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Pipetting: Mechanical vs. Transfer

Mechanical pipettes allow you to make more accurate measurements. Transfer pipettes are quick and require less technique. Must use mechanical pipette for volumes greater than 1 mL.

DNA Extraction

Add bacteria suspension to tube of lysozyme, shake until it dissolves. Add freeze dried M.luteus in spooling tube. Shake for five minutes. Add SDS to solution. Invert gently for five minutes - solution becomes more viscous as bacteria are lysed. Set tube in hot water bath for 30 min. Measure and chill ethanol. Allow tube to cool to room temp. Add spooling rod to tube. Add cold ethanol via pipette down side so layers don't mix (at 45 degree angle). Twirl for several minutes as DNA attaches. Immerse in ethanol for two minutes and let dry.

What is the function of lysozyme in DNA extraction? Where does it come from in nature?

Enzyme that destroys cell walls of bacteria.

Naturally found in egg whites.

What is the function of SDS in DNA extraction?

sodium dodecyl sulfate
disrupts cell membrane and interactions that hold macromolecules in place
lyses the nucleus

What is the function of ethanol in DNA extraction?

used to precipitate DNA as it is insoluble
the result is white snotty extraction

What is bacterial transformation?

the uptake of naked DNA molecules by bacterial cells

What is transduction?

involves transfer of genetic material as mediated by bacteriophages, or bacterial viruses

What is conjugation?

involves the transfer of genetic material from one bacterium to another and requires close proximity or contact of the bacteria cells

In what bacteria was transformation first discovered?

Streptococcus pneumoniae

transformation competence

ability of a bacterium to take up naked DNA molecules
some bacteria (like s. pneumo) are naturally competent while others are not but can be made competent in a lab

chemical transformation

requires treating bacteria cells with high concentration of calcium chloride to make them competent; after suspension in calcium chloride, it is subjected to heat shock so DNA molecules can be taken up by the cell

electrical transformation

also known as electroporation
more efficient but requires an electrical pulser to permeabilize the cell membrane and allow DNA to enter the cell

What are plasmids?

extrachromosomal DNA molecules that can replicate independent of the chromosome in the cell

they often carry genes, like antibiotic resistance, which aid is survival of the organism

antibiotic resistance can be spread through "horizontal gene transfer" and this can result in a whole community of bacteria with resistance to antibiotic

cloning vectors

plasmids can act as these
investigators insert a gene of interest in the plasmid to obtain amplification of the gene

What is the purpose of the "no plasmid" sample? What possible explanations are there for getting colonies from the "no plasmid" sample?

it served as a control
if plasmid were present in each sample, there would be no way to show the difference in growth between a transformed cell and not transformed cell
the no plasmid samples should only grow on the LB plate
it is unable to grow on plates with ampicillan because it doesn't have B-lactamase to cut it up
if colonies appeared on the "no plasmid" sample, then there's the possibility that cross contamination occurred

What factors are likely to influence the transformation efficiency of E. coli?

plasmid size
inadequate/too much heat shock
presence of contaminants
good or bad technique
need super cooled DNA?

How might humans contribute to the development of normal flora bacteria that are antibiotic resistant? Why is this problematic for an individual who is prone to pneumococcal infections?

depending too much on antibiotics
in one community, one bacterium that carries antibiotic resistance plasmid can transfer that genetic info to other bacteria by way of horizontal gene transfer
the result is the community of bacteria, comprised of different species, is resistant to a particular antibiotic

Why is bacterial transformation useful to geneticist and molecular biologists?

it helps them understand genetic diversity in bacteria
it can be useful in cloning or movie DNA around in different bacteria
plasmids and transformation could be used to genetically recombined a strain for medical purposes
transformation is important in understanding resistance to antiobiotics

What is the source of the green fluorescent protein gene? What is its function in nature?

bioluminescent jellyfish
in jellyfish, the gene provides a glow-in-the-dark feature in the deep ocean

How does the pGLO/ampicillin/arabinose switch system work?

