microbio lab final
Terms
undefined, object
copy deck
- who does rosie love?
- chetho
- who does chetho love?
- rosie
- who's anniversary is on 3-21-05?
- chetho n rosie
- who does capital cool love?
- conqueress o melee
- who does conqueress o melee love?
- capital cool
- culture medium (definition)
- a solution or suspension of nutrients that provides an appropriate physical and chemical environment to support the growth of microorganisms
- first media used?
-
broths (hay infusion and meat infusions)
-provided way to grow large #'s in single container
-difficult to isolate - how to isolate cells
- -grow on solid surface (agar-does not melt at boiling temp and stuff cant break it down)
- what is defined (synthetic) medium?
- medium in which the exact chemical composition is known
- what is undefined (complex) medium?
- extracts or digests are used, exact composition not known. provides widest array of nutrients, more used
- sterilized ?
- freed from all life forms (use autoclave=chamber using steam under pressure)
- What is the Germ Theory of Disease?
- developed by Robert Koch that diseases are caused by specific microorganisms
- aerosols?
- tiny droplets scattered in the air
- fomites?
-
previously touched objects that harbor microorganisms
(doorknobs, money, sickroom contents) - aseptic technique?
- methods taken to prevent contamination
- what are wet mounts for
- to observe live cells, to observe motility
- example of motile bacterium?
- Pseudomonas aeruginosa
- brownian movement?
- vibration of cells when struck by water
- streaming?
- movement of cells caused by flowing of water or medium used to make slide
- monotrichous?
- one flagellum
- amphitrichous
- many flagella located at opposite ends
- lophotrichous
- many flagella located at one end of the bacteria
- peritrichous
- many flagella located all over (like hair)
-
in a gram stain,
purple is Gram ____ and red is Gram _____ -
purple=gram +
red = gram - (usually rods) - why are gram positive stains purple?
- because they have thick, tightly cross-linked peptidoglycan cell membrane wall and it holds the crystal violet
- why dont gram negative hold the purple?
- they have only a little peptidoglycan in their cell wall so crystal violet can be washed out
- in a gram stain, what is the primary stain?
- crytal violet
- in a gram stain, what is the mordant and its purpose?
- iodine, helps the color to bind further
- what is the alcohol in a gram stain for?
- to decolorize the primary crystal violet stain
- what is the counterstain in a gram stain?
- safranin (red), it just stains whatever is decolorized by the alochol
- what is the acid-fast stain for?
- used to identify members of the genus Mycobacterium (Tuberculosis and leprosy)
- why use the acid-fast?
- because bacteria have mycolic acid in their cell wall so Gram stain can't penetrate
- what color acid-fast positive cells? negative?
-
acid-fast positive=bright pink
negative=blue - what is a capsule?
- slimy layer of polysaccharides, proteins or both that surround the cell, and inhibits phagocytosis
- if a capsule is present what does does the cell look like when stained? example?
- there is a clearing around the cell. example=Kebsiella pneumoniae has capsules
- what are endospores?
- thick coated survival structures that help a cell survive pH changes, heat, cold dessication, lack of nutrients, etc.
- examples of cells that form endospores?
- Bacillus and Clostridium (look like green circle inside red mother)
- selective media?
- has something added that will inhibit the growth of all but the desired type of organism (dyes, salt concentration)
- differential media?
- containes substances that will allow you to visually distinguih colonies with certain characteristics (carbohydrate fermentation or substrate break down)
- differential and selective?
- select for a desired group of organisms and allow you to distinguish which among that group has the metabolic ability you are seeking
-
crystal violet agar
selective or differential?
inhibits? -
selective
inhibits growth of Gram +
only gram - will grow -
blood agar
selective or differential?
what is it for?
what is alpha hemolytic?
beta?
gamma?
organisms used for?
related disease? -
differential
categorizes bacteria by how it breaks down red blood cells
alpha=partially break down rbc's
beta=completely break down
gamma=not broken down at all
--used for Streptococcus
-strep throat -
Mannitol Salt Agar
Selective or differential?
grows which bacteria?
turns yellow if what? example?
remains pink if what? example? -
Both S and D
Staphylococcus
yellow if ferments mannitol, example fermenter = s. aureus
pink if cannot ferment
example of no ferment= s. epidermis -
MacConkey Agar
s or d?
any pH indicator?
inhibits?
shows which metabolic activity?
group of bacteria? -
both s and d
neutral red
inhibits gram + (crystal violet)
if organism can ferment lactose, acid produced reacts with neutral red turns deep red
strong fermenters =dark pink (e. coli)
nonfermenters=colorless
Family Enterobacteriacea -
Eosin-Methylene Blue (EMB)
group of bacteria?
s or d?
tests for what?
results? -
family enterobacteriacea
both s and d
tests for Gram -, and identifies e. coli as strong lactose fermenter
-strong lactose fermenter (e.coli) turn metallic green
lesser fermenters=pinkish purple (e. aerogenes) non fermenters= colorless - psychrophiles?
- cold loving ~5 degrees C fridge
- thermophiles?
- heat loving ~>50 C volcanoes, hot springs
- mesophiles?
- room temp ~20 C
- psychrotrophs?
- mesophile in little colder temp (fridge)
- optimum temp?
- best temp for organism to grow
- buffer?
- included in media to make up for the pH changes caused by the organisms' own waste
- obligate aerobes?
- must have oxygen to survive
- anaerobes?
- do not need oxygen
- aerotolerant anaerobes?
- ignore oxygen with no harm to themselves
- obligate anaerobes?
