microbio lab exam 2 Flashcards
types of stains (suck dick and suck flaccid cock)
simple stain: methylene blue, CV, safranin
- if cells cells retain stain, and background white, positive stain
- if cells repel stain, and background colour, negative stain
differential stain: different cells types or cellular components stain differently (Gram stain)
acid fast stain: mycobacterium spp. are gram neutral non-motile rods, and cell walls with mycolic acids make cells impermeable to dyes
- use carbol fuchsin (red stain)
(if retain, cells are acid fast), cannot be removed with acidified organic solevents
- non-acid bacteria lose red with acid-alcohol, colourless, stained with methylene blue counterstain
schaeffer-fulton stain: endospore formation, Bacillus and Clostridium, endospores are impermeable to chemcials
- malachite green (water soluble), spore mother cells are decolourized with water, then counterstained with safranin
- spores bright green
- brownish red vegetative cells
red with green interior are cells containing mature endospores
flagella stain: deposition stain used to increase apparent diameter (stain precipitate accumulates around flagellum)
capsule stain: simple stains will not adhere
- stains bacteria and background more intensely than capsule itself
stains area around capsule with negative acidic stain
- capsule appears as white halo between the cell and darker background
types of media (double dick mass suck cock)
defined media: chemically defined with a known composition of all components to allow bacteria to grow
differential media: distinguish one type of microoragnism from another based on differences in metabolic abolieis of various cells growing on the same medium (can tell by colour)
- EMB: eosin methylene blue agar contains lactose and differentiates between lactose fermenters based on acid production
- e. coli appear as metallic green
- klebsiella aerogenes has fish eye
- these dyes kill gram positive baceria, selective for gram negative abcteira
- lactose broth: not seletive, liquid medium if acid form, pH indicator phenol red turns yellow, gases collected in inverted vial
minimal media: only a few chemically defined ingredients, does not promote growth of a lot of microbes
selective media
complex media (TSA): not chemcially defined
complex and defined media are both
non-selective and non-differential (do not select for certain organisms or use biochemical properties for differentiating bacterial growth)
oxidase test
assesses for presence of cyto C oxidase (essential enzyme for obligate aerobic respiration)
- last cytochrome in the ETC
uses TMPD
TMPD (reduced) + Cyto C (oxidized) -> TMPD (oxidized) + Cyto C (reduced)
reduced TMPD coloourless
oxidized TMPD blue
swab, Gram-negative colony, filter paper, 1drop TMPD
ONPG
tests for the presence of beta-galactosidase (intracelular enzyme present in cells that uselactose as a carbon source)
one of the products of ONPG hydroysis is o-nitrophenol, which is yello
ONPG (colourless) -> (B-galactosidase) -> o-nitrophenol (yellow) + galactose
urease test
tests for presence of urease (intracellular enzyme)
hydrolyzes urea for N source and produced ammonia (pH raises as ammonia produces NH4+ and OH-, phenol red turns purple
urea + 2H2O -> (urease) -> 2NH3 (ammonia) + CO2 + H2O
colourless to purple
inoculate a colony and put in medium
indole test
tests for presence of tryptophanase, metabolizes tryptophan and converts it to indole
pyruvic acid is a C and E source
indole (waste product) complexes with DMACA (blue)
tryptophan + h2O -> (tryptophanase) - > indole, pyruvate, ammonia
gram negative
swab and DMACA
bile-esculin test
tests an organisms ability to grow in the presence of bile and hydrolyze esculin (glycoside)
esculin hydrolysis produces glucose, E, and esculetin, which reacts with ferric salts in bile-esculin agar to produce a dark brown colour (positive)
BAP
some gram psotiive bacteira produce exoenzymes (extracellular enzymes) called hemolysins that lyze RBC (hemolysis)
parafilm
MSA
selective and differential used for the isolation and differntiation of gam potive staphylococci based on tolerance to high salt concetaions and the ability to ferment mannitol (carbohydate)
starch hydrolysis
tests for exoenzyme amylase
starch -> amylase -> glucose + maltose
iodine will react with starch to make dark colour
oxygen requirement test (obscene organisms fuck all mice)