promoter region between GFP and araC genes (the promoter causes transcription to happen - turns gene "on")
RNA polymerase is assembled
araC binds to the promoter in presence of arabinose and RNA polymerase = makes complex
when promoter is turned on, RNA polymerase allows mRNA to make protein
arabinose present - turns on araC
B-lactamase - antiobiotic resistance (allows bug to live in presence of ampicillin/penicillin)


plasmid that carries the gene that codes for fluorescent protein

4 major steps of bacterial transformation

1. pre-incubation
2. incubation
3. heat shock
4. recovery/growth


recipient cells will be exposed to positively charged calcium chloride ions. This stresses the bacteria in order to render its cell membrane and the cell wall permeable to the donor plasmid. This process makes the cell "competent" to uptake the plasmid.


The plasmid is added to the E. coli suspension, which will now be called E. coli + because it is being transformed. Another E. coli suspension (-) will act as the control because it will not get the plasmid (and not inherit the fluorescent gene).

Heat shock

the recipient cells plus plasmid and the control cells alone are briefly exposed to heat to maximize the uptake of the plasmid through the wall and membrane of the cell


cool to room temp for ten minutes to repair the damage done during transformation and then plated and incubated to form colonies

Calculate transformation efficiency

total # of cells growing on LB/amp \ amount of DNA spread on the agar plate = # of transformants per mg of DNA
1. count the # of cells on the plates
2. determine the amount of DNA: total amount of DNA (mg) = concentration (mg/ml) X volume (ml)
3. determine the fraction of DNA solution spread: volume of the solution spread on the plate divided by the total amount in the tube
4. determine the mass of DNA spread on the plates: total mass of DNA mg (3) X fraction spread (2)
5. # of colonies/ answer to 4 = # of transformants per mg of DNA

Bacterial transformation growth on plates


Restriction enzymes

restriction endonucleases act molecular scissors, cutting double stranded DNA molecules at specific nucleotide sequences and generating a mixture of smaller, but reproducible DNA fragments from a given molecule. This allows investigators to isolate and purify specific genes of interest.

How to restriction enzymes work?

they hydrolyze specific phosphodiester bonds in DNA molecules
the enzymes have an optimum temp of 37 degrees C

Gel electrophoresis

Following digestion, DNA fragments can be separated by agarose gel electrophoresis, which separates fragments on the basis of length. DNA is negatively charged and migrates toward the positive electrode when place in an electric field. Agarose, with the consistency of firm jello, acts a molecular sieve, separating DNA molecules of different sizes with some fragments migrating faster than larger ones.

How is DNA seen when using gel electrophoresis?

it must be stained. ethidium bromide is commonly used because it fluoresces under UV light but it is a mutagen and carcinogen so SYBR stain is used. It is used in place of ethidium bromide.

Lamda DNA

isolated from a bacteriophage, a virus which infects E. coli. The genome of this virus is 48,502 base pairs (called the ladder and used for comparison and calculation of cuts)

Why have bacteria evolved to produce restriction enzymes?

they are means of protection from infection by viruses. they act like microbial immune systems and selectively cut up foreign DNA

How are restriction enzymes named?

they are named after the bacteria from which they were isolated, including bacterial genus, species, and strain. the number at the end corresponds to the order it was identified in the bacteria.

Where do EcoRI and HINDIII come from?

Eschericha coli ad haemophilus influenzae

How is a bacterium's own DNA protected from the restriction endonucleases it makes?

bacteria's own DNA is methylated by the enzyme methylase to protect it. methylation refers to the addition of a methyl group. their DNA doesn't contain recognition sites for the restriction enzymes

Which bands should be closest to the well of the gel?

The largest bands should be closest to the wells of the gel as the smallest fragments travel the quickest through the gel


restriction fragment length polymorphism
bacterial restriction enzymes are used to cut a segment of DNA at different places, based on where the sequences for cut sites occur. Because different sequences of DNA have different cut sites, unique sets of fragments are produced for each type of DNA used. When run on a gel, these fragments produce finger-print like banding patterns which can be useful in identifying samples.

What is the purpose of the loading dye?

