues occur when pivot element is 0, small remedy for ill cond.
partial pivoting, switch pivot element, which is in column 1, with other element in that column which is the biggest in that solumn. Basically, just switch to highest clolumn 1 row.
complete pivoting: switch to biggest absolute value in whole matrix. so column and row
pivoting algorithm: for large matrices, exchanging rows becomes time consuming, just keep track of appropriate subscripts

not understanding multideminsional versoin of Newton_raphson method. need to relook at Taylor series expansion. seriously, wtf is this [Z] = crap stuff? non-linear equatoins look hard...
Also, complex systems have a few workarounds. 3 are recommended. Remember Fortran? Your ex-best friend??? Saving variables as complex is something special to it apparently.

Gauss-Jordan method is nothing more than Gauss elimination without the back substiution stage. you just forward eleminate, but instead of aiming for a triangular method, eleminate every column element except the one in question from that column. Just like the method by hand – typically.

There is also the concept of flops. Know definition, with GJ, more flops are needed than GE

===
chapter 10
LU Decomposition
only operations with matrix coeffs, also if you have many right sided bectors [B] you just need to solve one coeff matrix[A].

U is pretty stright forward. just forward elminate until you get a triangle matrix
to get L, you need to put in the f-factors and use L as its holder. diagonals are all 1's, and
f12 = a21/a11,
f31 = a31/a11,
f32 = a'32/a'22

[A] --> [L][U]
[U] = [a11,a12,a13;
0,a'22,a'23;
0,0,a''33]

[L] = [1,0,0;
f21,1,0
f31,f32,1]

SUB decompose(a,n)
DOFR k = 1, n -1
DOFOR i = K+1,n
factor = a_1,k / ak,c //???
a_i,k = factor
DOFOR j = k + 1, n
a_i,j = a,j - factor*a_kj
END DO
END DO
END DO
END Decompose

Croute Decomposition
aka. Doolittle decomposition, factorization

---
Calculating the inverse
Can do this with LU method
ch 10 or something 11 special matrices

Banded Matrices have values only in diagonal? There is BW and HBW, BW = 2*HBW+1
Everything below is a 0, and thus algorithms will try to pivot the hell out of them; however, this is useless.

Thomas algorithm is an efficient method
Cholesky decomposition : [A] = [L][L]T
This method requires positively defined matrices to avaoid round-off error some

Gauss-Seidel method
iteratin method used to approximate the soln, method converges on true soln
ex 11.3, try 0,0,0 as a starting guess
|ea,1| is a means to estimate error
Jacobi iteration
same as G-S method but rather than using the latest available x's, this technique uses an equation to compute a set of new x's on the basis of a set of old x's. So as new values are generated they are not immediately used but rather are retained for the next iteration. Not sure what this 'new eq' is. Seems like it's just using the initial guess, and saving new outputs. Start with 0 again?

Convergence criterion for GS method: similar in spirit to simple fixed-point iteration
  • 2 fundamental issues. Sometimes non-convergent, if it does, it does so slowly maybe
  • partial derivative thingy.. basically the absolute values of the slopes must be less than unity to ensure convergence, so if a 2x2, then |a12/a11| < 1 and |a21/a22| < 1, but this is a sufficient and not necessary condition for congergence...
overrelaxation is designed to accelerate the convergence of an already convergent system. Aka successive or simultaneous overrelaxation or SOR
//interesting fact, they're calling the error check a variable "sentinel"
--sleep...

Thursday, October 4, 2012

The Ambiguity of Voting UNLV 2012

A little ambiguity goes a long way.

Considering my lack of politician participation in about everything, I find myself wondering why I get involved sometimes. Of course, I would rather see some senators be seated over others, so I suppose there is an inclination to vote. And indeed, I am an advocate of a "fair vote". There's a case to make for this here.

My voting experience today consists of this little story:

It's to my knowledge that the undergraduate college at the University of Nevada, Las Vegas has moved from using an older platform known as OrgSync to one with greater functionality dubbed collegiatelink.net. I haven't explored it entirely yet, but all of our data from the previous site has been ported over. This was done during the summer or sometime.

One of the appealing functions of link.net was the secure voting and it's here where a question I have arises. At UNLV, we're on our 2nd day of student government elections. I just voted, and here are 3 screen shots I would like to share with you all.

photo 1.0

photo 1.1

photo 1.2

[At this point let me state that I've always been a fan of the "None of the Above" option, and that I very much dislike my state for vanquishing it this upcoming election.]

I wanted to know if anyone was only getting 2 pages in their "ballot". With that stated, notice how I'm forced to vote for 6 out of the 8 Rebel Yell advisory board members.. I'm being forced to select these people. To be honest, the main criteria was on how long their last names were. But, also notice how I was given the option to vote for these members twice.

Now notice how I'm actually voting for 5 senators, 2 from one, and 3 from another college. Apparently my self and others like me are more special. Not only does our voting get counted more than yours, but I was asked (on two separate occasions) to vote for these [RY] board members. Or more like, forced to vote for some of them twice! Now I don't remember, but now I'm wondering if this system would have allowed me to click on submit without having everything checked on as required. It's doubtful, seeing as "required" is placed up there.

What should I make of this? I'm not too sure at the moment. I'm an undergraduate majoring in two degrees within two different colleges at UNLV. Is this voting system really fair? And how many of these votes actually get counted? I believe today is a day to admire the intelligence of those that call themselves "ballot creators." It's a user error, or maybe we should blame link.net?