Microbiology EXAM #3
Who discovered the lysozyme?
Does the lysozyme work best against gram (+) or gram (-)bacteria? Why? And how does it work?
Gram (+) bacteria because of the muliple layers of peptidoglycan. It weakens the net by cutting the NAM-NAG chain. The cell wall keeps the membrane from bulging out. Lysozyme will weaken wall & evenually the membrane ruptures: cell lysis.
Why does no one take lysozymes as a drug?
B/c it's so much bigger than penicillin. The MW of it is 14,000 compared to Penicillin at 375.
T or F. The lysozyme is the most protective element of tears.
Describe how the lysozyme affects gram (+) and gram (-) bacteria.
Gram (+): lysozyme attacks the first layer of peptidoglycan.
Gram (-): lysozyme doesn't get down to peptidoglycan layer since it's single layered and has outer membrane layer.
What is the genus of pink eye and is it + or - bacteria? Describe how lysozymes affect it.
Hemophilus. Gram (-). Lysozyme can't get down into peptidoglycan layer b/o of outer membrane.
What is an antibiotic?
a soluble substance produced by microorganisms that can be used to inhibit the growth and/or kill a different microorganism and is effective in minute amounts.
What is a genus/species of a penicillin Fleming worked with?
What is ampicillin?
Belongs to a class of penicillin's that are used for treating bacterial infections. The cells begin to round up and they rupture; weakens the cell wall. It is a broad spectrum drug so it words on a variety of things instead of just gram (+) cocci.
It only interferes w/ the cross linking enzyme (the bridge), NOT (NAM or NAG). It doesn't affect the previously cross linked bridges and ONLY affects new cross links. The cross links won't form, bacterium will continue to grow but can't link them, net gets weaker, membrane ruptures, and everything goes out.
So, penicillin ONLY hurts growing bacteria. It won't destroy existing walls.
T or F. Lysozymes and penicillin have trouble getting into gram (-) bacteria wall?
What is the stucture of penicillin?
*** know this. Know the the 4 membered ring called B-Lactam ring. Changing R groups changes penicillin.
Is methicillin penicillanase-resistant? What is penicillanase?
Yes. Penicillinase is a bacterial product. Methicillin folds over so penicillinase can't break it. So, if it's penicillinase-resistant it breaks the B-lactam ring.
T or F. Natural penicillin G (+ cocci) & V are natural by mold.
T. Natural form is cheap but some are expensive.
T or F. Semisynthetic pencillin you put R group on it.
T. The R group wraps around to protect B-lactam ring.
What are two B-lactam antibiotics?
Penicillin and cephalosporin (1st generation (old), 2nd, 3rd (newest). Know cephalosporin structure just in case. Has 2 R groups to play w/ so it's more expensive.
What ramifications are there since the B-lactam ring is reactive?
1. The ring inactivates enzymes in bacteria. 2. It modifies human proteins.
What is transpeptidase?
Enzyme that forms cross-link. Penicillin attaches to enzyme- enzyme is too heavy to form crosslink so it can't make anymore. If bacterium has penicillinase, it won't hurt transpeptidase.
What is the term called when the immune system scans cells and notices they aren't normal cells and responds against it?
An allergy; only happens to 5% of people.
t or f. It is the B-lactam ring that is reactive, not penicillin.
What are 5 ways to kill bacterium (targets)?
1. cell wall drugs- peptidoglycan synthesis
2. cell membrane
3. protein synthesis
4. nucleic acid synthesis
5. metabolic pathways
Note* Humans have all EXCEPT cell walls so no side effects.
What is the genus/species of the antibiotic penicillin?
Penicillium chrysogenum. This produces penicillin.
What is the name of the main genus that produces the MOST antibiotics?
Streptomyces. This is the most common microbe.
What is Streptomyces habitat? Describe it.
Natural habitat is the soil, where they may constitute 1 to 20% of the culturable population. The odor of the moist earth is largely the result of Streptomycete production of volatile substances such as *geosmin*. It plays a major role in mineralization. They are flexible nutritionally and can aerobically degrade resistant substances such as pectin, lignin, chitin, keratin, and latex.
What are 4 important features of Streptomyces?
