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Microbiology Lab final


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three methods for genetic recombination
Conjugation, transformation and transduction
direct passage of DNA (microbe sex)
the uptake of naked DNA from the environment
is mediated by ciruses that inject genetic material into the cell.
Conjugation can only occur between
F+ to F- cells. F stands for Fertility Factor.

Once a F- recieves the DNA it becomes Hfr (High frequency recombinant) and can now act as a F+ and transfer DNA.
In conjugation the whole chromosome (is/is not) transfered
How did we show bacterial conjugation in lab.
We had Hfr cells who were sensative to streptomycin. and F- cells who required three amino acids. Then we provided a media without the three amino acids and with streptomycin (nothing should grow) unless conjugation occurs.
Before bacterial cells are to undergo transformation (uptake of naked DNA) they must first be made ____________. 2 exceptions
Bacillus subtilus and Streptococci pneumonia
How dowe makee.coli comptent?
place cells in COLD calcium chloride followed by heat shock. this process facilitates transfer of DNA across the membrane
In the bacterial transformation lab how did we prove that transformation occured?
The original "competent cells" were placed in two tubes (control- no plasmid) and experimental (with plasmid). recall the plasmid was for ampicillin resistance. Now both were plated on ampicilian plates and only the experimental e.coli grew because they had transformed and incorporated the ampicilin resistant plasmid into thier own DNA.
How did we extract a plasmid?

(many chemicals involved)
Cell must first be lysed (lysozyme)
The membrane must be disrupted (SDS, NaOH)

Now you have the plasmid you need it to reanneal (Potassium acetate)also the plasmid reanneals in soln while the rest of the cells DNA is caught in complex with K+ (recall DNA is -ve)

Lastly it is centrifuged and the soln (plasmid) is dried over isopropyl alcohol and later ethanol and allowed to sit in a buffer.
Restriction endonucleases
Naturally occuring bacterial enzymes that cut up any foreign DNA in the bacterial cell (serve as immune system)
What sequence do restriciton enzymes recognize and excise?
pallindromic ex. AATGCCGTAA
How are restriction enzymes named? (HIND III)
by the organism from which they were first isolated from

Haemophilus INfluenzae strain D 3rd isolated.
Why isnt water used for electrophoresis? what is instead used?
because deionized water does not conduct current and no movement will occur. A buffer.
Electrophoresis- DNA is negatively charged thus it
"runs to the red" positive electrode.
in electrophoresis how does one see the movement and what does the movement quantify?
Tracking dye is added to measure the displacement. it quantifies conformation, size and charge of the samples.
What results do we expect with electrophoresis of singly cut or doubly cut DNA?
The doubly cut fragments will have two marks in a single line. Also one should be much furthere than all other samples as it would be (at least 50% smaller than the rest of the samples). the singly cut should have one mark and not move to much from the negative electrode.
Restriction enzymes recognize what sequences?
palindromic (AATGCATT)
in gel electrophoresis why is water not used?
Because deionized water would not produce current and the DNA fragments would not move. Instead a buffer is used.
In gel electrophoresis how is it that we can see the DNA fragment moving across the gel?
We add a tracking dye (no longer use ethidium bromide)
what does the distance the DNA traveled quantify?
Recall DNA is negative and "runs to the red" (positive electrode)...

The distance it travels is dependent on its size, charge and conformation.
What results did we expect in the gel electrophoresis lab where we cut DNA singly or doubly with restriction endonucleases?
We would expect those that were cut twice (much smaller pieces) to have two different final stains in the same sequence and to have moved further than any of the singly cut fragments.
In the gel electrophoresis lab what was the point of adding a Lambda HIND III digest?
This is a standard fragment. the lengths are known and thus comparisons in displacement can be made relative to the known displaecments of Lambda HIND III.
How did we analyze food in MCB Lab? was this useful?
First we dilluted the food with water in a blender and plated extracts and counted the number of bacterial colonies. Not really useful because some of the bacteria are supposed to be there (f.e yogurt)
In the food analysis lab how did we confirm suspicion of pathogens in different foods?
we plated them for isolation of the suspicious organism:

E.coli---- EMB
S.aureus----- MSA
Bacillus----- heat and TSA
Clostridium---- heat + anaerobic conditions
Fungi------ Saboroud plate
In the food analysis lab why were foods suspected of having either bacillus or clostridium species heated before being plated?
because these are endosporing bacteria and by heating vegetative cells are killed leaving the organisms in question.

