1
Q
UV light
A
- used to control microbial growth of objects (nonliving things)
- adjacent thymine molecules in DNA cross to form thymine dimer
- leads to mutations during DNA replication
- kills microbes by causing mutations
2
Q
UV light experiment
A
-4 nutrient agar plates are inoculated for s. marcescens -inoculated for confluent growth
-swabbed on with cotton swab
-exposed to UV light
-plate 1 was exposed to UV light half covered for 1 min
-plate 2 was exposed to UV light hald covered with paper (5 mins)
-plate 3 is under UV light but covered with plastic lid (5 mins)
-plate 4 is control
-incubated
results:
-expect to see growth in 4, 3
-half growth in 2 under the paper
-complete growth under the paper and colonies on the other side bc of the short UV time for plate 1
-length of time is a factor
3
Q
slide smear
A
- place a drop of water on center of slide
- use aseptic procedure to pick up bacteria culture with inoculating loop
- mix bacteria in drop of water to make a suspension of cells
- spread suspension on the slide -> smear
- sterilize the loop
- air dry smear
- then heat fix smear by passing the slide through a flame of a bunsen burner quickly 4 times (so cells are attached to the slide and dont wash off)
4
Q
staining the slide
A
- pour methylene blue on the smear and cover the smear with methylene blue
- wait 1 minute for the stain to react with the smear
- rinse methylene blue off of the slide using water
- blot dry the smear by placing the slide between 2 sheets of paper towels
- once the slide is dry -> observe under microscope
- determine shape and arrangement of cells under 100x objective lens
5
Q
bacillus subtilis
A
- streptobacili arrangement
- rod shape
- rod shape shape chain
- also see some single bacili
6
Q
escherichia coli
A
- rod shape cell
- bacili
- no specific arrangement
- single cells
- no chains or clusters
7
Q
anitobiotics
A
- made by microbes
- antibiotics are found in nature
- purified by the pharmaceutical companies
- penicillin
- mold makes penicillium notatum -> purified into penicillin
- penicillin inhibits the formation of peptidoglycan
- penicillin is selectively toxic to bacteria (not toxic to host -> humans) bc humans have no peptidoglyan
- gram neg- resistant to penicillin
- gram +- sensitive to penicillin
8
Q
chloramphenicol
A
- produced by streptomyces
- inhibits protein synthesis on the 70S ribosomes
- eukaryotic cells have 80S ribosomes
- antibiotic selectively inhibits protein synthesis on the 70S ribosome
- high concentration and prolongs use can cause aplastic anemia
- tetracycline, streptomycin, erythromycin are made by streptomyces
- gram + and neg are sensitive to tetracycline and streptomycin and chloramphenicol
- gram neg is resistant to erythromycin and gram pos it sensitive -> bc erythromycin is large and cannot penetrate the outer membrane and interfere with ribosomes
9
Q
gentamicin
A
- antibiotic
- produced by micromonospora
- inhibits protein synthesis on the 70S ribosomes
- gram + and neg are sensitive to gentamicin
10
Q
antibiotic sensitivity test
A
11
Q
antibiotic sensitivity test
A
- done to see if a bacterium is sensitive or resistant to various antibiotics
- isolate the pathogen from the sample
- based on results the physician will find treatment
- done every day in clinical labs
- plates are inoculated with bacteria for confluent growth
- antibiotic discs are placed on the medium and the plates are incubated
- after incubation the plates are observed for the zone of inhibition (absence of growth) around the disc
- absence of growth -> sensitive to antibiotic
- antibiotic diffuses into the medium and comes into contact with bacteria (stops or kills bacteria if they are sensitive)
12
Q
influence of O2 on the growth of bacteria
A
- micrococcus luteus
- escherichia coli
- clostridium sporogenese
- inoculate each of the organisms on 2 plates of nutrient agar by using streaking for isolation procedure
- incubate one plate of each organism in the presence of O2
- incubate the other plate of each organism in the absence of O2
- look for growth after
13
Q
microccocus luteus
A
- expect to see growth on the plate incubated with O2
- do not expect growth on plate incubated with no O2
- obligate aerobe- needs O2 for growth
14
Q
Escherichia coli
A
- facultative anaerobe- growth with or without O2 but grows better with O2
- expect to see growth on both plates