1. Doesn't cause human disease (very rare); not pathogenic.
2. produces A LOT of the antibiotics we use
3. the antibiotic is good against fungi, molds, and human tumor cells.
4. antibiotic (Ivermectin) good to kill tapeworms, roundworms, and hookworms
The organism Streptomyces peucetius produces what antibiotic?
Daunorubincin- used to kill cancer like breast cancer.
what is the spectrum of activity? What two categories is it divided into?
It means which group of organisms does it work with?
1. Broad spectrum: (Tetracylines) works on a lot of microbes
2. Narrow spectrum: (Isoniazid) works on few microbes.
Penicillins are best used against gram (+) or gram (-) bacteria?
t or f. Broad spectrum drugs do have disadvantages because bacteria become resistant when you use drugs too much.
T. Antibiotic resistance is basic evolution: anytime the drugs are used, survivor germs can emerge stronger and spread. That's why antibiotics are supposed to be used carefully, picking the best one for each bacterial infection- & NOT using them against viruses that they can't fight.
What is the top concern for the medical community?
What are 2 reasons why microbes make antibiotics in the first place?
1. antibiotics are weapons that confer an advantage to the secreting organisms in their struggle for survival. NOT the major reason. It takes a lot of energy to make an antibiotic. For example, Tetracycline, is the end result of 72 seperate enzymatic steps.
2. Some research shows that these antibiotics are used naturally to communicate w/ relative. Used for ecological purposes!
How do the Pharmceutical companies get antibiotics?
A) We start w/ soil--> innoculate--> Isolate pure culture in slant.
B) antimicrobial activity testing: Put a single streak of bacteria and grow it. Put other bacteria perpendicular to line streak and see if they grow or don't (something is toxic).
C)Production and concentration of antibiotic material: if antimicrobial activity is present, the culture is grown in laboratory fermentation vessels to produce larger amounts of the antibiotic for further testing.
D) Toxcity testing on animals
E) Vials of purified antibiotic from a large piolet plant fermentator
When referring to gram (-) bacteria, they have an outer membrane layer that contains what?
Porins. Gram (+) bacteria don't have this. The bacteria can shut off pores/pump back out if they don't like something.
What is the long tails coming out of the membrane called? What is it composed of?
Lipopolysaccharide (LPS layer). It is composed of sugar molecules coming out of the membrane. Going into the membrane is the lipid portion (inside). Carbohydrates to the outside and lipids in the inside.
T or F. Different strains of bacteria have different O-chain sugars.
What is the chain called right in the middle of the O-chain and lipid part?
Core sugar chain.
What part of the lipopolysaccharide is very harmful?
The lipid A part. It is the killing feature of many gram (+) bacteria.
Lipid A is also called what? What happens if you inject LPS in the blood of a human?
gram (-) endotoxin. They would die of endotoxin shock.
Endotoxin comes from gram (-) bacteria. Where is it found?
It's present in LPS of outer membrane of cell wall and released only w/ destruction of cell. When you kill the bacteria, it releases endotoxins. We need to inhibit it from growing (don't kill it) and hope that your WBC's fight it off themselves.
what is endotoxin's effect on the body (Pharmocology)?
Causes fever, weakness, aches, and shock
What is endotoxin's heat stability and toxicity level?
It is stable in heat and can w/stand autoclaving (121C for 1 hr). It's toxcity is low but you can get a lot of this in a short period of time and this can kill you.
Endotoxin is fever-producing. Is this a major characteristic of gram (-) bacteria? What is the lethal dose?
Yes! The lethal dose is considerably more (easy to get a lot in a short period of time).
T or F. Proteins are the best antigens to make a vaccine against and gram (-) bacteria are hard to make a vaccine against since they aren't proteins. Ex. bladder infections or E.coli.
What are two examples of gram (-) bacteria diseases?
urinary tract infections and meningitis
What is the animal called where we get the reagent to test people for endotoxins? What is the animal's genus?
The horseshoe crab. The genus is Limulus. It is the only known animal known to have this reageant. they get the crabs blood and put them back in the ocean.
Name 2 tests that have microbial origin?
AMES mutation test and Horseshoe crab reagent (for endotoxins).
What is the test called to test for endotoxins?
LAL. Designed to determine whether endotoxins are present. If it clots, endotoxin is present.