- killed by oxygen
- facultative anaerobes?
- grow best with oxygen but can still grow without oxygen
- microaerophiles?
- only grow if provided with small amount of oxygen
-
Gas-pak? how does it work?
species that would grow in it? -
uses water activated packet to chemically release hydrogen to bind with the oxygen in the sealed jar and also release carbon dioxide and replace the oxygen
works 100% of except 2 hour it takes to rid jar of oxygen
clostridium (obligate anaerobe) would grow -
starch plate
checks for break down of what?
which shows what enzymatic activity? - break down starch into sugars shows amylase activity (clearing around growth)
- tributyrin
-
ability to break down lipids
lipase activity - skim milk plate
- ability to break down casin into amino acids = caseinase actiity
- nutrient gelatin
- ability to break down gelatin into amino acids =gelatinase activity
-
carbohydrate fermentation
(glucose, lactose, sucrose, mannitol) -
contain phenol red
show ability to break down specific carb
if can break down=acid (pyruvic) produced during fermentation turns media yellow
if gas produced=CO2 in durham tube - IMViC tests for what
- Enterobacteriacea, Gram -, faculative anaerobic rods
- 1% tryptone broth
-
tests for ability to produce indole (tryptophanase activity)
kovac's reagent gives red ring
enterobacteriacea - SIM
- hydrogen sulfide, indole, and motility (used for hydrogen sulfide)
-
2 causes of UTI's
cause of stomach ulcers
what tests for these? -
proteus vulgaris and proteus mirabilis (and e. coli)
helicobacter pylori
urea test=tests oranisms that hydrolyze urea into ammonia and water by urease - what is triple sugar iron agar for?
- identify enterobacteriacea
- litmus milk detects what? 2 purposes?
-
detects metabolic activities that occur in milk
serves as pH indicator and redox indicator - what does catalase do?
- splits hydrogen peroxide into water and oxygen
- what are the 3 ways genetic recombination can occur in prokaryotes?
- conjugation, transformation, and transduction
- conjugation?
- the direct passage of DNA into another cell
- transformation?
- taking up and incorporating naked DNA from the environment
- transduction?
- viruses inject genetic material into the cell
- F+
-
cell that has fertility plasmid (transfer F plasmid via conjugation)
do not require threonine, leucine, and thiamine
resistant to streptomycin - F-
-
cell that does not have fertility plasmid (only has chromosome)
do require t,l,t
resistant to streptomycin - HFr (high frequency recombinant)
-
has fertility plasmid incorporated into chromosome (transfers F plasmid and chromosomal DNA via conjugation)
do not require t,l,t
sensitive to streptomycin -
competent cell?
example of naturally competent? -
cell that has ability to take up naked DNA from environment
example= Bacillus subtilus and Streptococcus pneumoniae - ways to make cell competent?
-
1. electroporation=shock cells with electricity to put holes in membrane
2. CaCl2- treat cells with calcium chloride then treat with cold and heat shock (we used) -
plasmid extraction
why?
how? -
-to prove that the cell actually took up the plasmid and not just a mutation
1. lyse cell using lysozyme (also in tears and sweat), necessary for G (+) cells
2. Disrupt membrane using sodium hydroxide (base) and SDS (detergent). Detergent disrupts lipids of membrane rupturing membrane. High pH of NaOH disrupts base pairing in chromosomal DNA causing denaturation.
3. Plasmid reanneals using potassium acetate. Also, chromosomal DNA, lipids and proteins get trapped in complex (plasmid in supernatant and DNA, lipids, and proteins in pellet at bottom of microcentrifuge tube)
4. Supernatant added to isopropyl alcohol to remove water and precipitate plasmid - Restriction endonuclease
- naturally occurring enzymes in bacterial cells that cut nd destroy foreign DNA (cells protect their own DNA by methylation)
- restriction enzymes
- recognize pallindromic sequences
- gel electrophoresis
- movement of charged fragments through a gel due to an electrical current
- DNA is ____ charged so moves how?
- negatively charged so moves toward positive electrode
- what is tracking dye for?
- added to sample so movement can be visualized
- what is staining dye for?
- added to gel while liquid so DNA can be viewed uner UV
- to check for S. aureus in food what do you do?
- streak on Mannitol salt agar plate
- to check for fungi?
- streak on Sabouraud dextrose agar plate (sab)
- check for spores in bacillus and clostridium?
-
heat and spread on NA plates
(clostridium is anaerobe so put in gas pak) - check for e. coli?
- eosin-methylene blue plate (EMB)
- what do acids and alcohols do in fermentation?
- act as preservatives and prevent growth of other microbes
- what is non-iodized salt used for in saurkraut
- iodine would inhibit microbial growth
- fermentation aerobic or anaerobic?
- anaerobic
- microbial succession?
-
different microbial species acting sequentially
1. leuconostoc and lactobacillus then 2. streptococcus (acid-loving) -
production of wine
used what organism?
preservatives?
why regulate temp in initial stages? -
used saccharomyces cervisiae
sulfites=preservatives
regulation of temp in initial stages essential since fermentation could be stopped before all the sugar has been fermented -
protozoe eukary or prokaryotes?
2 stages in life cycle?
what is the one foundin cat feces and can cross the placenta? -
eukaryotes (have nucleus)
motile trophozoite and non-motile cyst
toxoplasma - fungi, eukary or prokaryotes?
- eukaryotes
- mycoses?
- fungal infection
- thallus
- body of fungus
- hyphae
- filaments of fungus