- expect to see larger colonies on the plate incubated with O2
- expect to see smaller colonies on the plate incubated without O2
- why does it grow better in O2 -> O2 helps the bacteria make more ATP (38)
- without O2 it only makes 2 ATP
- ATP helps cell make macromolecules to grow faster
15
Q
clostridium sporogenes
A
- obligate anaerobe- grows only without O2
- O2 is toxic bc the bacteria doesnt have the enzymes to neutralize the toxin
- expect to see growth on the plate incubated without O2
- do not expect growth on plate incubated with O2
16
Q
growing obligate anaerobes
A
- anaerobic jar to growth obligate anaerobes like costridium sporogenes
- place plates in anaerobic jar
- place envelope with chemicals
- methylene blue- O2 detector
- blue in presence of O2
- clear in absence of O2
- chemicals react -> release Hydrogen
- hydrogen combines with O2 -> water (removes O2)
- check oxygen indicator after 5-10 min
17
Q
endospore staining bacillus subtilis culture: prep
A
- smear has to be prepared:
- drop of water on slide -> using aseptic procedure pick up culture and put it in the water
- mix the bacteria in the drop of water
- spread the suspension onto the slide
- air dry
- heat fix- pass through flame 4 times quickly (so cells get attached to slide and dont wash off)
18
Q
endospore staining bacillus subtilis culture
A
- cover the smear with a piece of paper towel
- pour malachite green on to the smear that is covered with the paper towel
- wait 30 mins
- rinse the malachite green off of the slide with water
- cover the smear with safranin
- wait 1 min
- rinse safranin off of the slide
- wait for the slide to dry
- observe under 100x objective lens
- pink -> vegetative cells
- green -> endospores
19
Q
biochemical tests
A
- indirectly checking for enzyme by directly looking for the presence of the product or the disappearance of the substrate
- enzyme profile/activity of bacteria
- each species has a unique set of enzymes (bc it has unique set of genes)
- therefore, the enzyme profile helps us to identify bacteria
- give the bacteria the substrate
- if the bacteria makes the enzyme -> the enzyme will convert substrate to products
20
Q
substrate
A
-is a substance with which the enzyme reacts
21
Q
exoenzymes: starch hydrolysis test
A
- exoenzyme- enzymes produced in the cell and then released into the environment
- once the enzyme is outside it breaks down macromolecules into smaller molecules in the environment
- smaller molecules cross the plasma membrane and they are used by the cell for energy or to make structure of the cell
- exoenzyme: amylase
- substrate- starch
- product- maltose
- amylase breaks down starch and produces maltose
- use 2 plates of starch plates
- one plate gets inoculated with B. subtilis
- the other plate is inoculated with E. coli
- we make a single line inoculation
- after incubation we expect a single line of growth across the medium
- we look for the disappearance of the substrate (starch)
- add iodine- indicator for starch
- if medium turns purple -> starch is present and bacteria did not use it
- bacteria is negative for starch hydrolysis -> negative for making amylase
- if there is clear area around the growth -> no starch in the medium
- bacteria broke down the starch -> bacteria is positive for starch hydrolysis -> amylase is present
22
Q
results of starch hydrolysis test
A
- positive for starch hydrolysis -> b. subtilis
- negative for starch hydrolysis -> e. coli
- e. coli- amylase is not present and starch is present in the medium -> purple
- b. subtilis- amylase is present -> clear around the starch strip
23
Q
nitrate reduction test
A
- we want to see if the bacteria can use nitrate
- 3 possibilities:
- some bacteria make the enzyme nitrate reductase and convert nitrate to nitrite
- some bacteria make 2 enzymes: nitrate reductase which converts nitrate to nitrite -> NITRITE reductase then converts nitrite to nitrogen gas
- some bacteria dont make either enzymes -> bacteria cant use nitrate
24
Q
nitrate reduction test procedure
A
- 3 tubes of nitrate broth
- one tube for each bacteria:
- e. coli
- m. luteus
- p. aeruginosa
- inoculate and incubate
- we add 2 chemicals (nitrite indicators) to see if nitrite is present
- if nitrite is present the chemicals will react and turn red (nitrate reductase must be present)
- add a pinch of zinc to the tubes that didnt turn red