It is a state law that requires mandatory meningitis shots when students live on campus at college. Where does the bacteria live in and what is the damage after being infected?
Lives in the human throat ( only place we know it is carried). A person may lose fingers, toes, legs, arms, and have brain damage.
Out of the 5 strains of meningitis, how many does the vaccine prevent?
4 of the 5.
What is the genus/species of meningitis and is it gram - or gram + pair of cocci?
Neisseria meningitidis. Gram (-). Also had pili (hair-like structures)
Why is this gram (-) bacteria rare?
B/c it packages up endotoxin and releases it into blook w/out being killed. It can get into the cerebral spinal fluid in the brain and if too much swelling occurs it causes death.
What is the generation time when E. coli grows the fastest? What is generation time?
20 minutes! The generation time is starting w/ 1 cell which takes 20 min. to produce 2 cells. After 20 min. more (40 min total) those 2 cells each produce 2 cells = 4cells. After another 20 min. more (60 min total) = 8 cells.
What is the optimum generation time for Staphlococcus aureus? (the time is can grow the fastest)
Describe the synthetic medium for growth of E. coli.
It can grow on a single carbon source like glucose. It's capable of growing on simple medium but won't grow fast. If you want your bacteria to grow fast, already give it amino acids (20), different types of vitamins (7), and lots of carbon sources.
How many tons of E.coli would grow in 24 hours from 1 bacterium?
T or F. When a bacterium is growing in a culture medium, its exponential phase is relatively short. it can be limited by nutrient depletion, oxygen deprivation, the accumulation of inhibitory products.
what is the 4 phases the bacteria grow?
1. Lag phase: just getting use to the medium
2. Exponential growth phase: double in growth
3. stationary phase: run out of something or build up of waste products
4. Death phase
What are ways to count bacteria?
1. direct microscopic count of bacteria
2. coulter counter: as cells pass through device, they trigger an elctronic sensor that tallies their #'s. This is pretty expensive. We used the spectromatemeter in lab which measured optical density.
Know page 40 in lecture handout.
Know 3 reasons why curve C levels off after the logarithmic phase?
1. ran out of nutrients. 2. build up of waste products. 3. oxygen deprivation
What ink is used to perform a capsule stain?
T or F. The capsule is a sugar coating over the cell wall and it can be well uniformed or sloppy mucoid form (slime layer).
What is the difference in the slime layer?
More than one bacteria is embedded in the slime. The slime is also called glycocalyx or extracellular polysaccharide.
T or F. Capsules are composed of protein. The immune system doesn't do a good job against these capsule surrounded bacteria. It's hard to get rid of them compared to sugar coated bacteria.
F! Capsules are composed of sugar. It's hard to get rid of them compared to protein coated bacteria.
What is the vaccine called against Streptococcus pneumoniae? What is it composed of?
Pneumovax(R) 23 *Side note, the (R) is the the suppose to be the circled r for copyright purposes. It is a mixture of purified capsular polysaccrides from the 23 most prevalent or invasive pneumoncoccal types of S. pneumonia. It isn't a required vaccine and is mainly given to children and the elderly. So, basically the vaccine is giving you capsules from 23 different types of S. pneumoniae.
What is the cause of meningitis?
A yeast that has a capsule. The capsule greatly enhances the organism to kill you.
Why does the yeast have a capsule?
It lives in the soil where predatory organisms, like amoebas, are present.
In the slime capsule, is 1 or many bacteria embedded in the slime?
More than 1 bacteria is embedded in the slime.
WBC's (macrophages) look for bacteria to engulf. If they attach and engulfment takes place what phenotype is this? what is the phenotype where no engulfment takes place?
Non-mucoid phenotype. Mucoid phenotype.
Do we drink the treated water that comes out of the waste plant?
No, even though it is treated.
Study sewage processes in lab exercise #4
What is the aeration tank?
Intentionally grow microbes in here to eat what's left. They also intentionally pump air so there is oxygen and they can efficiently break stuff down.
Ater the aeration tank, where is the water sent to?
The settling tank. The stuff settling is called activated sludge (purely microbes).
What comes after the settling tank?
The sludge digester-microbes are killed and they get rid of them in places like landfills.
Concerning the waste water treatment in San Angelo, how many gallons are pumped per day and what is the cost for the electrical bills?