Stops the function of the enzyme
gives DNA weight to go into well of gel

What is the purpose of heat shock in gel electrophoresis?

to make the DNA linear instead of circular


catalase negative
oxidase negative
cells in pairs and chains
weak to no growth on MSA
use Lancefield type kits to determine which type
also classified by hemolysis
cause pneumonia, meningitis, etc.
normal flora of mouth and pharynx


catalase positive
oxidase negative
cells in irregular clusters
S. epidermis : makes no acid on mannitol salt plate (no color change, just growth)
S. aureus : acid produced on mannitol salt plate (color change of yellow)
gram positive
abscesses, boils, carbuncles, etc.
nosocomial hospital acquired
S. aureus can coagulate plasma = pathogenicity and is B hemolytic

Strep Groups

A : S. pyogenes
B : S. agalactiae
C : S. equis
D : S. faecalis

Describe the selective and differential properties of mannitol salt agar (MSA) for the isolation and identification of staph.

phenol red changes to yellow = acid production
S. aureus ferments mannitol and produces acid
S. epidermis is unable to ferment mannitol to produce acid
S. saprophyticus mostly ferments mannitol to produce acid

Why are staphylococci among the leading causes of nosocomial infections?

most human populations are carriers of staph (90%) antibiotic resistance is increasingly becoming a problem (MRSA) Staph is an opportunistic pathogen

What precautions are taken in nosocomial settings to prevent staph infections?

hand washing, equipment sterilization, use of alcohol swabs, personnel uniforms, use of microbials, isolation to prevent transmission, wearing gloves, etc.

When bacteria from a throat swab are streaked on blood agar, why is the agar stabbed several times with the loop?

to mimic anaerobic respiration by creating a "pocket" of lower content of oxygen. some microorganisms release enzymes that will only work under anaerobic conditions

Which strep species includes cells that are arranged predominantly in pairs rather than chains?

s. pyogenes

Vaginal swabs are taken from pregnant women in their third trimester. Which strep species is the focus of investigation

s. agalactiae (group B strep)

MSA media

selective (only grows staph) and differential (changes color with acid production)

What is a fomite?

an inanimate object
attempted to isolate a strain of staph from this

Staph saprophyticus

causative agent of UTIs
non-coagulase forming
ferments mannitol to produce acid without gas

Staph aureus

occur singly, in pairs, or in clusters
salt tolerant
ferments mannitol to produce acid
coagulase positive and will cause serum to form a clot
a-toxin produced that causes a wide clear zone of B-hemolysis on blood agar


blood agar plates
inoculated throat swabs
used to classify strep
Beta: completely destroy RBCs to produce clear zone
Alpha: greenish or brownish zone around colonies (partial hemolysis)

Coagulase Test

definitive test for confirming the ID of S. aureus
S. epidermis and S. saprophyticus are always coagulase negative
Inoculate small tube of plasma with several loopfuls of organism. Any degree of coagulation, from a loose clot to a solid immovable clot, it considered a positive result, even if it takes 24 hours.

Lancefield Test

Classification of strep based on immunological groups related to carb antigens associated with the cells

CAMP test

used to identify Group B Strep
if unknown produces an enlarged arrow-head shaped hemolytic zone at the juncture where the unknown meets the S. aureus streak, the organism is S. agalactiae.
The only problem is that if the plate in incubated anaerobically, S. pneumoniae cause give a positive result as well.

Bacitracin test

used to identify Group A Strep
place bacitracin disk on agar and any zone of inhibition around the disks should be considered a positive test result. S. pyogenes is positve for this characteristic.

Two limitations of the bacitracin test

1. the disks must be the differential type, not the sensitibity type (sensitivity disk have too high a concentration of the antibiotic)
2. the test should not be applied to the alpha-hemolytic strep (a-hemolytic strep are sensitive to this type of disk)

S. aureus vs. S. epidermis

S. aureus : ferments mannitol salt and produces acid; coagulase +
S. epidermis : ferments mannitol but doesn't produce acid; coagulase -

Reductase test

milk that contains large numbers of actively growing bacteria will have a lowered oxidation-reduction potential due to the exhaustion of dissolved O2 by microorganisms
the fact that methylene blue loses its color (becomes reduced)


methylene blue reduction time
the time it takes for methylene blue to become colorless
the shorter the MBRT, the lower the quality of milk
an MBRT of 6 hours is very good (30 = poor quality)

What bacteria are present in raw milk?