- if the medium becomes red after adding zinc -> zinc converted nitrate to nitrite (reduction) -> this means nitrate wasnt used and there are no enzymes present
- if the medium doesnt become red after adding zinc -> we know the bacteria made 2 enzymes (nitrate and nitrite reductase) -> no nitrate or nitrite in the tube bc nitrogen gas is present in this tube
25
red after adding nitrite indicators
- nitrite is present
- nitrate reductase is present
- e. coli
26
no red after adding nitrite indicators but turns red after adding zinc
- neither of the enzymes are present
- nitrate was present
- zinc converts the nitrate to nitrite
- reduction
- m. luteus
27
no red after adding nitrite indicators and zinc
- 2 enzymes are present
- nitrate and nitrite are not present
- nitrogen gas is present
- p. aeruginosa
28
differential medium
-helps us to differentiate one group of bacteria from another
29
blood agar: beta hemolysis
- common differential medium
- has 5% sheeps RBCs
- blood agar is red
- differentiates hemolytic bacteria from nonhemolytic bacteria
- some bacteria make the enzyme beta hemolysin which breaks down RBCs -> there will be clearing of RBCs around the colonies on the blood agar
- beta hemolysin- complete hemolysis
- when beta hemolytic bacteria grow on blood agar complete clearing of RBCs round their colonies
30
alpha hemolysis
- some bacteria make the enzyme alpha hemolysis
- this enzyme converts hemoglobin to methemoglobin (green)
- when this bacteria grows we expect to see green colonies
- partial hemolysis
31
gamma hemolysis
- no hemolysis
- no color change in medium
- some bacteria dont make any enzymes that cause damage to the RBCs
- stays red
32
selective medium
- allows the growth of some bacteria
- this medium prevents the growth of certain other bacteria
- NaCl agar- facultative halophiles (grow with or without salt) such as S. aureus will grow while other bacteria such as e. coli cannot grow
- 6.5% NaCl in the NaCl agar
- looking for growth and colonies
- NaCl agar is selective for S. aureus
33
MacConkey agar
- selective and differential medium
- selective for G- bacteria (only gram neg will grow)
- it has crystal violet and bile salts that prevent the growth of G+
- differential medium bc it has lactose in it
- lactose differentiates lactose fermenters from non-lactose fermenters
- fermenters make pink colonies
- non-fermenters make colorless or white colonies
- uninoculated macconkey agar is pinkish purple on its own
34
lactose fermenter
- e. coli
- gram negative
- pink and present on MacConkey agar plate -> therefore it is G- and lactose fermenter
35
lactose nonfermenter
- p. vulgaris
- present but colorless/white on the macconkey agar
- this means the bacteria is G- and lactose nonfermenter
36
triple sugar iron agar composition
- differential medium
- TSI medium
- three sugars
- .1% glucose
- 1% lactose
- 1% sucrose
- peptone
- iron
- phenol red- pH indicator
37
triple sugar iron agar
- TSI medium
- slant
- inoculate needle is used
- if the entire medium is yellow -> bacteria fermented one or both of the disaccharides (sucrose and lactose) in addition to glucose -> E. coli
- if there is space at the bottom is also tells us the bacteria has reduced a lot of gas (makes breaks in the agar as well)
- if entire medium is red -> none of the sugars were fermented -> P. aeruginosa (negative for fermentation)
- red slant and rest of the medium is yellow -> only glucose is fermented (not disaccharides) -> s. marcescens
- yellow slant and most of the medium black -> bacteria fermented a disaccharide in addition to glucose and also produced hydrogen sulfide (black) -> p. vulgaris
38
hydrogen sulfide rxn
- amino acid cystine- substrate
- cystine desulfhydrase- enzyme
- hydrogen sulfide- product
- medium has iron- iron is the indicator for hydrogen sulfide
- hydrogen sulfide combine with iron to make a black precipitate
39
IMViC test
- I- indole
- M- methyl red
- V- voges (proskeauer)
- C- citrate
- differentiate E. coli from E. cloacae (enterobacter cloacae) and K. pneumoniae (kelbsiella pneumoniae)
- these 3 bacteria have similar enzyme bacteria -> when it comes to IMViC test they are different
- these three bacteria will be used in all of these tests
40
indole test
- we are looking to see if the bacteria can make tryptophanase enzyme
- we give the bacteria the substrate -> tryptophan (amino acid) with tryptone broth