13.2 million gallons. $580,000 for electrical costs.
T or F. Activated sludge is forcibly aerated w/ compressed air, and stirred vigorously. The bacterial cells grow as large flocs in digester and the flocs settle to the bottome of the tank. The protozoa then eat the bacteria, #'s of protozoa go up, bacteria #'s go down. The effluent, low in organic matter, is chlorinated and dumped.
What is the slimy coated bacteria called?
What can best eat the zooglea?
Crawling and stalked ciliates.
Where was the activated sludge process invented? Describe it.
England in 1913. No other process has more advantages. It is a biological process that is a very simple concept. Formed by aerating a degradable waste for a period of time until a large mass of settable solids form. It is by far the most widespread and the largest industrial application of continous microbial culture.
what are the 3 differences compared to industrial growth?
1. the objective of activated sludge process is to degrade (not create). All others you are trying to make something. (Ex. yogurt; penicillin)
2. Act. sludge is the most largest contaminated culture.
3. No one ever made any money out of activated sludge.
What are the 3 essential requirements for act. sludge process?
1. mixed population of aerobic microbes must be able to degrade the noxious components of the waste.
2. pop. must be able to grow in the envir. of the aeration tank.
3. must grow in such a form that they will settle out.
Describe the formation of a floc.
As bacteria begin growing, they develop into chains or clumps. They are active and motile and it's diff. for them to settle. They haven't yet developed the slime layer which aids in their sticking together. When more slime is produced, the clumps grow bigger and bigger until they form a floc. When aeration is started, the food/microbes ratio is very large. The bacteria grow 1st then protozoa 2nd.
Describe the growth of microorganisms.
We depend upon bacteria to use organic material and protozoa use bacteria for food. Crawling ciliates & stalked ciliates don't use as much energy. Filamentous bacteria stick to zooglea (slime). Amoeba-->flagellates-->free-swimming-->crawling & stalked.
Microbes produce adhesive material that make them stick. What can stick to your cells and are sticky when they reach a surface?
What is the slime material called that a whole colony lives in?
Who was the first to realize biofilms were important?
J. William Costerton. "If biofilm bacteria were simply sticky cells that stick to a surface it would be important but found at least 500 times more resistant in film than out of film, need to get them before they do that or antibiotics won't help it."
What is a biofilm? What is free planktonic growth?
1. matrix enclosed bacterial populations adherent to each other and/or surfaces or interfaces.
2. free surface attachment
What is (attached) sessile growth? What is the extracellular polysaccharide?
1. attached to a surface
2. extensive slime layers composed largely of carbs & H2O.
What is pioneer organism? What is quorom sensing?
1. one that came to surface 1st. After a while many join- consortium: join pioneer.
2. regulatory pathways in bacteria that respond to population density; ability of microbes to know they have enough members & know they can make a community- like a meeting.
What is a classic biofilm example?
plaque on teeth. If you brush everyday you don't get enough for form a biofilm. Once formed biofilm is almost impossible to get off.
In your bathroom, vast amounts of bacteria can be found on what?
Shower curtain-has a film of scum on it.
The steps how bacteria adhere to a surface.
Planktonic cells-(everybody for themselves) There is a surface that 1 of them have an attraction for (Glass, metal, etc.) Net charge of (+),bacteria w/ net (-) charge.
Cells attach to surface (+ to-). Particular genes turn on when they are on a surface (those genes don't work when not on a surface). Cell can produce pili (hair-like protein on surface of bacteria) enable bacteria to solidfy attachment to surface.
Show capsular material. Product of genes that bacteria start to activate in contact w/ a surface. Building up adherence
Irreversible adherence. They have an altered phenotype.
Community development. Have a stronger protection as a community.
T or F. When a biofilm reaches a maximum density (quorum), cells communicate through signaling inducer molecules & turns on quorum-dependent proteins.
F. minimum density
What are some human infections involving biofilms?
contacts, catheters, tooth decay
What is the inherited genetic disease where bacteria gets into the lungs and starts releasing a biofilm. Describe it.