Also for CLOSTRIDIUM it was put in GASPAK where hydrogen is released and reacts with oxygen to form water and produces the anoxic conditions required for clostridium species to grow.
in the food analysis lab why did we used Sabouroud plates?
Sabouroud plates were used for foods expected to have fungal growth.

this is because the fungi love the carbohydrates of the plate and the acidic conditions.
in the food analysis lab why did we swab our noses and then perform a dilution and finally plate it? (hint: plated on MSA)
Wer were testing for Staph Aureus which is part of the normal flora but can be deadily if you are shedding it.
Fermentation is an industrial procedure done by microbes. (Mushroom pizza)
Dough rises because of fungi. Mushroom are fungi. the cheese is made by steptococci and lactobacillus from milk.
What is absolutely KEY to fermentation reactions? what did we make by fermentative process?
They are aaerobic processes (No O2)

Sauerkraut (fermented cabbage)
What technique other than fermentation was used to produce saukraut (Where microbes were used after one another to do different steps)?
Microbial succession. Lactobacillus and finally acid loving streptococci.
Production of wine is a fermentative process carried out by which organism
Sacchromyces cerevisae
Protozoans are (Prokaryotes/Eukaryotes)
protozoans are classified by
Protozoans have two life cycles
1.) Motile trophozoite
2.) non-motile cyst
What is the problem in treating patients against protozoans?
They are eukaryotes so standard antibiotics do not work. infact any pharmaceuticals that damage the parasitic cells may also damage eukaryotic host cells as well.
Entamoeba histolytica
amoebic dysentary (bloody diarrhea)

Asymptomatic carriers are the primary cause of spread
Giardia Lamblia
Cells coat intestinal lining and prevent nutrient uptake.
Cryptospodidium parvum
waterborne protozoan that usually only causes problems in immunosuppressed individuals.
Naeglaria fowleri
Found in lakes and moist soil

Enters through the nose and rapidly infects the brain and meninges.
Toxoplasma gondii
parasite from feces that can cross the placenta and causes brain damage to the fetus.
Pneumocystis carinii
Part of normal flora. but causes pneumonia in immunosupressed individuals.
Babesia species
Cause Texas cattle fever (transmitted by ticks)
Leishmania species
form ulcerative lesions on the skin may also attack internal organs. transfered by sand flies.
Trypsonoma Cruzi
causes Chagas' disease (from bug feces entering the eye. the disease is characterized by relaxing involuntary muscles such as the colon and esophagus.
Fungal infections are called? difficulty in treating fungal infections?
Fungi are eukaryotes and like protozoans antibiotics dont work and medication that does may harm the hosts cells.
the three fungal species we learned about were:
Amantia- death angel mushroom

Aspergillus niger- release black spores that cause bridge-painters disease.

Penicillium- gives blue cheese flavor and medication
to truly get an accurate view of a fungus, we should view _________________. so in the fungi lab we did what slide preparation? how?
THALLUS (whole body)

Henrici slide prep:
petri plate is lined with moist paper upon which a microscope slide is placed and a drop of Aspergillus niger is placed bounded on three sides by petroleum jelly and finally a cover slip is placed on top.
In the standard analysis of water it is unfeasable to detect all organsisms instead what is done?
The water is tested for indicator organisms whose presence suggests that water may be contaminated (E.coli)
The standard analysis of water tests the water supply for colliforms by a three-test profcess...
1.) Presumptive test
2.) Confirmed test
3.) Completed test
In the standard analysis of water, the presumptive test is the first of the tests. what does it test for? and what do its results suggest?
It tests for the fermentation of lactose. dilutions of water supply are made and based on the amount of gas produced from carbohydrate fermentation and the dilution facor one can estimate the # of colonies from the MPN table (Most probable number)
The second of the three tests for the standard analysis of water is the CONFIRMED test. What is done on this test?
On this test, we chose the tube with the largest dillution factor (but still w/ lactose fermentation) and plate it for isolation on EMB (recall e.coli will be metallic green).
The last of the three standard analysis of water tests is the COMPLETED TEST. In this test what is done? results?
Here a metallic green colony from the comfirmed test is used to innoculate a lactose fermentation tube and a nutrient agar tube for gram staining. this last step confirms that the organism is a gram-negative lactose fermenting rod.
An alternative method for testing water is the memrane filtration analysis of water where
water to be tested is passed through a membrane with holes large enough to allow water to pass but to small for bacteria to pass. the filter papers are then plated to check for bacterial growth.
In membrane filtration analysis of water a total coliform count was obtained by using what media?
TCC- all gram negative organisms