- 3 tubes of tryptone broth are each inoculated with the three bacteria
- indole is the product
- we add dropperful of kovacs reagent and react with indole -> top layer becomes red or bright pink
- top layer is somewhat orange for negative test
- if the top becomes red or bright pink -> indole is present -> tryptophanase is present -> positive
- e. coli is positive
- e. cloacae is negative
- k. pneumoniae is negative
41
methyl red test
- some bacteria ferment glucose and release acids
- 3 tubes of MRVP broth -> inoculate with the 3 bacteria
- incubate
- add 10 drops of methyl red
- if medium is red -> positive for acid (fermentation took place)
- various enzymes depending on what kind of acid is produced
- yellow color for negative
- example of an enzyme -> lactate dehydrogenase
- e. coli is positive -> acids and fermentation -> red
- e. cloacae is negative -> glucose is still present -> no fermentation -> yellow color
- k. pneumoniae is positive
42
voges-proskauer test
- 3 tube of MRVP medium (glucose in it) -> inoculate with 3 bacteria
- incubation
- we want to see if the medium has acetyl methyl carbinol (neutral)
- add 10 drops of barritts reagent A and B
- shake the tube every few minutes for 20 minutes
- if medium is red -> positive for making acetyl methyl carbinol -> enzyme is present (acetolactate dehydrogenase)
- enzyme is acetolactate dehydrogenase, substrate is glucose, and product is acetyl methyl carbinol
- e. coli is negative -> yellow, glucose is present, no enzyme present
- e. cloacae is positive -> positive for enzyme and acetyl methyl carbinol
- k. pneumoniae is negative
43
citrate test
- want to see if the bacteria use citrate as the only source of carbon
- citrate medium has the pH indicator -> bromthymol blue
- green -> neutral
- blue -> basic
- yellow -> acidic
- when bacteria use citrate ammonium ions are released
- environment becomes basic -> blue
- if blue -> ammonium ions are present bc citrate was used -> citrase is present
- enzyme is citrase
- e. coli is negative -> green -> no ammonium ions -> negative for using citrate and the enzyme citrase
- e. cloacae is positive -> basic -> citrate was used and released ammonium ions -> citrase is present
- k. pneumoniae is positive
44
urea hydrolysis test
- some bacteria make urease and break down urea
- ammonium ions are leased medium becomes basic
- pH indicator becomes fuchsia when it is basic
- urea broth has substrate (urea)
- if substrate has the urease enzyme it will use urea and release ammonium ions
- p. vulgaris (proteus vulgaris) -> strong positive -> urease and a lot of ammonium ions is present -> basic
- k. pneumoniae- weak positive -> has enzyme urease but not a lot -> broke down some urea and release some ammonium ions
- e. coli is negative -> no urease -> urea is present
- e. cloacae is negative
45
motility
- allows us to see is bacteria can swim in the environment
- motility medium is semisolid
- stab inoculation
- use an inoculating needle
- distinct red line of growth in the medium:
- growth is concentrated along the stab line
- these bacteria have no choice but to stay along the line bc they have no flagella to move away
- growth is red because of tetrazolium chloride in the medium. it reacts with the cells and color the cells red (bacteria is not red)
- if the test is positive there is diffused growth
- growth is not concentration along stab line -> entire medium is red if bacterium is strong positive for motility
- bacteria have the flagella and help them to move into the medium and grow throughout the medium
- e. coli- weak positive
- e. cloacae- strong positive
- k. pneumoniae- negative
46
samples used in restriction enzyme analysis of lambda DNA
- intact lambda phage DNA
- Pstl digest of Lambda phage DNA
- EcoRI digest of Lambda phage DNA
- Hindlll digest of Lambda phage DNA
- Pstl, EcoRI, Hindlll -> restriction enzymes
- lambda phage infects E. coli -> its DNA is about 49,000 base pairs long (e. coli has 4 mil)
47
gel electrophoresis
- slab of gel made up of a substance known as agarose
- on one end of the gel there are well -> where we place the sample that is in a dye
- gel will be immersed in a buffer in chamber
- connect the chamber to a power supply and turn on the power
- DNA fragments are separated based on the size
- 40 minutes -> take out gel
- stain gel blue
- this is done to stain the DNA bands such that the bands are visible
- gel is porous