E. coli and Strep lactis
they are both strong reducers

Types of enumeration

reductase test: qualitative
dilutions: quantitative
plate counts: quantitative
spectrophotometry: qualitative
filtration: quantitative
MPN: qualitative


measures the absorbance or optical density which tells about the turbidity of the solution under question
living and dead cells contribute to the culture turbidity, which is a disadvantage of the method
denser the bacteria (the more turbid), the more absorbance (lower transmittance)
{wavelength is 550 nm}

Optical Density

as light passes through the culture, it will be absorbed by the bacterial cells and the light emerging from the culture will be proportionately decreased by the number of cells present
galvanometer measures OD

Reductase Pros and Cons

Pros: simple to perform
Cons: only qualitative

Diltuations Pros and Cons

Pros: Gives orignial concentration (CFU/mL)
Cons: time consuming

Plate Counts Pros and Cons

Pros: easy to see, quantitative
Cons: takes a lot of resources and grows on certain media

Spectrophotometer Pros and Cons

Pros: good starting place
Cons: live and dead cells contribute; require galvanometer

Filtration Pros and Cons

Pros: more rapid than MPN; requires less technique
Cons: only gives coliform #; have to use other media type to determine which type

MPN Pros and Cons

Pros: used to determine clean drinking water; time saving vs. standard plate count; easy to use
Cons: assume that coliforms are E. coli; limited to testing where statistical tables have been set up


Most Probable Number
indicator bacteria = coliforms, which are found in the intestines of warm-blooded animals and ferment lactose to produce acid and gas
the presence of these bacteria indicate the potential for disease
old school method for estimating the number of bacteria in a sample without a plate count


Standard Plate Count
a sample is diluted in a series of dilution banks and plated
the number of bacteria in the original sample is determined by multiplying the number of colonies by the dilution factor
reported in colony forming units (CFUs)
only numbers between 30 and 300 CFUs are considered valid

SPC pros and cons

pros: determines only the number of viable cells
cons: biased because it uses specific conditions and media that may exclude certain bacteria in the count

Membrane Filter Method

known volumes of water are filtered through filters
most bacteria, including coliforms, are larger than the pore diameters in the filter paper and so they are retained on the filter paper
the filter disk containing the bacterial cells is placed in Endo broth
any coliforms that are present on the filter will ferment lactose in the Endo broth, producing acid cause the media to form a metallic sheen
non coliform bacteria will not produce the metallic sheen
gram + bacteria are inhibited from growing

Calculate MPN from membrane filtration

(coliform count x 100)/amount of water filtered


gram - rods
ferment lactose
intestinal tracts of animals
facultative anaerobes
nonendospore forming

mTEC media

selects for gram - bacteria
thermotolerant E. coli
yellow-yellowish-brown colonies
associated with humans or warm blooded animals (indicator bacteria)

mEntero media

selects for gram + bacteria
Enterococcus - strep faecelis
pink to red colonies
associated with humans or warm blooded animals (indicator bacteria)

EMB media

selects for gram - bacteria
E. coli result in green metallic sheen
lactose-fermenting bacteria are purple (not E. coli)
fecal coliforms
confirmed test

mEndo media

selects for gram - bacteria
lactose fermenting bacteria result in gold metallic sheen
selective/differential media for E. coli
confirmed test

UV light

shorter the wavelength = more damaging
260 nm is the most harmful because it is when DNA maximally absorb UV light

Pyrimidine dimers

form when DNA absorbs UV light
covalent bond between 2 adjacent C or T
deform DNA, DNA polymerase cannot replicate

SOS System

enzymatically removes dimers and inserts new pyrimidine molecules (if lots of dimers have formed then it may not work)

Physical methods to control microbial growth

heat; filtration; cold; high pressure; dessication; osmotic pressure; radiation

Physical control: HEAT

boiling for ten minutes kills bacteria through protein inactivation
autoclaving kills endospores if repeated
hot-air sterilization