Cystic fibrosis pneumonia. When P. aeruginosa gets into the body it turns on a gene to enable it to produce slimy materia when it gets in fibrosis patient. People could live w/ this condition if it wasn't for microbes. The people die of microbial infection. This disease leads to very stick mucous and comes from a recessive trait from both parents. The bacteria attach to mucous in lungs when breathed in. Antibiotics don't work when P. aeruginosa produces slime from gene b/c they can't get through the slime (biofilm). Anitibiotics work only when the bacteria is in the planktonic phase-not biofilm stage. Eventually your WBC's/immune system kills your lung tissue.
What is the seaweed secretions of compunds called that ward off bacteria?
What is the antibiotic called from a microbe that works against P.aeruginosa when it's at free living form (not biofilm form).
T or F. Furanone by itself isn't toxic. It keeps bacteria from communicating w/ each other so the tobramycin can kill them when the two make a compund.
What is the procedure called to figure out how old something is?
T or F. If something has a biofilm, it can alter your results if you are testing something below the film. You can't trust carbon dating if it has a biofilm on it because they are VERY hard to remove.
What is the term for hair-like structures that are smaller in diameter/length than flagella and there is a lot more of them?
Pili- The British used the term fimbriae. The pili help bacteria attach to a surface better.
What are 3 important virulence factors concerning pili?
1. they mediate horizontal gene transfer
2. adhesion to host mammalian cells
3. can extend & retract; get bacteria from 1 plane to another.
What is a prime example of a bacteria having pili?
Neisseria gonorrhoeae (cocci non-motile). They move by pili attaching to male sperm.
E. coli mainly lives in the GI Tract and is the #1 cause of what?
Bladder infections. Cystitis is a term for bladder infections.
What was the group of 60 students called that drank E. coli w/ pili or no pili?
Sensational sixty. The E.coli strain that have the pili can attach to the urinary tract and these students had the symptoms- not the E.coli w/ no pili.
What juice causes pili to curl up and can prevent urinary tract infections. What is another fruit that prevents urinary infections?
Cranberry juice. Also, blueberries do too. They don't cure they just prevent.
What are the 2 kinds of pili? Describe.
1. sex pilus: allow bacteria to transfer its genes to another bacteria by bacterial conjugation. The sex pilus doesn't pass genes. It hooks the bacteria and reels it in so bacteria are wall-to-wall and gene transfer can happen.
2. non-sex pili: adherence structures. Horizontal gene tranfer. Examples of this is transplanting tissue or gene therapy in humans.
What did engineers find out about pili?
Electricty runs through pili. The bacteria send electricial signals to each other when pili touch. More pili means more electricity that can be sent.q
What is the 2 arrangements of flagella? Flagella are organelles of locomotion; no cilia.
1. polar arrangement- flagella coming off short end of bacteria.
2. peritrichous- flagella come off all sides of bacteria. E. coli is peritrichous. Important to not E. coli has both flagella and pili.
What enviroments do bacteria w/ flagella live in?
Liquid enviroments. This is why E. coli is a common cause of bladder infections b/c it has flagella. Bacteria don't wave their flagella. They are actually rigid structures. They are the only known structure to to rotate on a bearing!!!!
What is the term that means movement in response to a chemical (away or towards)?
Chemotaxis. Bacteria have sensors that they can move to or away from things.
Know prokaryotic and eukaryotic. What does the term geonome mean?
Pro- bacteria w/out nucleus.
Eu- w/ nucleus
Geonome- all of the genes you have
Pro- all genes they have are on 1 circular molecule of DNA
Eu- genes spread out b/w several DNA molecules (multiple linear DNA molecules)
Pro- no coating of protein (no histones)
Eu- have complexed w/ histones (coated w/ histones)
Amount of DNA
T or F. Bacteria have a nucleoid (region of DNA) and no nucleus. People want to mess w/ bacteria that have plasmids and not circular DNA b/c there is no end and start to DNA arranged in a circle.
How many genes in regular bacteria, E.coli, and humans?
Reg. bacteria: 3000 genes
E.coli: 4289 genes
4,600,000 base pairs
Humans: 23,000 genes/set of chromosomes
3,000,000,000 base pairs
Humans have alot more DNA than bacteria.
Describe a plasmid
Circular, double stranded, extrachromosomal DNA.
1-20 copies per cell
Self-replicating by the same mechanism that any other DNA uses to replicate. They don't depend on chromosome to tell them when to replicate. Generally, plasmids carry genes that code for functions not essential for cell growth; the chromosome carries the genes that code for essential functions.