- DNA fragments migrate through the gel
- electric current push the DNA
- DNA is negatively charged bc of the phosphates
- migrate from negative to postiive
- smaller fragments migrate faster
- each band has many copies of a fragment
48
purpose of gel electrophoresis
- see the DNA
- become familiar with restriction enzymes
- electrophoresis
- analysis of DNA bands
49
applications of gel electrophoresis
- used to identify bacteria and viruses based on the DNA finger printing of these organisms
- genetic screening- electrophoresis is the first step
50
restriction enzymes
- molecular scissors
- recognize specific sequence cut DNA
- make cuts in the DNA
- each enzyme recognizes different sequence
51
restriction enzyme analysis of lambda DNA
- Lambda phage DNA is treated with three restriction enzymes: EcoRI, Hindlll, Pstl
- each restriction enzyme recognizes a different sequence -> makes different cuts
- lanes:
- 1,5- uncut lambda DNA
- 2,6- Pstl cut lambda DNA
- 3,7- EcoRI cut lambda DNA
- 4,8- HindIII cut lambda DNA
- lane 1 and 5 only have one band bc there is one piece of DNA -> no fragments -> also large band (higher up)
- lane 2 and 6- have many bands (10) -> there are many recognition sites for PstI
- lane 3 and 7- 4 DNA bands
- lane 4 and 8- 5 DNA bands
- DNA finger print of lambda phage
- can identify species and bacteria bc they all have unique print
52
staphylococcus: blood agar
- s. aureus and s, epidermidis look the same under the microscope-> both cocci, gram +, and exist in grape like clusters -> cant tell apart
- bc of this we use various selective differential media
- blood agar: differential medium
- tells us if bacteria is hemolytic or non
- staphylococcus aureus- beta hemolytic -> clearing of RBC colonies -> golden brown color
- makes the enzyme beta hemolysin -> breaks down
- staphylococcus epidermidis- gamma hemolytic -> no damage to RBC -> stays red
- no enzyme is present to destroy RBC
53
staphylococcus: mannitol salt agar
- mannitol salt agar:
- mannitol- carbohydrate
- salt- 7.5%
- pH indicator- phenol red
- differentiates and is selective for s. aureus and s. epidermidis
- selective for staphylococcus bc they are facultative halophils -> only they will grow -> salt aspect makes medium selective
- mannitol and the pH indicator makes the medium differential
- mannitol is fermented and acids are released -> pH indicator becomes yellow
- mannitol is fermented and no acids are released -> neutral -> red color
54
streptococcus
- 3 species:
- streptococcus pyogenes
- streptococcus lactis
- streptococcus faecalis (enterococcus faecalis)
- all look the same under microscope: gram +, exist in chains, cocci
- use differential and selective medium to differentiate them
- we use: blood agar 37oC, NaCl agar 37oC, and brain heart infusion agar (BHIA) 45oC
- bacitracin disc (antibiotic disc) is placed in section 1 of the plate for blood agar
55
streptococcus: blood agar
- bacitracin disc is placed in section 1 of streaking for isolation
- incubate at 37oC
- s. pyogenes- beta hemolytic -> clearing of RBCs -> sensitive to bacitracin disc (clearing)
- s. lactis and s. faecalis -> gamma hemolytic (no enzyme or clearing) -> resistant to bacitracin (no clearing around disc)
56
streptococcus: NaCl agar
- 6% NaCl
- 37oC incubation
- only s. faecalis will grow
- s. lactis and s. pyogenes cannot grow
57
streptococcus: BHIA
- most bacteria will grow bc its a nutrient rich medium
- only s. faecalis will grow
- selective factor is the temperature -> 47oC
- s. pyogenes and s. lactis will die at this temperature
58
streptococcus and staphylococcus: catalase test
- used to differentiate staphylococcus and streptococcus
- hydrogen peroxide (H2O2) is converted to oxygen and water via catalase
- place a drop of hydrogen peroxide on a slide
- add bacteria to the drop using aseptic procedure
- if the bacteria is positive for catalase -> bubbles will be seen in the sample
- if bacteria is negative for catalase -> no rxn
- staphylococcus will be positive
- streptococcus will be negative
59
indirect ELISA
- serological test
- done to see if the patients serum has antibodies against a specific antigen
- that tells us if the patient has been exposed to the pathogen
- plastic plates with well in them are used
60
serological test
- antigen antibody rxns
| - based on the fact antibodies are specific for the antigen
61
indirect ELISA test ex.