Physical control: FILTRATION

filters should have pores with 0.2 micrometer diameter

Physical control: COLD

refrigeration prevents food spoilage and inhibits bacterial growth (but doesn't kill cells)
deep freezing processes slow enough to have microorganisms killed by the formation of ice crystals


inhibits bacterial growth but doesn't kill cells

Phyiscal control: HIGH PRESSURE

can kill cells
often used in conjunction with heat (like autoclaving)

Physical control: DESSICATION

drying up or removing water
used to preserve foods such as fruits, cereals, grains, and milk

Physical control: OSMOTIC PRESSURE

hypertonic environment where water moves out of cells thereby desicatting them
salting fish or meats is an example

Physical control: RADIATION

ionizing radiations; gamma rays; called X rays: short wavelength); causes electrons and protons to eject from molecules thus leaving ions and free radicals
non-ionizing includes UV light; kills microorganisms by interacting with it's DNA, causes thymine-thymine dimers


1928 - Fleming discovered, produced by Penicillium (mold that killed bacteria around it)
1940 - Florey and Chain performed first clinical trials using this antibiotic
development of more antibiotics during WWII

Selective toxicity

ability to kill harmful microbes without damaging the host

broad spectrum antibiotics

kill gram positive and negatives strains


agent that kills bacteria


stopping bacterial growth

How to antibiotics kill bacteria?

1. inhibit cell wall synthesis (ex: penicillins and bacitracin)
2. inhibition of protein synthesis (ex: tetracyclines)
3. inhibition of nucleic acid replication and transcription (ex: quinolones)
4. injury to plasma membrane (ex: polymyxin B)
5. inhibition of synthesis of essential metabolites (ex: sulfa)

Antibiotics that inhibit translation

bind to ribosomes

penicillin inhibitors

penicillinase and B-lactam rings


occurs when the effect of two drugs together is greater than the effect of either alone


occurs when the effect of two drugs together is less than the effect of either alone

Chemical control: Antisepsis

removal (inhibition) of pathogens from (on) living tissue

Chemical control: disinfection

removal of vegetative microbes from an inanimate object

chemical control: biocide/germicide

kills microbes

Chemical control: bacteriostasis

inhibiting, not killing, microbes

Types of disinfectants: PHENOLS

disrupts plama membranes
ex: Lysol

Types of disinfectants: HALOGENS

iodine, chlorine
oxidizing agents
damage all cell components, primarily attacks proteins
iodine and ethanol - tincture (iodophor) prevents staining
bleach is hypochorous acid(HOCl)


Zaphiran an Cepacol antiseptics
cation is a charged molecule that denatures proteins, dirsupts membranes
strongly bacteriocidal to gram positive bacterias
odorless, colorless, stable, and easily diluted

Types of disinfectant: ALCOHOLS

isopropanol - denatures proteins, dissolves lipids, and dehydrates

Types of disinfectants: OXIDIZING AGENTS

peroxygens (O3, H2O2, benzoyl peroxide)
produce highly reactive oxygen radicals (oxidize cellular components)
H2O2 is a strong oxidizng agent that damages human cells and slows healing

Types of antiseptics: SOAPS

surface-active agents or surfactants
minimal activity, allows mechanical removal of microbes, breaks up oil on hands

Type of antiseptics: BIGUANIDES

Chlorhexidine: sticks to human membranes without damage, disrupts plasma membranes of bacteria, and often part of surgical scrubs

MHA plates

Mueller-Hinton Agar used for antibiotic testing


compounds that kill or inhibit microorganisms


compounds that are produced by microorganisms that inhibit or kill other microorganisms


antibiotics chemically altered to make them more effective


antimicrobials chemically synthesized in a laboratory

zone of inhibition

no growth occurring around an antibiotic disk

Kirby Bauer Method of testing antibiotics

takes into consideration all variables
MHA agar inoculated with cotton swab
high potency antibiotic disks are placed on the agar with mechanical dispenser or sterile foreceps
the zones of inhibition are measured to the nearest mm
significane of the zones (resistant, intermediate, and sensitive) are determined by chart


substances that inhibit microbial growth or kill microorganisms and are gentle enough to be applied to living tissue (ex: alcohol and betadine) doesn't destroy endospores


substances applied to inantimate objects to kill microorganisms; usually more harsh that antiseptics (more damaging to living tissues)
ex: steriliants/sporocides - kill endospores too - ethylene oxide
ex: sanitizers - reduce microbial growth #s to safe level, but don't completely eliminate


agent only inhibits growth but doesn't kill bacteria


agent that kills bacterial cells

Why are gram - bugs more difficult to treat with an antibiotic?