Media: M-endo (fuchsin and sodium sulfite) hinders gram positive species.
in the membrane filtration analysis of water a FCC (fecal coliform count) was obtained by using what media
M-FC agar plate. this plate contains aniline blue to inhibit gram positive and bile salts to assure any growth is a fecal organism.
in the membrane filtration analysis of water a FSC (fecal streptococci count) was obtained by using what media
KF agar- which contains sodium azide and inhibits growth of all genus except streptococci which will change the plate from purple to yellow.
what is a bacteiopage?
bacteria-eating virus (filterable particles)
how are viruses seen?
electron microscopy
What makes up a virus? why are they "not alive"?
virus is a piece of genetic material (ss/ds RNA/DNA) in a protein coat. They are not considered "live" because they cannot reproduce or function independently of a host.
Viral life cycle (5 stages)
A- Adsorption- attach to cell
I- infection- inject DNA/RNA
R- replication- take over processes
M- maturation- assemble its parts
R- release- lyse the host cell
in the plaque assay technique why is soft agar used for detecting viruses?
so nonmotile viruess can find hosts (bacerial e.coli cells)
Why dont antibiotics work against viruses
antibiotics for the most part attack cell walls. recall a virus is just capsid enclosed genome segment.
Osmosis is
the flow of water thorugh a semi-permeable membrane from an area of high solute concentration to low solute concentration.
the enviornement is said to be hypertonic to the cell when
there is more solute (less water) outside the cell than inside the cell. in this case water will move out of the cell and cause shrinkage of PLASMOLYSIS
when the environment is said to hypotonic to the cell...
there is more solute inside the cell than in the environment. in this case, water will move from the environment into the cell causing the cell to enlarge called PLASMOPTYSIS.
when the cell and the environment have equal amounts of solute and solvent this state is referred to as
3 methods of physically controlling microbial growth
1.) osmotic pressure
2.) heat
3.) UV light
Why does heat kill bacteria?
it causes permanent denaturation of proteins and enzymes.
how does UV light kill bacteira?
it is a form of radiation. it causes THYMINE DIMERS to the bacterial DNA. this causes frameshift mutation in the whole genome and ultimately death.
UV light kills bacteria by producing thymine dimers throughout its genome. what are two ways this is corrected?
1.) photoreactivation- when light of a particular wavelength stimulates enzymes to repair the dimers.
2.) excision repair- whereby restriction enzymes excise the damaged DNA and replace it with the correct sequence.
In testing the effects of osmotic pressure on microbial growth we used e.coli and s. aureus. in what conditions did these grow that the other couldnt?
E.coli grew in high glucose concentration.

S. aureus grew in high salt concentrations.
In testing the effects of heat on microbial growth nothing grew at 100 degrees but 2 organisms grew at 70 what were they?
Bacillus cereus formed some spores at 70 degrees and Aspergillus niger produced alot of spores at 70 degrees.
substances that kill bacteria are called
substances that inhibit bacterial growth
Harsh strong chemicals designed for use on inanimate objects
milder chemicals used to inhibit bacterial growth without hurting the skin
3 main ways antobiotics kill bacteria
1.) inhibit cell wall synthesis
2.) interupt ribosomal activity
3.) destroy cell membrane
What is a major problem now a days with using antibiotics? solution?
Alot of strains are resistant to them.