What is binary fission, mitosis, and cytokinesis?
binary fission: Bacterial reproduction happens by this. 1 cell turns into 2.
mitosis: division of the nucleus. Doesn't occur into bacteria b/c they don't have a nucleus
cytokinesis: cell division
What are 5 different plasmids?
1. F plasmids: have the gene for making pili
2. resistance (R) plasmids: have genes resistant to antibiotics and to heavy metals
3. bacteria-killing proteins
4. virulence plasmids: cause disease signs and symptoms
5: tumor-inducing plasmids cause tumors in plants
help transfer genes
Steps in plasmid replication
a. replication independent of chromosome
b. plasmids can stop replicating but cells can keep replicating. Why stop? B/c it cost that cell energy.
c. conjugation:cell-to-cell transfer
d. integration of plasmid into chromosome
You can transfer plasmids into non-relatives and they depend upon living cells to replicate.
What is the selfish gene theory?
Spreading independently of the species they originated in confirm their place as evolution's unit of replication.
Describe the DNA of E.coli.
lac stand for lactose- has to do w/ some of our experiments in lab like the H2O analysis. Lac ables the bacteria to break down lactose.
Lac operon (A,Y,Z,I)- lactose degradation.
Z gene- makes B-Galactosidase
T or F. E. Coli has an area on it's chromosome coded w/ lac and when people get older, they can turn off the lactose gene & E.coli will ferment lactose & give you all sorts of gas- lactose intolerance. Lactose is a sugar derived from milk products.
What is lactose made out of?
Galactose + Glucose (Disaccharide)
What is the objective of glycolysis? Do humans and bacteria both do glycolysis? Describe the glycolysis cycle.
To attain energy! (2 ATP)
Start w/ glucose and make pyruvate acid. From this point on it depends on the bacteria's genes to take pyruvate acid and made whatever end products from it. If you start w/ the same sugar you will get diff. end products from diff. bacteria.
Describe human glycolysis.
Humans-->sugar-->breaks down to H2O and CO2 (bacteria have a lot more diff. end products)
In the lab experiment why did the blue and white colonies both grow together?
b/c blue colonies broke down ampicillin so white colonies could then also grow.
What gene shows the blue color?
lac Z gene - reporter gene
What is one end product of E. coli?
H2! If we get gas in our lab experiment lactose broth, we can say it's probably E.coli b/c not many organisms can break down lactose. So it is a (+) presumptive test. And it is a (+) confirmed test if coliform colonies form.
E. coli is an example of coliform bacteria. Describe the total coliform test and fecal coliform test.
Total coliform test: all ferment lactose @ 35C. Uses enzyme name B-Galactosidase.
Fecal Coliform: If incubated @ elevated temp. of 44.5C designate fecal coliforms. Less numerous but inlcude E.coli.
What is the best indicator of fecal contamination?
E.coli. Gram (-) rod shaped bacteria
What are the regulations for drinking water? What are indicator organisms?
Fecal coliform in drinking H2O = 0 per 100ml H2O.
Allowable levels in recreation h2O = 200 colonies per 100ml H2O.
Just indicate there may be fecal material. They predict a health risk
1. organsim is associated w/ intestinal sources of pathogenic organism
2. must occur in high #s than pathogen
3. High resistant to envir. stresses
4. must not proliferate
5. reliable and inexpensive methods for detection
Describe San Marcos lake levels
It was shut off to the public because fecal coliform levels b/w 800 and 1700 colonies per 100 ml H2O. TX health standards are 200 colonies or less. Note that India has high fecal matter
Describe the operon
Nobel peace prize in 1965 by Monod. Opened up the field of gene regulation. Monod was analyzing the properties of the B-galactisidase gene. The operon is a collection of linked genes under common coordinate control. The lactose operon is the most intensely studied genetic regulatory system and is useful in genetic engineering.
List the 3 genes that come on/go off.
1. B-galactosidase: splits lactose into galactose and glucose
2. permease: transport protein
3. transacetylase. Note that the first three go together.
4. repressor: control gene. In charge of other 3 genes.
When lactose is present it can pull the repressor off. It no lactose, repressor stays blocked.