- we want to find out if pt has antibodies for HIV (positive for HIV)
- get sample from pt (blood)
- attach HIV protein to the wall of the well
- the well is a tiny cup that holds all the reagents
- pts serum is then added
- if the pts serum has pts antibodies for HIV it will attach to HIV protein
- antigen antibody complex is formed
- well is then rinsed to removed any free floating antibodies
- secondary antibodies are then added -> antibodies against human antibodies
- they get the secondary antibodies by injecting human antibodies into animals -> animal immune system makes antibodies against human antibodies
- attach enzyme molecules to the secondary antibodies -> enzyme linked antibodies
- secondary antibodies are antibodies against human antibodies AND enzyme molecules are attached
- secondary antibodies attach to the antibody in the antigen antibody complex
- pt antibody is sandwiched between antibody and secondary
- well is rinsed for any free floating secondary antibodies
- colorless substrate is then added
- if there is enzyme -> converts colorless substrate to blue product -> pt is positive for HIV -> pt serum has antibodies specific for HIV
- secondary antibody was able to attach to antibody antigen complex
- if pt is not blue -> negative for HIV -> no antibodies for HIV antigen
- quick
62
molds
- belong in the kingdom fungi
- eukaryotic
- multicellular
- made up of filamentous structure
- produce asexual spores
63
Rhizopus nirgicans
- common black bread mold
- asexual spores -> sporangiospores
- enclosed in a sac called sporangium
- spores are black
64
penicillium notatum
- produces antibiotic penicillin
- some species are used in cheese production
- asexual spores -> conidiospores
- formed in chains
- not enclosed in sac
- looks like fingers
65
yeast
- kingdom fungi
- unicellular
- reproduce by budding (axesual)
- circular or oval
- ex. saccharomyces cerevisiae
- beneficial yeast -> bread, wine
- ferments sugar to release alcohol and CO2 in absence of O2
66
candida albicans
- yeast
- normal flora
- opportunist
- if one undergoes prolonged antibacterial therapy -> overgrows and causes thrush, vaginitis
- pseudohyphae- cell is elongated (tubular)
67
yogurt
- streptococcus lactis
- lactobacillus bulgaricus
- lactobacillus acidophilus
- live gram positive bacteria
- casein milk protein
- warm the milk and add a spoon of yogurt -> room temp
- inoculate the milk
- ferments lactose and release acids
- acids coagulate -> milk becomes solid
- we call the solid substance yogurt
68
genetic transformation of bacteria and gene regulation
- transform E. coli cells with plasmid
- plasmid has ampicillin resistant gene
- green fluorescent protein (GFP) gene
- natural source of the gene is jelly fish -> aequorea victoria
- GFP gene is attached to the promoter of the gene that codes for enzyme for arabinose (carbohydrate) fermentation
- promoter comes from bacteria
- GFP doesnt have its own promoter
- DNA from different sources are inserted into the plasmid
- genetically engineered plasmid
69
pGLO pasmid
- genetically engineered plasmid
- ampicillin resistant gene- selection marker
- allows us to select the cells that have picked up the plasmid
- has GFP gene -> attached to the arabinose promoter that comes from bacteria cell
70
resistance and regulation of gene expression experiment
- e. coli cells that are sensitive to ampicillin are placed in tube
- pGLO plasmids are added to the cells
- tube is incubated at room temp for 10 mins
- some e. coli will come in contact with plasmid and pick it up and some wont get the chance
- suspension is plated onto different medium
- suspension is plated onto:
- nutrient agar with ampicillin and no arabinose -> GFP gene is not expressed
- nutrient agar with ampicillin and arabinose -> GFP is expressed
- only the cells that picked up the plasmid would grow on both these plates
- ampicillin is selective for plasmid -> resistant cells (cells with pGLO plasmid) grow -> transformation is successful
- colonies with arabinose have GFP -> arabinose activates GFP gene -> RNA polymerase attached to promoter of GFP gene -> mRNA -> translation
- just bc the cell has the gene it doesnt mean it is expressed -> environment plays a major role
Microbiology
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