Synergism- using a combination of antibiotics seems to be effective.
how did we test the antibiotic effect on bacterial growth?
Kirby-Bauer paper disc technique. On Mueller-Hinton platea lawn of bacteria is grown and papers with antibiotics diffuse into the plate causing zones of inhibition which determine its effectiveness against the bacteria.
How is an effective dosage of antibiotics actually determined?
By using the Minimum Inhibitory concentration technique. where dilutions are done of the antibiotic and all tubes are innoculated. the tube with no growth and the minimum amount of antibiotics= correct dossage.
what were some of the antibiotics used in lab
WHy might alcohols test poorly as antibiotics?
while they do kill antibiotics they evaporate to quickly to ward off pathogens for any length of time.
Citrate test is only used for gram negative rods of which family?
Eneterobactericae... particularly it is to distinguish between E. aerogenes and E. coli.
If it is plate dilution take # of colonies X plate dilution

if it does not say plate dillution then take the dilution factor x .1 (pippete) X the number of colonies
ignore .1 ml were plated.
Microbes that are harmless maybe even beneficial and that live on the body and will not come off regardless of showering and scrubbing
Normal flora
Blood agar was used to grow bacteria that were part of normal flora. what is the advantage of using this media?
Blood agar is highly nutritious to bacteria. it grows the most fastidious bacteria.
In the normal flora lab we used the Sabouraud plate to grow FUNGAL species. Why use this media?
Fungi like the high sugar content and the low pH of the media.
In the normal flora lab we used the Chocolate agar plate to grow species. Why use this media?
Chocolate agar is media used for the most delicate bacteria for which even blood agar is to harsh (has been heated to detoxify heavy metals).
In the normal flora lab we saw chocolate agar used for the first time. What was the organism we were selecting for with this plate?
Neisseria species.
In the normal flora lab we saw the chocolate agar plate for the first time and incubated it in a closed container with a candle. What was the purpose?
Chocolate agar grows Neisseria species which thrive in microenviornments with high CO2 content and no O2 (acheived by the burning candle).
In the normal flora lab how is the presence of Neisseria sicca on the chocolate agar plate confirmed?
the species has the enzyme to OXIDIZE Cytochrome C. when in contact with the reagent tetramethyl-p-phenylene diamine, the reagent will turn dark blue-black if cytochrome oxidase is present.
Two organisms of ORAL FLORA that have been linked to both gum disease and heart disease (endocarditis)?
Strep salivarius and Strep mitis
The degree of dental caries depends on ... (what test is done?)
the acids in the mouth... snyder test
The snyder test is a tube containing...
acidic medium and glucose it is innoculated with saliva and the indicator will turn yellow as the microbes ferment glucose. the quicker the fermentation the more likely the patient has dental caries. NOT TRULY ACCURATE.
In the oral flora lab, besides the snyder test we used the TSY20B media plate (?) to isolate which bacteria?
Strep mutans.

TSY= yeast extract
20= 20% sucrose
B= bacitracin (inhibits other strep species)
Staph epidermidis have caused problems where doing what?
In hospitals on plastic prosthetics and heart valve replacements. Virtually impossible to eliminate from the O.R.
Staph saphrophiticus causes what in humans
Staph aureus causes what in humans
A list of things including:

septicimia, endocarditis, acne, food poisoning
What are three properties of Staph aureus that make it standout as a staph species?
1.) it can hydrolyze DNA as a nutrient source.
2.) can coagulate blood "coagulate-positive"
3.) rapid resistance to antibiotics.
Staph saprophyticus (recall causes UTI's) its test results are exactly the same as ___________________ except that it...
Staph epidermitis... except that unlike epidermitis it is resistant to novobiocin.

S. aureus- sensative
S. epidermidis- sensative
S. saprophyticus- resistant
DNA agar plate was used in lab to isolate which species? why is it used?
Staph aureus;
it can hydrolize DNA as a nutrient.
Mueller-hinton plate was used in the lab. why was it used?
it was used as a rich medium to grow almost any bacteria. it was actually used to determine antibiotic resistance.
Steptococci species are broken down into how many groups.
How were strep species originally classified into groups. Now how are they classified?
They used to be classified according to their hemolytic ability on blood agar plates. Now, they are classified by the antigens on their cell surfaces.
Group A steptococci

- example organism
- how do we identify it?
Strep pyogenes

strep throat, tonsilitis, scarlet fever

Group is sensative to bacitracin (recall: would not grow on TSY20B plates)
Group B Strep species

- identified by what test?
they produce beta hemolysis on a particular Staph aureus species.

CAMP speceis produce an arrowhead shape on blood agar and completely hemolyze over s. aureus' hemolysis.
Group C strep

- cause
- can be identified by?
sore throats and fever.