obligate aerobes: MUST utilize oxygen as terminal e- acceptor (grow top of tube) (Pseudomonas and Mycobacterium)
obligate anaerobes: will not grow in the presence of oxygen (bottom of tube) (Bifidobacterium)
facultative anaerobes: preferably use oxygen for ATP creation, but can switch to fermentation in anaerobic conditions -> turbid, grow throguhout length of tube, more dense near surface (Staphylococcus, Bacillus, Listeria, Proteus, Klebsiella, E.coli)
Aerotolerant anaerobes: tolerate and grow in the presence of oxygen but can’t respire, growth is unifrom through medium (Enterococcus, Streptococcus)
Microaerophiles: require lower concentration of oxygen, grow away from the oxic zone. (still at top)Many require a higher carbon dioxide concentration (Campylobacter, Helicobacter pylori)
MAC (MacConkey’s Agar)
has CV and bile salts that inhibit the growth of most gram postitive bacteria
differentiates enteric bacteria based on their ability to ferment lactose and produce acid
lower pH acid production reddish/pink colonies
E. coli is a strong lactose fermenter, matte dark pink colonies with pink halo
klebsiella aerogenes weak lactose germentor, pink colonies no pink halo, gelatinous due to capsule producion
lactose non-fermentors are white colonies
Gram staining
CV
dH20
iodine
dH20
ethanol
dH20
safranin
dH20
Microscope
open iris aperture (left)
4X
condenser (close field iris diaphargm, use condenser focus knob above course and fine knob) octagon
10X
100X
immersion oil
BAP results
gamma: no lysis, no clearing or discolouration
alpha: impartial hemolysis, greenish opaque halos (oxidation of iron in hemoglobin due to exposure to hydrogen peroxide produced by bacteria, RBC not lysed)
beta: lysis of blood cells, clear zone (clearing, surrounding agar becomes translucent due to lysis of RBC)
cell arrangement, make sure to all shape too
e.x. diplococcus
e.x. streptococcus
TSA
trypticase soy agar, complex media
growth: non-selective, non-differential
MSA
mannitol salt agar, gram +ve, high salt, mannitol fermentation
mannitol fermenter: growth: yellow medium
mannitol non-fermenter: growth, red medium
salt intolerant: no growth
BAP
blood agar plat
gram +ve, blood hemolysis
gamma: no clearing
alpha: greening
beta: clearing
EMB
eosin methylene blue
gram -ve, lactose fermentation
rapid lactose fermenter: metallic green/black
slow lactose fermenter: fish-eye, pink brown
non-fermenter: pale, colourless
MAC
MacConkey’s
gram-ve, lactose fermentation, selects for enteric
string lactose fermenter: matte pink with halo
weak lactose fermenterL gelatinous pink, no halo
non-fermenter: pale
MRS
De Man, Rogosa and Sharpe
selects fo Lactobacillus
lactobaccilus growth (white to yellow)
BSM
(Bifidus Selective Medium), selects for bifidobaterium
bifidobacterium growth (light to dark pink/brown)
starch agar
gram -ve, starch hydrolysis, growth; non-selective, differential
PCA (Plate count agar)
complex media, growth: non-selective, non-differential
KF (Kenner - Fecal)
fecal streptococci, E. faecalis grwoth, fermentaiton (yellow)
compound light microscopes have 3 lens systems:
condesner lens: focuses light from light source to specimen
objective lens magnifies image of specimen
ocular lens magnifies the image and inverts it for better viewing
gram positive bacteria
have exposed, thick peptidoglycan layer that absorbs the crystal violet dye
the iodine reacts with CV and attaches it more tightly to peptidoglycan
ethanol removes background stain
safranin counterstains the cell
CV overpowers CV
gram negative bacteria
have a lipopolysaccharide outer membrane and thin peptidoglycan layer
outer membrane acts as a barrier and prevents CV and iodine complex from securely attaching to peptidoglycan layer underneath
ethanol washes away CV from cells
counterstain safranin is applied to visualize bacteria
form (CIPSFR)
circular, irregular, punctiform, spindle, filamentous, rhizoid
surface texture
smooth, rough glistening, dull
elevation
flat, raised, convex, pulvinate, umbonate (pimple)
margin (6) (Fleecu)
entire, undulate, lobate, filamentous, erose (serrated), curled
example explanation for pink gram stain, diplobacilli
the bacterium is gram negative. Cells appear pink due to the presence of an outer membrane that prevents CV-I complex from attaching to the thin-peptidoglycan layer underneath, allowing the ethanol to wash this stain away and cells retain the safranin (pink) counterstain