they have an extra membrane to keep the antibiotic out

broad vs. narrow spectrum antibiotics

broad work on gram + and gram -
narrow work against a particular group

What kind of antibiotics are better to use for diagnosis and treatment?

narrow spectrum because if broad spectrum were used, it gives the bug the chance to build up resistance and once a bug it resistant, it is always resistant

Describe the damaging effects of UV radiation on living cells

has mutagenic properties
when DNA absorbs UV light, pyrimidine dimes (covalent bonds between thymine or cytosine molecules) form. The DNA becomes deformed and DNA polymerase is unable to replicate strains, so the genes cannot be transcribed.

Why does exposure to UV radiation cause death in vegetative cells but not endospores?

this is because DNA of endospores is protected by acid-soluble proteins that alter it's conformation (protecting it from photochemical damage) Also, a photo-product is made by UV light in endospores that functions in DNA repair

At which wavelength is UV radiation more germicidal?

260 nm because this is the wavelength at which DNA maximally absorb UV light

What limited protection do cells have against the damaging effects of UV radiation?

One repair mechanism is the SOS system which uses enzymes to remove dimers and insert new pyrimidine molecules. This system may prove noneffective, however, if exposure to UV causes massive number of dimers.

Factors that influence the size of zone of inhibition of an antibiotic

diffusibility of agent, size of inoculum, type of media, etc.

Why are gram positive bacteria typically more resistant than gram negative bacteria to antibiotics, that disrupt plasma membrane>

gram + have a thicker cell wall (more peptidoglycan)
more cell wall to penetrate means more resistant to things that are trying to penetrate or disrupt the cell wall

Why are certain gram negative bugs more resistant than gram positive bugs to antibiotics that attack cytoplasmic targets?

they have two layers to get through (LPS and peptidoglycan) to the cytoplasm whereas gram positive only have one layer (peptidoglycan)

Factors that influence the size of zone of inhibition produced by a chemical

different modes of action
different chemical properties (ex: solubility)
amount inoculated
diffusion rate of chemical
sensitivity of microorganism to chemical

Why do Bacillus and Serratia respond differently to UV radiation?

Bacillus produces spores which are not killed by UV radiation


enzyme-linked immunosorbent assay
if antibodies are being detected, then a portion of the virus is attached to the solid surface to act as the antigen. serum (of the patients tested) will be added and if it contains antibodies to the antigen, it will bind. another antibody that recognizes HIV is bound/linked in an enzyme. in the final step, a substance that reacts with the enzyme on the antibody is added to produce a colored product (blue).

What does a color change (blue) indicate on an ELISA test?

the test is positive
if the patient doesn't have antibodies to the certain antigen, no reaction will occur and therefore no color change will be observed (because there is no sandwich effect)

What is the purpose of an ELISA test?

detects antibodies in blood to determine if exposure to disease has occurred

What are antigen?

molecules that cause the body to mount immune response (may include component of infectious disease)

What are antibodies?

proteins that recognize antigen and bind very tightly to it...they flag invaders for destruction by other cells of the immune system

How much of the blood is made up of antigens?

15% and they are very specific, recognizing only one antigen

What must be present for immunoassay to be valid?

positive and negative controls to guard against experimental error

What types of compounds in bacterial cells can serve as antigens?

macromolecules such as proteins, polysaccharides, lipoproteins, and nucleoproteins

What is a serotype of an organism?

refers to the distinct variations between bacteria
it is use for classification based on which types of antigens are present

What is the importance of running controls?

negative controls illustrate no change while positive controls show what is expected to happen to warrant a positive result. comparison to controls lets one know whether or not experimentation was successful

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