Sensative to synergism with the combination of (trimethoprim and sulfamethoxozole).
Group D strep

- some are enteric organisms
- what media do we use to test for these?
Bile esculin plates. the high salt assures only strep will grow and may or may not be able to hydrolyze esculin.
Strep pneumoniae

-on blood agar has what hemolysis?
- identified by what stain
-identified by what test
- alpha hemolysis (most are betta)

- Quellung
- Mouse virulence test

- in what conditions does it thrive?
intestinal distress

gram negative rod

low oxygen concentration, high temps
Vibrio cholerae

-why is it so dangerous
- massive diarrhea
-produces an exotoxin causing loss of fluids
Borrelia burgdorferi

pennicillin or tetracycline
Clostridium botolinum

-dangerous because
gangreen and tetanus
produces exotoxin
large amounts of antitoxin
Neisseria meningenitis
and Neisseria gonorrhoeae

- morphology
meningitis or gonorrhea
gram negative cocci (paired)
antibiotics (silver nitrate)
the enterotube is an example of a ________________ system. it is primarily used for organisms of the family _____________________.
Multitest system.
On day two of the Enterotube lab we used what reagent and why?
Kovac's reagent for the indole test.
Why might the enterotube not give accurate results?
The amount of organism getting to each compartment can not be assured.
if a patient comes to you with acne and asks you to run an enterotube on a culture from the acne. would you do it?
NO. enterotube tests for enterobactericae species. These enteric species cause problems such as food poisoning and diahrrhea but not acne. Acne is most commonly cauased by staph species (specifically s. aureus)
In the use of the bactercult tube lab we tested for organisms which commonly cause what?
A bactercult tube consists of what?
plastic container with media on the walls. The media has lactose, urea and phenol red indicator
The bactercult tube results gave organisms divided into three classes. what were they?
Group 1- fermented lactose and acidified the media (yellow)

Group 2- do both or neither (orange)

Group 3- break down urea (pink)
Besides grouping into one of three classes, the bactercult test also had another test. what was it? results?
A colony count.

< 25- no UTI
25-50= suspicion of UTI
> 50 = indicates infection
2 types of immunological rxns
1.) agglutination- where INSOLUBLE antigen complexes with antibodies

2.)Precipitin rxns- where soluble antigen and antibodies result in ppt.
"foreign body"
proteins made by the immune system whose function is to bind to and destroy antigens
Why are we "immune to reinfection"?
because once the body has undergone an immune response memory cells "remember" the strain and upon infection, antibodies are quickly made.
What is the ouchterlony plate and what is its purpose?
A plate of agar with wells where antigens and antibodies are placed. The solutions diffuse through the agar and when they meet they either produce a ppt (precipitin rxns) or not. This proves whether an antibody is affective against the antigen placed in a well.
The process of converting proteins to Ammonia is called
The conversion of ammonia to nitrite and the subsequent conversion to nitrate is called
the conversion of nitrate to gaseous divalent nitrogen is called
Two forms of nitrogen fixation. what are they? and give a modern organism?
1.) Symbiotically- rhizobium

2.) Non-symbiotically- azotobacter
How does symbiotic nitrogen fixation occur?
First microbe infects a plant and the symbiosis will producenitrogenous compounds for the plant to use.

Then a nodule is formed and the bacteria enters the plant. Now a bacteroid and is able to fix nitrogen.
Non-symbiotic nitrogen fixers
usually live around roots but do not require roots to fix nitrogen. they contain specialized cells called heterocysts which contain enzymes for fixing nitrogen.
in the nitrogen cycle lab we add Nessler's reagent during the second lab this is to test for the presence of... which would indicate...
Ammonia. LIGHT (little ammonia) DARK (alot)

The amount of nitrogen fixation that went on.
what component of air is bound by leghemoglobin?
Oxygen in nitrogen fixating bacteroids.
What bodily fluid should be tested with the bactercult tube?
Urine (it is a test for UTIs)
List four ways S. aureus is different from S. epidermitis
1.) aureus coagulates blood
2.) it grows on DNA plate
3.) it is topographically gold
4.) MSA aureus- yellow
what genus and species is selected for using TSY20B plate?
Which of the following are true of Kirby bauer technique
antibiotics on the dispensed discs are standard concentrations

antibiotics are delivered to the bacteria via diffusion

(antibiotic testing is determined on Mueller-Hinton plate)
what 2 genetic elements are necessary for a plasmid to have in order for transformation to occur
1.) Origin of replication
2.) selectable marker

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